WO2012052391A1 - Polypeptide ayant l'activité catalytique de jmjd3 - Google Patents

Polypeptide ayant l'activité catalytique de jmjd3 Download PDF

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Publication number
WO2012052391A1
WO2012052391A1 PCT/EP2011/068087 EP2011068087W WO2012052391A1 WO 2012052391 A1 WO2012052391 A1 WO 2012052391A1 EP 2011068087 W EP2011068087 W EP 2011068087W WO 2012052391 A1 WO2012052391 A1 WO 2012052391A1
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sequence
seq
jmjd3
polypeptide
residues
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PCT/EP2011/068087
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English (en)
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Chun-Wa Chung
Julie Mosley
Pamela Joan Thomas
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Glaxo Group Limited
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Publication of WO2012052391A1 publication Critical patent/WO2012052391A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/50—Molecular design, e.g. of drugs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
    • G01N2333/91011—Methyltransferases (general) (2.1.1.)

Definitions

  • the present disclosure provides, inter alia, polypeptides that comprise JMJD3 (jumonji domain containing 3) catalytic activity, nucleic acid sequences encoding the same, and crystal structure and uses thereof. Also provided herein are polypeptides that comprise a novel GATA-like motif, sequences encoding the motif, and uses thereof.
  • Chromatin is the complex combination of DNA and protein that makes up chromosomes. It is found inside the nuclei of eukaryotic cells and is divided between heterochromatin (condensed) and euchromatin (extended) forms.
  • the major components of chromatin are DNA and proteins, including histones.
  • the basic building blocks of chromatin are nucleosomes, each of which is composed of 146 base pairs of DNA wrapped around a histone octamer consisting of 2 copies of each H2A, H2B, H3 and H4.
  • chromatin The functions of chromatin are to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis, and to serve as a mechanism to control expression and DNA replication.
  • Chromatin contains genetic material serving as instructions to direct cell functions. Changes in chromatin structure are regulated by modifications on histone and DNA methylation. Epigenetic mechanisms do not change the DNA sequence but allow the genes to be expressed differently. Epigenetic modifications include numerous mechanisms including DNA methylation and post-translational modification of N-terminal tails of histone proteins such as methylation, acetylation, phosphorylation and ubiquitination.
  • Histone methylation is an abundant epigenetic modification of core histones found in eukaryotic organisms that has been linked to a number of cellular processes including DNA repair, cell cycle progression, cell differentiation and regulation of gene expression. This modification is catalysed by the specific histone methyltransferases (HMTs), lysine methyl transferase and arginine methyl transferase, which introduce methyl groups at lysine (K) or arginine (R) residues respectively.
  • HMTs histone methyltransferases
  • K lysine methyl transferase
  • arginine methyl transferase which introduce methyl groups at lysine (K) or arginine (R) residues respectively.
  • histone methylation has been found to occur at six major sites, including histone H3 lysine 4 (H3K4), H3K9, H3K27, H3K36, H3K79 and H4K20.
  • lysine residue can be methylated to different degrees to include mono-, di- or trimethyl moieties, which may have different functional consequences.
  • lysine methylation at H3K4, H3K36 and H3K39 is associated with regions of transcriptionally active chromatin, whereas methylation at H3K9, H3K27 and H4K20 is associated with transcriptionally silenced regions (Martin C. and Zhang Y., Nature Rev. Mol. Cell Biol. 2005, 6, 838-849).
  • H3K9 promoter methylation is considered a repressive mark for euchromatic genes (Nielsen et al., Nature 2001 ,412, 561 -565; Shi et al., Nature 2003, 422, 735-738) and is also one of the landmark modifications associated with heterochromatin (Nakayama et al., Science 2001 , 292, 1 10-1 13) but some studies have also identified association of H3K9 trimethylation (H3K9me3) with actively transcribed genes (Vakoc C. et al, Mol. Cell 2005, 19. 381 -391).
  • Histone methylation appears thus to be regulated by a complex network that involves a large number of site-specific methylases, demethylases, and methyl recognition proteins, which play an important role in controlling the expression of genetic information through transcriptional changes and chromatin structure alterations. Since levels of lysine methylation are known to change during processes such as transcriptional regulation, it was proposed that specific enzymatic activity might remove the methyl groups (Bannister et ai, 2002 Cell 109, 801 -806).
  • the Jumonji protein is the founding member of a group of proteins characterised by a novel structural motif, the JmjC domain.
  • This is an extensive group of demethylase enzymes which can be defined into several families according to sequence similarity within the JmjC domain and the presence of other domains in the full length protein.
  • the JmjC domain of several members of this family has been shown to possess lysine demethylation activity, which is dependent on iron (Fe (II)) and a- ketoglutarate as co-factors (Klose RJ et ai, Nat Rev Genet. 2006 Sep; 7(9); 715-27).
  • the JmjC-domain-containing histone demethylases JHDMs
  • JHDMs can remove all three histone lysine-methylation states.
  • JMJD3 (KDM6B) is one of the approximately 30 JmjC family members found in humans, and functions as a specific demethylase of lysine 27 of histone H3 (H3K27). JMJD3 can demethylate both the tri- and dimethylated H3K27-repressive histone marks, thereby facilitating gene transcription. This was first demonstrated in C. Elegans embryogenesis, where JMJD3 was shown to regulate gonadal development through modulation of HOX gene expression (Agger K et al, Nature 2007 Oct;
  • JMJD3 has been demonstrated to regulate the differentiation state of the epidermis (Sen GL et al, Genes Dev. 2008 Jul; 22; 1865-1870) and to activate the tumour suppressor, INK4A-Arf, in response stress induced signals (Agger K et al, Genes Dev. 2009 Apr; 23; 1 171 -1 176).
  • JMJD3 also appears to be involved in more acute, externally-driven, inflammatory processes.
  • JMJD3 is rapidly induced through an NF-kB- dependent mechanism in response to bacterial products and inflammatory stimuli (De Santa F et al, Cell 2007 Sept; 130; 1083-1094).
  • depletion experiments in these cells have demonstrated that JMJD3 participates directly in the inflammatory transcriptional response, although it remains unclear whether this is achieved through demethylation of H3K27me3 at target gene promoters (De Santa F et al, EMBO J. 2009 Sept; 28; 3341 -3352).
  • This disclosure also relates to (i) crystals of the polypeptides that contain JMJD3 catalytic activity (e.g., human H3K27 demethylase activity) which are suitable for structure determination of the polypeptide using X-ray crystallography and (ii) computational methods using the structural coordinates of the polypeptide to screen for and design compounds that modulate, e.g., decrease (e.g., inhibit) or increase, JMJD3 catalytic activity (e.g., human H3K27 demethylase activity) of the polypeptide.
  • JMJD3 catalytic activity e.g., human H3K27 demethylase activity
  • this disclosure relates to methods for identifying compounds that bind to the polypeptide and/or modulate JMJD3 catalytic activity (e.g., human H3K27 demethylase activity), e.g., in a high throughput setting.
  • JMJD3 catalytic activity e.g., human H3K27 demethylase activity
  • a novel GATA-like Zn++ binding domain and polypeptides containing the same are also provided herein.
  • this disclosure provides methods for identifying compounds that bind to the polypeptide and/or modulate Zn++ binding by the polypeptide, e.g., in a high throughput setting.
  • the disclosure relates to a polypeptide (e.g., an isolated polypeptide), wherein the polypeptide comprises a JMJD3 sequence, wherein the polypeptide possesses a catalytic activity of JMJD3 (e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)); and wherein the JMJD3 sequence consists of:
  • the JMJD3 sequence can consist of a sequence at least 85% identical to residues 253-1682 of SEQ ID NO:1 ; 281 -1682 of SEQ ID NO:1 ; residues 400-1682 of SEQ ID NO:1 ; residues 600-1682 of SEQ ID NO:1 ; residues 1000-1682 of SEQ ID NO:1 ; residues 1 100-1682 of SEQ ID NO:1 ; residues 1 140- 1682 of SEQ ID NO:1 ; and so forth.
  • the JMJD3 sequence can consist of a sequence at least 85% identical to residues 247-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 253-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 281 -1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 400-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 600-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 1000-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 1 100-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 1 140-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); and so forth.
  • a sequence that is at least 85% identical to a sequence in (i)-(iv) can be, e.g., at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the recited sequence.
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site. In some embodiments, the polypeptide is in soluble form.
  • the polypeptide is in crystal form. In some embodiments, the crystal has the structure deposited in Protein Data Bank (PDB) ID: 2XUE.
  • the polypeptide is suitable for crystallization, i.e., preferably the polypeptide fragment is crystallizable.
  • the crystals obtainable from the polypeptide described herein are suitable for structure determination of the polypeptide using X-ray crystallography.
  • the crystals are radiation stable enough to permit more than 85% diffraction data completeness at resolution of preferably 3.5 A or better to be collected upon exposure to monochromatic X-rays.
  • the polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • PDCA pyridine 2,4 dicarboxylic acid
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • the disclosure relates to an isolated nucleic acid encoding an isolated polypeptide described herein.
  • the disclosure relates to a recombinant vector comprising the isolated nucleic acid.
  • the disclosure relates to a recombinant host cell comprising the isolated nucleic acid or the recombinant vector.
  • the disclosure relates to a method for making a polypeptide comprising a JMJD3 sequence (e.g., a polypeptide described herein), the method comprising culturing the recombinant host cell under conditions permitting expression of the polypeptide from the nucleic acid or recombinant vector.
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.).
  • the disclosure relates to a method for making a polypeptide comprising a JMJD3 sequence (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c-myc tag, etc.).
  • the disclosure relates to a nucleic acid (e.g., an isolated nucleic acid), wherein the nucleic acid comprises a JMJD3 nucleic acid sequence, wherein the encoded polypeptide possesses a catalytic activity of JMJD3 (e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)); and wherein the JMJD3 nucleic acid sequence consists of:
  • nucleic acid e.g., with respect to (iii) and (iv), is in the proper reading frame to encode a polypeptide that possesses a catalytic activity of JMJD3 (e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)).
  • the JMJD3 nucleic acid sequence consists of (i) a sequence at least 85% identical to nucleotides 3797-5422 of SEQ ID NO:2. In some embodiments, the JMJD3 nucleic acid sequence consists of a sequence 100% identical to nucleotides 3797-5422 of SEQ ID NO:2.
  • the JMJD3 nucleic acid sequence consists of (ii) a sequence at least 85% identical to nucleotides 3797-5422 with a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2. In some embodiments, the JMJD3 nucleic acid sequence consists of a sequence 100% identical to nucleotides 3797-5422 with a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the JMJD3 nucleic acid sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of nucleotides 1 133 to 3796 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2. In some embodiments, the JMJD3 nucleic acid sequence consists of a sequence 100% identical to a sequence that initiates at any of nucleotides 1 133 to 3796 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2.
  • the JMJD3 nucleic acid sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of nucleotides 1 1 15 to 3796 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2, wherein the sequence comprises a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the JMJD3 nucleic acid sequence consists of a sequence 100% identical to a sequence that initiates at any of nucleotides 1 1 15 to 3796 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2, wherein the sequence comprises a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the nucleic acid comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 nucleic acid sequence). In some embodiments, the nucleic acid comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 nucleic acid sequence) at 5' or 3' end.
  • the heterologous sequence can include a sequence that encodes a peptide used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a sequence encoding a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the nucleic acid comprises a sequence encoding a 6- His tag, e.g., at its 3' end.
  • the nucleic acid comprises a sequence encoding a FLAG tag, e.g., at its 5' end. In some embodiments, the nucleic acid comprises a sequence encoding a 6-His tag and a FLAG tag, e.g., at its 5' or 3' end. In some embodiments, the nucleic acid comprises a sequence encoding peptides used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the encoded polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • the encoded polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • the disclosure relates to a recombinant vector comprising the isolated nucleic acid. In another aspect, the disclosure relates to a recombinant host cell comprising the isolated nucleic acid or the recombinant vector.
  • the disclosure relates to a method for making a polypeptide comprising a JMJD3 sequence (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.).
  • a tag e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.
  • the disclosure relates to a method for making a polypeptide comprising a JMJD3 sequence (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c-myc tag, etc.).
  • the disclosure relates to a method for identifying a compound which modulates (e.g., increases or decreases) JMJD3 catalytic activity (e.g., H3K27 demethylase activity), the method comprising:
  • JMJD3 e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)); and wherein the JMJD3 sequence consists of:
  • test compound by a method selected from the group consisting of:
  • the structure coordinates are deposited in Protein Data Bank (PDB) ID: 2XUE.
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence). In some embodiments, the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus.
  • the polypeptide comprises a FLAG tag, e.g., at its amino terminus.
  • the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus.
  • the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the method further comprises contacting the test compound with the polypeptide or a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide) (e.g., and with a JMJD3 substrate e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27) (e.g., a peptide with the sequence ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (e.g., that is methylated, e.g., di- or tri- methylated, at the residue corresponding to K27); or histone 3 (e.g., that is methylated, e.g., di- or tri- methylated, at K27)); the substrate can
  • test compound modulates JMJD3 catalytic activity of the polypeptide (e.g., as compared to a control).
  • a co-factor such as Fe(ll) and/or 20G, can optionally be included (or may, e.g., be naturally occurring in the host cell).
  • the control can be the amount (e.g., percentage) of JMJD3 catalytic activity in the absence of a test compound under identical conditions.
  • a decrease in JMJD3 catalytic activity indicates that the test compound decreases (e.g., inhibits) JMJD3 catalytic activity.
  • an increase in the JMJD3 catalytic activity indicates that the test compound increases (e.g., promotes) JMJD3 catalytic activity.
  • the JMJD3 catalytic activity is determined by measuring demethylase activity (e.g., demethylation of di- or tri-methylated H3K27; e.g., the amount of mono-, di-, or tri-methylated H3K27).
  • demethylase activity e.g., demethylation of di- or tri-methylated H3K27; e.g., the amount of mono-, di-, or tri-methylated H3K27.
  • the demethylase activity is determined by MALDI-TOF, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP).
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule.
  • the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • the disclosure relates to a method for identifying compounds which modulate (e.g., increase or decrease) JMJD3 catalytic activity (e.g., H3K27 demethylase activity), the method comprising:
  • a polypeptide e.g., an isolated polypeptide
  • the polypeptide comprises a JMJD3 sequence
  • the polypeptide possesses a catalytic activity of JMJD3
  • the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)
  • the JMJD3 sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide)
  • the substrate can be naturally occurring in the host cell; and
  • test compound e.g., H3K27 demethylase activity
  • a co-factor such as Fe(ll) and/or 20G, can optionally be included (or may, e.g., be naturally occurring in the host cell).
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in JMJD3 catalytic activity indicates that the test compound decreases (e.g., inhibits) JMJD3 catalytic activity.
  • an increase in the JMJD3 catalytic activity indicates that the test compound increases (e.g., promotes) JMJD3 catalytic activity.
  • the JMJD3 catalytic activity is determined by measuring demethylase activity (e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27).
  • demethylase activity e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27.
  • the demethylase activity is determined by MALDI-TOF, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP).
  • MALDI-TOF enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • Western blotting immunofluorescence
  • immunohistochemistry immunoprecipitation
  • immunoprecipitation chromatin immunoprecipitation
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule.
  • the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • a polypeptide e.g., an isolated polypeptide
  • the polypeptide comprises a JMJD3 sequence
  • the polypeptide possesses a catalytic activity of JMJD3
  • the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)
  • the JMJD3 sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide),
  • a JMJD3 substrate e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the residue corresponding to K27) (e.g., a peptide with the sequence ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (e.g., that is methylated, e.g., di- or tri- methylated, at the residue corresponding to K27); or histone 3 (e.g., that is methylated, e.g., di- or tri- methylated, at K27)); the substrate can be naturally occurring in the host cell)
  • test compound modulates JMJD3 catalytic activity, e.g., as compared to a control.
