WO2022025502A1 - Method for screening histone h2b epigenetic regulator targeting phf6 - Google Patents

Method for screening histone h2b epigenetic regulator targeting phf6 Download PDF

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WO2022025502A1
WO2022025502A1 PCT/KR2021/009223 KR2021009223W WO2022025502A1 WO 2022025502 A1 WO2022025502 A1 WO 2022025502A1 KR 2021009223 W KR2021009223 W KR 2021009223W WO 2022025502 A1 WO2022025502 A1 WO 2022025502A1
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phf6
histone
protein
domain
ubiquitination
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PCT/KR2021/009223
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French (fr)
Korean (ko)
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백성희
오성룡
부경진
박대찬
이지민
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서울대학교산학협력단
아주대학교산학협력단
강원대학교산학협력단
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Priority to JP2022563022A priority Critical patent/JP2023521927A/en
Publication of WO2022025502A1 publication Critical patent/WO2022025502A1/en

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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
    • 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

Definitions

  • This application is a field related to a drug screening technology based on a molecular mechanism that elucidates the histone H2B epigenetic regulation of PHF6.
  • Histone modification is one of the important biological phenomena that regulate gene expression by causing chromatin changes in cells. Chromatin is largely bound to four types of histone proteins, H2A, H2B, H3 and H4, and it is known that various types of modifications such as acetylation exist at the N-terminus of each histone protein. These modifications activate or repress specific gene expression, which is referred to as epigenetic regulation. Epigenetic regulatory proteins/genes involved in these histone modifications are functionally classified into writers, erasers, and readers.
  • the leader is a protein with the function of recognizing histone modifications, and its importance is emerging. Unlike writers and erasers, it is the most important to understand the interaction between different types of histone modifications (histone modification crosstalk). This is because it is the person who becomes
  • histone modifications are essential concepts in understanding the complex system in which the expression of genes in vivo must be precisely regulated.
  • WO 2013-138237 relates to a method and composition for the treatment, diagnosis or prognosis of acute leukemia, and discloses PHF6 as one of the markers for judging the prognosis of leukemia. It is necessary to discover targets based on the epigenetic regulatory mechanism of histone proteins.
  • An object of the present application is to provide a drug screening method based on the epigenetic regulation mechanism of histone H2B by PHF6, which is known as a key mutant gene in hereditary diseases such as leukemia and BFL syndrome.
  • the present application provides a PHF6 (PHD finger-containing protein 6) protein and a histone 2B (H2BK12-Ac) protein in which the 12th lysine residue is modified by acetylation; contacting the PHF6 protein and the H2BK12-Ac protein in the presence of a test substance expected to modulate the recognition of the H2BK12-Ac by the PHF6; measuring ubiquitination (H2BK120-ub) of the 120th lysine residue of the histone protein; And, as a result of the measurement, when there is a change in the ubiquitination level of the 120th lysine residue of the histone protein in the cells treated with the test substance compared to the control group not treated with the test substance, the test substance is treated with the PHF6 activity It provides a method for screening a histone epigenetic modulator by PHF6, comprising the step of selecting a modulatory candidate.
  • a change in level means an increase or decrease compared to an untreated control, and substances capable of affecting the increase or decrease of H2BK120-ub may be usefully used as histone epigenetic modulators.
  • a person skilled in the art will be able to select a decrease or an increase according to the specific purpose.
  • Epigenetic modulators selected and developed according to the method herein can be developed for the treatment of leukemia.
  • PHF6 protein and histone 2B derived from mammals including humans may be used, and those skilled in the art will be able to select appropriate ones in consideration of experimental conditions and the like. In one embodiment, those of human origin are used.
  • the method according to the present application can be performed in eukaryotic cells (over)expressing the protein used in the method of the present application, for example, non-human embryonic stem cells or non-human animal models.
  • the epigenetic regulation of histone 2B protein by PHF6 involves the PHD1 and PHD2 domains of the PHF6 protein, the H2BK12-Ac is recognized by the PHD2 domain, and the ubiquitination of the H2BK120 is by the PHD1.
  • the present application provides a PHD1 domain of PHF6; or providing a PHF6 protein and a histone 2B protein in which the PHD2 domain is deleted or mutated (loss of function due to mutation such as point, deletion, substitution, etc.); contacting the PHF6 protein in which the PHD1 domain or the PHD2 domain is deleted or mutated in the presence of a substance expected to promote binding of the histone 2B protein; measuring the ubiquitination level of the 120th lysine residue of the histone 2B protein; And when the ubiquitination of the 120th lysine residue of histone 2B protein is increased when the test substance is treated as compared to the test substance and the untreated control as a result of the measurement, selecting the test substance as a candidate for regulating PHF6 activity, A method for screening histone epigenetic modulators by PHF6 is provided.
  • the PHD1 domain; Alternatively, the PHF6 protein and histone 2B protein in which the PHD2 domain is deleted or mutated may be provided as eukaryotic cells or animal models other than humans.
  • the eukaryotic cells are embryonic stem cells of animals other than humans.
  • Epigenetic modulators selected and developed according to the method herein can be developed for the treatment of leukemia.
  • FIG. 1 shows a defect occurs in the blastocyst differentiation stage of embryonic stem cells due to Phf6 deficiency.
  • A Schematic diagram of Phf6 gene knockout (KO) construction using the CRISPR-Cas9 gene editing system. Guide RNA sequences and altered sequences for the Phf6 gene are indicated.
  • B Immunoblot analysis showing PHF6 deficiency and Phf6 knockdown by shRNA in Phf6 KO embryonic stem cells. Nanog as a positive control, and ⁇ -actin as a loading control.
  • C Growth curve of Phf6 KO embryonic stem cells, growth curve of WT embryonic stem cells, growth rate curve of shPhf6, and growth curve of shNS.
  • E Schematic of DEG analysis between WT and Phf6 KO ZHBTc4 during DOX treatment-induced blastocyst lineage reprogramming and RA treatment-induced neuroectoderm differentiation.
  • 3) is indicated by the red dotted line.
  • Placental development is the most significant biological function in cluster 1.
  • E Representative gene set analysis of DEG (GSEA). The gene sets involved in embryonic placental development and trophoblast giant cell differentiation were significant. The median value of (C) was used as a reference for pattern analysis of gene expression.
  • F Down-regulated blastocyst differentiation gene family in Phf6 KO embryonic stem cells compared to WT. Color bars represent the log2 value of the expression change in each comparison. Genes extracted from GSEA were selected.
  • G qRT-PCR analysis of blastocyst genes in WT and Phf6 KO ESCs with and without DOX treatment. The mRNA levels of each gene were determined relative to Gapdh and compared relative to WT-DOX. Statistical significance was calculated by ANOVA test (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001).
  • FIG. 3 shows that PHF6 activates the expression of blastocyst marker genes for blastocyst lineage determination.
  • B-C Immunostaining of CDX2 and PHF6 in the outer cell layer of WT EBs. Magnifications X100 (B) and X200 (C).
  • FIG. 4 PHF6 recognizes H2BK12Ac and regulates single ubiquitination of H2BK120.
  • A Far western analysis of histone proteins using GST or GST-PHF6 proteins. Analysis was performed on histone extracts obtained from ZHBTc4 embryonic stem cells. Histone extracts were separated into H3, H2B, H2A and H4 according to their size on SDS-PAGE.
  • B Top five histone variants showing the highest affinity for GST-PHF6. Screening was performed using the Histone Peptide Array Kit.
  • C Confirmation of the recognition ability of GST-PHF6 for the top five modified histone peptide candidates. Histone peptide pulldown analysis was performed.
  • E In vitro peptide binding assays were performed with GST-PHF6 WT or E223S mutants.
  • PHF6 is an acetylation-dependent E3 ubiquitin ligase for histone H2B of K120.
  • A Flow diagram of the in vitro ubiquitination assay.
  • B In vitro ubiquitination assay using single-nucleosomes containing H2B WT or K12R mutations. Single-nucleosomes were extracted from DOX-treated Phf6 KO ZHBTc4.
  • C In vitro ubiquitination assay of single-nucleosomes containing H2B-Flag WT with GST-PHF6 WT, C82A or E223S mutations.
  • (G) In vitro GST-pulldown analysis using GST, GST-PHF6 WT or mutants of His-UBCH3.
  • E CHIP analysis of the promoter of the Phf6 independent expression gene Msx2 by DOX treatment using IgG, PHF6, H2BK120ub, H3K4me3 and H2BK12Ac antibodies in WT and Phf6 KO. Statistical significance was calculated by ANOVA test (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001).
  • E-F Generation of pLVX-PHF6 WT or MTs stable cells by lentiviral infection in Phf6 KO ESCs. Immunoblot analysis of histone H2BK120ub and PHF6 levels of stable structures of Phf6 KO ESCs of Phf6 WT or MT by DOX treatment (E).
  • FIG. 7 is a schematic diagram of the function of PHF6 on the expression of blastocyst genes.
  • the schematic model shows that PHF6 functions as a transcriptional activator of blastocyst genes during blastocyst reprogramming.
  • PHF6 recognizes H2BK12Ac through its second extended PHD domain and ubiquitinates the H2BK120 residue through the first extended PHD domain on the promoter of the blastocyst genes (top).
  • the level of H2BK120ub on the promoter of blastocyst genes does not increase, indicating that it leads to failure of transcriptional activation of blastocyst genes (below).
  • PHF6 PHF finger protein 6
  • H2BK12 histone 2B, 12th lysine residue
  • ubiquitin of H2BK120 histone 2B, 120th lysine residue
  • the present application provides a PHF6 protein and a modified histone 2B (H2BK12-Ac) protein in which the 12th lysine residue is acetylated; contacting said protein in the presence of a test substance expected to modulate recognition of said H2BK12-Ac of said PHF6; measuring the ubiquitination (H2BK120-ub) of the 120th lysine residue of H2B; And, as a result of the measurement, when there is a change in the ubiquitination level of the 120th lysine residue of H2B in the cells treated with the test substance compared to the control not treated with the test substance, the test substance is adjusted to the PHF6 activity It relates to a method for screening a histone epigenetic modulator by PHF6, comprising the step of selecting a candidate substance.
  • PHF PHD finger-containing protein 6
  • PHF6 has been the most studied in leukemia, and mutations in PHF6 are known in several leukemia reports (Van Vlierberghe, P. et al. (2010) PHF6 mutations in T-cell acute lymphoblastic leukemia. Nat Genet , 42, 338-342) .; Van Vlierberghe, P. et al. (2011) PHF6 mutations in adult acute myeloid leukemia. Leukemia , 25, 130-134.).
  • PHF6 performs a key function of the H2BK12 acetylation-H2BK120 ubiquitination interaction.
  • two reader domains called PHD2 and 1 included in PHF6 recognize H2BK12 acetylation and H2BK120 ubiquitination, respectively. was found to be involved in (see FIG. 7 and the like).
  • the PHD (Plant Homeodomain) domain is the domain most known for its ability to recognize K4me2/3 of histone H3. is known However, the PHD domain has greater diversity per gene compared to other domains. It is also known that half of the PHD domain captures three zinc cations, and one PHD domain is directly connected to capture four zinc cations. There are also known cases.
  • PHF6 whose function has been identified herein includes two extended PHD (extended PHD) domains to which half of the PHD is further attached.
  • the human PHF6 protein sequence herein is represented by the sequence of SEQ ID NO: 1.
  • PHD1 domains are 14 to 132, and PHD2 is 209 to 330.
  • the gene sequence encoding it can be readily determined by those skilled in the art.
  • the human PHF6 gene sequence is represented by SEQ ID NO:2.
  • histone proteins are basic proteins that pack DNA in eukaryotic cells to make structural units called nucleosomes.
  • H2A, H2B, H3 and H4 constitute the core of the nucleosome, and are H1/H5 linker histones. Core histones exist as dimers.
  • the PHF6 PHD2 domain recognizes the acetylated 12th residue of H2B.
  • the PHD1 domain of PHF6 is an E3 ubiquitin ligase that ubiquitinates the 120th lysine residue of H2B.