  • a co-factor such as Fe(ll) and/or 20G, can optionally be included (or may, e.g., be naturally occurring in the host cell).
  • the control can be the amount (e.g., percentage) of JMJD3 catalytic activity in the absence of a test compound under identical conditions.
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence). In some embodiments, the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus.
  • the polypeptide comprises a FLAG tag, e.g., at its amino terminus.
  • the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus.
  • the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • PDCA pyridine 2,4 dicarboxylic acid
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in JMJD3 catalytic activity indicates that the test compound decreases (e.g., inhibits) JMJD3 catalytic activity.
  • an increase in the JMJD3 catalytic activity indicates that the test compound increases (e.g., promotes) JMJD3 catalytic activity.
  • the JMJD3 catalytic activity is determined by measuring demethylase activity (e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27).
  • demethylase activity e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27.
  • the demethylase activity is determined by MALDI-TOF, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP).
  • MALDI-TOF enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • Western blotting immunofluorescence
  • immunohistochemistry immunoprecipitation
  • immunoprecipitation chromatin immunoprecipitation
  • test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule.
  • the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • test compound comprises a chemical compound.
  • a method of evaluating a test compound comprising:
  • a polypeptide e.g., an isolated polypeptide
  • the polypeptide comprises a JMJD3 sequence
  • the polypeptide possesses a catalytic activity of JMJD3
  • the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)
  • the JMJD3 sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide);
  • the substrate can be naturally occurring in the host cell); and
  • the test compound modulates JMJD3 catalytic activity (e.g., as compared to a control).
  • a co-factor such as Fe(ll) and/or 20G, can optionally be included (or may, e.g., be naturally occurring in the host cell).
  • the control can be the amount (e.g., percentage) of JMJD3 catalytic activity in the absence of a test compound under identical conditions.
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 140 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence). In some embodiments, the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus.
  • the polypeptide comprises a FLAG tag, e.g., at its amino terminus.
  • the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus.
  • the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • PDCA pyridine 2,4 dicarboxylic acid
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in JMJD3 catalytic activity indicates that the test compound decreases (e.g., inhibits) JMJD3 catalytic activity.
  • an increase in the JMJD3 catalytic activity indicates that the test compound increases (e.g., promotes) JMJD3 catalytic activity.
  • the JMJD3 catalytic activity is determined by measuring demethylase activity (e.g., demethylation of di- or tri-methylated H3K27; e.g., the amount of mono-, di-, or tri-methylated H3K27).
  • the demethylase activity is determined by MALDI-TOF, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP).
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule. In some embodiments, the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • the disclosure relates to a polypeptide (e.g., an isolated polypeptide), wherein the polypeptide comprises a JMJD3 sequence, wherein the polypeptide possesses a catalytic activity of JMJD3 (e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)); and wherein the JMJD3 sequence consists of:
  • the JMJD3 sequence can consist of a sequence at least 85% identical to residues 1 178-1682 of SEQ ID NO:1 ; and so forth.
  • the JMJD3 sequence can consist of a sequence at least 85% identical to residues 1 178-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); and so forth.
  • a sequence that is at least 85% identical to a sequence in (i)-(iv) can be, e.g., at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the recited sequence.
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 181 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 180 of SEQ ID NO:1 (e.g., at residue 1 178) and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide is in soluble form.
  • the polypeptide is in crystal form. In some embodiments, the crystal has the structure deposited in Protein Data Bank (PDB) ID: 2XUE.
  • PDB Protein Data Bank
  • the polypeptide is suitable for crystallization, i.e., preferably the polypeptide fragment is crystallizable.
  • the crystals obtainable from the polypeptide described herein are suitable for structure determination of the polypeptide using X-ray crystallography.
  • the crystals are radiation stable enough to permit more than 85% diffraction data completeness at resolution of preferably 3.5 A or better to be collected upon exposure to monochromatic X-rays.
  • the polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • the disclosure relates to an isolated nucleic acid encoding an isolated polypeptide described herein.
  • the disclosure relates to a recombinant vector comprising the isolated nucleic acid. In another aspect, the disclosure relates to a recombinant host cell comprising the isolated nucleic acid or the recombinant vector.
  • the disclosure relates to a method for making a polypeptide comprising a JMJD3 sequence (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.).
  • a tag e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.
  • the disclosure relates to a method for making a polypeptide comprising a JMJD3 sequence (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c-myc tag, etc.).
  • the disclosure relates to a nucleic acid (e.g., an isolated nucleic acid), wherein the nucleic acid comprises a JMJD3 nucleic acid sequence, wherein the encoded polypeptide possesses a catalytic activity of JMJD3 (e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)); and wherein the JMJD3 nucleic acid sequence consists of: (i) a sequence at least 85% identical to nucleotides 3917-5422 of SEQ ID NO:2;
  • nucleic acid e.g., with respect to (iii) and (iv), is in the proper reading frame to encode a polypeptide that possesses a catalytic activity of JMJD3 (e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)).
  • the JMJD3 nucleic acid sequence consists of (i) a sequence at least 85% identical to nucleotides 3917-5422 of SEQ ID NO:2. In some embodiments, the JMJD3 nucleic acid sequence consists of a sequence 100% identical to nucleotides 3917-5422 of SEQ ID NO:2.
  • the JMJD3 nucleic acid sequence consists of (ii) a sequence at least 85% identical to nucleotides 3917-5422 with a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2. In some embodiments, the JMJD3 nucleic acid sequence consists of a sequence 100% identical to nucleotides 3917-5422 with a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the JMJD3 nucleic acid sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of nucleotides 1 133 to 3916 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2. In some embodiments, the JMJD3 nucleic acid sequence consists of a sequence 100% identical to a sequence that initiates at any of nucleotides 1 133 to 3916 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2.
  • the JMJD3 nucleic acid sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of nucleotides 1 1 15 to 3916 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2, wherein the sequence comprises a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the JMJD3 nucleic acid sequence consists of a sequence 100% identical to a sequence that initiates at any of nucleotides 1 1 15 to 3916 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2, wherein the sequence comprises a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the nucleic acid comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 nucleic acid sequence). In some embodiments, the nucleic acid comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 nucleic acid sequence) at 5' or 3' end.
  • the heterologous sequence can include a sequence that encodes a peptide used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a sequence encoding a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the nucleic acid comprises a sequence encoding a 6- His tag, e.g., at its 3' end.
  • the nucleic acid comprises a sequence encoding a FLAG tag, e.g., at its 5' end. In some embodiments, the nucleic acid comprises a sequence encoding a 6-His tag and a FLAG tag, e.g., at its 5' or 3' end. In some embodiments, the nucleic acid comprises a sequence encoding peptides used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the encoded polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • PDCA pyridine 2,4 dicarboxylic acid
  • the encoded polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • the disclosure relates to a recombinant vector comprising the isolated nucleic acid.
  • the disclosure relates to a recombinant host cell comprising the isolated nucleic acid or the recombinant vector.
  • the disclosure relates to a method for making a polypeptide comprising a JMJD3 sequence (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.).
  • a tag e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.
  • the disclosure relates to a method for making a polypeptide comprising a JMJD3 sequence (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c-myc tag, etc.).
  • the disclosure relates to a method for identifying a compound which modulates (e.g., increases or decreases) JMJD3 catalytic activity (e.g., H3K27 demethylase activity), the method comprising:
  • JMJD3 e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)); and wherein the JMJD3 sequence consists of:
  • test compound by a method selected from the group consisting of:
  • the structure coordinates are deposited in Protein Data Bank (PDB) ID: 2XUE.
  • PDB Protein Data Bank
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 181 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence). In some embodiments, the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus.
  • the polypeptide comprises a FLAG tag, e.g., at its amino terminus.
  • the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus.
  • the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the method further comprises contacting the test compound with the polypeptide or a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide) (e.g., and with a JMJD3 substrate e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27) (e.g., a peptide with the sequence ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (e.g., that is methylated, e.g., di- or tri- methylated, at the residue corresponding to K27); or histone 3 (e.g., that is methylated, e.g., di- or tri- methylated, at K27)); the substrate can
  • test compound modulates JMJD3 catalytic activity of the polypeptide (e.g., as compared to a control).
  • a co-factor such as Fe(ll) and/or 20G, can optionally be included (or may, e.g., be naturally occurring in the host cell).
  • the control can be the amount (e.g., percentage) of JMJD3 catalytic activity in the absence of a test compound under identical conditions.
  • a decrease in JMJD3 catalytic activity indicates that the test compound decreases (e.g., inhibits) JMJD3 catalytic activity.
  • an increase in the JMJD3 catalytic activity indicates that the test compound increases (e.g., promotes) JMJD3 catalytic activity.
  • the JMJD3 catalytic activity is determined by measuring demethylase activity (e.g., demethylation of di- or tri-methylated H3K27; e.g., the amount of mono-, di-, or tri-methylated H3K27).
  • demethylase activity e.g., demethylation of di- or tri-methylated H3K27; e.g., the amount of mono-, di-, or tri-methylated H3K27.
  • the demethylase activity is determined by MALDI-TOF, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP).
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule. In some embodiments, the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • the disclosure relates to a method for identifying compounds which modulate (e.g., increase or decrease) JMJD3 catalytic activity (e.g., H3K27 demethylase activity), the method comprising:
  • a polypeptide e.g., an isolated polypeptide
  • the polypeptide comprises a JMJD3 sequence
  • the polypeptide possesses a catalytic activity of JMJD3
  • the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)
  • the JMJD3 sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide)
  • the substrate can be naturally occurring in the host cell; and
  • test compound e.g., H3K27 demethylase activity
  • a co-factor such as Fe(ll) and/or 20G, can optionally be included (or may, e.g., be naturally occurring in the host cell).
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 181 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence). In some embodiments, the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus.
  • the polypeptide comprises a FLAG tag, e.g., at its amino terminus.
  • the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus.
  • the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in JMJD3 catalytic activity indicates that the test compound decreases (e.g., inhibits) JMJD3 catalytic activity.
  • an increase in the JMJD3 catalytic activity indicates that the test compound increases (e.g., promotes) JMJD3 catalytic activity.
  • the JMJD3 catalytic activity is determined by measuring demethylase activity (e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27).
  • demethylase activity e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27.
  • the demethylase activity is determined by MALDI-TOF, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP).
  • MALDI-TOF enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • Western blotting immunofluorescence
  • immunohistochemistry immunoprecipitation
  • immunoprecipitation chromatin immunoprecipitation
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule.
  • the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • a polypeptide e.g., an isolated polypeptide
  • the polypeptide comprises a JMJD3 sequence
  • the polypeptide possesses a catalytic activity of JMJD3
  • the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)
  • the JMJD3 sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide),
  • a JMJD3 substrate e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the residue corresponding to K27) (e.g., a peptide with the sequence ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (e.g., that is methylated, e.g., di- or tri- methylated, at the residue corresponding to K27); or histone 3 (e.g., that is methylated, e.g., di- or tri- methylated, at K27)); the substrate can be naturally occurring in the host cell)
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 181 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • PDCA pyridine 2,4 dicarboxylic acid
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in JMJD3 catalytic activity indicates that the test compound decreases (e.g., inhibits) JMJD3 catalytic activity.
  • an increase in the JMJD3 catalytic activity indicates that the test compound increases (e.g., promotes) JMJD3 catalytic activity.
  • the JMJD3 catalytic activity is determined by measuring demethylase activity (e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27).
  • demethylase activity e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27.
  • the demethylase activity is determined by MALDI-TOF, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP).
  • MALDI-TOF enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • Western blotting immunofluorescence
  • immunohistochemistry immunoprecipitation
  • immunoprecipitation chromatin immunoprecipitation
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule.
  • the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • a method of evaluating a test compound comprising:
  • a polypeptide e.g., an isolated polypeptide
  • the polypeptide comprises a JMJD3 sequence
  • the polypeptide possesses a catalytic activity of JMJD3
  • the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)
  • the JMJD3 sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide); (e.g., and with a JMJD3 substrate (e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the residue corresponding to K27) (e.g., a peptide with the sequence ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (e.g., that is methylated, e.g., di- or tri- methylated, at the residue corresponding to K27); or histone 3 (e.g., that is methylated, e.g., di- or tri- methylated, at K27)); the substrate can be naturally occurring in the host cell); and
  • test compound modulates JMJD3 catalytic activity (e.g., as compared to a control).
  • a co-factor such as Fe(ll) and/or 20G, can optionally be included (or may, e.g., be naturally occurring in the host cell).
  • the control can be the amount (e.g., percentage) of JMJD3 catalytic activity in the absence of a test compound under identical conditions.
  • the JMJD3 sequence consists of (i) a sequence at least 85% identical to residues 1 181 -1682 of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (ii) a sequence at least 85% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the JMJD3 sequence consists of a sequence 100% identical to residues 1 181 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iii) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the JMJD3 sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1 180 (e.g., at residue 1 178) of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the JMJD3 sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the JMJD3 sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide is not inhibited by pyridine 2,4 dicarboxylic acid (PDCA).
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in JMJD3 catalytic activity indicates that the test compound decreases (e.g., inhibits) JMJD3 catalytic activity.
  • an increase in the JMJD3 catalytic activity indicates that the test compound increases (e.g., promotes) JMJD3 catalytic activity.
  • the JMJD3 catalytic activity is determined by measuring demethylase activity (e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27).
  • demethylase activity e.g., demethylation of di- or tri-methylated H3K27; e.g. , the amount of mono-, di-, or tri-methylated H3K27.
  • the demethylase activity is determined by MALDI-TOF, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP).
  • MALDI-TOF enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • Western blotting immunofluorescence
  • immunohistochemistry immunoprecipitation
  • immunoprecipitation chromatin immunoprecipitation
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule.
  • the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • the disclosure relates to a polypeptide (e.g., an isolated polypeptide), wherein the polypeptide comprises a GATA-like domain sequence, wherein the polypeptide is capable of binding Zn++; and wherein the GATA-like domain sequence consists of:
  • the JMJD3 sequence can consist of a sequence at least 85% identical to residues 253-1682 of SEQ ID NO:1 ; 281 -1682 of SEQ ID NO:1 ; residues 400-1682 of SEQ ID NO:1 ; residues 600-1682 of SEQ ID NO:1 ; residues 1000-1682 of SEQ ID NO:1 ; residues 1 100-1682 of SEQ ID NO:1 ; residues 1 140- 1682 of SEQ ID NO:1 ; residues 1300-1682 of SEQ ID NO:1 ; residues 1400-1682 of SEQ ID NO:1 ; residues 1500-1682 of SEQ ID NO:1 ; residues 1540-1682 of SEQ ID NO:1 ; and so forth.
  • the JMJD3 sequence can consist of a sequence at least 85% identical to residues 247-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 253-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 281 -1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 400-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 600-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 1000-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 1 100-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 1 140-1682 of SEQ ID NO:1 (with a deletion of residues 1637-1675); residues 1300-1682 of SEQ ID NO:1 (with a deletion of residues)
  • a sequence that is at least 85% identical to a sequence in (i)-(v) can be, e.g., at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the recited sequence.
  • the GATA-like domain sequence consists of (i) a sequence at least 85% identical to residues 1543-1626 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1543-1626 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of (iii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (v) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1.
  • the GATA-like domain sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1.
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the GATA-like domain sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the GATA-like domain sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • a cleavage site e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site. In some embodiments, the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • the polypeptide is in soluble form. In some embodiments, the polypeptide is in crystal form. In some embodiments, the crystal has the structure deposited in Protein Data Bank (PDB) ID: 2XUE.
  • the polypeptide is suitable for crystallization, i.e., preferably the polypeptide fragment is crystallizable.
  • the crystals obtainable from the polypeptide described herein are suitable for structure determination of the polypeptide using X-ray crystallography.
  • the crystals are radiation stable enough to permit more than 85% diffraction data completeness at resolution of preferably 3.5 A or better to be collected upon exposure to monochromatic X-rays.
  • the disclosure relates to an isolated nucleic acid encoding an isolated polypeptide described herein.
  • the disclosure relates to a recombinant vector comprising the isolated nucleic acid.
  • the disclosure relates to a recombinant host cell comprising the isolated nucleic acid or the recombinant vector.
  • the disclosure relates to a method for making a polypeptide comprising a GATA- like domain (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.).
  • a tag e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.
  • the disclosure relates to a method for making a polypeptide comprising a GATA- like domain (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c-myc tag, etc.).
  • the disclosure relates to the use of a polypeptide (e.g., isolated polypeptide) that contains the GATA-like domain described herein as a dominant negative protein to decrease (e.g., inhibit) JMJD3 activity.
  • the polypeptide can comprise the GATA-like domain (e.g., residues 1543- 1626 of SEQ ID NO:1 , (or a sequence at least 85% identical thereto)) with an additional heterologous sequence, e.g., of about 10, about 20, about 30, about 40, or about 50 additional residues, e.g., at the amino or carboxy terminus of the GATA-like domain.
  • the GATA-like domain is capable of binding Zn++.
  • the disclosure relates to a nucleic acid (e.g., an isolated nucleic acid), wherein the nucleic acid comprises a GATA-like domain nucleic acid sequence, wherein the encoded polypeptide is capable of binding Zn++; and wherein the GATA-like domain nucleic acid sequence consists of:
  • nucleic acid e.g., with respect to (iv) and (v), is in the proper reading frame to encode a polypeptide that possesses a catalytic activity of JMJD3 (e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)).
  • the GATA-like domain nucleic acid sequence consists of (i) a sequence at least 85% identical to nucleotides 5003-5254 of SEQ ID NO:2. In some embodiments, the GATA-like domain nucleic acid sequence consists of a sequence 100% identical to nucleotides 5003-5254 of SEQ ID NO:2.
  • the GATA-like domain nucleic acid sequence consists of (ii) a sequence at least 85% identical to nucleotides 3797-5422 of SEQ ID NO:2. In some embodiments, the GATA-like domain nucleic acid sequence consists of a sequence 100% identical to nucleotides 3797-5422 of SEQ ID NO:2.
  • the GATA-like domain nucleic acid sequence consists of (iii) a sequence at least 85% identical to nucleotides 3797-5422 with a deletion of nucleotides 5285-5401 (del 5285- 5401) of SEQ ID NO:2. In some embodiments, the GATA-like domain nucleic acid sequence consists of a sequence 100% identical to nucleotides 3797-5422 with a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the GATA-like domain nucleic acid sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of nucleotides 1 133 to 5003 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2. In some embodiments, the GATA-like domain nucleic acid sequence consists of a sequence 100% identical to a sequence that initiates at any of nucleotides 1 133 to 5003 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2.
  • the GATA-like domain nucleic acid sequence consists of (v) a sequence at least 85% identical to a sequence that initiates at any of positions 1 1 15 to 5003 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2, wherein the sequence comprises a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the GATA-like domain nucleic acid sequence consists of a sequence 100% identical to a sequence that initiates at any of positions 1 1 15 to 5003 of SEQ ID NO:2 and terminates at nucleotide 5422 of SEQ ID NO:2, wherein the sequence comprises a deletion of nucleotides 5285-5401 (del 5285-5401) of SEQ ID NO:2.
  • the nucleic acid comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the GATA-like domain nucleic acid sequence).
  • the nucleic acid comprises a heterologous sequence (e.g., non naturally occurring with the GATA-like domain nucleic acid sequence) at 5' or 3' end.
  • the heterologous sequence can include a sequence that encodes a peptide used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a sequence encoding a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • a cleavage site e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the nucleic acid comprises a sequence encoding a 6-His tag, e.g., at its 3' end. In some embodiments, the nucleic acid comprises a sequence encoding a FLAG tag, e.g., at its 5' end. In some embodiments, the nucleic acid comprises a sequence encoding a 6-His tag and a FLAG tag, e.g., at its 5' or 3' end. In some embodiments, the nucleic acid comprises a sequence encoding peptides used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the encoded polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • the disclosure relates to a recombinant vector comprising the isolated nucleic acid.
  • the disclosure relates to a recombinant host cell comprising the isolated nucleic acid or the recombinant vector.
  • the disclosure relates to a method for making a polypeptide comprising a GATA- like domain (e.g., a polypeptide described herein), the method comprising
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.).
  • a tag e.g., poly-His (e.g., 6-His), Flag, or c- myc tag, etc.
  • the disclosure relates to a method for making a polypeptide comprising a GATA- like domain (e.g., a polypeptide described herein), the method comprising culturing a recombinant host cell containing a nucleic acid or recombinant vector that encodes a polypeptide that comprises a JMJD3 sequence under conditions permitting expression of the polypeptide from the nucleic acid or recombinant vector.
  • the method can further comprise isolating the polypeptide from the host cell.
  • the polypeptide can be isolated, for example, from a cell lysate.
  • the isolating can be performed, e.g., by affinity chromatography (e.g., utilizing a tag (e.g., poly-His (e.g., 6-His), Flag, or c-myc tag, etc.).
  • a tag e.g., poly-His (e.g., 6-His), Flag, or c-myc tag, etc.
  • the disclosure relates to an antibody directed against a polypeptide described herein (e.g., an epitope thereof).
  • the antibody can bind to the GATA-like domain.
  • the disclosure relates to a method for identifying compounds which modulate (e.g., increase or decrease) GATA-like domain Zn++ binding, the method comprising:
  • polypeptide e.g., an isolated polypeptide
  • the polypeptide comprises a GATA-like domain sequence, wherein the polypeptide is capable of binding Zn++; and wherein the GATA-like domain sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide)
  • the Zn++ can be naturally occurring in the host cell
  • the GATA-like domain sequence consists of (i) a sequence at least 85% identical to residues 1543-1626 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1543-1626 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of (iii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (v) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1.
  • the GATA-like domain sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1.
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the GATA-like domain sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the GATA-like domain sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • a cleavage site e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in Zn++ binding by the polypeptide indicates that the test compound decreases (e.g., inhibits) Zn++ binding.
  • an increase in the Zn++ binding by the polypeptide indicates that the test compound increases (e.g., promotes) Zn++ binding.
  • Zn++ binding is determined by measuring the level of free Zn++ (e.g., an increase (e.g., as compared to a negative control) in free Zn++ in the presence of a test compound indicates that the compound decreases Zn++ binding by the polypeptide; e.g., a decrease (e.g., as compared to a negative control) in free Zn++ in the presence of a test compound indicates that the compound increases Zn++ binding by the polypeptide).
  • the level of free Zn++ is determined by a spectrophotometric assay.
  • test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule.
  • the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • a method of evaluating (e.g., in vitro) a test compound for its ability to modulate (e.g., decrease or increase) GATA-like domain Zn++ binding comprising:
  • polypeptide e.g., an isolated polypeptide
  • the polypeptide comprises a GATA-like domain sequence, wherein the polypeptide is capable of binding Zn++; and wherein the GATA-like domain sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide)
  • the incubating occurs in conditions that are favorable for GATA-like domain Zn++ binding in the absence of a test compound;
  • test compound modulates GATA-like domain Zn++ binding, e.g., as compared to a control.
  • the control can be the amount (e.g., percentage) of GATA-like domain Zn++ binding in the absence of a test compound under identical conditions.
  • the GATA-like domain sequence consists of (i) a sequence at least 85% identical to residues 1543-1626 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1543-1626 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of (iii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (v) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1.
  • the GATA-like domain sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1.
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the GATA-like domain sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the GATA-like domain sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • a cleavage site e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in Zn++ binding by the polypeptide indicates that the test compound decreases (e.g., inhibits) Zn++ binding.
  • an increase in the JMJD3 catalytic activ Zn++ binding by the polypeptide indicates that the test compound increases (e.g., promotes) Zn++ binding.
  • Zn++ binding is determined by measuring the level of free Zn++ (e.g., an increase (e.g., as compared to a negative control) in free Zn++ in the presence of a test compound indicates that the compound decreases Zn++ binding by the polypeptide; e.g., a decrease (e.g., as compared to a negative control) in free Zn++ in the presence of a test compound indicates that the compound increases Zn++ binding by the polypeptide).
  • an increase e.g., as compared to a negative control
  • a decrease e.g., as compared to a negative control
  • the level of free Zn++ is determined by a spectrophotometric assay.
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule. In some embodiments, the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • a method of evaluating a test compound comprising:
  • a test compound contacting (e.g., in vitro) a test compound with (1) a polypeptide (e.g., an isolated polypeptide), wherein the polypeptide comprises a GATA-like domain sequence, wherein the polypeptide is capable of binding Zn++; and wherein the GATA-like domain sequence consists of:
  • a recombinant host cell comprising the polypeptide (e.g., a recombinant host cell described herein; e.g., containing a nucleic acid encoding the polypeptide)
  • the Zn++ can be naturally occurring in the host cell
  • test compound modulates GATA-like domain Zn++ binding (e.g., as compared to a control).
  • the control can be the amount (e.g., percentage) of GATA-like domain Zn++ binding in the absence of a test compound under identical conditions.
  • the GATA-like domain sequence consists of (i) a sequence at least 85% identical to residues 1543-1626 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1543-1626 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of (ii) a sequence at least 85% identical to residues 1 141 -1682 of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1 141 -1682 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (iii) a sequence at least 85% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of a sequence 100% identical to residues 1 141 -1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 . In some embodiments, the GATA-like domain sequence consists of (iv) a sequence at least 85% identical to a sequence that initiates at any of residues 253 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of a sequence 100% identical to identical to a sequence that initiates at any of residues 253 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of (v) a sequence at least 85% identical to a sequence that initiates at any of residues 247 to 15430 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the GATA-like domain sequence consists of a sequence 100% identical to a sequence that initiates at any of residues 247 to 1543 of SEQ ID NO:1 and terminates at residue 1682 of SEQ ID NO:1 , wherein the sequence comprises a deletion of residues 1637-1675 of SEQ ID NO:1 .
  • the polypeptide comprises a heterologous sequence (e.g., a sequence that does not naturally occur with the GATA-like domain sequence).
  • the polypeptide comprises a heterologous sequence (e.g., non naturally occurring with the GATA-like domain sequence) at its amino or carboxyl terminus.
  • the heterologous sequence can include a sequence used to detect or purify the polypeptide, e.g., a poly His (e.g., 6-His), FLAG, or c-myc tag, and/or can include a cleavage site, e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • a cleavage site e.g., a protease cleavage site, e.g., a thrombin, Factor Xa, PreScission protease, or TEV protease cleavage site.
  • the polypeptide comprises a 6-His tag, e.g., at its carboxy terminus. In some embodiments, the polypeptide comprises a FLAG tag, e.g., at its amino terminus. In some embodiments, the polypeptide comprises a 6-His tag and a FLAG tag, e.g., at its amino or carboxy terminus. In some embodiments, the polypeptide comprises a sequence used to detect or purify the polypeptide and a cleavage site, e.g., a 6-His tag and a TEV protease cleavage site.
  • the polypeptide comprises Cys (cysteine) at residues 1575, 1578, 1602, and 1605 of SEQ ID NO:1 .
  • a decrease in Zn++ binding by the polypeptide indicates that the test compound decreases (e.g., inhibits) Zn++ binding.
  • an increase in Zn++ binding by the polypeptide indicates that the test compound increases (e.g., promotes) Zn++ binding.
  • Zn++ binding is determined by measuring the level of free Zn++ (e.g., an increase (e.g., as compared to a negative control) in free Zn++ in the presence of a test compound indicates that the compound decreases Zn++ binding by the polypeptide; e.g., a decrease (e.g., as compared to a negative control) in free Zn++ in the presence of a test compound indicates that the compound increases Zn++ binding by the polypeptide).
  • an increase e.g., as compared to a negative control
  • a decrease e.g., as compared to a negative control
  • the level of free Zn++ is determined by a spectrophotometric assay.
  • the test compound is comprised in a compound library/ members of a compound library are evaluated.
  • the test compound comprises a nucleic acid, a protein, or a small molecule.
  • the test compound comprises RNAi.
  • the RNAi is selected from the group consisting of: miRNA, siRNA, esiRNA, and shRNA.
  • the test compound comprises a chemical compound.
  • the disclosure relates to a polypeptide (e.g., isolated polypeptide) that possesses JMJD3 catalytic activity (e.g., H3K27 demethylase activity), e.g., wherein the polypeptide contains residues 1 141 -1682 of SEQ ID NO:1 (or a sequence at least 85% identical thereto) or residues 1 141 - 1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 (or a sequence at least 85% identical thereto), but not the full length JMJD3 sequence. Nucleic acids encoding the same are also described.
  • the disclosure relates to a polypeptide (e.g., isolated polypeptide) that possesses JMJD3 catalytic activity (e.g., H3K27 demethylase activity), e.g., wherein the polypeptide contains residues 1 181 -1682 of SEQ ID NO:1 (or a sequence at least 85% identical thereto) or residues 1 181 - 1682 with a deletion of residues 1637-1675 (del 1637-1675) of SEQ ID NO:1 (or a sequence at least 85% identical thereto), but not the full length JMJD3 sequence. Nucleic acids encoding the same are also described.
  • the disclosure relates to a polypeptide (e.g., isolated polypeptide) that contains the GATA-like domain and binds Zn++, e.g., wherein the polypeptide contains residues 1543-1626 of SEQ ID NO:1 (or a sequence at least 85% identical thereto), but not the full length JMJD3 sequence. Nucleic acids encoding the same are also described.
  • a polypeptide described herein is suitable for crystallization, e.g., the polypeptide fragment is crystallizable.
  • the crystals obtainable from a polypeptide described herein are suitable for structure determination of the polypeptide using X-ray crystallography. In some embodiments, the crystals are radiation stable enough to permit more than 85% diffraction data completeness at resolution of e.g., 3.5 A or better to be collected upon exposure to monochromatic X- rays.
  • FIG. 1 is a diagram of the overall architecture of JMJD3 showing residues 1 178 to 1488 of the catalytic domain (top domain of diagram- residues 1 141 -1488) and the GATA-like insert (in darker shading) (residues 1543-1626) within the four helical domain (bottom domain of diagram- residuesl 489-1636) plus residues 1676 and 1677 after the deletion 1637-1675 (del 1637-1675).
  • FIG. 2 is a series of six panels showing the co-ordination of 2-OG in active sites of JMJD3, PHF8 (PDB ID: 3K30), JMJD2A (PDB ID: 2GP5), and FBX1 1 (PDB ID: 2YU1). Also shown is PDCA in the active site of Jmjd2A (PDB ID: 2VD7) and finally PDCA in Jmjd2A (PDB ID: 2VD7) superposed on 2- OG in JMJD3.
  • FIG. 3 is a series of three structures of C-terminal helical domains of PHF8 (center), FBX1 1 (right) and JMJD3 (left) with the Zn-binding GATAL insert.
  • FIG. 4 is a diagram showing the topology of the zinc-binding domain in JMJD3.
  • the two strands that are "added” to the treble clef fold are highlighted in darker shading on the left side of the diagram.
  • FIG. 5 is a diagram showing JMJD3.
  • the JmjC domain is at the left; helical domain at the bottom right; and GATAL at the top right of the structure.
  • the FOG fragment (labelled) is positioned consistent with its corresponding interactions with the GATA1 protein in the GATA-1 /FOG complex (PDB ID: 1 Y0J).
  • FIG. 6 is a bar graph showing the quantification of the respective H3K27 methyl states on an
  • JmjD3t JMJD3 1 141 -1682 (del. 1637-1675) substrate preference for the tri- and di- methyl valencies. All data is mean of 3 independent experiments.