  • Ubiquitin is a protein composed of 76 amino acids, and gene transcription is promoted in chromatin wound on histones bound to ubiquitin.
  • H2B NCBI The gene IDs of human and mouse H2B NCBI are: 8347 and 68024, respectively.
  • H2B protein and gene sequences are known from the NCBI as follows: human protein sequence of H2B: NP_001368918.1, human gene sequence of H2B: NM_001381989.1; Mouse protein sequence: NP_001277309.1, gene sequence: NM_001290380.1.
  • the PHF6 protein and the modified histone 2B (H2BK12-Ac) protein in which the 12th residue is acetylated can be provided as an isolated protein or a cell or animal model expressing the protein. .
  • the protein may be used after being isolated/purified from eukaryotic cells.
  • the isolated histone protein is post-translational modification (PTM) produced after protein synthesis in eukaryotic cells, and includes acetylation at the N-terminal region of the histone protein.
  • PTM post-translational modification
  • the H2B 12th lysine residue is an acetylated protein. If necessary, it can be used separately from prokaryotic cells.
  • the isolated protein may be acetylated in vitro using an acetylation enzyme if necessary. (David Kuninger et al., J Biotechnol , 15; 131(3): 253-260.).
  • eukaryotic cells may be used.
  • the eukaryotic cells may be established eukaryotic cells or animal embryonic stem cells.
  • the cell used in the method according to the present application is a cell derived from a mammal, including a human or mouse, and is a eukaryotic cell overexpressing PHF6.
  • the eukaryotic cell may be ZHBTc4 (mouse embryonic stem cell), E14Tg2A.4 (mouse embryonic stem cell), HEK293T (embryonic kidney), or Hela (cervix cancer cell line).
  • the cell line is commercially available. Methods for overexpressing a specific gene in a specific cell line are known and can be used, for example, by transfecting a plasmid encoding human PHF6 protein into a target cell line.
  • the cell line is not limited thereto, and various cells capable of achieving the object according to the present disclosure may be used.
  • the method according to the present disclosure includes measuring the degree of ubiquitination of the histone protein in a protein or cell treated with a test substance.
  • Methods for measuring ubiquitination of histone H2B are known, for example, reference may be made to those described in the Examples herein (Zhang et al. Nat. Commun 8:14799 (2017)).
  • the test substance can be selected as a histone epigenetic modulator candidate. have.
  • the cells used in the method according to the invention are part of an animal model, for example a mouse.
  • a mouse is used.
  • the cells used in the method according to the present disclosure may be provided as organoids containing such cells or as non-human animal models.
  • Such an animal model may be prepared using the examples herein or methods known in the art.
  • an organoid is a mini-organ produced in vitro in a three-dimensional environment representing the microstructure of an actual organ. Cells with self-renewal and differentiation ability are created in a three-dimensional culture environment.
  • a method for preparing an organoid may refer to a known (Organoid Culture handbook, Ambiso).
  • H2BK12 histone 2B, lysine residue 12
  • ubiquitination of H2BK120 histone 2B, lysine residue 120
  • the present application provides a PHD1 domain of PHF6; or providing a PHF6 protein and a histone 2B protein in which the PHD2 domain is deleted or mutated; contacting the protein with the PHD1 domain or the PHD1 domain included in the PHF6 protein in the presence of a substance expected to promote binding of the histone 2B protein; measuring the ubiquitination level of the 120th lysine residue of the histone 2B protein; And when compared with the test substance and the untreated control as a result of the measurement, when the treatment increases ubiquitination at the 120th position of the histone 2B protein, it relates to a screening method for a histone epigenetic modulator by PHF6.
  • the PHF6 protein in which all or part of the PHD2 domain is deleted or mutated can be prepared by those skilled in the art by a genetic method based on the examples herein and the human gene and protein sequences disclosed herein.
  • the PHD2 domain mutation refers to a loss-of-function mutation, but in particular, a glutamic acid rich motif (see FIG. 4 ), which is important for H2BK12Ac recognition, is mutated to another amino acid by deletion or substitution.
  • the protein used in the method may be provided in the form of a eukaryotic cell, an animal model, or an organoid expressing the protein, for which reference may be made to the aforementioned bar.
  • the method for measuring ubiquitination used in the above method may refer to the aforementioned bar.
  • test substance used in the method according to the present application modulates the recognition of H2BK12-Ac of PHF6 identified herein and is expected to result in modulation of H2BK120 ubiquitination linked thereto, for example, low molecular weight compounds, high molecular weight compounds, compounds mixtures of these (e.g., natural extracts or cell or tissue cultures), or biopharmaceuticals (e.g., proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzyme and DNAzyme) or sugars and lipids, etc. including, but not limited to.
  • biopharmaceuticals e.g., proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzyme and DNAzyme
  • sugars and lipids etc. including, but not limited to.
  • a low molecular weight compound may be used as a test substance.
  • the test substance may be obtained from a library of synthetic or natural compounds, and methods for obtaining a library of such compounds are known in the art.
  • Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and natural compound libraries are available from Pan Laboratories (USA) and It can be purchased from MycoSearch (USA).
  • Test substances can be obtained by various combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution Required synthetic library methods, "1-bead 1-compound” library methods, and synthetic library methods using affinity chromatography selection can be obtained.
  • Methods for synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem.
  • compounds having a low molecular weight therapeutic effect may be used.
  • a compound having a weight of about 1000 Da, such as 400 Da, 600 Da, or 800 Da may be used.
  • Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and template-based small molecule compounds such as benzodiazepines, hydantoins, biaryls, carbocycles and polycyclic compounds (such as naphthalene, phenothi azine, acridine, steroids, etc.), carbohydrates and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely exemplary.
  • the present invention is not limited thereto.
  • biologics can be used for screening.
  • Biologics refer to cells or biomolecules
  • biomolecules refer to proteins, nucleic acids, carbohydrates, lipids, or substances produced using cell systems in vivo and in vitro.
  • the biomolecule may be provided alone or in combination with other biomolecules or cells.
  • Biomolecules include, for example, polynucleotides, peptides, antibodies, or other proteins or biological organisms found in plasma.
  • an increase or decrease that can select a candidate substance in the method according to the present application can be appropriately selected by those skilled in the art in consideration of the results disclosed herein and common knowledge of those skilled in the art, for example, but not limited thereto, with the test substance and the test substance.
  • a mouse embryonic stem cell line capable of conditionally inducing Oct4 deficiency is known.
  • Cell cultures were briefly maintained in a litter of mouse embryonic fibroblasts in the first 1-2 passages. After stabilization in this state, embryonic stem cells were cultured using a culture dish coated with 0.1% gelatin.
  • the culture medium was 15% fetal bovine serum (FBS; Hyclone), 0.055 mM beta-mercaptoethanol, 2 mM L-glutamine, 0.1 mM ratio in Dulbecco's modified Eagle medium (DMEM; Welgene).
  • FBS fetal bovine serum
  • DMEM Dulbecco's modified Eagle medium
  • Non-essential amino acids 5,000 units/ml penicillin/streptomycin (GIBCO), and 1,000 units/ml leukemia inhibitory factor (LIF) (Chemicon) were added in a humidified incubator at 37°C. cultured. All cell lines were routinely tested for mycoplasma contamination.
  • GIBCO penicillin/streptomycin
  • LIF leukemia inhibitory factor
  • siRNA sequences for knockdown of CBP and p300 were designed at http://gesteland.genetics.utah.edu/siRNA_scales.
  • the siRNA sequence for knockdown of RNF20 was used as previously reported.
  • the information of the antibody used in the experiment is as follows.
  • Novus anti-PHF6 (#68262); Abcam anti-nanog (ab21624), anti-H3 (ab1791), anti-H2B (ab1790), anti-H4 (ab10158), anti-H3K4me3 (ab8580), anti-H3K4me1 (ab8895), anti-H3K27me3 (ab6002) ), anti-H3K27Ac (ab4729), anti-H3K9me3 (ab8898), anti-H2BK12Ac (ab195494), anti-RNF40 (ab191309) and anti-Cdx2 (ab157524); Anti-USP44 (sc-377203), anti-p300 (sc-584), anti-CBP (sc-1211), anti-Oct4 (sc-5279), anti-GAPDH (sc-25778) and anti-GST from Santacruz (sc-459); anti-RNF20 from Bethly (A300-7
  • shRNA shRNA knockdown cells To construct short hairpin RNA (shRNA) shRNA knockdown cells, lentiviral shRNA was transfected into HEK293T cells together with viral packaging plasmids (psPAX2 and pMD2.G). One day after transfection, when the cells were in a state of 60 to 70% growth, the medium was changed and maintained for 24 hours for virus collection. After virus collection, the medium was filtered and mixed with a 4X Lenti-X collector (#631232, TAKARA) and incubated overnight at 4°C. Then, the collected virus was mixed with polybrene to infect the cells.
  • the shRNA sequence is as follows:
  • Phf6 Knock Out (KO) mouse ESC generation Phf6 KO mouse embryonic stem cells were produced using the CRISPR-Cas9 system.
  • a single guide RNA (sgRNA) design was performed from GPP sgRNA Designer (CRISPRko). The selected sgRNA was cloned into the pRGEN-U6 vector and transfected into ZHBTc4. Phf6 KO colonies were obtained after single colony selection by puromycin treatment, and after confirming by immunoblotting, frame-shift mutations were confirmed by Sanger-sequencing.
  • Embryo body (EB) formation ZHBTc4 embryonic stem cells were started at 1000 cells/drop by the hanging-drop method. After maintaining in the droplet state for 2 days, each droplet was transferred to an uncoated sterile cover glass and a round-bottom 96-well plate for incubation. Medium was added or changed daily, and plate movement was kept to a minimum to minimize physical impact on EBs. All EBs were cultured using LIF-free medium.
  • AP staining For AP staining, wild-type (WT) and Phf6 KO ZHBTc4 were cultured on 0.1% gelatin-coated cover glass in 12 well plates. 10 4 cells per well were initially seeded. After 2 days, AP staining was performed using an alkaline phosphatase detection kit (# SCR004, Millipore). Staining was performed according to the protocol provided. Briefly, each cell was fixed with 4% paraformaldehyde for 1-2% and reacted with the solution (naphthol/fast red violet mix) for 15-20 min at room temperature. After the reaction, the solution was washed and an image slide was made.
  • WT wild-type
  • Phf6 KO ZHBTc4 Phf6 KO ZHBTc4 were cultured on 0.1% gelatin-coated cover glass in 12 well plates. 10 4 cells per well were initially seeded. After 2 days, AP staining was performed using an alkaline phosphatase detection kit (# SCR00
  • a JuLI Stage real-time cell history recorder (NanoEnTeK) was used.
  • 10 4 cells were seeded in 0.1% gelatin-coated 12-well culture dishes, and the degree of cell growth was recorded every 12 hours. After the recording was complete, the cell growth curves were analyzed by the software provided by JuLI.
  • JuLI For embryoid body formation, each drop was transferred to each well of a 96-well round plate and cell morphology was recorded every 12 h. Then, images were taken with the provided software. To calculate the cell area outside the embryonic core, the area size was calculated from the images taken on day 7 using the ImageJ program.
  • Embryo bodies for immunofluorescence imaging were grown on coverslips (1 embryo per coverslip) prior to experiments. Embryo bodies were fixed with 2% paraformaldehyde mixed in PBS for 10 minutes, and then washed twice with DPBS at room temperature. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS (PBS-T) for 5 min at room temperature. Blocking was performed with 10% FBS in 0.1% PBS-T for 30 minutes. For staining, cells were incubated with primary antibody for 4 h at room temperature, washed 4 times with 0.1% PBS-T, and incubated with fluorescently labeled secondary antibody (Invitrogen) for 1 h. Cells were washed and fixed by VECTASHIELD (H-1200, Vector Laboratories) with DAPI (Sigma). Fluorescence was captured on a Zeiss LSM700 confocal microscope (Carl Zeiss).
  • Lysis Cells were briefly rinsed with cold PBS prior to lysis . Cells were lysed in a protease inhibitor-added lysate (50 mM Tris-HCl pH 8.0, 200 mM NaCl and 0.5% NP-40), and then the cells were lysed by sonication using a Branson Sonifier 450 (outputs 4, 10 to 12 pulses). Lysates were quantified by the Bradford method and analyzed by SDS-PAGE.
  • protease inhibitor-added lysate 50 mM Tris-HCl pH 8.0, 200 mM NaCl and 0.5% NP-40
  • E. coli GST-PHF6 protein was purified using GST beads. Histone extracts were separated from ZHBTc4 embryonic stem cells by SDS-PAGE. After separation of histone octamers in the order of H3, H2B, H2A and H4 according to size, 0.1 mg/ml of purified GST-PHF6 protein was added to a buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.6], 10% Glycerol, 0.1% Tween-20, 50 mM ZnCl 2 , 2% skim milk powder and 1 mM DTT) were reacted with histone extracts. After binding of GST-PHF6 to the histone-separated membrane, immunoblot analysis was performed using an anti-GST antibody.
  • a buffer 100 mM NaCl, 20 mM Tris-HCl [pH 7.6], 10% Glycerol, 0.1% Tween-20, 50 mM ZnCl 2 , 2% skim milk
  • In Vitro Histone Peptide Binding Array Histone Peptide Binding Array Kit was purchased from ActiveMotif (#13005). The following assays were performed according to the provided protocol. Briefly, the kit was blocked with TTBS (10 mM Tris-HCl [pH 7.5], 150 mM NaCl and 0.05% Tween-20) mixed with 5% milk overnight at 4°C. The kit was then incubated with 0.3 ⁇ g of eluted GST-PHF6 in binding buffer (100 mM KCl, 20 mM HEPES [pH 7.9], 1 mM EDTA, 10% glycerol and 0.1 mM DTT) for 1 h at room temperature. Then, the GST primary antibody and secondary antibody were sequentially incubated for 1 hour at room temperature in the kit. Washing was performed three times with TTBS between each step.
  • TTBS Tris-HCl [pH 7.5], 150 mM NaCl and 0.05% Tween-20
  • PHF6 WT and E223S mutants were expressed as GST fusion proteins in Rosetta (DE3) E. coli cells.
  • Cells were harvested after induction with 0.25 mM isopropyl 1-thio- ⁇ -D-galactopyranoside (IPTG) followed by overnight growth at 20 °C, 500 mM NaCl and 1 mM phenylmethane sulfonyl fluoride (PMSF) This was dissolved using Emulciplex C3 (Avestin) in supplemented phosphate buffered saline (PBS).
  • IPTG isopropyl 1-thio- ⁇ -D-galactopyranoside
  • PMSF phenylmethane sulfonyl fluoride
  • Each GST-PHF6 (WT and E223S) was purified on a HiTrap SP cation exchange column, a Superdex 200 size exclusion column, pre-equilibrated with gel filtration buffer (20 mM HEPES-NaOH [pH 7.5] and 150 mM NaCl).
  • GST was expressed in Rosetta (DE3) E. coli cells and purified similarly as described above, except that a HiTrap Q anion exchange column was used instead of a HiTrap SP column.
  • MST MicroScale Thermophoresis
  • Binding measurements were performed with a Nanotemper Monolith NT.115pico.
  • H2B peptide (1-20) WT and peptides with K12Ac modifications were purchased from Genscript and GST-PHF6 (WT and E223S MT) and GST, respectively, were labeled with the dye NT-647 (Cy5) (Lumiprobe). Labeled GST and GST-PHF6 were used at a concentration of ⁇ 90 nM.
  • H2B peptide Dilute each H2B peptide from ⁇ 880 mM to 26 nM in MST buffer (20 mM HEPES-NaOH [pH 7.5], 150 mM NaCl, 0.05% Tween-20 and 0.5 mg/mL BSA), with labeled protein at room temperature for 10 min. cultured in MST measurements were performed at 22°C with either 40% MST power and 25% LED power (GST-PHF6 WT) or 12% LED power (GST-PHF6 E223S and GST). Each data set was analyzed using MO (Nanotemper Technology).
  • H2B-Flag WT/K12R was transfected into Phf6 KO embryonic stem cells, and H2B-Flag containing mono-nucleosomes was purified by MNase digestion. After purification, these nucleosomes were mixed with E1, E2, E3 enzymes, ubiquitin, 50 mM ZnCl2 and 10x buffer (500 mM Tris-HCl [pH 7.5], 20 mM ATP, 10 mM MgCl2, 2 mM DTT) in 50 ⁇ l. Then, it was incubated for 1 hour at 37°C. To stop the reaction, sampling buffer was added and the sample boiled at 100° C. for 10 minutes. E1, E2 (UBCH3, UBCH6) and ubiquitin were purchased from Boston Biochems. Purified GST-PHF6 WT/mutant was considered E3 ligase.
  • ubiquitin-conjugated His-UBCH3 was bead-bound in 1 ml volume of pull-down-assay buffer (20 mM Tris-HCl [pH 7.8], 125 mM NaCl, 10% glycerol, 0.1% NP-40 and protease inhibitor). GST alone or with GST-PHF6 WT or mutants were incubated overnight at 4°C. After the reaction, the beads were washed 4 times with the same buffer and the beads were boiled with the sampling buffer.
  • ChIP assays Chromatin-Immunoprecipitation (ChIP) and MNase-ChIP assays ChIP assays were performed essentially as reported.
  • ChIP assay cells were crosslinked with 1% formaldehyde for 15 min at room temperature. Nuclear pellets were sonicated in RIPA buffer.
  • MNase-ChIP nuclear pellets were dissolved in MNase lysis buffer and reacted with MNase at 37°C for 10 min instead of sonication. Backcrosslinking was performed overnight at 65°C.
  • immunoprecipitated beads were mixed with 2X sampling buffer and boiled at 100 °C for 45 min. DNA was isolated by purification column (#28105, QIAGEN). Eluted DNA was detected by qRT-PCR. All reactions were performed in triplicate.
  • the ChIP-qRT-PCR primers used in this study are shown in Table 1.
  • Quantitative RT-PCR total RNA was extracted using Trizol (Invitrogen) and reverse transcription was performed on 1-2 ⁇ g of total RNA using the M-MLV cDNA Synthesis Kit (Enzynomics). The amount of mRNA was detected by BioRad CFX384 or ABI Prism 7500 system with SYBR TOPreal qPCR 2X PreMix (Enzynomics). The amount of mRNA was calculated using the ddCt method and Gapdh and ⁇ -actin were used as controls. All reactions were performed in triplicate. Table 1 shows the qRT-PCR primers used in this study.
  • RNA sequencing analysis Total RNA was extracted from WT and Phf6-deficient cells with and without DOX treatment, respectively. Then, strand mRNA-seq library was prepared according to Illumina's TruSeq protocol. After generating raw data on the HiSeq platform, reads were preprocessed by Trimmomatic (v0.36) to remove poor quality adapters and bases. Next, reads were aligned using STAR (v2.5.3) and TPM (transcripts per million) was calculated as RSEM (v1.3.0).
  • DEGs differentially expressed genes
  • DESeq2 v1.18.1
  • DESeq2 v1.18.1
  • DESeq2 v1.18.1
  • DESeq2 v1.18.1
  • RA-treated DOX-treated
  • k-means clustering was performed for the binding of four sets of DEGs in R-(v 3.4.3).
  • GSEA gene set enrichment analysis
  • Pearson correlation coefficient per gene used for ranking became An enrichment score was then calculated for a set of genes in the Molecular Signature Database (MSigDB) v6.2.
  • MSigDB Molecular Signature Database
  • Phf6-deficient embryonic stem cells PHF6-deficient embryonic stem cells
  • FIG. 1A ZHBTc4 mouse embryonic stem cell line, which is a stem cell designed to be depleted of Oct4 by tetracycline (Tc) or doxycycline (DOX) treatment. It is known that embryonic stem cells are reprogrammed to an early blastocyst lineage after inhibition of Oct4 expression.
  • the ZHBTc4 embryonic stem cell system has the advantage of being able to identify the entire lineage of early stage embryos in vitro.
  • Phf6 KO ESCs Phf6-deficient ZHBTc4 embryonic stem cell line
  • Example 3 It was identified that PHF6 activates the expression of early blastocyst lineage determining genes for blastocyst lineage determination
  • Example 4 PHF6 was identified as an epigenetic regulator that regulates H2BK120ub levels through H2BK12Ac recognition
  • PHF6 has a function as a transcriptional regulator by recognizing which histone modifications.
  • histones directly recognized by PHF6 were identified through the Far-western experiment technique together with GST-PHF6 recombinant protein after extracting histones from ZHBTc4 ESCs.
  • GST-PHF6 recombinant protein selectively recognized histones H2B and H3 ( FIG. 4A ).
  • screening was performed using a histone peptide array kit to identify specific modifications of H2B and H3 recognized by PHF6.
  • H2BK12 acetylation was known as a marker of transcriptional activation. Therefore, we hypothesized that the acetyl recognition function of PHF6 is important for regulating the expression of lineage-specific genes.
  • Various histone modifications including H2BK120ub, H3K4me3, H3K27me3, H3K9me3 and H2BK12Ac were investigated in WT and Phf6 KO embryonic stem cells. Among them, it was confirmed that the level of ubiquitination (H2BK120ub) of H2B residue 120 was significantly reduced in Phf6 KO compared to that of WT ESCs upon DOX treatment ( FIG. 4G ).
  • H2BK12R acetylation-deficient mutant
  • H2BK120R ubiquitination-deficient mutant did not affect H2BK12 acetylation (Fig. 4I).
  • PHF6 regulates H2BK120 ubiquitination through antecedent H2BK12 acetylation recognition.
  • Proteins bearing PHD typically act as linker proteins that recruit other enzymes for alteration of histone modification state. Therefore, we confirmed the possibility that PHF6 modulates H2BK120 ubiquitination by recruiting E3 ubiquitin ligase or de-ubiquitinase known for H2BK120 ubiquitination.
  • RNF20/40 was known as an E3 ligase for H2BK120 ubiquitination during the differentiation of embryonic stem cells into neuroectoderm
  • USP44 was known to function as a de-ubiquitinase.
  • H2BK120 ubiquitination following knockdown of Phf6, Rnf20 and Rnf40 in the presence or absence of DOX treatment was investigated. compared. As a result, it was confirmed that knockdown of Rnf20/40 rather than Phf6 reduced H2BK120 ubiquitination even in the absence of DOX treatment ( FIG. 4L ). However, knockdown of Phf6 resulted in a decrease in H2BK120 ubiquitination only upon DOX treatment (Fig. 4L).
  • Example 5 It was established that PHF6 functions as an E3 ubiquitin ligase for the 120th lysine (H2BK120) of the histone H2B protein.
  • FIG. 6B ChIP analysis was performed on the promoters of Cdx2 and Gata2 in WT and Phf6 KO embryonic stem cells with or without DOX treatment. As a result, it was confirmed that DOX treatment increased the recruitment of PHF6 along with the H2BK120ub level in the promoter of these genes in WT embryonic stem cells, but this increase was not seen in Phf6 KO embryonic stem cells ( FIG. 6C ). In addition, in the ChIP analysis of the promoter of Msx2, a gene expressed with or without Phf6, it was confirmed that there was no significant change in PHF6 recruitment and H2BK120ub levels between WT and Phf6 KO embryonic stem cells (Fig. 6D).
  • H3K4me3 following H2BK120ub in the promoter region was also confirmed.
  • H3K4me3 level in the Cdx2 and Gata2 promoters increased with the increase of H2BK120ub and Phf6 depending on DOX treatment ( FIG. 6C ).
  • the level of H3K4me3 in the promoter of Msx2, which is a gene independent of PHF6, also increased regardless of the presence or absence of PHF6 FIG. 6D ).

Abstract

A screening method, described in the present specification, for targeting an epigenetic regulation mechanism of PHF6 causing the acetylation of H2BK12 and the ubiquitination of H2BK120, which are two independently known histone modifications involved in transcriptional regulation, to operate as a single functional unit may be useful for developing a therapeutic agent for a disease caused by an abnormality of the PHF6 gene.