  • FIG. 7 is a series of three diagrams: (i) GATA-1 (right) and DNA (left) showing the long C-terminal tail of GATA-1 that binds to the DNA minor groove; (ii) The GATAL of JMJD3 in the same orientation to highlight that instead of a long C-terminal tail, JMJD3 has a C-terminal region that forms one of the additional strands absent in GATA-1 ; (iii) an overlay of the two GATA domains in the presence of DNA is shown.
  • the GATA-1 domain is from PDB ID: 2DFV.
  • JmjC domain-containing proteins as histone demethylases that mediate the reversal of methylation at histone H3K4, H3K9 and H3K36.
  • the JMJD3 family of proteins is a subfamily of JmjC domain-containing proteins that has been shown to demethylate H3K27 (e.g., K27 on human histone 3).
  • the polypeptides, nucleic acids, crystal structures, and assays described herein can be utilized to screen for and identify compounds that modulate (e.g., increase or decrease) JMJD3 catalytic activity.
  • Polypeptides, nucleic acids, crystal structures, and assays described herein can also be used to screen for and identify compounds that modulate (e.g., increase or decrease) Zn++ binding by the JMJD3 GATA- 1 ike domain.
  • JMJD3 JMJD3
  • nucleotide and amino acid sequences for human JMJD3 are provided below. All sequence numbers are based on the numbering of residues taken from NCBI reference sequence entries NM_001080424 (nucleotide sequence) and NP_001073893 (amino acid sequence).
  • the human JMJD3 amino acid sequence is as follows. The amino acid sequence for the GATA-like zinc binding domain is highlighted in bold and underlined in the sequence below (taken from NP_001073893).
  • the human JMJD3 nucleotide sequence is provided below.
  • the nucleotide sequence for the zinc binding domain is highlighted in bold and underlined in the sequence below (taken from
  • NM_001080424 Also marked in this sequence are the methionine (atg) that is the start of the JMJD3 sequence and the stop codon (tga) for JMJD3, which are both highlighted in bold and underlined and italicized.
  • JMJD3 possesses catalytic (e.g., demethylase) activity.
  • JMJD3 can demethylate both tri- and dimethylated H3K27 (H3K27 demethylase activity) (e.g., K27 on human histone 3).
  • the polypeptides described herein contain a JMJD3 catalytic activity (e.g., H3K27 demethylase activity) and can contain amino acids (residues) 1 141 -1682 of the sequence shown in NP_001073893 or SEQ ID NO:1 .
  • the polypeptides can be encoded by sequences containing nucleotides 3797-5422 of the sequence shown in NM_001080424 or SEQ ID NO:2.
  • polypeptides described herein contain a JMJD3 catalytic activity (e.g., H3K27 demethylase activity) and can contain amino acids 1 141 -1682 (residues) and a deletion of amino acids (residues) 1637-1675 (del 1637-1675) of the sequence shown in NP_001073893 or SEQ ID NO:1.
  • the polypeptides can be encoded by sequences containing nucleotides 3797-5422 (del 5285-5401) of the sequence shown in NM_001080424 or SEQ ID NO:2.
  • polypeptides described herein contain a JMJD3 catalytic activity (e.g., H3K27 demethylase activity) and can contain amino acids (residues) 1 181 -1682 of the sequence shown in NP_001073893 or SEQ ID NO:1 .
  • polypeptides can be encoded by sequences containing nucleotides 3917-5422 of the sequence shown in NM_001080424 or SEQ ID NO:2; the polypeptides described herein contain a JMJD3 catalytic activity (e.g., H3K27 demethylase activity) and can contain amino acids 1 181 -1682 (residues) and a deletion of amino acids (residues) 1637-1675 (del 1637-1675) of the sequence shown in NP_001073893 or SEQ ID NO:1 .
  • JMJD3 catalytic activity e.g., H3K27 demethylase activity
  • the polypeptides can be encoded by sequences containing nucleotides 3917-5422 (del 5285- 5401) of the sequence shown in NM_001080424 or SEQ ID NO:2. JMJD3 binds to 2-oxo-glutarate (20G).
  • the polypeptides described herein can bind to 20G and can contain amino acids 1 141 -1682 or amino acids 1 141 -1682 with a deletion of amino acids 1637-1675 (del 1637-1675) of the sequence shown in NP_001073893 or SEQ ID NO:1 .
  • polypeptides described herein can bind to 20G and can contain amino acids 1 181 -1682 or amino acids 1 181 -1682 with a deletion of amino acids 1637-1675 (del 1637-1675) of the sequence shown in NP_001073893 or SEQ ID NO:1 .
  • JMJD3 binds iron (Fe(ll)).
  • the polypeptides described herein can bind iron and can contain amino acids 1 141 -1682 or amino acids 1 141 -1682 with a deletion of amino acids 1637-1675 (del 1637-1675) of the sequence shown in NP_001073893 or SEQ ID NO:1.
  • the polypeptides described herein can bind iron and can contain amino acids 1 181 -1682 or amino acids 1 181 -1682 with a deletion of amino acids 1637-1675 (del 1637-1675) of the sequence shown in NP_001073893 or SEQ ID NO:1 .
  • JMJD3 (and JMJD3 polypeptides described herein) can bind to other divalent ions, such as Co(ll) and Ni(ll), in addition to Fe(ll).
  • the catalytic site containing beta helix contains amino acids 1 181 -1488 of the sequence shown in NP_001073893 or SEQ ID NO:1 .
  • the catalytic site containing beta helix can be encoded by nucleotides 3917-4840 of the sequence shown in NM_001080424 or SEQ ID NO:2.
  • Polypeptides comprising this catalytic site beta helix are part of the disclosure, and can be used in the methods described herein for identifying compounds which modulate (e.g., increase or decrease) JMJD3 catalytic activity (e.g., H3K27 demethylase activity).
  • Polypeptides containing the JMJD3 GATA-like domain are also described herein.
  • the polypeptides contain amino acids 1543-1626 of the sequence shown in NP_001073893 or SEQ ID NO:1 .
  • the polypeptides can bind Zn++.
  • the conserved zinc-binding cysteine motif within the GATA-like domain is CxxC.CxxC and contains Cys1575, Cys 1578, Cys1602 and Cys1605.
  • the GATA-like domain can be encoded by nucleotides 5003-5254 of the sequence shown in NM_001080424 or SEQ ID NO:2.
  • the GATA-like domain structure consists of a single treble-clef zinc finger fold elaborated with two extra strands.
  • the structure is similar to the zinc binding domain in the erythroid transcription factor GATA-1 .
  • Calculations of "homology” or "sequence identity" between two sequences are performed as follows.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
  • the length of a reference sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence.
  • the reference sequence may be the length of a sequence described herein (e.g., 100% of the length of a JMJD3 sequence or GATA-like domain sequence described herein).
  • the polypeptides may comprise additional amino acids not derived from JMJD3, e.g., tags, enzyme cleavage sites, etc. Such additional amino acids are not considered in an alignment used to determine percent identity, i.e., the additional amino acids are excluded from the calculation of the percent identity.
  • the percent identity is obtained when aligning the sequence of the polypeptide at least over a length of 375, 400, 425, 450, 475, 500, 510, 525, or 540 amino acids for the polypeptide that possesses JMJD3 catalytic activity; or at least over a length of 60, 65, 70, 75, 80, or 83 amino acids for the polypeptide that possesses the GATA-like domain.
  • the term "substantially identical” is used herein to refer to a first amino acid or nucleic acid (e.g., nucleotide) sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleic acid sequence such that the first and second amino acid or nucleic acid sequences have (or encode proteins having) similar activities, e.g., a catalytic activity (e.g., H3K27 demethylase activity), a binding preference or specificity, or other activity.
  • a catalytic activity e.g., H3K27 demethylase activity
  • the second polypeptide encoded by or containing the sequence can have the same specificity and/or activity, and/or shares at least one substrate specificity and/or has at least 50%, at least 25%, or at least 10% of the activity relative to the same substrate.
  • Sequences similar or homologous e.g., have at least about 85% sequence identity
  • the sequence identity can be 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
  • nucleic acid sequences hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the other sequence.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • Specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1 % SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); (2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 60°C; (3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 65°C; and (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1 % SDS at 65°C.
  • SSC sodium chloride/sodium citrate
  • Very high stringency conditions are the preferred conditions and the ones that should be used unless otherwise specified.
  • the disclosure includes nucleic acids that hybridize with low, medium, high, or very high stringency to a nucleic acid described herein or to a complement thereof, e.g., nucleic acids encoding a binding protein described herein.
  • the nucleic acids can be the same length or within 30, 20, or 10% of the length of the reference nucleic acid.
  • the nucleic acid can correspond to a sequence described herein.
  • a polypeptide of the disclosure may have mutations (e.g., at least one, two, three, four, five, six, seven, eight, nine, or ten, and/or less than 15, 10, 5, or 3) relative to a sequence described herein (e.g., conservative or non-essential amino acid substitutions), which do not have a substantial effect on polypeptide function (e.g., the polypeptide has at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 100% of the activity of the reference polypeptide).
  • the polypeptide comprises amino acid substitutions, insertions, or deletions, e.g., less than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions, as compared to the sequence of amino acids 1 141 -1682 of SEQ ID NO:1 , and wherein the polypeptide possesses a catalytic activity of JMJD3, e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)).
  • the His at position 1390 (with reference to the full length JMJD3 sequence of SEQ ID NO:1) is needed for JMJD3 catalytic activity. Mutation of this residue to Ala (H1390A) abolishes catalytic activity.
  • the polypeptide comprises a sequence at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of amino acids 1 141 -1682 (del 1637-1675) of SEQ ID NO:1 , and wherein the polypeptide possesses a catalytic activity of JMJD3, e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)).
  • the His at position 1390 (with reference to the full length JMJD3 sequence of SEQ ID NO:1) is needed for JMJD3 catalytic activity. Mutation of this residue to Ala (H1390A) abolishes catalytic activity.
  • the polypeptide comprises amino acid substitutions, insertions, or deletions, e.g., less than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions, as compared to the sequence of amino acids 1 181 -1682 of SEQ ID NO:1 , and wherein the polypeptide possesses a catalytic activity of JMJD3, e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)).
  • the His at position 1390 (with reference to the full length JMJD3 sequence of SEQ ID NO:1) is needed for JMJD3 catalytic activity. Mutation of this residue to Ala (H1390A) abolishes catalytic activity.
  • the polypeptide comprises a sequence at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of amino acids 1 181 -1682 (del 1637-1675) of SEQ ID NO:1 , and wherein the polypeptide possesses a catalytic activity of JMJD3, e.g., the polypeptide is capable of demethylating H3K27 (lysine 27 (K27) of histone 3 (H3)).
  • the His at position 1390 (with reference to the full length JMJD3 sequence of SEQ ID NO:1) is needed for JMJD3 catalytic activity. Mutation of this residue to Ala (H1390A) abolishes catalytic activity.
  • the polypeptide comprises amino acid substitutions, insertions, or deletions, e.g., less than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions, as compared to the sequence of amino acids 1543-1626 of SEQ ID NO:1 , and wherein the polypeptide is capable of binding Zn++.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • amino acid residues of a polypeptide described herein may include one or more conservative substitutions.
  • one or more of the amino acids that line the active site of JMJD3 (identified above) are kept invariant or replaced by only conservative substitutions.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of the polypeptide, without abolishing or more preferably, without substantially altering a biological activity, whereas changing an "essential" amino acid residue results in a substantial loss of activity.
  • His1390, Glu1392, and His 1470 form a triad that binds iron.
  • the His at position 1390 is needed for JMJD3 catalytic activity and is an essential amino acid. Mutation of this residue to Ala (H1390A) abolishes catalytic activity.
  • Glu1392 and His1470 are also needed for JMJD3 catalytic activity and are essential amino acids. Mutation of either of these residues is expected to abolish catalytic activity.
  • polypeptides lacking catalytic activity may contain a GATA-like domain (described herein) and be able to bind to Zn++. As a result, these polypeptides are useful, e.g., in assays for evaluating a test compound for its ability to modulate Zn++ binding by the GATA-like domain.
  • Cys1575, Cys 1578, Cys1602 and Cys1605 are essential amino acids for the GATA-like domain.
  • An isolated polypeptide refers to a preparation at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of which is made up of the polypeptide.
  • the isolated polypeptide can make up at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the preparation.
  • An isolated nucleic acid or polynucleotide refers to a preparation at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of which is made up of the nucleic acid or polynucleotide.
  • the isolated nucleic acid or polynucleotide can make up at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the preparation.
  • the polypeptide according to the present disclosure is purified to an extent to be suitable for crystallization, for example, it is 85% to 100%, 90% to 100%, or 95% to 100% pure.
  • a polypeptide described herein e.g., a polypeptide containing a JMJD3 sequence, is capable of binding to Fe(ll).
  • a polypeptide described herein e.g., a polypeptide containing a JMJD3 sequence, is capable of binding to 2-oxo-glutarate (20G).
  • a polypeptide described herein e.g., a polypeptide containing the GATA-like domain
  • a polypeptide described herein is capable of binding Zn++.
  • a polypeptide described herein e.g., a polypeptide containing a JMJD3 sequence
  • PDCA pyridine 2,4 dicarboxylic acid
  • the polypeptide consists of amino acids 1 141 -1682 (del 1637-1675) of SEQ ID NO:1 and has the structure defined by the structure coordinates deposited on October 19, 2010 in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) and assigned PDB ID 2XUE.
  • nucleic acids containing sequences that encode polypeptides described herein.
  • the nucleic acid can include nucleotides 3797-5422, 3917-4840, or 5003-5254 of SEQ ID NO:2, or sequences at least 85% identical (e.g., 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical) thereto (e.g., over at least 85% of the length of these nucleotides).
  • sequences at least 85% identical to a sequence disclosed herein encode polypeptides that possess an activity described herein (e.g., JMJD3 catalytic ativity, 20G binding, Fe(ll) binding, or Zn++ binding).
  • the molecular biology methods applied for obtaining such isolated nucleotide fragments are generally known to the person skilled in the art (for standard molecular biology methods see Sambrook et al., Eds., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), which is incorporated herein by reference).
  • RNA can be isolated from a human cell and cDNA generated applying reverse transcription polymerase chain reaction (RT-PCR) using either random primers (e.g., random hexamers of decamers) or primers specific for the generation of the fragments of interest.
  • RT-PCR reverse transcription polymerase chain reaction
  • the fragments of interest can then be amplified by standard PCR using fragment specific primers.
  • the isolated polynucleotides are derived from SEQ ID NO: 2.
  • SEQ ID NO:2 encodes the full-length JMJD3 and, thus, polynucleotides coding for the fragments may comprise deletions at the 3'- and/or 5'-ends of the polynucleotide such that the polynucleotide does not encode the full-length protein.
  • a recombinant vector includes any vector known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC).
  • the vectors include expression as well as cloning vectors.
  • Expression vectors include plasmids as well as viral vectors, and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems.
  • Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.
  • the present disclosure relates to a recombinant vector comprising an isolated nucleic acid described herein. Techniques used for the incorporation of nucleic acid sequences of interest into vectors are known (see, e.g., Sambrook et al., 1989, above).
  • Such vectors include any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC).
  • the vectors may be expression vectors suitable for prokaryotic or eukaryotic expression.
  • the plasmids may include an origin of replication (ori), a multiple cloning site, and regulatory sequences such as a promoter (constitutive or inducible), transcription initiation site, ribosomal binding site, transcription termination site, polyadenylation signal, and/or selection marker such as antibiotic resistance or auxotrophic marker based on complementation of a mutation or deletion.
  • the polynucleotide sequence of interest is operably linked to the regulatory sequences.
  • the vector includes nucleotide sequences encoding epitope-, peptide-, or protein-tags that facilitate purification or detection of the polypeptide of interest.