Description

피에이치에프6를 표적으로 하는 히스톤 에이치2비 에피제네틱 조절제 스크리닝 방법Screening method for histone H2B epigenetic modulators targeting PF6
본원은 PHF6의 히스톤 H2B 에피제네틱 조절을 규명한 분자 기전에 근거한 약물 스크리닝 기술과 관련된 분야이다.This application is a field related to a drug screening technology based on a molecular mechanism that elucidates the histone H2B epigenetic regulation of PHF6.
히스톤 변형(histone modification)은 세포의 크로마틴(chromatin) 변화를 야기함으로써 유전자 발현을 조절하는 중요한 생물학적 현상 중 하나이다. 크로마틴에는 크게 H2A, H2B, H3 및 H4의 4 종류의 히스톤 단백질에 결합되어 있으며, 각 히스톤 단백질의 N-말단에는 아세틸화와 같은 여러 종류의 변형이 존재하는 것으로 알려져 있다. 이러한 변형은 특정 유전자 발현을 활성화하거나 억제하며, 이를 에피제네틱 조절이라고 한다. 이러한 히스톤의 변형에 관여하는 에피제네틱 조절 단백질/유전자는 기능적으로 라이터(writer), 이레이저(eraser), 그리고 리더(reader)로 분류된다.Histone modification is one of the important biological phenomena that regulate gene expression by causing chromatin changes in cells. Chromatin is largely bound to four types of histone proteins, H2A, H2B, H3 and H4, and it is known that various types of modifications such as acetylation exist at the N-terminus of each histone protein. These modifications activate or repress specific gene expression, which is referred to as epigenetic regulation. Epigenetic regulatory proteins/genes involved in these histone modifications are functionally classified into writers, erasers, and readers.
이 중 리더는 히스톤의 변형을 인지하는 기능을 가진 단백질로 그 중요성이 대두되고 있는데, 이는 라이터나 이레이저와 달리 서로 다른 종류의 히스톤 변형 간의 상호 작용(histone modification crosstalk) 현상을 이해하는 데 가장 핵심이 되는 인자이기 때문이다.Among them, the leader is a protein with the function of recognizing histone modifications, and its importance is emerging. Unlike writers and erasers, it is the most important to understand the interaction between different types of histone modifications (histone modification crosstalk). This is because it is the person who becomes
이러한 히스톤 변형 간 상호 작용은 생체 내 유전자들의 발현이 정교하게 조절되어야 하는 복잡계를 이해하는데 필수적 개념이다. 또한 이들 히스톤 변형 간 상호 작용이 망가지게 되는 경우 암을 비롯한 여러 질병들을 야기하게 된다는 보고들이 있지만, 알려져 있는 히스톤 변형의 종류에 비해 이들 간의 상호 작용은 많이 알려져 있지 않다 (Wright, D.E., et al. Front Biosci (Landmark Ed), 17, 1051-1078; Chen, S. et al.. Cell Res, 22, 1402-1405.).The interaction between histone modifications is an essential concept in understanding the complex system in which the expression of genes in vivo must be precisely regulated. In addition, there are reports that if the interaction between these histone modifications is broken, it causes various diseases including cancer, but the interaction between them is not known much compared to the known types of histone modifications (Wright, DE, et al. Front Biosci (Landmark Ed) , 17, 1051-1078; Chen, S. et al. Cell Res , 22 , 1402-1405.).
WO 2013-138237은 급성 백혈병의 치료, 진단 또는 예후용 방법 및 조성물에 관한 것으로 PHF6를 백혈병 예후와 관련된 판단을 위한 마커의 하나로 개시하고 있다. 히스톤 단백질의 에피제네틱 조절 기전에 근거한 표적의 발굴이 필요하다.WO 2013-138237 relates to a method and composition for the treatment, diagnosis or prognosis of acute leukemia, and discloses PHF6 as one of the markers for judging the prognosis of leukemia. It is necessary to discover targets based on the epigenetic regulatory mechanism of histone proteins.
본원에서는 백혈병 및 BFL 증후군과 같은 유전병 질환들에서 핵심 돌연변이 유전자로 알려져 있는 PHF6에 의한 히스톤 H2B의 에피제네틱 조절 기전에 근거한 약물 스크리닝 방법을 제공하고자 한다.An object of the present application is to provide a drug screening method based on the epigenetic regulation mechanism of histone H2B by PHF6, which is known as a key mutant gene in hereditary diseases such as leukemia and BFL syndrome.
한 양태에서 본원은 PHF6 (PHD finger-containing protein 6) 단백질 및 12번째 라이신 잔기가 아세틸화로 변형된 히스톤 2B (H2BK12-Ac) 단백질을 제공하는 단계; 상기 PHF6 단백질 및 H2BK12-Ac 단백질을 상기 PHF6의 상기 H2BK12-Ac의 인식을 조절할 것으로 기대되는 시험물질의 존재 중에서 접촉하는 단계; 상기 히스톤 단백질의 120번째 라이신 잔기의 유비퀴틴화 (H2BK120-ub)를 측정하는 단계; 및 상기 측정결과, 상기 시험물질로 처리되지 않은 대조군과 비교하여, 상기 시험물질로 처리된 세포에서 상기 히스톤 단백질의 120번째 라이신 잔기의 유비퀴틴화 수준에 변화가 있는 경우, 상기 시험물질을 상기 PHF6 활성 조절 후보물질로 선별하는 단계를 포함하는, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법을 제공한다. In one embodiment, the present application provides a PHF6 (PHD finger-containing protein 6) protein and a histone 2B (H2BK12-Ac) protein in which the 12th lysine residue is modified by acetylation; contacting the PHF6 protein and the H2BK12-Ac protein in the presence of a test substance expected to modulate the recognition of the H2BK12-Ac by the PHF6; measuring ubiquitination (H2BK120-ub) of the 120th lysine residue of the histone protein; And, as a result of the measurement, when there is a change in the ubiquitination level of the 120th lysine residue of the histone protein in the cells treated with the test substance compared to the control group not treated with the test substance, the test substance is treated with the PHF6 activity It provides a method for screening a histone epigenetic modulator by PHF6, comprising the step of selecting a modulatory candidate.
본원에 따른 방법에서 수준의 변화는 처리되지 않은 대조군과 비교하여 증가 또는 감소를 의미하는 것으로, H2BK120-ub의 증가 또는 감소에 영향을 줄 수 있는 물질은 히스톤 에피제네틱 조절제로서 유용하게 사용될 수 있다. 당업자라면 구체적 목적에 따라 감소 또는 증가를 선택할 수 있을 것이다. In the method according to the present application, a change in level means an increase or decrease compared to an untreated control, and substances capable of affecting the increase or decrease of H2BK120-ub may be usefully used as histone epigenetic modulators. A person skilled in the art will be able to select a decrease or an increase according to the specific purpose.
본원의 방법에 따라 선별, 개발된 에피제네틱 조절제는 백혈병 치료용으로 개발될 수 있다. Epigenetic modulators selected and developed according to the method herein can be developed for the treatment of leukemia.
본원에 방법에서 PHF6 단백질 및 히스톤 2B은 인간을 포함하는 포유류 유래의 것이 사용될 수 있으며, 당업자라면 실험 조건 등을 고려하여 적절한 것을 선택할 수 있을 것이다. 일 구현예에서는 인간 유래의 것이 사용된다. In the method herein, PHF6 protein and histone 2B derived from mammals including humans may be used, and those skilled in the art will be able to select appropriate ones in consideration of experimental conditions and the like. In one embodiment, those of human origin are used.
일 구현예에서 본원에 따른 방법은 본원의 방법에 사용되는 단백질을 (과)발현하는 진핵세포 예를 들면 인간을 제외한 동물의 배아줄기세포 또는 인간을 제외한 동물모델에서 수행될 수 있다. In one embodiment, the method according to the present application can be performed in eukaryotic cells (over)expressing the protein used in the method of the present application, for example, non-human embryonic stem cells or non-human animal models.
본원에 따른 PHF6에 의한 히스톤 2B 단백질의 에피제네틱 조절은 PHF6 단백질의 PHD1 및 PHD2 도메인이 관여하며, 상기 H2BK12-Ac는 상기 PHD2 도메인에 의해 인식되고, 상기 H2BK120의 유비퀴틴화는 상기 PHD1에 의한 것이다. According to the present application, the epigenetic regulation of histone 2B protein by PHF6 involves the PHD1 and PHD2 domains of the PHF6 protein, the H2BK12-Ac is recognized by the PHD2 domain, and the ubiquitination of the H2BK120 is by the PHD1.
이에 다른 양태에서 본원은 PHF6의 PHD1 도메인; 또는 PHD2 도메인이 결실 또는 돌연변이 (점, 결실, 치환 등의 돌연변이에 의한 기능상실 돌연변이)된 PHF6 단백질 및 히스톤 2B 단백질을 제공하는 단계; 상기 PHD1 도메인 또는 상기 PHD2 도메인이 결실 또는 돌연변이된 PHF6 단백질과 상기 히스톤 2B 단백질의 결합을 촉진시킬 것으로 기대되는 물질의 존재 중에서 상기 단백질을 접촉하는 단계; 상기 히스톤 2B 단백질의 120번째 라이신 잔기의 유비퀴틴화 수준을 측정하는 단계; 및 상기 측정 결과 시험물질과 처리되지 않은 대조군과 비교하여, 처리된 경우 히스톤 2B 단백질의 120번째 라이신 잔기의 유비퀴틴화가 증가한 경우, 상기 시험물질을 상기 PHF6 활성 조절 후보물질로 선별하는 단계를 포함하는, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법을 제공한다. Accordingly, in another aspect, the present application provides a PHD1 domain of PHF6; or providing a PHF6 protein and a histone 2B protein in which the PHD2 domain is deleted or mutated (loss of function due to mutation such as point, deletion, substitution, etc.); contacting the PHF6 protein in which the PHD1 domain or the PHD2 domain is deleted or mutated in the presence of a substance expected to promote binding of the histone 2B protein; measuring the ubiquitination level of the 120th lysine residue of the histone 2B protein; And when the ubiquitination of the 120th lysine residue of histone 2B protein is increased when the test substance is treated as compared to the test substance and the untreated control as a result of the measurement, selecting the test substance as a candidate for regulating PHF6 activity, A method for screening histone epigenetic modulators by PHF6 is provided.
일 구현예에서 상기 PHD1 도메인; 또는 PHD2 도메인이 결실 또는 돌연변이된 PHF6 단백질 및 히스톤 2B 단백질은 진핵세포 또는 인간을 제외한 동물모델로서 제공될 수 있다. In one embodiment the PHD1 domain; Alternatively, the PHF6 protein and histone 2B protein in which the PHD2 domain is deleted or mutated may be provided as eukaryotic cells or animal models other than humans.
일 구현예에서 상기 진핵세포는 인간을 제외한 동물의 배아줄기세포이다.In one embodiment, the eukaryotic cells are embryonic stem cells of animals other than humans.
본원은 기존에 독립적으로 알려진 전사 조절에 관여하는 두 히스톤 변형 즉 H2BK12 (히스톤 2B, 12번째 라이신 잔기)의 아세틸화 및 H2BK120 (히스톤 2B, 120번째 라이신 잔기)에 대한 유비퀴틴화가 PHF6에 의해 기능 단위로 작동하는 것을 규명하였다. 이러한 기전에 근거하여 개발된 PHF6에 의한 히스톤 에피제네틱 기능을 조절할 수 있는 물질은 PHF6의 문제로 인한 다양한 질환의 치료제 개발에 효과적으로 사용될 수 있다. Herein, two histone modifications involved in transcriptional regulation previously known independently, namely, acetylation of H2BK12 (histone 2B, lysine residue 12) and ubiquitination to H2BK120 (histone 2B, lysine residue 120), are converted into functional units by PHF6. found to work. A substance capable of regulating the histone epigenetic function by PHF6 developed based on this mechanism can be effectively used in the development of therapeutic agents for various diseases caused by the problem of PHF6.