  • Such tags include, but are not limited to, hemagglutinin- (HA-), FLAG-, c-myc, poly-His (e.g., 6-His (6H)), glutathione-S-transferase- (GST-), maltose-binding-protein- (MBP-), NusA-, and thioredoxin tags, or fluorescent protein-tags such as (enhanced) green fluorescent protein ((E)GFP), (enhanced) yellow fluorescent protein ((E) YFP), red fluorescent protein (RFP) derived from Discosoma species (DsRed) or monomeric (mRFP), cyan fluorescence protein (CFP), and the like.
  • HA- hemagglutinin-
  • FLAG- hemagglutinin-
  • c-myc poly-His
  • poly-His e.g., 6-His (6H)
  • GST- glutathione-S-transferase-
  • the tags can be cleaved off the polypeptide, for example, using a protease such as thrombin, Factor Xa, PreScission, TEV protease, and the like.
  • a protease such as thrombin, Factor Xa, PreScission, TEV protease, and the like.
  • the tag can be removed with a TEV protease and its recognition site.
  • the recognition sites for such proteases are well known to the person skilled in the art.
  • the seven amino acid consensus sequence of the TEV protease recognition site is Glu-X-X-Tyr-X-Gln-Gly/Ser (SEQ ID NO:3), wherein X may be any amino acid and "Gly/Ser" indicates that the amino acid is either Gly or Ser; for example, the sequence can be Glu-Asn-Leu-Tyr-Phe- Gln-Gly (SEQ ID NO:4).
  • the vector includes a functional sequence (e.g., a signal sequence) that leads to secretion of the polypeptide into the culture medium of the recombinant host cells or into the periplasmic space of bacteria.
  • the signal sequence usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • the protein is either secreted into the growth media (gram- positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
  • a recombinant host cell refers to a host cell that comprises a polynucleotide/nucleic acid that encodes a polypeptide of interest, i.e., a polypeptide described herein.
  • This nucleic acid may be found inside the host cell (i) freely dispersed as such, (ii) incorporated in a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA.
  • the recombinant cell can be used for expression of a nucleic acid of interest or for amplification of the nucleic acid or the recombinant vector of the disclosure.
  • the recombinant host cell includes the progeny of the original cell into which the polynucleotide/nucleic acid or the recombinant vector has been introduced (e.g., via transformation, transfection, or infection).
  • a recombinant host cell may be a bacterial cell such as an E.
  • mammalian cells examples include Chinese hamster ovary (CHO) cells, green African monkey kidney (COS) cells, human embryonic kidney (HEK293) cells, HELA cells, and the like.
  • the disclosure provides a recombinant host cell comprising the isolated nucleic acid or the recombinant vector.
  • the recombinant host cells may be prokaryotic cells such as archea and bacterial cells or eukaryotic cells such as yeast, plant, insect, or mammalian cells.
  • the host cell is a bacterial cell such as an E. coli cell.
  • Methods for introducing an isolated nucleic acid or recombinant vector into a host cell are known in the art.
  • bacterial cells can be readily transformed using, for example, chemical transformation, e.g., the calcium chloride method, or electroporation.
  • Yeast cells may be transformed, for example, using the lithium acetate transformation method or electroporation.
  • Other eukaryotic cells can be transfected, for example, using commercially available liposome-based transfection kits such as
  • LIPOFECTAMINETM (Invitrogen), commercially available lipid-based transfection kits such as Fugene (Roche Diagnostics), polyethylene glycol-based transfection, calcium phosphate precipitation, gene gun (biolistic), electroporation, or viral infection.
  • the recombinant host cell expresses the nucleic acid.
  • expression leads to soluble polypeptide.
  • the polypeptide may be purified using protein purification methods well known to the person skilled in the art, optionally taking advantage of the above-mentioned tags.
  • Antibody refers to both monoclonal and polyclonal antibodies, i.e., any immunoglobulin protein or portion thereof which is capable of recognizing an antigen or hapten, i.e., a polypeptide possessing JMJD3 catalytic activity (e.g., H3K27 demethylase activity) or the JMJD3 sequence or a peptide thereof, or a polypeptide containing the GATA-like domain described herein or the GATA-like domain sequence or a peptide thereof.
  • Antigen-binding portions of the antibody are also included in the meaning of antibody and may be produced, e.g., by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • the present disclosure provides an antibody directed against a polypeptide described herein (e.g., a polypeptide containing a JMJD3 sequence (e.g., with JMJD3 catalytic activity) and/or a GATA-like domain).
  • the antibody may be a monoclonal or polyclonal antibody or a portion(s) (e.g., antigen-binding portion) thereof.
  • Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments (e.g., three CDRs from an antibody heavy chain and/or three CDRs from an antibody light chain (e.g., from the same antibody as the heavy chain)), single-chain antibodies (scFv), chimeric antibodies such as humanized antibodies, diabodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.
  • CDR complementarity determining region
  • a polyclonal antibody may be generated by immunizing an animal such as mouse, rat, rabbit, goat, sheep, pig, cattle, or horse with the antigen of interest optionally in combination with an adjuvant such as Freund's complete or incomplete adjuvant, RIBI (muramyl dipeptides), or ISCOM (immunostimulating complexes) according to standard methods known in the art.
  • Monoclonal antibodies may be generated by methods known in the art, e.g., using hybridoma technology or by screening antibody libraries (e.g., phage or yeast display libraries of antibodies, e.g., human antibodies). Methods of selecting, cloning, and expanding hybridomas are known in the art.
  • Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g., PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification.
  • test compound refers to an agent that is being tested for its ability to modulate (e.g., increase or decrease) JMJD3 catalytic activity (e.g., H3K27 demethylase activity), Fe(ll) binding, 20G binding, or GATA-like domain Zn++ binding .
  • JMJD3 catalytic activity e.g., H3K27 demethylase activity
  • the test compound can be any agent including, but not restricted to, a peptide, a peptoid, a polypeptide (such as an antibody), a lipid, a metal, a nucleotide, a nucleotide analog, a nucleoside, a nucleic acid, an organic compound, an inorganic compound, a small organic or inorganic molecule, a chemical compound (e.g., a low molecular compound, or a high molecular compound), a pharmacological agent, an element, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, an analytical probe, a polyamine, and combinations and derivatives thereof.
  • a test compound can be naturally occurring or synthetically prepared. It can be isolated from a microorganism, an animal, or a plant, and can be produced recombinantly, or synthesized, e.g., by a chemical method.
  • nucleic acid compounds that may be used include RNAi, such as shRNA, siRNA, esiRNA, miRNA, oligo DNA, oligo RNA, a ribozyme, and antisense nucleic acid, such as antisense RNA.
  • the test compound can have a formula weight of less than about 10,000 grams per mole, less than 5,000 grams per mole, less than 1 ,000 grams per mole, or less than about 500 grams per mole.
  • the test compound of the disclosure may comprise a mixture of substances (e.g., purified or partially purified), for example, the test compound can be an extract (e.g., an extract derived from a marine organism, plant, animal, soil, or a phage display library), or the product of mixed combinatorial syntheses.
  • the mixture can be tested, e.g., in a method described herein, and the component that modulates (e.g., increases or decreases) JMJD3 catalytic activity (e.g., H3K27 demethylase activity), Fe(ll) binding, 20G binding, or GATA-like domain Zn++ binding can be purified from the mixture in a subsequent step.
  • JMJD3 catalytic activity e.g., H3K27 demethylase activity
  • Fe(ll) binding 20G binding
  • GATA-like domain Zn++ binding can be purified from the mixture in a subsequent step.
  • a test compound can be obtained, for example, using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries (such as libraries of peptides or polypeptides (e.g., antibodies), e.g., phage display or eukaryotic cell (e.g., yeast) display libraries) or small molecule libraries, synthetic library methods requiring deconvolution, the "one-bead one- compound” library method, and synthetic library methods using affinity chromatography selection.
  • biological libraries such as libraries of peptides or polypeptides (e.g., antibodies), e.g., phage display or eukaryotic cell (e.g., yeast) display libraries
  • small molecule libraries synthetic library methods requiring deconvolution
  • the "one-bead one- compound” library method and synthetic library methods using affinity chromatography selection.
  • Test compounds can be derived or selected from libraries of synthetic or natural compounds.
  • synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ChemBridge Corporation (San Diego, CA, USA), ICCB Known Bioactives library (Enzo Life Sciences, Plymouth Meeting, PA, USA), Aldrich (Milwaukee, Wl, USA) ComGenex (Princeton, N.J., USA), Asinex (Moscow, RU), Tripos, Inc. (St. Louis, MO, USA), Otava (Kyiv, UA), Light Biologicals (Shirley, NY, USA), and ChemStar, Ltd. (Moscow, RU).
  • a natural compound library is, for example, available from TimTec LLC (Newark, DE, USA). Libraries of natural compounds in the form of bacterial, fungal, plant and animal cell and tissue extracts can be used. Test compounds can be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures. A collection of compounds made using combinatorial chemistry is a combinatorial library.
  • RNAi e.g., miRNA, siRNA, esiRNA, or shRNA
  • libraries can also be prepared, and are available from, for example, Sigma-Aldrich (St. Louis, MO, USA), System Biosciences (Mountain View, CA, USA), Open Biosystems (Huntsville, AL, USA), and Applied Biosystems/Ambion (Austin, TX, USA).
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991); and Houghton et al., Nature 354:84-88 (1991)).
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No.
  • WO 93/20242 random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 1 14:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer. Chem. Soc.
  • PCT/US96/10287 carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and
  • test compounds of the present disclosure can also be obtained from biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological libraries include libraries of nucleic acids and libraries of proteins.
  • nucleic acid libraries encode a diverse set of proteins (e.g., natural and artificial proteins; others provide, for example, functional RNA and DNA molecules such as nucleic acid aptamers or ribozymes.
  • a peptoid library can be made to include structures similar to a peptide library. (See also Lam (1997) Anticancer Drug Des. 12:145).
  • a library of proteins may be produced by an expression library or a display library (e.g., a phage or yeast display library).
  • a test compound may optionally comprise a detectable label.
  • labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds, and bioluminescent compounds. High throughput screening methods can be used to evaluate test compounds.
  • High throughput screening methods involve providing a library (e.g., a combinatorial chemical, RNAi (e.g., miRNA, siRNA, esiRNA, or shRNA), or peptide library) containing a large number of test compounds (library members). Such libraries are then screened in one or more assays, as described herein, to identify those library members (e.g., particular chemical species or subclasses) that display a desired characteristic activity.
  • a library e.g., a combinatorial chemical, RNAi (e.g., miRNA, siRNA, esiRNA, or shRNA), or peptide library) containing a large number of test compounds (library members).
  • libraries are then screened in one or more assays, as described herein, to identify those library members (e.g., particular chemical species or subclasses) that display a desired characteristic activity.
  • Test compounds and/or libraries thereof can be screened in one or more assays, as described herein, to identify those test compounds and/or library members (particular chemical species or subclasses) that display a desired characteristic activity, e.g., have an effect (e.g., inhibitory effect) on JMJD3 catalytic activity (e.g., H3K27 demethylase activity), Fe(ll) binding, 20G binding, or GATA-like domain Zn++ binding.
  • JMJD3 catalytic activity e.g., H3K27 demethylase activity
  • Fe(ll) binding 20G binding
  • GATA-like domain Zn++ binding e.g., GATA-like domain Zn++ binding.
  • the compounds thus identified can serve as conventional "lead compounds,” e.g., which are varied (e.g., by derivatization), or can themselves be used as modulators of JMJD3 catalytic activity (e.g., H3K27 demethylase activity), Fe(ll) binding, 20G binding, or GATA-like domain Zn++ binding, respectively.
  • JMJD3 catalytic activity e.g., H3K27 demethylase activity
  • Fe(ll) binding e.g., 20G binding, or GATA-like domain Zn++ binding, respectively.
  • An assay for identifying and/or selecting test compounds which modulate (e.g., increase or decrease) JMJD3 catalytic activity can include contacting a solution containing JMJD3 catalytic activity (e.g., containing a polypeptide described herein that contains a JMJD3 catalytic activity) and a JMJD3 substrate (e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., with at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 residues on either side of the methylated lysine (e.g., the peptide is methylated, e.g., di- or tri- methylated at the position corresponding to K27)) (e.g., a peptide with the sequence
  • ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (the bold underlined K corresponds to K27); or histone 3 (e.g., human histone 3) (e.g., that is methylated, e.g., di- or tri- methylated, at K27)) with a test compound and determining the ability of the test compound to modulate (e.g., increase or decrease) JMJD3 catalytic activity (e.g., H3K27 demethylase activity).
  • a co-factor, such as Fe(ll) and/or 20G, can optionally be included.
  • Determining the ability of the test compound to modulate JMJD3 catalytic activity can be accomplished, for example, by MALDI-TOF to detect and measure demethylation of the substrate (e.g., conversion of a trimethylated substrate lysine to a dimethylated lysine) by the polypeptide.
  • JMJD3 binds 20G and Fe(ll).
  • Compounds that interfere with Fe(ll) or 20G binding can decrease (e.g., inhibit) catalytic activity (e.g, H3K27 demethylase activity) and are identifiable by the assays described herein.
  • Compounds that promote Fe(ll) or 20G binding can increase catalytic activity (e.g, H3K27 demethylase activity) and are identifiable by the assays described herein.
  • Controls can also be included in the assay.
  • the amount of demethylation after exposure to a test compound can be compared to the amount of demethylation in the absence of the test compound under identical assay conditions, e.g., identical buffer conditions, reaction time, co-factors, and temperature.
  • a solution containing JMJD3 catalytic activity e.g., containing a polypeptide described herein that contains a JMJD3 catalytic activity
  • a JMJD3 substrate e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is di- or tri- methylated at the position corresponding to K27)
  • the solutions can be evaluated to determine the amount of demethylation under each condition.
  • the test compound modulates JMJD3 demethylation. If a greater amount of demethylation is measured in the solution containing the test compound as compared to the amount of demethylation in the solution that does not contain the test compound, the test compound increases JMJD3 catalytic (demethylase) activity. If a lesser amount of demethylation is measured in the solution containing the test compound as compared to the amount of demethylation in the solution that does not contain the test compound, the test compound decreases (e.g., inhibits) JMJD3 catalytic (demethylase) activity.
  • the difference in amount of demethylase activity can be a statistically significant difference (e.g., statistical significance by a T test (e.g., student's T test) (e.g., with a probability (p) value of p ⁇ 0.05 or 0.01)), or can be a difference of about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%, or greater.
  • a reference e.g., a reference value, e.g., obtained from the literature, a prior assay, and so forth. Appropriate correlations and art known statistical methods can be used to evaluate an assay result(s).
  • the compound that modulates JMJD3 catalytic activity decreases (e.g., inhibits) the activity.
  • the compound decreases the JMJD3 catalytic activity (e.g., H3K27 demethylase activity) of the polypeptide by 50%, 60%, 70%, 80%, 90%, or up to 100%, as compared to the activity of the polypeptide without the compound but otherwise with the same reaction conditions, e.g., buffer conditions, reaction time, co-factors, and temperature.
  • the compound specifically decreases or inhibits the activity of the polypeptide but does not decrease or inhibit the activity of another demethylase, e.g., another Jumonji protein (e.g., JMJD2A, JMJD2B, JMJD2C, JMJD2D, or JMJD6), to the same extent, or not at all.
  • another demethylase e.g., another Jumonji protein (e.g., JMJD2A, JMJD2B, JMJD2C, JMJD2D, or JMJD6)
  • the disclosure provides a method for identifying a compound which modulates (e.g., increases or decreases) JMJD3 catalytic activity (e.g., H3K27 demethylase activity) of a polypeptide (that contains a JMJD3 catalytic activity) described herein, the method comprising the steps of
  • the modulating compound decreases (e.g., inhibits) JMJD3 catalytic activity (e.g., H3K27 demethylase activity).