본원의 방법에 따라 선별, 개발된 에피제네틱 조절제는 백혈병 치료용으로 개발될 수 있다.Epigenetic modulators selected and developed according to the method herein can be developed for the treatment of leukemia.
도 1은 Phf6 결핍으로 배아줄기세포의 배반포 분화 단계에서 결함이 발생한다. (A) CRISPR-Cas9 유전자 가위 시스템을 사용한 Phf6 유전자 녹아웃 (KO) 제작의 개략도. Phf6 유전자에 대한 가이드 RNA 서열 및 변경된 서열이 표시되어 있다. (B) Phf6 KO 배아줄기세포에서의 PHF6 결핍 및 shRNA에 의한 Phf6 녹다운을 나타내는 면역 블롯 분석. 양성 대조군으로서 Nanog, 및 로딩 대조군으로서 β-actin. (C) Phf6 KO 배아줄기세포의 성장 곡선과 WT 배아줄기세포의 성장 곡선 및 shPhf6의 성장률 곡선과 shNS의 성장 곡선. (D) WT, Phf6 KO, shNS 및 shPhf6 배아줄기세포에서 AP 염색에 의해 측정된 분화능의 비교. 이미지는 100배 확대율로 촬영되었다. (E) DOX 처리로 유도된 배반포 계통 리프로그래밍 및 RA 처리로 유도된 신경 외배엽 분화 동안 WT와 Phf6 KO ZHBTc4 간의 DEG 분석의 개략도. (F) WT와 Phf6 KO ZHBTc4 사이의 상위 50% 유전자 (n = 7,389)의 비교 발현. 컷오프 ((Phf6 KO 대 WT) | = 3의 폴드 변화)를 빨간색 점선으로 나타내었다. Phff6 KO에서 유의미하게 하향 조절 된 유전자는 붉은 점 (n = 15)으로, 상향 조절 된 유전자는 푸른 점(n = 1)으로 나타내었다. (G) DOX 처리와 RA 처리 사이의 화학적 의존성 유도 유전자들의 유전자 온톨로지 (GO) 분석. 발현 차이가 3배 이상 큰 유전자는 화학적 처리에 의한 유의하게 상향 조절된 유전자로 선택되었다. 유전자 온톨로지의 유의미성 = log10 (조정 된 P-값 = 0.1). (H) 약물 처리에서 발현이 증가한 유전자군에서 Phf6 의존적으로 발현 된 유전자의 GO 분석. 유전자 온톨로지의 유의미성 = log10 (조정 된 P- 값 = 0.05).1 shows a defect occurs in the blastocyst differentiation stage of embryonic stem cells due to Phf6 deficiency. (A) Schematic diagram of Phf6 gene knockout (KO) construction using the CRISPR-Cas9 gene editing system. Guide RNA sequences and altered sequences for the Phf6 gene are indicated. (B) Immunoblot analysis showing PHF6 deficiency and Phf6 knockdown by shRNA in Phf6 KO embryonic stem cells. Nanog as a positive control, and β-actin as a loading control. (C) Growth curve of Phf6 KO embryonic stem cells, growth curve of WT embryonic stem cells, growth rate curve of shPhf6, and growth curve of shNS. (D) Comparison of differentiation capacity measured by AP staining in WT, Phf6 KO, shNS and shPhf6 embryonic stem cells. Images were taken at 100x magnification. (E) Schematic of DEG analysis between WT and Phf6 KO ZHBTc4 during DOX treatment-induced blastocyst lineage reprogramming and RA treatment-induced neuroectoderm differentiation. (F) Comparative expression of the top 50% genes (n = 7,389) between WT and Phf6 KO ZHBTc4. The cutoff (fold change of (Phf6 KO vs. WT) | = 3) is indicated by the red dotted line. Genes significantly downregulated in Phff6 KO are indicated by red dots (n = 15) and genes upregulated by blue dots (n = 1). (G) Gene ontology (GO) analysis of genes inducing chemical dependence between DOX treatment and RA treatment. Genes with a greater than 3-fold difference in expression were selected as genes that were significantly upregulated by chemical treatment. Significance of gene ontology = log10 (adjusted P-value = 0.1). (H) GO analysis of Phf6-dependently expressed genes in a group of genes whose expression was increased upon drug treatment. Significance of gene ontology = log10 (adjusted P-value = 0.05).
도 2는 RNA-seq 분석 결과, PHF6는 배반포 분화 유전자들의 전사 활성 인자이다. (A) DOX 처리 유무에 따른 WT 및 Pfh6 KO 배아줄기세포에서 발현된 유전자의 k-평균 군집 히트 맵 (n = 1,775, k = 6). 유전자는 발현 유사성에 기초하여 6개의 클러스터로 분류되었다. (B) WT 및 Phf6 KO 배아줄기세포에 대한 DOX 반응의 차이. DOX 반응은 배아줄기세포에서 DOX 처리에 의한 유전자의 평균 z-점수 차이를 사용하여 계산하였다. (C) 클러스터 1 유전자의 Z-점수 센트로이드. 중간값은 빨간색으로 강조 표시하였다. (D) 클러스터 1 유전자군의 온톨로지 (GO) 분석에 따른 기능 분석. 태반 발달이 클러스터 1에서 가장 유의미한 생물학적 기능이다. (E) DEG의 대표적인 유전자 세트 분석 (GSEA). 배아 태반의 발달 및 영양 아세포 거대 세포 분화에 관여하는 유전자 세트는 유의미성을 나타내었다. (C)의 중간값을 유전자 발현의 패턴 분석의 기준으로 사용하였다. (F) WT과 비교하여 Phf6 KO 배아줄기세포에서 하향-조절된 배반포 분화 유전자군. 색상 막대는 각 비교에서 발현 변화값의 로그2 값을 나타낸다. GSEA로부터 추출된 유전자들이 선택되었다. (G) DOX 처리의 유무에 의한 WT 및 Phf6 KO ESC에서 배반포 유전자의 qRT-PCR 분석. 각 유전자의 mRNA 수준은 Gapdh에 대한 상대적인 값으로 결정되었고 WT-DOX에 기초하여 상대적으로 비교되었다. 통계적 유의성은 ANOVA 테스트에 의해 계산되었다 (* P <0.05, ** P <0.01, *** P <0.001).2 is a result of RNA-seq analysis, PHF6 is a transcriptional activator of blastocyst differentiation genes. (A) K-mean cluster heat map of genes expressed in WT and Pfh6 KO embryonic stem cells with and without DOX treatment (n = 1,775, k = 6). Genes were classified into six clusters based on expression similarity. (B) Differences in DOX response to WT and Phf6 KO embryonic stem cells. The DOX response was calculated using the mean z-score difference of genes by DOX treatment in embryonic stem cells. (C) Z-score centroid of cluster 1 genes. Median values are highlighted in red. (D) Functional analysis according to the ontology (GO) analysis of the cluster 1 gene group. Placental development is the most significant biological function in cluster 1. (E) Representative gene set analysis of DEG (GSEA). The gene sets involved in embryonic placental development and trophoblast giant cell differentiation were significant. The median value of (C) was used as a reference for pattern analysis of gene expression. (F) Down-regulated blastocyst differentiation gene family in Phf6 KO embryonic stem cells compared to WT. Color bars represent the log2 value of the expression change in each comparison. Genes extracted from GSEA were selected. (G) qRT-PCR analysis of blastocyst genes in WT and Phf6 KO ESCs with and without DOX treatment. The mRNA levels of each gene were determined relative to Gapdh and compared relative to WT-DOX. Statistical significance was calculated by ANOVA test (*P<0.05, **P<0.01, ***P<0.001).
도 3은 PHF6는 배반포 계통 결정을 위해 배반포 마커 유전자의 발현을 활성화한다. (A) 배아체(EB) 분화 7일차에 WT 및 Phf6 KO EB의 형태학적 특징. 각각의 EB를 성장시키고 개별적으로 관찰하였다. 배율 X10. 외부 세포층의 평균 면적 (mm2)을 WT (n = 11, 왼쪽)와 Phf6 KO (n = 9, 오른쪽) EB 사이에서 비교 하였다. 통계적 유의성은 ANOVA 테스트에 의해 계산되었다 (* P <0.05, ** P <0.01, *** P <0.001). (B-C) WT EB의 외부 세포층에서 CDX2 및 PHF6의 면역 염색. 배율 X100 (B) 및 X200 (C). (D) WT 및 Phf6 KO EB 분화 동안 배반포 계통 마커 유전자들의 qRT-PCR 분석. WT 및 Phf6 KO EB를 LIF없이 유지하고 각각 표시된 날짜에 샘플링하였다. 각 유전자의 mRNA 수준은 Gapdh 또는 b-actin에 대한 상대적인 값으로 결정되었고 WT 0일을 기준으로 상대적으로 비교되었다. 통계적 유의성은 ANOVA 테스트에 의해 계산되었다 (* P <0.05, ** P <0.01, *** P <0.001 ).Figure 3 shows that PHF6 activates the expression of blastocyst marker genes for blastocyst lineage determination. (A) Morphological characteristics of WT and Phf6 KO EBs at day 7 of embryonic body (EB) differentiation. Each EB was grown and observed individually. magnification X10. The average area (mm2) of the outer cell layer was compared between WT (n = 11, left) and Phf6 KO (n = 9, right) EBs. Statistical significance was calculated by ANOVA test (*P<0.05, **P<0.01, ***P<0.001). (B-C) Immunostaining of CDX2 and PHF6 in the outer cell layer of WT EBs. Magnifications X100 (B) and X200 (C). (D) qRT-PCR analysis of blastocyst lineage marker genes during WT and Phf6 KO EB differentiation. WT and Phf6 KO EBs were maintained without LIF and sampled on the indicated days, respectively. The mRNA levels of each gene were determined as relative values for Gapdh or b-actin and compared relative to day 0 of WT. Statistical significance was calculated by ANOVA test (*P<0.05, **P<0.01, ***P<0.001).