  • the disclosure provides a method for identifying a compound which modulates (e.g., increases or decreases) Zn++ binding of a polypeptide described herein, the method comprising the steps of
  • the modulating compound decreases (e.g., inhibits) Zn++ binding.
  • the co-ordinates disclosed herein may be used for building homology models of variants of JMJD3, orthologues of JMJD3, and homologues of JMJD3, such as UTX and UTY.
  • Such predictive models are valuable, e.g., in light of the higher costs associated with the expression, purification and testing of the many diverse polypeptides that are related to JMJD3 by at least 30% identity.
  • Construction of a homology model may be achieved through the use of software that is capable of generating three- dimensional co-ordinates of polypeptides or portions thereof from a set of one or more polypeptide structure coordinates.
  • An example for such a computer program is MODELER (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815 as implemented in the Insight II Homology software package (Insight II (97.0), Molecular Simulations Incorporated, San Diego, CA)).
  • JMJD3 catalytic activity e.g., H3K27 demethylase activity
  • Zn++ binding e.g., Zn++ binding.
  • This process may begin by a visual inspection of, for example, a three-dimensional computer model of the polypeptide based on the structural coordinates deposited in, e.g., PDB ID: 2XUE (deposited on October 19, 2010 in the
  • test compounds may then be positioned in a variety of orientations or docked onto the polypeptide (e.g., onto the active site or the GATA-like domain). Docking may be accomplished using software such as Cerius, Quanta, and Sybyl (Tripos Associates, St. Louis, MO), followed by energy minimization and molecular dynamics with standard molecular dynamics force fields such as OPLS-AA, CHARMM, and AMBER.
  • suitable compounds can be designed or assembled into a single compound or complex.
  • This manual model building is performed using software such as Quanta or Sybyl.
  • Useful programs aiding the skilled person in connecting individual compounds or fragments include, for example, (i) CAVEAT (Bartlett et al., 1989, in Molecular Recognition in Chemical and Biological Problems, Special Publication, Royal Chem. Soc. 78:182-196; Lauri and Bartlett, 1994, J. Comp. Aid. Mol. Des. 8:51 -66; CAVEAT is available from the University of California, Berkley, CA), (ii) 3D Database systems such as ISIS (MDL Information Systems, San Leandro, CA; reviewed in Martin, 1992, J. Med. Chem.
  • Another approach enabled by this disclosure is the computational screening of small molecule databases for compounds that can bind in whole or part to the polypeptide (e.g., the catalytic site or the GATA-like domain).
  • the quality of fit of such compounds to the polypeptide may be judged either by shape complementarity or by estimated interaction energy (Meng et al., 1992, J. Comp. Chem. 13:505-524).
  • a potential modulator for the polypeptide e.g., an inhibitor of a JMJD3 catalytic activity (e.g., H3K27 demethylase activity) or Zn++ binding
  • a potential modulator for the polypeptide may be designed de novo on the basis of the 3D structure of the polypeptide, e.g., deposited in PDB ID: 2XUE (deposited on October 19, 2010 in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB)).
  • RCSB Research Collaboratory for Structural Bioinformatics
  • PDB ID Protein Data Bank
  • a molecule designed or selected as binding to the polypeptide may be further computationally optimized so that in its bound state it preferably lacks repulsive electrostatic interaction with the region it binds.
  • Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole- dipole and charge-dipole interactions.
  • the sum of all electrostatic interactions between the compound and the region in a bound state preferably make a neutral or favorable contribution to the enthalpy of binding.
  • Examples of suitable programs include (i) Gaussian 92, revision C (Frisch, Gaussian, Incorporated, Pittsburgh, PA), (ii) AMBER, version 4.0 (Kollman, University of California, San Francisco, CA), (iii) QUANTA/CHARMM (Molecular
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will approximate the same size, shape, hydrophobicity and charge as the original group. It should be understood that components known in the art to alter conformation should be avoided.
  • substituted chemical compounds may then be analyzed for efficiency of fit to the polypeptide by the same computer methods described in detail above.
  • the compound may be synthesized and optionally the compound or a pharmaceutically acceptable salt thereof may be formulated with one or more pharmaceutically acceptable excipient(s) and/or carrier(s).
  • the above-described method may comprise the further step of (e) synthesizing the compound and optionally formulating the compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s).
  • the ability of the compound or of a pharmaceutically acceptable salt thereof or of a formulation thereof to modulate, e.g., decrease, JMJD3 catalytic activity (e.g., H3K27 demethylase activity) or Zn++ binding may be tested in vitro or in vivo comprising the further step of (f) contacting the compound with the polypeptide (e.g., and with a JMJD3 substrate (e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27) (e.g., a peptide with the sequence ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (the bold underlined K corresponds to K27); or histone 3 (e.g., human histone 3) (e.g., that is methylated, e.g., di- or tri- methylated, at K27
  • a co-factor such as Fe(ll) and/or 20G
  • the quality of fit of such compounds to the polypeptide may be judged either by shape complementarity or by estimated interaction energy (Meng et al., 1992, J. Comp. Chem. 13:505-524).
  • Methods for synthesizing compounds are well known to the person skilled in the art or compounds may be commercially available. For example, MALDI-TOF, ELISA, Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation (ChIP) can be used to measure demethylase activity.
  • the disclosure provides a method for identifying compounds which modulate (e.g., decrease, e.g., inhibit) JMJD3 catalytic activity (H3K27 demethylase activity), the method comprising (i) contacting a polypeptide described herein (that contains a JMJD3 catalytic activity) (e.g., and with a JMJD3 substrate (e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27) (e.g., a peptide with the sequence ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (the bold underlined K corresponds to K27); or histone 3 (e.g., human histone 3) (e.g., that is methylated, e.g., di- or tri- methylated, at K27)) or a re
  • a co-factor such as Fe(ll) and/or 20G
  • the polypeptide and a substrate thereof e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27)
  • a peptide with the sequence e.g., a peptide with the sequence
  • ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (the bold underlined K corresponds to K27); or histone 3 (e.g., human histone 3) (e.g., that is methylated, e.g., di- or tri- methylated, at K27)) are contacted in the presence or absence of varying amounts of a test compound and incubated for a certain period of time, for example, for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 30, 40, 60, or 90 minutes.
  • the reaction conditions are chosen such that the polypeptide is catalytically active in the absence of the test compound.
  • the substrate is then analyzed for demethylation, for example, by MALDI-TOF.
  • ELISA Western blotting, immunofluorescence, immunohistochemistry,
  • chromatin immunoprecipitation can also be used to measure demethylase activity (e.g., assays are available from Active Motif (Carlsbad, CA), and Epigentek (Brooklyn, NY)). Demethylation can also be measured, e.g., using a histone assay (e.g., incubating the polypeptide with core histones, e.g., as described in Miller et al., Genes & Dev. 2008. 22: 2980- 2993 or Lee at al., Science 2007: 318: 447 - 450), or by a fluorescent assay.
  • a histone assay e.g., incubating the polypeptide with core histones, e.g., as described in Miller et al., Genes & Dev. 2008. 22: 2980- 2993 or Lee at al., Science 2007: 318: 447 - 450
  • Fluorescent assays to measure H3K27 demethylase activity are available, e.g., from Abnova (Taipei, Taiwan), Active Motif (Carlsbad, CA, USA), and Epigentek (Brooklyn, NY, USA).
  • Antibodies specific to mono-, di- or tri- methylated H3K27 are also available (e.g., from Epigentek (Brooklyn, NY, USA); Abeam (Cambridge, UK)) and can be used to detect and measure levels of mono-, di- or tri-methylated H3K27 as a measure of JMJD3 catalytic activity.
  • Such antibodies can be used, e.g., in ELISA, Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, and chromatin
  • the interaction between the polypeptide (e.g., and a substrate thereof (e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27) e.g., a peptide with the sequence
  • ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (the bold underlined K corresponds to K27); or histone 3 (e.g., human histone 3) (e.g., that is methylated, e.g., di- or tri- methylated, at K27)) and a test compound may be analyzed in form of an enzyme-linked immunosorbent assay (ELISA)-based experiment.
  • the polypeptide may be immobilized on the surface of an ELISA plate and contacted with the test compound.
  • a co-factor, such as Fe(ll) and/or 20G, can be included in the assay.
  • Binding of the test compound may be verified, for example, for proteins, polypeptides, peptides, and epitope-tagged compounds by antibodies specific for the test compound or an epitope tag on the compound. These antibodies might be directly coupled to an enzyme or detected with a secondary antibody coupled to the enzyme that - in combination with the appropriate substrates - carries out chemiluminescent reactions (e.g., horseradish peroxidase) or colorimetric reactions (e.g., alkaline phosphatase). In another embodiment, binding of compounds that cannot be detected by antibodies might be verified by labels directly coupled to the test compounds.
  • chemiluminescent reactions e.g., horseradish peroxidase
  • colorimetric reactions e.g., alkaline phosphatase
  • Such labels may include enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds.
  • the test compounds might be immobilized on the ELISA plate and contacted with the polypeptide according to the disclosure. Binding of the polypeptide may be verified by a polypeptide-specific antibody (e.g., specific for a mono-, di-, or tri-methylated H3K27) and chemiluminescence or colorimetric reactions as described above. The effects of a test compound on the interaction between the polypeptide and its substrate can be analyzed in like manner.
  • ELISA can be used to detect and/or measure the interaction between the polypeptide and its substrate in the presence and absence of the test compound.
  • the effects of the test compound on the level of interaction can be analyzed (e.g., determining if the level of interaction decreases or increases in the presence of the test compound).
  • the polypeptide (e.g., and a substrate thereof (e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27) (e.g., a peptide with the sequence ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (the bold underlined K corresponds to K27); or histone 3 (e.g., human histone 3) (e.g., that is methylated, e.g., di- or tri- methylated, at K27))
  • a substrate thereof e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27)
  • a substrate thereof e.g., a peptide corresponding to H3K27 and adjacent sequence (
  • a recombinant host cell described herein is contacted with a test compound.
  • test compounds e.g., polypeptides
  • FRET fluorescence resonance energy transfer
  • directly labeled test compounds may be added to the medium of the recombinant host cells. The potential of the test compound to penetrate membranes and bind to the polypeptide may be, for example, verified by immunoprecipitation of the polypeptide and verification of the presence of the label.
  • the ability of the test compound to modulate (e.g., decrease, e.g., inhibit) JMJD3 catalytic activity (H3K27 demethylase activity) of the polypeptide is assessed.
  • the purified polypeptide and a substrate thereof e.g., a peptide corresponding to H3K27 and adjacent sequence (e.g., that is methylated, e.g., di- or tri- methylated at the position corresponding to K27) (e.g., a peptide with the sequence
  • ATKAARKSAPATGGVKKPHRYRPG (SEQ ID NO:5) (the bold underlined K corresponds to K27); or histone 3 (e.g., human histone 3) (e.g., that is methylated, e.g., di- or tri- methylated, at K27)) are contacted in the presence or absence of varying amounts of the test compound and incubated for a certain period of time, for example, for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 30, 40, 60, or 90 minutes.
  • a co-factor, such as Fe(ll) and/or 20G can be included in the assay.
  • the reaction conditions are chosen such that the polypeptide is catalytically active in the absence of the test compound.
  • the substrate is then analyzed for demethylation, for example, by MALDI-TOF.
  • ELISA Western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, and chromatin immunoprecipitation (ChIP) can also be used to measure demethylase activity (e.g., assays are available from Active Motif (Carlsbad, CA, USA), Epigentek (Brooklyn, NY, USA)).
  • Demethylation can also be measured, e.g., using a histone assay (e.g., incubating the polypeptide with core histones, e.g., as described in Miller et al., Genes & Dev. 2008.
  • Fluorescent assays to measure H3K27 demethylase activity are available, e.g., from Abnova (Taipei, Taiwan), Active Motif (Carlsbad, CA, USA), and Epigentek (Brooklyn, NY, USA).
  • Antibodies specific to mono-, di- or tri-methylated H3K27 are also available (e.g., from Epigentek (Brooklyn, NY, USA); Abeam (Cambridge, UK)) and can be used to detect and measure levels of mono-, di- or tri-methylated H3K27 as a measure of JMJD3 catalytic activity.
  • Such antibodies can be used, e.g., in ELISA, Western blotting, immunofluorescence,
  • the experiments may be applied in a multi-well plate format and are suitable for high throughput screening of test compounds regarding their ability to modulate (e.g., decrease, e.g., inhibit) JMJD3 catalytic activity (H3K27 demethylase activity).
  • the above-described methods for identifying compounds which modulate (e.g., decrease, e.g., inhibit) JMJD3 catalytic activity (H3K27 demethylase activity) are performed in a high-throughput setting.
  • the disclosure provides a method for identifying compounds which modulate (e.g., decrease, e.g., inhibit) Zn++ binding by the GATA-like domain, the method comprising (i) contacting a polypeptide containing the GATA-liked domain, e.g., a polypeptide described herein (e.g., and Zn++) or a recombinant host cell with a test compound and (ii) analyzing the ability of the test compound to modulate (e.g., decrease, e.g., inhibit) Zn++ binding by the polypeptide.
  • the polypeptide and Zn++ are contacted in the presence or absence of varying amounts of a test compound and incubated for a certain period of time, for example, for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, , 1 1 , 12, 13, 14, 15, 20, 30, 40, 60, or 90 minutes.
  • the reaction conditions are chosen such that the polypeptide binds Zn++ in the absence of the test compound.
  • the reaction is then analyzed for Zn++ binding, e.g., levels of Zn++ (e.g., free Zn++) that is not bound to the GATA-like domain.
  • Assays to measure Zn++ binding to the GATA-like domain include zinc determination according to Hunt et al, 1985, Anal. Biochem. 146, 150-157.
  • the A500 can be monitored using a Beckman DU600
  • Zn++ detecting reagents are also commercially available, e.g., from Invitrogen (Carlsbad, CA, USA).
  • the interaction between the GATA-like domain e.g., a polypeptide containing the GATA-liked domain
  • a test compound may be analyzed in form of an enzyme-linked immunosorbent assay (ELISA)-based experiment.
  • the polypeptide may be immobilized on the surface of an ELISA plate and contacted with the test compound. Binding of the test compound may be verified, for example, for proteins, polypeptides, peptides, and epitope-tagged compounds by antibodies specific for the test compound or an epitope tag on the compound.
  • antibodies might be directly coupled to an enzyme or detected with a secondary antibody coupled to the enzyme that - in combination with the appropriate substrates - carries out chemiluminescent reactions (e.g., horseradish peroxidase) or colorimetric reactions (e.g., alkaline phosphatase).
  • chemiluminescent reactions e.g., horseradish peroxidase
  • colorimetric reactions e.g., alkaline phosphatase
  • binding of compounds that cannot be detected by antibodies might be verified by labels directly coupled to the test compounds.
  • labels may include enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals,
  • test compounds might be immobilized on the ELISA plate and contacted with the polypeptide according to the disclosure.
  • ELISA can be used to detect and/or measure the interaction between the polypeptide and Zn++ in the presence and absence of the test compound. The effects of the test compound on the level of interaction can be analyzed (e.g., determining if the level of interaction decreases or increases in the presence of the test compound).
  • the GATA-like domain e.g., a polypeptide containing the GATA-liked domain
  • a polypeptide array may be incubated with a peptide array and binding of the polypeptide to specific peptide spots corresponding to a specific peptide sequence may be analyzed, for example, by polypeptide-specific antibodies, antibodies that are directed against an epitope-tag fused to the polypeptide, or by a fluorescence signal emitted by a fluorescent tag coupled to the polypeptide.
  • the effects of a peptide from the array on the interaction between the polypeptide and Zn++ can be analyzed in like manner.
  • a recombinant host cell described herein is contacted with a test compound.
  • test compounds e.g., polypeptides
  • FRET fluorescence resonance energy transfer
  • directly labeled test compounds may be added to the medium of the recombinant host cells.
  • the potential of the test compound to penetrate membranes and bind to the polypeptide may be, for example, verified by immunoprecipitation of the polypeptide and verification of the presence of the label.
  • the ability of the test compound to modulate e.g., decrease, e.g., inhibit) Zn++ binding of the polypeptide is assessed.
  • the purified polypeptide and Zn++ are contacted in the presence or absence of varying amounts of the test compound and incubated for a certain period of time, for example, for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 30, 40, 60, or 90 minutes.
  • the reaction conditions are chosen such that the polypeptide can bind Zn++ in the absence of the test compound.
  • the effects of the test compound on Zn++ binding can be analyzed (e.g., determining if the level of binding decreases or increases in the presence of the test compound).
  • the experiments may be applied in a multi-well plate format and are suitable for high throughput screening of test compounds regarding their ability to modulate (e.g., decrease, e.g., inhibit) Zn++ binding by the GATA-like domain.
  • the above-described methods for identifying compounds which modulate (e.g., decrease, e.g., inhibit) Zn++ binding by the GATA-like domain are performed in a high-throughput setting.
  • the term “crystal” or “crystalline” means a structure (such as a three- dimensional solid aggregate) in which the plane faces intersect at definite angles and in which there is a regular structure (such as internal structure) of the constituent chemical species.
  • the term “crystal” can include any one of: a solid physical crystal form such as an experimentally prepared crystal, a crystal structure derivable from the crystal (including secondary and/or tertiary and/or quaternary structural elements), a 2D and/or 3D model based on the crystal structure, a representation thereof such as a schematic representation thereof or a diagrammatic representation thereof, or a data set thereof for a computer.
  • the crystal is usable in X-ray crystallography techniques.
  • the crystals used can withstand exposure to X-ray beams and are used to produce diffraction pattern data necessary to solve the X-ray crystallographic structure.
  • a crystal may be characterized as being capable of diffracting X-rays in a pattern defined by one of the crystal forms depicted in T. L. Blundell and L. N. Johnson, "Protein Crystallography", Academic Press, New York (1976).
  • the term "unit cell” refers to a basic parallelepiped shaped block. The entire volume of a crystal may be constructed by regular assembly of such blocks. Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal.
  • space group refers to the arrangement of symmetry elements of a crystal.
  • the capital letter indicates the lattice type and the other symbols represent symmetry operations that can be carried out on the contents of the asymmetric unit without changing its appearance.
  • structure coordinates refers to a set of values that define the position of one or more amino acid residues with reference to a system of axes.
  • the term refers to a data set that defines the three- dimensional structure of a molecule or molecules (e.g., Cartesian coordinates, temperature factors, and occupancies).
  • S cortural coordinates can be slightly modified and still render nearly identical three-dimensional structures.
  • a measure of a unique set of structural coordinates is the root mean square deviation of the resulting structure.
  • Structural coordinates that render three-dimensional structures (in particular, a three- dimensional structure of an enzymatically active center) that deviate from one another by a root mean square deviation of less than 3 A, 2 A, 1 .5 A, 1 .0 A, or 0.5 A may be viewed by a person of ordinary skill in the art as very similar.
  • root mean square deviation means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object.
  • root mean square deviation defines the variation in the backbone of a variant of the polypeptide from the backbone of the polypeptide as defined by the structure coordinates of the polypeptide.
  • constructing a computer model includes the quantitative and qualitative analysis of molecular structure and/or function based on atomic structural information and interaction models.
  • modeling includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry, and other structure-based constraint models.
  • fitting program operation refers to an operation that utilizes the structure coordinates of a chemical entity, an enzymatically active center, a binding pocket, molecule or molecular complex, or portion thereof, to associate the chemical entity with the enzymatically active center, the binding pocket, molecule or molecular complex, or portion thereof. This may be achieved by positioning, rotating or translating the chemical entity in the enzymatically active center to match the shape and electrostatic complementarity of the enzymatically active center. Covalent interactions, non-covalent interactions such as hydrogen bond, electrostatic, hydrophobic, van der Waals interactions, and non- complementary electrostatic interactions such as repulsive charge-charge, dipole-dipole and charge- dipole interactions may be optimized.
  • Crystals can be grown by any method known to the person skilled in the art including, but not limited to, hanging and sitting drop techniques, sandwich-drop, dialysis, and microbatch or microtube batch devices. It would be readily apparent to one of skill in the art to vary the crystallization conditions disclosed above to identify other crystallization conditions that would produce crystals of the polypeptide alone or in complex with a compound.
  • Such variations include, but are not limited to, adjusting pH, protein concentration and/or crystallization temperature, changing the identity or concentration of salt and/or precipitant used, using a different method for crystallization, or introducing additives such as detergents (e.g., TWEEN 20 (monolaurate), LDOA, Brij 30 (4 lauryl ether)), sugars (e.g., glucose, maltose), organic compounds (e.g., dioxane, dimethylformamide), lanthanide ions, or poly-ionic compounds that aid in crystallizations.
  • detergents e.g., TWEEN 20 (monolaurate), LDOA, Brij 30 (4 lauryl ether)
  • sugars e.g., glucose, maltose
  • organic compounds e.g., dioxane, dimethylformamide
  • lanthanide ions e.g., lanthanide ions
  • poly-ionic compounds that aid in crystallizations.
  • micro- crystals are crushed to yield a stock seed solution.
  • the stock seed solution is diluted in series.
  • a needle, glass rod or strand of hair a small sample from each diluted solution is added to a set of equilibrated drops containing a protein concentration equal to or less than a concentration needed to create crystals without the presence of seeds.
  • the aim is to end up with a single seed crystal that will act to nucleate crystal growth in the drop.
  • Protein crystallization screens can be used, e.g., to identify or select crystallization conditions. Such screens are commercially available, e.g., MORPHEUSTM Screen (Molecular Dimensions, Suffolk, UK); Crystal Screen, Crystal Screen 2, and Crystal Screen HT (Hampton Research, Aliso Viejo, CA, USA); Wizard I, Wizard II, Wizard III, and Wizard IV screens (Emerald BioSystems, Bainbridge Island, WA, USA); and BIOXTALTM screen kit (XtalQuest, Beijing, CN).
  • X-ray diffraction data is first acquired, often using cryoprotected (e.g., with 20% to 30% glycerol) crystals frozen to 100 K, e.g., using a beamline at a synchrotron facility or a rotating anode as an X-ray source.
  • cryoprotected e.g., with 20% to 30% glycerol
  • the phase problem is solved by a generally known method, e.g., multiwavelength anomalous diffraction (MAD), multiple isomorphous replacement (MIR), single wavelength anomalous diffraction (SAD), or molecular replacement (MR).
  • MIR multiple isomorphous replacement
  • SAD single wavelength anomalous diffraction
  • MR molecular replacement
  • the substructure may be solved using SFDELXD (Schneider and Sheldrick, 2002, Acta Crystallogr. D. Biol. Crystallogr.
  • Crystallogr. 53: 240-255 The skilled person can use the structure coordinates, e.g., PDB ID: 2XUE (deposited on October 19, 2010 in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB)), as input for secondary analysis, including the determination of electrostatic surface potential, which aids in the determination of side groups in test compounds, which are likely to interact with a surface area of the polypeptide of a given electrostatic potential.
  • RCSB Research Collaboratory for Structural Bioinformatics
  • PDB Protein Data Bank
  • the structure coordinates generated for the polypeptide it is necessary to convert the structure coordinates into a three-dimensional shape. This is achieved through the use of commercially available software that is capable of generating three-dimensional graphical representations of molecules or portions thereof from a set of structure coordinates.
  • Such a three-dimensional graphical representation can be used with suitable programs including (i) Gaussian 92, revision C (Frisch, Gaussian, Incorporated, Pittsburgh, PA), (ii) AMBER, version 4.0 (Kollman, University of California, San Francisco, CA), (iii) QUANTA/CHARMM (Molecular Simulations Incorporated, San Diego, CA), (iv) OPLS-AA (Jorgensen, 1998, Encyclopedia of Computational Chemistry, Schleyer, Ed., Wiley, New York, Vol. 3, pp. 1986-1989), and (v) Insight ll/Discover (Biosysm Technologies Incorporated, San Diego, CA) to generate graphic representations of, e.g., electrostatic potential.
  • the structural information can be combined with information on the conservation of residues at the various amino acid positions to highlight those residues at the surface of the polypeptide.
  • incorpororation of selenomethionine into proteins in place of methionine aids the structure elucidation of proteins by X-ray crystallography using SAD or MAD.
  • the incorporation of heavy atoms such as selenium helps solving the phase problem in X-ray crystallography. Examples
  • Jumonji (Jmj) enzymes are histone demethylases critical for epigenetic mechanisms of cell regulation.
  • a crystal structure of the C-terminus of JMJD3 1 141 -1682 (del. 1637-1675) reveals that the catalytic domain of JMJD3 is associated with a zinc-binding domain (GATA-like domain) of unusual topology. This provides structural insights into mechanisms of specificity for this hitherto structurally unexplored branch of the Jmj phylogenetic tree important for biological function and drug discovery.
  • JMJD3 (KDM6B), one of the approximately 30 Jumonji (Jmj) family members found in humans, functions as a specific demethylase of lysine-27 of histone H3 (H3K27) (Hong, S.H. et al. Proc. Nat. Acad. Sci. l/S/4104, 18439-18444 (2007)).
  • JMJD3 at key cell fate decision points in T lymphocytes (Miller, S. A. et al. Genes Dev. 22 2980-2993 (2008)) and macrophages. It appears to be involved in acute, externally-driven, inflammatory processes.
  • JMJD3 is rapidly induced through an NF-kB-dependent mechanism in response to bacterial products and inflammatory stimuli (De Santa, F. et al. Ce// 130, 1083-1094 (2007)). JMJD3 has also been demonstrated to regulate the differentiation state of the epidermis (Sen, G.L. Genes Dev. 22 1865-1870 (2008)), to activate the tumour suppressor, INK4A-Arf (Sen, G.L. Genes Dev. 22 1865- 1870 (2008); Agger, K. et al. Genes Dev. 23, 1 171 -1 176 (2009)) and to be upregulated in prostate cancer (Xiang, Y. et al. Cell Res. 17, 850-857 (2007)).
  • Jmj demethylase enzymes have a catalytic (JmjC) domain of the cupin fold that contains a conserved motif of two histidines and either a glutamate or aspartate residue. These key residues coordinate the catalytic iron(ll) and co-factor, 2-oxoglutarate (2-OG), required for activity (Cloos, P. et al. Genes Dev. 22, 1 115-1 140 (2008)).
  • Jumonji enzymes possess one or more histone reader domains (e.g., PHD, Vietnamese, Fbox) that have been shown to be important both for determining substrate specificity and for localising the enzymes to their site of action.
  • the small subfamily of demethylases comprising JMJD3, UTX and UTY have an unique domain architecture amongst the Jmj family and do not contain a recognised reader domain. Much of their N-terminal region consists of sequences of low complexity, amongst which several TPR motifs, implicated in protein-protein interactions, can be detected. At the C-terminus, the catalytic core is followed by a signature for a treble-cleft zinc-finger domain (TCZ) (Cloos, P. et al. Genes Dev. 22, 1 1 15-1 140 (2008)) that is not found in other Jumonji enzymes. The function and structure of this domain is unknown.
  • TZ treble-cleft zinc-finger domain
  • JmjD3t a C-terminal construct of JMJD3 1 141 - 1682 (del. 1637-1675), henceforth referred to as JmjD3t, that contains the JmjC and TCZ domains.
  • This truncate demonstrates activity in vitro and in cells (data not shown and FIG. 6).
  • Substrate profiling of this enzyme alongside JmjD2A (as a 6His-tev-FLAG-JmjD2A 1 -350 fragment), with a panel of 198 histone peptides, showed that the preferred substrate for JmjD2A is H3K9Me3, whereas for JmjD3t H3K27Me3 was found to be most rapidly demethylated.
  • JmjD3t could tolerate posttranslational modifications on the adjacent residues: dimethylation of R26 and phosphorylation of S28.
  • JmjD3t was also shown to effectively demethylate di- as well as tri- methylated H3K27, in contrast to a H1390A catalytically deficient mutant (data not shown).
  • This purified crystallographic truncate therefore, has the same substrate specificity as reported for the full- length enzyme (Burgold, T. PLoS ONE 3 e3034 (2008)).
  • JmjD3t shows a protein of two domains, the expected catalytic domain and a four helical bundle within which we have observed a Zn-binding region we have termed a GATA-like (GATAL) domain (FIG. 1).
  • JMJD3 Despite the low sequence homology between JMJD3 and other JmjC domains the spatial distribution of the central beta-strands of its catalytic domain are similar to those observed for JmjD2A, FBX1 1 , and PHF8 (FIG. 3).
  • the surrounding loops and helices differ significantly and prevented the structure solution of JMJD3 by simple molecular replacement methods.
  • the structure was solved by SAD using SelenoMethionine labelled protein, as detailed below.
  • 2-OG makes an extensive network of hydrogen-bonding interactions.
  • the acid of the keto-acid makes direct interactions with the catalytic metal, Ser1398 and Asn1400, while the ketone completes the bi-dentate chelation of the iron.
  • the carboxylate of 2-OG makes four hydrogen bonds to Asn1400, a water, Thr1387 and Lys1381 . It is notable that the precise details of the interactions made by this common co-factor differs markedly amongst the Jmjs (FIG. 2) and these differences influence substrate and inhibitor selectivity.
  • PDCA pyridine-2,4-dicarboxylic acid
  • the GATAL domain of JMJD3 is an 84 residue insert (1543-1626) between helices 3 and 4 of a C- terminal helical bundle which is also found in PHF8 and FBX1 1 . It is not clear whether this helical domain is more generally found in JmjCs such as JmjD2A because the crystal structures are often truncated prior to the end of this helical region (FIG. 3). Whilst zinc-binding domains (ZBD) have been seen in the C-terminal region of a number of JmjC domain containing proteins, such as the PHD domain found in PHF8, the architecture of the ZBD in JMJD3 is distinct.
  • ZBD zinc-binding domains
  • the conserved zinc-binding cysteine motif within the GATAL domain is CxxC.CxxC and in JMJD3 consists of Cys1575, Cys1578, Cys1602 and Cys1605. Structurally this domain is related to the erythroid transcription factor, GATA, which also binds a single zinc ion. Therefore we will refer to this as a GATA-like (GATAL) domain.
  • GATA erythroid transcription factor
  • the topology of this fold is illustrated in FIG. 4.
  • the domain consists of a single treble-clef zinc-finger (Grishin, N.V. Nucleic Acids Res. 29, 1703-1714 (2001 )) fold elaborated with two extra strands.
  • Treble-clef domains embellished with additional structural elements have also been observed in the ubiquitin-binding domain ZnF UBP (Seigneurin-Berny, D. et al. Mol. Cell. Biol. 21 , 8035-8044 (2001 )), although the additional strands in ZnF UBP zinc fingers have a different topology to that found in JMJD3.
  • GATA-type zinc-fingers bind DNA and have also been observed to be involved in protein-protein interactions (Gamsjaeger, R., et al. Trends in Biochemical Sciences 32, 63-7014 (2007)).
  • this GATAL domain may have a role in specificity, by directly interacting with the substrate and/or localising JMJD3 to its chromatin target complex.
  • Comparison of the GATAL domain with that from GATA-1 shows that the long C-terminal tail of GATA-1 that binds to the DNA minor groove is absent in JMJD3 (FIG. 7). Instead, the region C-terminal to the helix of the treble clef forms one of the additional strands of the GATAL domain. This suggests that a DNA-binding function for this domain is unlikely.
  • GATA domains are also known to be mediators of protein-protein interactions (Gamsjaeger, R. et al. Trends in Biochemical Sciences 32, 63-70 (2007)) as exemplified by the interaction between GATA-1 and Friend of GATA (FOG) (Chu Kong Liew et al. Proc. Nat. Acad. Sci. USA 102, 583-588 (2005)).