도 4는 PHF6은 H2BK12Ac를 인지하고 H2BK120의 단일 유비퀴틴화를 조절한다. (A) GST 또는 GST-PHF6 단백질을 사용한 히스톤 단백질의 Far western 분석. ZHBTc4 배아줄기세포로부터 얻은 히스톤 추출물에 대해 분석을 수행하였다. 히스톤 추출물은 SDS-PAGE 상의 크기에 따라 H3, H2B, H2A 및 H4로 분리되었다. (B) GST-PHF6에 가장 높은 친화력을 나타내는 상위 5개의 히스톤 변형. 스크리닝은 히스톤 펩티드 어레이 키트를 사용하여 수행되었다. (C) 상위 5개의 변형 된 히스톤 펩티드 후보들에 대한 GST-PHF6의 인지 능력을 확인. 히스톤 펩티드 풀다운 분석을 수행하였다. (D) 다양한 종에서 PHF6의 첫번째 확장 PHD 도메인 (ePHD1) 및 두번째 확장 PHD 도메인 (ePHD2)의 아미노산 서열. 붉은색으로 표시된 아미노산은 ePHD2에서 음으로 하전된 아미노산들이 모여있는 영역을 나타낸다. 아미노산 정렬은 ClustalX로 수행하였다. (E) 시험 관내 펩티드 결합 분석을 GST-PHF6 WT 또는 E223S 돌연변이로 수행하였다. (F) 음성 대조군으로서 GST-PHF6 WT 및 E223S 돌연변이 (왼쪽 축) 및 GST (오른쪽 축)를 갖는 H2B 펩티드 (1-20) (비 변형 및 K12Ac)의 MST 결합 곡선. 오차 막대는 3개의 독립적인 실험의 표준 편차를 나타낸다. 측정된 KD 값은 각 결합 곡선에 대해 표시되었다. * N.B. 바인딩이 없음을 나타낸다. (G) DOX 처리로 WT 및 Phf6 KO 배아줄기세포에서 각각의 표기된 항체를 사용하여 면역 블롯 분석을 수행하였다. (H) DOX 처리 후 p300 또는 CBP을 siRNA로 녹다운 후 표기된 항체를 사용하여 면역 블롯 분석을 수행하였다. (1) H2B K12R / K120R 돌연변이를 이용한 K12Ac와 K120ub의 상관 관계 비교. 이들 H2B WT 및 각 돌연변이를 함유하는 단일-뉴클레오솜을 추출하기 위해 MNase 효소를 사용하였고, 이들 단일-뉴클레오좀의 히스톤 변형 상태를 면역 블롯 분석으로 확인하였다. (J) DOX 처리로 WT와 Phf6 KO 사이의 면역 블롯에 의한 RNF20 및 RNF40 단백질 수준. (K) 공동-면역 침전 분석을 수행하여 DOX 처리 유무에 따른 배아줄기세포에서 PHF6과 USF44, RNF20 또는 RNF40과의 상호 작용을 확인. (L) DOX 처리의 유무에 따라 Phf6, Rnf20 및 Rnf40의 녹다운 사이의 H2BK120ub 수준의 면역 블롯. (M) DOX 처리를 사용한 Phf6, Rnf20 및 Rnf40 녹다운 배아줄기세포에서 Cdx2 및 Gata2의 qRT-PCR 분석. 각 유전자의 mRNA 수준은 Gapdh에 대한 상대적인 값으로 결정되었고 shNS + DOX에 기초하여 상대적으로 비교되었다. 통계적 유의성은 ANOVA 테스트에 의해 계산되었다 (* P <0.05, ** P <0.01, *** P <0.001). (N) DOX 처리와 함께 Phf6, Rnf20 및 Rnf40 녹다운 ESC에서 H2BK120ub 항체를 사용하여 Cdx2 및 Gata2의 프로모터에 대해 CHIP 분석을 수행 하였다. 통계적 유의성은 ANOVA 테스트에 의해 계산되었다 (* P <0.05, ** P <0.01, *** P <0.001).Figure 4 PHF6 recognizes H2BK12Ac and regulates single ubiquitination of H2BK120. (A) Far western analysis of histone proteins using GST or GST-PHF6 proteins. Analysis was performed on histone extracts obtained from ZHBTc4 embryonic stem cells. Histone extracts were separated into H3, H2B, H2A and H4 according to their size on SDS-PAGE. (B) Top five histone variants showing the highest affinity for GST-PHF6. Screening was performed using the Histone Peptide Array Kit. (C) Confirmation of the recognition ability of GST-PHF6 for the top five modified histone peptide candidates. Histone peptide pulldown analysis was performed. (D) Amino acid sequences of the first extended PHD domain (ePHD1) and the second extended PHD domain (ePHD2) of PHF6 in various species. Amino acids indicated in red indicate regions in which negatively charged amino acids are clustered in ePHD2. Amino acid alignment was performed with ClustalX. (E) In vitro peptide binding assays were performed with GST-PHF6 WT or E223S mutants. (F) MST binding curves of H2B peptides (1-20) (unmodified and K12Ac) with GST-PHF6 WT and E223S mutations (left axis) and GST (right axis) as negative controls. Error bars represent the standard deviation of three independent experiments. Measured KD values are displayed for each binding curve. * N.B. Indicates that there is no binding. (G) Immunoblot analysis was performed using each indicated antibody in WT and Phf6 KO embryonic stem cells with DOX treatment. (H) After DOX treatment, p300 or CBP was knocked down with siRNA, followed by immunoblot analysis using the indicated antibody. (1) Comparison of correlation between K12Ac and K120ub using H2B K12R/K120R mutations. MNase enzyme was used to extract single-nucleosomes containing these H2B WTs and each mutation, and the histone modification status of these single-nucleosomes was confirmed by immunoblot analysis. (J) RNF20 and RNF40 protein levels by immunoblot between WT and Phf6 KO with DOX treatment. (K) Co-immunoprecipitation analysis was performed to confirm the interaction of PHF6 with USF44, RNF20 or RNF40 in embryonic stem cells with and without DOX treatment. (L) Immunoblot of H2BK120ub levels between knockdowns of Phf6, Rnf20 and Rnf40 with and without DOX treatment. (M) qRT-PCR analysis of Cdx2 and Gata2 in Phf6, Rnf20 and Rnf40 knockdown embryonic stem cells using DOX treatment. The mRNA levels of each gene were determined relative to Gapdh and compared relative to the basis of shNS + DOX. Statistical significance was calculated by ANOVA test (*P<0.05, **P<0.01, ***P<0.001). (N) CHIP analysis was performed on the promoters of Cdx2 and Gata2 using the H2BK120ub antibody in Phf6, Rnf20 and Rnf40 knockdown ESCs with DOX treatment. Statistical significance was calculated by ANOVA test (*P<0.05, **P<0.01, ***P<0.001).
도 5는 PHF6은 K120의 히스톤 H2B에 대한 아세틸화-의존적 E3 유비퀴틴 리가아제다. (A) 시험관 내 유비퀴틴화 분석의 흐름도. (B) H2B WT 또는 K12R 돌연변이가 포함된 단일-뉴클레오솜을 사용한 시험관 내 유비퀴틴화 분석. 단일-뉴클레오솜은 DOX 처리한 Phf6 KO ZHBTc4로부터 추출하였다. (C) GST-PHF6 WT, C82A 또는 E223S 돌연변이와 함께 H2B-Flag WT이 포함된 단일-뉴클레오솜에 대한 시험 관내 유비퀴틴화 분석. (D) DOX 처리에 의한 Phf6 KO ZHBTc4에서의 PHF6 WT, C82A 또는 E223S 돌연변이의 발현 수준. (E) PHF6의 첫번째 확장 PHD의 개략도. 흑색 문자는 아연 포획을 위한 PHD 코어 아미노산이다; 적색 문자는 종 간 보존 된 E3 리가아제 활성에 필요한 새로운 잔기 후보이다. (F) PHF6 돌연변이들을 이용한 시험관 내 유비퀴틴화 분석. (G) His-UBCH3의 GST, GST-PHF6 WT 또는 돌연변이를 이용한 시험관 내 GST-풀다운 분석. (H) PHF6에서 각 도메인의 기능 개략도.Figure 5. PHF6 is an acetylation-dependent E3 ubiquitin ligase for histone H2B of K120. (A) Flow diagram of the in vitro ubiquitination assay. (B) In vitro ubiquitination assay using single-nucleosomes containing H2B WT or K12R mutations. Single-nucleosomes were extracted from DOX-treated Phf6 KO ZHBTc4. (C) In vitro ubiquitination assay of single-nucleosomes containing H2B-Flag WT with GST-PHF6 WT, C82A or E223S mutations. (D) Expression levels of PHF6 WT, C82A or E223S mutants in Phf6 KO ZHBTc4 by DOX treatment. (E) Schematic diagram of the first extended PHD of PHF6. Black letters are PHD core amino acids for zinc capture; Red letters are new residue candidates required for cross-species conserved E3 ligase activity. (F) In vitro ubiquitination assay using PHF6 mutants. (G) In vitro GST-pulldown analysis using GST, GST-PHF6 WT or mutants of His-UBCH3. (H) Functional schematic of each domain in PHF6.
도 6은 PHF6에 의한 H2B에서의 아세틸화- 유비퀴틴화 연결은 배반포 유전자 발현에 중요하다. (A) PHF6와 결합한 단일-뉴클레오솜을 확인하기 위한 H2BK12Ac 및 H3K4me1 항체를 사용한 MNase-ChIP 분석의 면역 블롯. Flag 항체를 사용하여 염색질의 면역 침전을 수행 하였다. Flag-PHF6 WT, M125A 및 E223S는 Phx6 KO 배아줄기세포에서 2일 동안 DOX 처리로 과발현 되었다. IP : IgG 및 IP : Flag에서 각 항체의 블로팅은 동일한 노출 상태에서 비교하였다. 1% Input이 사용되었다. (B) pLVX-PHF6 WT 및 돌연변이들의 과발현 후 H2BK120ub 및 PHF6 수준의 면역 블롯 분석. (C) WT과 Phf6 KO에서 IgG, PHF6, H2BK120ub, H3K4me3 및 H2BK12Ac 항체를 사용하여 Phf6 의존적으로 발현된 유전자인 Cdx2 및 Gata2의 프로모터에 대해 ChIP 분석. 통계적 유의성은 ANOVA 테스트에 의해 계산되었다 (* P <0.05, ** P <0.01, *** P <0.001). (D) WT 및 Phf6 KO에서 IgG, PHF6, H2BK120ub, H3K4me3 및 H2BK12Ac 항체를 사용하여 DOX 처리에 의해 Phf6 비의존적 발현 유전자인 Msx2의 프로모터에 대한 CHIP 분석. 통계적 유의성은 ANOVA 테스트에 의해 계산되었다 (* P <0.05, ** P <0.01, *** P <0.001). (E-F) Phf6 KO ESC에서 렌티 바이러스 감염에 의한 pLVX-PHF6 WT 또는 MTs 안정 세포의 생성. DOX 처리에 의한 Phf6 WT 또는 MT의 안정한 구조의 Phf6 KO ESC의 히스톤 H2BK120ub 및 PHF6 수준의 면역 블롯 분석 (E). DOX 처리에 의한 Phf6 WT 또는 돌연변이들을 Phf6 KO 배아줄기세포에서 지속 발현시킨 후, 배반포 마커 유전자들 발현의 qRT-PCR 분석 (F). 각 유전자의 mRNA 수준은 Gapdh에 대한 상대적인 값으로 결정되었고 Phf6 WT 안정 세포에 기초하여 비교되었다. 통계적 유의성은 ANOVA 테스트에 의해 계산되었다 (* P <0.05, ** P <0.01, *** P <0.001).6 shows that the acetylation-ubiquitination linkage in H2B by PHF6 is important for blastocyst gene expression. (A) Immunoblot of MNase-ChIP assay using H2BK12Ac and H3K4me1 antibodies to identify single-nucleosomes bound to PHF6. Immunoprecipitation of chromatin was performed using Flag antibody. Flag-PHF6 WT, M125A and E223S were overexpressed in Phx6 KO embryonic stem cells with DOX treatment for 2 days. Blotting of each antibody in IP:IgG and IP:Flag was compared under the same exposure conditions. 1% input was used. (B) Immunoblot analysis of H2BK120ub and PHF6 levels after overexpression of pLVX-PHF6 WT and mutants. (C) ChIP analysis of promoters of Phf6-dependently expressed genes, Cdx2 and Gata2, using IgG, PHF6, H2BK120ub, H3K4me3 and H2BK12Ac antibodies in WT and Phf6 KO. Statistical significance was calculated by ANOVA test (*P<0.05, **P<0.01, ***P<0.001). (D) CHIP analysis of the promoter of the Phf6 independent expression gene Msx2 by DOX treatment using IgG, PHF6, H2BK120ub, H3K4me3 and H2BK12Ac antibodies in WT and Phf6 KO. Statistical significance was calculated by ANOVA test (*P<0.05, **P<0.01, ***P<0.001). (E-F) Generation of pLVX-PHF6 WT or MTs stable cells by lentiviral infection in Phf6 KO ESCs. Immunoblot analysis of histone H2BK120ub and PHF6 levels of stable structures of Phf6 KO ESCs of Phf6 WT or MT by DOX treatment (E). qRT-PCR analysis of the expression of blastocyst marker genes after sustained expression of Phf6 WT or mutants by DOX treatment in Phf6 KO embryonic stem cells (F). The mRNA levels of each gene were determined relative to Gapdh and compared based on Phf6 WT stable cells. Statistical significance was calculated by ANOVA test (*P<0.05, **P<0.01, ***P<0.001).