  • the GATAL domain of JMJD3 may therefore be an additional recognition site for the histone substrate or a docking site of a yet unidentified partner protein. Inspection of the surface of JMJD3 shows that there is a groove in the surface of the zinc-binding domain that is aligned with the active site.
  • a GATAL domain is predicted to be present in the structures of the homologous proteins UTX and UTY in which the zinc chelating cysteines are completely conserved. A number of residues that appear critical for the structural integrity of this domain and its interface with the catalytic core are also fully conserved. Interestingly, there is significant variability of the residues that line the GATAL groove. Given that JMJD3, UTY and UTX are all H3K27 demethylases, it is intriguing to speculate that this variation may contribute to specificity beyond the active site and the ability of these proteins to carry out distinct functions.
  • JMJD3 subfamily of H3K27 demethylases The distinct active site geometry of JMJD3 and the presence of an integral zinc-binding domain provides novel insights into specificity determinants within and outside the catalytic domain with implications for its biological function and drug discovery.
  • JMJD3 DNA encoding the catalytic domain (residues 1 141 -1682) and lacking residues 1637-1675 (a splice variant deletion; GenBank Accession No. BC009994) was cloned into the pFB-HTb vector (Invitrogen) using BamHI and Xhol restriction sites, which encodes for a protein with an N-terminal 6His (6H) tag followed by a TEV-protease cleavage site.
  • pFB1 -HTb-JMJD3-6H 1 141 -1682 (del 1637- 1675) was transposed into the baculovirus genome using the BAC-to-BAC technology (Invitrogen).
  • Bacmid DNA was transfected into Spodoptera frugiperda (Sf9) cells using Cellfectin II (Invitrogen), and expression was performed at a 1 L scale in Excel 420 media (SAFC Biosciences).
  • the culture at a cell concentration of 3.8x10 6 cells/ml, was infected with P1 recombinant Baculovirus at a nominal multiplicity of infection of 3 and incubated for 48 hours.
  • the cells were removed from the media by centrifugation at 2500g for 20 minutes, and the cell pellet was frozen for subsequent purification.
  • the Baculovirus Expression Vector System (BEVS) was used to produce recombinant protein with Seleno-Methionine incorporated to enable phasing of the x-ray diffractions.
  • Spodoptera frugiperda (Sf9) cells were cultivated in suspension using Excell 420 media (SAFC Biosciences) on a shaker incubator (Kuhner) at 27°C, 80 RPM. In mid exponential growth, the cells were removed from the growth media by centrifugation at 250g for 10 minutes on a bench top centrifuge (Heraeus). The cells were inoculated into the expression media, "Methionine Free" Excell 420 (SAFC Biosciences), at a cell concentration of 1 .5x10 6 cells/ml.
  • the culture was incubated for 27 hours using the described conditions to metabolise the residual amounts of methionine present from hydrolysates in serum free insect media.
  • the culture at a cell concentration of 3.8x10 6 cells/ml, was infected with P2 recombinant Baculovirus at a nominal multiplicity of infection of 3 and incubated for 19 hours.
  • the pellet from the Baculovirus culture was resuspended in buffer A (20mM Tris pH 8.5, 300mM NaCI, 10% glycerol, 1 ul/ml Protease Inhibitor Cocktail Set III (Calbiochem 539134) and 10uM Fe(NH4)2(S04)2).
  • Eluted JMJD3 protein from the HisTRAP column was concentrated fourfold (Amicon Ultrafree-15 30kDa, Millipore UFC903024) and loaded onto a HiLoad 26/20 Superdex 200 prep grade size exclusion column (GE Healthcare 17-1069-01), equilibrated with buffer B (20mM Tris pH 8.0, 150mM NaCI, 5% glycerol, 0.5mM TCEP, 2mM a-Ketoglutaric acid sodium salt (Sigma K1875) and 10uM Fe(NH4)2(S04)2). Fractions containing JMJD3 were pooled and concentrated (Ultrafree-15 30kDa).
  • the pellet from the Baculovirus culture was resuspended buffer A (20mM Tris pH 8.5, 300mM NaCI, 10% glycerol, 1 ul/ml Protease Inhibitor Cocktail Set III (Calbiochem 539134) and 10uM
  • Eluted JMJD3 protein from the HisTRAP column was diluted 20-fold with buffer C (20mM Tris pH 8.5, 10% glycerol and 10uM Fe (NH4)2(S04)2) and applied to a HiTRAP Q HP column (GE Healthcare 17-1 154-01). The column was washed with ten columns of buffer C and eluted using a linear 0-1 M NaCI gradient over twenty column volumes. The JMJD3 protein was eluted between 350mM and 450mM NaCI.
  • JMJD3 protein eluted from the HiTRAP Q column was concentrated threefold (Amicon Ultrafree-15 30kDa, Millipore UFC903024) and loaded onto a HiLoad 26/20 Superdex 200 prep grade size exclusion column (GE Healthcare 17-1069-01), equilibrated with buffer B (20mM Tris pH 8.0, 150mM NaCI, 5% glycerol, 0.5mM TCEP, 2mM a-Ketoglutaric acid sodium salt (Sigma K1875) and 10uM Fe(NH4)2(S04)2). Fractions containing JMJD3 were pooled and concentrated (Ultrafree-15 30kDa). Protein identity was confirmed by peptide mass fingerprinting.
  • JMJD3 1 141 -1682 with no internal deletion was expressed as a 6H-Tev-Flag construct.
  • JMJD3 DNA encoding the catalytic domain (residues 1 141-1682) was amplified by PCR to contain a Flag tag at the N-terminus and the PCR product cloned into the pFB-HTb vector (Invitrogen) using BamHI and Xhol restriction sites, which encodes for a protein with an N-terminal 6His tag followed by a TEV- protease cleavage site then a Flag tag.
  • pFB1 -HTb-Flag-JMJD3 1 141 -1682 was transposed into the baculovirus genome using the BAC-to-BAC technology (Invitrogen). Bacmid DNA was transfected into Spodoptera frugiperda (Sf9) cells using Cellfectin II (Invitrogen), and expression was performed at a 1 L scale in Excel 420 media (SAFC Biosciences). The culture, at a cell concentration of 3.8x10 6 cells/ml, was infected with P1 recombinant Baculovirus at a nominal multiplicity of infection of 3 and incubated for 48 hours. The cells were removed from the media by centrifugation at 2500g for 20 minutes, and the cell pellet was frozen for subsequent purification. Purification of JMJD3-6H 1141 -1682
  • the column was washed with ten column volumes of buffer A, followed by ten column volumes of buffer A containing 20mM Imidazole. Bound protein was eluted from the column using a linear gradient of 20-250mM Imidazole over twenty column volumes. The JMJD3 protein was eluted between 130mM and 200mM Imidazole.
  • Eluted JMJD3 protein from the HisTRAP column was concentrated (Amicon Ultrafree-15 30kDa, Millipore UFC903024) and half was loaded onto a HiLoad 26/20 Superdex 200 prep grade size exclusion column (GE Healthcare 17-1069-01), equilibrated with buffer B (20mM HEPES pH 8.0, 150mM NaCI, 5% glycerol, 1 mM DTT and 10uM Fe(NH4)2(S04)2). The protein precipitated on the column.
  • the remaining protein was loaded onto a HiLoad 26/20 Superdex 200 prep grade size exclusion column (GE Healthcare 17-1069-01), equilibrated with buffer C (20mM Tris pH 8.0, 150mM NaCI, 5% glycerol and 1 mM DTT). The protein precipitated on the column.
  • Protein identity was confirmed by peptide mass fingerprinting and predicted molecular weight confirmed by mass spectrometry. (Observed the following MWs: 65581 Da (-8 Da), could be minus N-terminal Methionine, +1 P04 and +1 acetylation; 65694 Da (+105 Da), could be minus N-terminal Methionine and +3P04; 65808 Da (+219 Da), could be +1 acetylation and +2P04 and 65888 Da (+299 Da), could be + 1 acetylation and +3P04).
  • Eluted JMJD3 protein from the HisTRAP column was concentrated threefold (Amicon Ultrafree-15 30kDa, Millipore UFC903024) and half was loaded onto a HiLoad 26/20 Superdex 200 prep grade size exclusion column (GE Healthcare 17-1069-01), equilibrated with buffer D (20mM Tris pH 8.0, 150mM NaCI, 5% glycerol, 1 mM DTT and 1 ul/ml Protease Inhibitor Cocktail Set III (Calbiochem 539134)). The protein precipitated on the column.
  • Protein was supplied post SEC (size exclusion chromatography)/concentration at 10mg/mL in 20mM Tris pH 8.0, 150mM NaCI, 5% glycerol, 0.5mM TCEP, 10uM Fe(NH4)2(S04)2 and 2mM 2-OG.
  • Protein was mixed with an equal volume of Morpheus screen (Molecular Dimensions) condition #A12 (0.03M MgCI2, 0.03M CaCI2, 0.1 M tris HCI/bicine pH 8.5, 12.5%v/v MPD, 12.5%w/v PEG1 K, 12.5%w/v Peg3350) to a total drop volume of 300nL in a sitting drop MRC plate. Crystals grew at 20°C over a period of one week to an approximate size of 120x70x10 microns and were harvested into mother liquor plus 15% glycerol before flash freezing.
  • Morpheus screen Molecular Dimensions
  • X-ray diffraction data were collected at the European Synchrotron Radiation Facility at beam line ID14-4. Datasets were collected at 100 K using an ADSC Q315 CCD detector with an X-ray beam transmission of 50% and an oscillation range of 1 °. A fluorescence energy scan was taken in the vicinity of the selenium edge. The crystal used for data collection was sufficiently long that a strategy was adopted to try to ensure a highly redundant peak dataset could be collected for use in a SAD experiment, as well as further datasets for use in a MAD experiment. Datasets were collected at four different wavelengths using a different part of the crystal for each wavelength.
  • Data collection statistics are given in Table A for the two datasets that were used in the crystal structure determination.
  • the structure was solved using the Single-wavelength Anomalous Dispersion (SAD) technique.
  • SAD Single-wavelength Anomalous Dispersion
  • AUTOSHARP Vonrhein, Clemens; Blanc, Eric; Roversi, Pietro; Bricogne, Gerard.
  • Bond angles (°) 1.089 a Data for the highest resolution shell are given in parentheses.
  • JMJD3t JMJD3-6H 1 141 -1682 (del 1637-1675) shows a protein of two domains, the expected catalytic domain and a four helical bundle within which we have observed a Zn-binding region we have termed a GATA-like (GATAL) domain.
  • GATA-like domain Within the active site the catalytic metal is chelated by H1390, E1392, and H1470.
  • 2-oxoglutarate (2-OG) co-factor crystallised within JMJD3 reveals that 2-OG makes an extensive network of hydrogen-bonding interactions.
  • the acid of the keto-acid makes direct interactions with the catalytic metal, Ser1398 and Asn1400, while the ketone completes the bi-dentate chelation of the iron.
  • the carboxylate of 2-OG makes four hydrogen bonds to Asn1400, a water, Thr1387, and Lys1381 . It is notable that the precise details of the interactions made by this common cofactor differ markedly amongst the Jmjs and these differences are likely to influence substrate and inhibitor selectivity.
  • Additional polypeptides that were prepared and crystallized include:
  • JMJD3 1 141 -1682 (del 1637-1675) (PreScission protease cleaved protein to remove C- terminal 6H tag)
  • JMJD3 1 141 -1682 (del 1637-1675) (TEV cleaved protein to remove C-terminal 6H tag)
  • the domain is inserted into a bundle of four helices which are seen in a number of JMJc domain structures, for example FBXL1 1 (PDB accession code 2YU1).
  • the domain is formed by extension of one of the helices in the bundle, the zinc binding treble clef fold and some additional beta strands.
  • the topology of the additional beta strands is not the same as seen in the ZnF-UBP domain type.
  • the extent of the domain is defined from a superposition of the structure of JMJD3 with the structure of FBXL1 1 (PDB accession code 2YU1).
  • the first residue of the domain is taken as the first residue within the extended helix that is not superimposed with a helical residue in 2YU1 , that is serine 1543 in JMJD3.
  • the last residue of the domain is defined as the residue before the first match of residue position and conformation in the final helix of the four helix bundle, glutamate 1626 in JMJD3.
  • This domain may have a role in specificity, either by directly interacting with the substrate or localising JMJD3 to its chromatin target complex.
  • the domain is proximal to the active site in the catalytic domain, and could interact with the H3 histone tail and/or JMJD3 partner proteins or DNA.
  • FDH formaldehyde dehydrogenase
  • the H3 and H3, H4 N terminal libraries were purchased from Alta Bioscience, University of Birmingham, UK.
  • JMJD3 a 6His- tev-FLAG-JMJD3 1 141 -1682 (del 1637-1675) was used and prepared as described above.
  • JMJD2A a 6His-tev-FLAG-JMJD2A 1 -350 fragment was used.
  • Human embryonic kidney (HEK) 293 cells were maintained at 37°C in DMEM/F-12 medium
  • HEK cells were cultured to 80% confluency prior to being transduced in suspension at a multiplicity of infection of 100 with either FLAG-tagged JMJD3 1 141 -1682 wild type or FLAG-tagged JMJD3 1 141 -1682 demethylase dead (H1390A) BacMam viruses.
  • Cells for immuno-fluorescence and cellular imaging were seeded onto cover slips and Greiner poly-D-lysine coated 384 well plates respectively. 24 hours post-transduction, the respective cell lines expressing the ectopic JMJD3 wild type and JMJD3 H1390A mutant forms were fixed with 4% paraformaldehyde for 15 minutes.
  • JMJD3 MALDI-TOF mass spectrometric assay monitors demethylation of a histone H3 peptide containing tri-methylated K27 by recombinant Jumonji D3 (JMJD3) demethylase enzyme.
  • JMJD3 Jumonji D3
  • JMJD3-6H 1 141 -1682 (del 1637-1675) protein was prepared as detailed above.
  • ATKAARKSAPATGGVKKPHRYRPG (the bold underlined lysine corresponds to K27 in human histone 3 and is tri-methylated) (SEQ ID NO:5): ATKAAR-K(TriMe)- SAPATGGVKKPHRYRPG (SEQ ID NO:6)
  • the peptide was supplied by Cambridge Research Biochemicals.
  • the protected peptide was assembled on a solid-phase synthesiser using preloaded Wang resin and utilising standard Fmoc synthesis protocols.
  • the crude peptide was obtained after cleavage from the resin with a mixture of trifluoroacetic acid (TFA), triisopropylsilane and water (95:2.5:2.5) for 3 hours at room temperature and was then purified using a C18 reverse-phase column utilising a 0.1 %TFA-buffered
  • the trimethyl lysine was incorporated into the sequence during assembly as Fmoc-Lys(TriMe)-OH. This was made by reacting a suitably protected alpha-nitrogen lysine with an excess of methyl chloride or bromide to form the quaternary salt.
  • reaction was quenched by the addition of 10ul of a 1 % TFA solution to all wells except column 18 (to which this was previously added) using a Multidrop COMBI®dispenser.

Abstract

L'invention concerne des polypeptides présentant l'activité catalytique de JMJD3 et contenant des séquences nucléotidiques et d'acides aminés les codant, l'invention concernant également les utilisations et la structure cristalline de ces polypeptides.
PCT/EP2011/068087 2010-10-19 2011-10-17 Polypeptide ayant l'activité catalytique de jmjd3 WO2012052391A1 (fr)

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CN110964765A (zh) * 2018-09-30 2020-04-07 中国科学院大连化学物理研究所 一种利用甲醛还原nad类似物的方法
US11524011B2 (en) 2017-09-01 2022-12-13 The Johns Hopkins University H3K27 demethylase inhibitors in pediatric and juvenile osteoporosis

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