도 7은 배반포 유전자의 발현에 대한 PHF6의 기능에 대한 모식도이다. 도식 모델은 PHF6이 배반포 리프로그래밍 동안 배반포 유전자들의 전사 활성 인자로서 기능한다는 것을 보여준다. PHF6은 두번째 확장 PHD 도메인을 통해 H2BK12Ac를 인식하고, 배반포 유전자들의 프로모터상의 첫번째 확장 PHD 도메인을 통해 H2BK120 잔기를 유비퀴틴화한다 (위). 그러나, PHF6이 결핍되었을 때, 배반포 유전자들의 프로모터상의 H2BK120ub 수준은 증가하지 않아, 배반포 유전자들의 전사 활성화의 실패로 이어지는 것을 나타낸다 (아래).7 is a schematic diagram of the function of PHF6 on the expression of blastocyst genes. The schematic model shows that PHF6 functions as a transcriptional activator of blastocyst genes during blastocyst reprogramming. PHF6 recognizes H2BK12Ac through its second extended PHD domain and ubiquitinates the H2BK120 residue through the first extended PHD domain on the promoter of the blastocyst genes (top). However, when PHF6 is deficient, the level of H2BK120ub on the promoter of blastocyst genes does not increase, indicating that it leads to failure of transcriptional activation of blastocyst genes (below).
본원은 PHF6 (PHD finger protein 6)이 기존에 독립적으로 알려진 전사 조절에 관여하는 두 히스톤 변형 즉 H2BK12 (히스톤 2B, 12번째 라이신 잔기)의 아세틸화 및 H2BK120 (히스톤 2B, 120번째 라이신 잔기)의 유비퀴틴화를 하나의 기능 단위로 작동하게 하는 에피제네틱 조절자라는 발견에 근거한 것이다. Herein, PHF6 (PHD finger protein 6) is involved in the previously known independent transcriptional regulation of two histone modifications, namely, acetylation of H2BK12 (histone 2B, 12th lysine residue) and ubiquitin of H2BK120 (histone 2B, 120th lysine residue). It is based on the discovery that anger is an epigenetic regulator that makes it act as a functional unit.
이에 한 양태에서 본원은 PHF6 단백질 및 12번째 라이신 잔기가 아세틸화된 변형된 히스톤 2B (H2BK12-Ac) 단백질을 제공하는 단계; 상기 단백질을 상기 PHF6의 상기 H2BK12-Ac의 인식을 조절할 것으로 기대되는 시험물질의 존재 중에서 접촉하는 단계; 상기 H2B의 120번째 라이신 잔기의 유비퀴틴화 (H2BK120-ub)를 측정하는 단계; 및 상기 측정결과, 상기 시험물질로 처리되지 않은 대조군과 비교하여, 상기 시험물질로 처리된 세포에서 상기 H2B의 120번째 라이신 잔기의 유비퀴틴화 수준에 변화가 있는 경우, 상기 시험물질을 상기 PHF6 활성 조절 후보물질로 선별하는 단계를 포함하는, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법에 관한 것이다. Accordingly, in one aspect, the present application provides a PHF6 protein and a modified histone 2B (H2BK12-Ac) protein in which the 12th lysine residue is acetylated; contacting said protein in the presence of a test substance expected to modulate recognition of said H2BK12-Ac of said PHF6; measuring the ubiquitination (H2BK120-ub) of the 120th lysine residue of H2B; And, as a result of the measurement, when there is a change in the ubiquitination level of the 120th lysine residue of H2B in the cells treated with the test substance compared to the control not treated with the test substance, the test substance is adjusted to the PHF6 activity It relates to a method for screening a histone epigenetic modulator by PHF6, comprising the step of selecting a candidate substance.
PHF (PHD finger-containing protein) 6는 후술하는 바와 같이 두 개의 확장된 PHD 도메인을 가진 단백질로 그 구조가 알려져 있다. PHF6는 백혈병에서 가장 많이 연구가 되었는데, 여러 백혈병 보고에서 PHF6의 돌연변이가 알려져 있고 (Van Vlierberghe, P. et al. (2010) PHF6 mutations in T-cell acute lymphoblastic leukemia. Nat Genet, 42, 338-342.; Van Vlierberghe, P. et al. (2011) PHF6 mutations in adult acute myeloid leukemia. Leukemia, 25, 130-134.). 이 돌연변이로 인한 PHF6의 히스톤 변형 인지 기능 및 전사 조절 기능이 망가진 것이 백혈병을 유발하는 주요 원인 중 하나로 알려져 있다( Meacham, C.E.et al. (2015) A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth. Genes Dev, 29, 483-488.). 또한 PHF6의 돌연변이는 Borjeson-Forssman-Lehmann (BFL) 증후군에서도 많이 알려져 있다 (Crawford, J. et al. (2006) Mutation screening in Borjeson-Forssman-Lehmann syndrome: identification of a novel de novo PHF6 mutation in a female patient. J Med Genet, 43, 238-243.; Berland, S. et al. (2011) PHF6 Deletions May Cause Borjeson-Forssman-Lehmann Syndrome in Females. Mol Syndromol, 1, 294-300.; Zweier, C. et al. (2013) A new face of Borjeson-Forssman-Lehmann syndrome? De novo mutations in PHF6 in seven females with a distinct phenotype. J Med Genet, 50, 838-847.). PHF6는 전사 조절 인자로서의 기능은 알려져 있었지만, 어떠한 히스톤 변형을 인지하는지에 대한 분자적 기전에 대해서는 밝혀진 바가 없었다.The structure of PHF (PHD finger-containing protein) 6 is known as a protein having two extended PHD domains, as will be described later. PHF6 has been the most studied in leukemia, and mutations in PHF6 are known in several leukemia reports (Van Vlierberghe, P. et al. (2010) PHF6 mutations in T-cell acute lymphoblastic leukemia. Nat Genet , 42, 338-342) .; Van Vlierberghe, P. et al. (2011) PHF6 mutations in adult acute myeloid leukemia. Leukemia , 25, 130-134.). It is known that one of the main causes of leukemia is the loss of histone modification cognitive function and transcriptional regulation function of PHF6 due to this mutation ( Meacham, CE et al. (2015) A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth. Genes Dev , 29, 483-488.). Mutation of PHF6 is also well known in Borjeson-Forssman-Lehmann (BFL) syndrome (Crawford, J. et al. (2006) Mutation screening in Borjeson-Forssman-Lehmann syndrome: identification of a novel de novo PHF6 mutation in a female patient J Med Genet , 43, 238-243.; Berland, S. et al . (2011) PHF6 Deletions May Cause Borjeson-Forssman-Lehmann Syndrome in Females. Mol Syndromol , 1, 294-300.; Zweier, C. et al. (2013) A new face of Borjeson-Forssman-Lehmann syndrome?De novo mutations in PHF6 in seven females with a distinct phenotype. J Med Genet , 50, 838-847.). Although PHF6 has been known to function as a transcriptional regulator, the molecular mechanism for recognizing which histone modifications has not been elucidated.
본원에서는 PHF6이 H2BK12 아세틸화-H2BK120 유비퀴틴화 상호 작용의 핵심 기능을 수행하는 것을 규명하였다 구체적으로 PHF6에 포함된 PHD2 및 1으로 불리는 두 개의 리더(reader) 도메인이 각각 H2BK12 아세틸화 인식 및 H2BK120 유비퀴틴화에 관여함을 규명하였다 (도 7 등 참조).In this paper, it was identified that PHF6 performs a key function of the H2BK12 acetylation-H2BK120 ubiquitination interaction. Specifically, two reader domains called PHD2 and 1 included in PHF6 recognize H2BK12 acetylation and H2BK120 ubiquitination, respectively. was found to be involved in (see FIG. 7 and the like).
에피제넥틴 reader인 PHD(Plant Homeodomain) 도메인은 기존에 히스톤 H3의 K4me2/3를 인지하는 기능이 가장 많이 알려져 있는 도메인으로, 아연 양이온을 두 개 캡처하는 구조를 가짐으로써 reader로의 기능을 획득하는 것으로 알려져 있다. 그러나 PHD 도메인은 다른 도메인들에 비해 유전자마다 다양성이 큰 편에 속하는데, PHD 도메인 절반이 더 있어서 아연 양이온을 3개 캡처하는 경우도 알려져 있고, PHD 도메인 한 개가 바로 연결되어서 아연 양이온을 4개 캡처하는 케이스도 알려져 있다. 본원에서 기능이 규명된 PHF6는 PHD의 절반이 더 붙어있는 2개의 확장된 PHD(extended PHD) 도메인을 포함한다. 이러한 PHF6 서열 및 이에 포함된 서열 자체는 공지된 것으로, 예를 들면 인간 및 마우스 PHF6에 대한 정보는 다음과 같은 ID로 찾을 수 있다: 인간 PHF6 Gene ID : 84295; 마우스 PHF6 Gene ID: 70998 DB (NCBI 데이터베이스). 본원에서 인간 PHF6 단백질 서열은 서열번호 1의 서열로 표시된다. 상기 서열번호 1의 서열에서 PHD1 도메인은 14 부터 132 까지이고, PHD2는 209 부터 330 이다. 단백질 서열이 알려지면, 이를 코딩하는 유전자 서열은 당업자라면 쉽게 결정할 수 있을 것이다. 일 구현예에서 인간 PHF6 유전자 서열은 서열번호 2로 표시된다. The PHD (Plant Homeodomain) domain, an epigenectin reader, is the domain most known for its ability to recognize K4me2/3 of histone H3. is known However, the PHD domain has greater diversity per gene compared to other domains. It is also known that half of the PHD domain captures three zinc cations, and one PHD domain is directly connected to capture four zinc cations. There are also known cases. PHF6 whose function has been identified herein includes two extended PHD (extended PHD) domains to which half of the PHD is further attached. These PHF6 sequences and the sequences contained therein are known per se, for example, information on human and mouse PHF6 can be found with the following IDs: Human PHF6 Gene ID: 84295; Mouse PHF6 Gene ID: 70998 DB (NCBI database). The human PHF6 protein sequence herein is represented by the sequence of SEQ ID NO: 1. In the sequence of SEQ ID NO: 1, PHD1 domains are 14 to 132, and PHD2 is 209 to 330. Once the protein sequence is known, the gene sequence encoding it can be readily determined by those skilled in the art. In one embodiment, the human PHF6 gene sequence is represented by SEQ ID NO:2.
일반적으로 히스톤 단백질은 진핵세포에서 DNA를 패킹하여 뉴클레오솜이라는 구조적 유니트를 만들어주는 염기성 단백질이다. 히스톤에는 H1/H5, H2A, H2B, H3, 및 H4의 다섯 종류의 패밀리가 있으며, H2A, H2B, H3 및 H4는 뉴클레오솜의 코어를 구성하며, H1/H5 링커 히스톤이다. 코어 히스톤은 다이머로 존재한다. 본원에 따른 PHF6 PHD2 도메인은 H2B의 아세틸화된 12번째 잔기를 인식한다. PHF6의 PHD1 도메인은 E3 유비퀴틴 라이게이즈로서 H2B의 120번째 라이신 잔기를 유비퀴틴화 시킨다. 유비퀴틴은 76개의 아미노산으로 이루어진 단백질로, 유비퀴틴이 결합된 히스톤에 감긴 크로마틴에서는 유전자 전사가 촉진된다.In general, histone proteins are basic proteins that pack DNA in eukaryotic cells to make structural units called nucleosomes. There are five families of histones: H1/H5, H2A, H2B, H3, and H4. H2A, H2B, H3 and H4 constitute the core of the nucleosome, and are H1/H5 linker histones. Core histones exist as dimers. The PHF6 PHD2 domain according to the present application recognizes the acetylated 12th residue of H2B. The PHD1 domain of PHF6 is an E3 ubiquitin ligase that ubiquitinates the 120th lysine residue of H2B. Ubiquitin is a protein composed of 76 amino acids, and gene transcription is promoted in chromatin wound on histones bound to ubiquitin.
인간 및 마우스의 H2B NCBI의 유전자 ID는 각각 : 8347 및 68024 이다. H2B 단백질 및 유전자 서열은 NCBI에 다음과 같이 공지되어 있다: H2B의 인간 단백질 서열: NP_001368918.1, H2B의 인간 유전자 서열: NM_001381989.1; 마우스 단백질 서열: NP_001277309.1, 유전자 서열: NM_001290380.1.The gene IDs of human and mouse H2B NCBI are: 8347 and 68024, respectively. H2B protein and gene sequences are known from the NCBI as follows: human protein sequence of H2B: NP_001368918.1, human gene sequence of H2B: NM_001381989.1; Mouse protein sequence: NP_001277309.1, gene sequence: NM_001290380.1.
본원에 따른 방법에서 후보물질을 선별하기 위해 PHF6 단백질 및 12번째 잔기가 아세틸화된 변형된 히스톤 2B (H2BK12-Ac) 단백질은 분리된 단백질 또는 상기 단백질을 발현하는 세포 또는 동물모델로서 제공될 수 있다. In order to select candidates in the method according to the present application, the PHF6 protein and the modified histone 2B (H2BK12-Ac) protein in which the 12th residue is acetylated can be provided as an isolated protein or a cell or animal model expressing the protein. .
일 구현예에서 상기 단백질은 진핵세포로부터 분리/정제되어 사용될 수 있다. 분리된 히스톤 단백질은 진핵세포에서 단백질 합성 후 생성되는 후번역변형 (PTM)으로 히스톤 단백질의 N-말단 부위에 아세틸화를 포함한다. 본원에서는 H2B 12번째 라이신 잔기가 아세틸화된 단백질이다. 필요한 경우 원핵세포로부터 분리되어 사용될 수 있다. 이 경우 분리된 단백질은 필요에 따라 아세틸화 효소를 이용하여 인비트로에서 아세틸화 될 수도 있다 (David Kuninger et al., J Biotechnol, 15; 131(3): 253-260.).In one embodiment, the protein may be used after being isolated/purified from eukaryotic cells. The isolated histone protein is post-translational modification (PTM) produced after protein synthesis in eukaryotic cells, and includes acetylation at the N-terminal region of the histone protein. Herein, the H2B 12th lysine residue is an acetylated protein. If necessary, it can be used separately from prokaryotic cells. In this case, the isolated protein may be acetylated in vitro using an acetylation enzyme if necessary. (David Kuninger et al., J Biotechnol , 15; 131(3): 253-260.).
다른 구현예에서는 진핵세포가 사용될 수 있다. 상기 진핵세포는 확립된 진핵세포 또는 동물의 배아줄기세포가 사용될 수 있다.In other embodiments, eukaryotic cells may be used. The eukaryotic cells may be established eukaryotic cells or animal embryonic stem cells.
일 구현예에서 본원에 따른 방법에 사용되는 세포는 인간 또는 마우스를 포함하는 포유류 유래의 세포로 PHF6를 과발현하는 진핵세포 이다. 예를 들면 진핵세포는 ZHBTc4 (mouse embryonic stem cell), E14Tg2A.4 (mouse embryonic stem cell), HEK293T (embryonic kidney), 또는 Hela (cervix cancer cell line) 등이 사용될 수 있으며. 상기 세포주는 시중에서 구입할 수 있다. 특정 세포주에서 특정 유전자를 과발현하는 방법은 공지된 것으로 예를 들면 인간 PHF6 단백질을 코딩하는 플라스미드를 목적하는 세포주에 전달이입하여 사용할 수 있다. 하지만 세포주는 이로 제한하는 것은 아니며, 본원에 따른 목적을 달성할 수 있는 다양한 세포가 사용될 수 있다. In one embodiment, the cell used in the method according to the present application is a cell derived from a mammal, including a human or mouse, and is a eukaryotic cell overexpressing PHF6. For example, the eukaryotic cell may be ZHBTc4 (mouse embryonic stem cell), E14Tg2A.4 (mouse embryonic stem cell), HEK293T (embryonic kidney), or Hela (cervix cancer cell line). The cell line is commercially available. Methods for overexpressing a specific gene in a specific cell line are known and can be used, for example, by transfecting a plasmid encoding human PHF6 protein into a target cell line. However, the cell line is not limited thereto, and various cells capable of achieving the object according to the present disclosure may be used.
본원에 따른 방법은 시험물질로 처리된 단백질 또는 세포에서 상기 히스톤 단백질의 유비퀴틴화 정도를 측정하는 단계를 포함한다. 히스톤 H2B의 유비퀴틴화를 측정하는 방법은 공지되어 있으며, 예를 들면 본원 실시예에 기재된 것을 참조할 수 있다 (Zhang et al. Nat. Commun 8:14799 (2017)). 상기 측정하는 단계에서 상기 시험물질로 처리된 단백질 또는 세포에서, 처리되지 않은 세포와 비교하여, 상기 히스톤 2B의 유비퀴틴화 수준에 변화가 있는 경우 상기 시험물질을 히스톤 에피제네틱 조절제 후보물질로 선별할 수 있다.The method according to the present disclosure includes measuring the degree of ubiquitination of the histone protein in a protein or cell treated with a test substance. Methods for measuring ubiquitination of histone H2B are known, for example, reference may be made to those described in the Examples herein (Zhang et al. Nat. Commun 8:14799 (2017)). When there is a change in the ubiquitination level of histone 2B in the protein or cell treated with the test substance in the measuring step, compared to the untreated cell, the test substance can be selected as a histone epigenetic modulator candidate. have.
다른 구현예에서 본원에 따른 방법에 사용되는 세포는 동물모델, 예를 들면 마우스의 일부이다. 본원에 따른 방법에서 마우스가 사용된다.In another embodiment the cells used in the method according to the invention are part of an animal model, for example a mouse. In the method according to the invention a mouse is used.
본원에 따른 방법에 사용되는 세포는 이러한 세포를 포함하는 오가노이드 또는 인간을 제외한 동물모델로서 제공될 수 있다. 이러한 동물모델은 본원 실시예 또는 당업계 공지된 방법을 이용하여 제조될 수 있다. 본원에서 오가노이드란 실제 장기의 미세구조를 나타내는 3차원 환경의 인비트로에서 생산된 미니 장기이다. 이는 자가 재생 및 분화 능을 갖는 세포가 3차원 배양 환경에서 만들어진다. 오가노이드를 제조하는 방법은 공지 (Organoid Culture handbook, Ambiso) 된 것을 참고할 수 있다. The cells used in the method according to the present disclosure may be provided as organoids containing such cells or as non-human animal models. Such an animal model may be prepared using the examples herein or methods known in the art. As used herein, an organoid is a mini-organ produced in vitro in a three-dimensional environment representing the microstructure of an actual organ. Cells with self-renewal and differentiation ability are created in a three-dimensional culture environment. A method for preparing an organoid may refer to a known (Organoid Culture handbook, Ambiso).
본원은 PHF6이 기존에 독립적으로 알려진 전사 조절에 관여하는 두 히스톤 변형 즉 H2BK12 (히스톤 2B, 12번째 라이신 잔기)의 아세틸화 및 H2BK120 (히스톤 2B, 120번째 라이신 잔기)의 유비퀴틴화를 PHD1 및 PHD2의 도메인을 통해 기능 단위로 작동하게 하며, PHD2의 H2BK12-Ac 인식이 H2BK120-Ub에 선행되어야 한다는 발견에 근거한 것이다. 특히 본원에서는 백혈병에서 발견되는 PHF6의 돌연변이가 PHD2 도메인의 H2BK12-Ac 인식 부위에 존재하는 것을 밝혔고, H2BK120의 유비퀴틴화에 영향을 끼지는 규명하였다. 이에 PHD2 도메인과 독립적으로 PHF6가 PHD1 도메인을 통해 H2BK120 유비퀴틴화를 촉진/증가/향상 시킬 수 있는 물질은 PHF6의 특히 PHD2 돌연변이로 인한 백혈병의 치료제로 개발될 수 있다.In the present application, the acetylation of H2BK12 (histone 2B, lysine residue 12) and ubiquitination of H2BK120 (histone 2B, lysine residue 120), two histone modifications involved in transcriptional regulation of which PHF6 is previously known independently, were analyzed by PHD1 and PHD2. It operates as a functional unit through a domain, and is based on the discovery that H2BK12-Ac recognition of PHD2 should precede H2BK120-Ub. In particular, in the present application, it was revealed that the mutation of PHF6 found in leukemia exists in the H2BK12-Ac recognition site of the PHD2 domain, and it was investigated whether it affects the ubiquitination of H2BK120. Therefore, a substance in which PHF6 can promote/increase/enhance H2BK120 ubiquitination through the PHD1 domain independently of the PHD2 domain can be developed as a therapeutic agent for leukemia caused by PHD2 mutation in PHF6.
이에 다른 양태에서 본원은 PHF6의 PHD1 도메인; 또는 PHD2 도메인이 결실 또는 돌연변이된 PHF6 단백질 및 히스톤 2B 단백질을 제공하는 단계; 상기 단백질을 상기 PHD1 도메인 또는 상기 PHF6 단백질에 포함된 PHD1 도메인과 상기 히스톤 2B 단백질과의 결합을 촉진시킬 것으로 기대되는 물질의 존재 중에서 접촉하는 단계; 상기 히스톤 2B 단백질의 120번째 라이신 잔기의 유비퀴틴화 수준을 측정하는 단계; 및 상기 측정 결과 시험물질과 처리되지 않은 대조군과 비교하여, 처리된 경우 히스톤 2B 단백질의 120번째의 유비퀴틴화가 증가한 경우, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법에 관한 것이다.Accordingly, in another aspect, the present application provides a PHD1 domain of PHF6; or providing a PHF6 protein and a histone 2B protein in which the PHD2 domain is deleted or mutated; contacting the protein with the PHD1 domain or the PHD1 domain included in the PHF6 protein in the presence of a substance expected to promote binding of the histone 2B protein; measuring the ubiquitination level of the 120th lysine residue of the histone 2B protein; And when compared with the test substance and the untreated control as a result of the measurement, when the treatment increases ubiquitination at the 120th position of the histone 2B protein, it relates to a screening method for a histone epigenetic modulator by PHF6.
PHF6의 PHD1 도메인; 또는 PHD2 도메인이 전부 또는 일부 결실 또는 돌연변이된 PHF6 단백질은 본원 실시예 및 본원에 개시된 인간 유전자 및 단백질 서열을 근거로 유전적 방법에 의해 당업자라면 제조할 수 있을 것이다. PHD2 도메인 돌연변이는 기능상실 돌연변이를 의미하나 특히 H2BK12Ac 인식에 중요한 글루탐산 풍부 모티브 (도 4 참조)가 결실 또는 치환 등에 의해 다른 아미노산으로 돌연변이 된 것이다.PHD1 domain of PHF6; Alternatively, the PHF6 protein in which all or part of the PHD2 domain is deleted or mutated can be prepared by those skilled in the art by a genetic method based on the examples herein and the human gene and protein sequences disclosed herein. The PHD2 domain mutation refers to a loss-of-function mutation, but in particular, a glutamic acid rich motif (see FIG. 4 ), which is important for H2BK12Ac recognition, is mutated to another amino acid by deletion or substitution.
상기 방법에 사용되는 단백질은 상기 단백질을 발현하는 진핵세포, 동물모델 또는 오가노이드 형태로 제공될 수 있으며, 이에 대해서는 앞서 언급한 바를 참조할 수 있다. The protein used in the method may be provided in the form of a eukaryotic cell, an animal model, or an organoid expressing the protein, for which reference may be made to the aforementioned bar.
또한 상기 방법에 사용되는 유비퀴틴화 측정방법도 앞서 언급한 바를 참조할 수 있다. Also, the method for measuring ubiquitination used in the above method may refer to the aforementioned bar.
본원에 따른 방법에 사용되는 시험물질은 본원에서 규명된 PHF6의 H2BK12-Ac의 인식을 조절하여 이와 연결된 H2BK120 유비퀴틴화의 조절을 가져올 것으로 기대되는 물질, 예를 들면 저분자량 화합물, 고분자량 화합물, 화합물들의 혼합물 (예컨대, 천연 추출물 또는 세포 또는 조직 배양물), 또는 바이오의약품 (예컨대, 단백질, 항체, 펩타이드, DNA, RNA, 안티센스 올리고뉴클레오타이드, RNAi, 앱타머, RNAzyme 및 DNAzyme) 또는 당 및 지질 등을 포함하나 이로 한정하는 것은 아니다. The test substance used in the method according to the present application modulates the recognition of H2BK12-Ac of PHF6 identified herein and is expected to result in modulation of H2BK120 ubiquitination linked thereto, for example, low molecular weight compounds, high molecular weight compounds, compounds mixtures of these (e.g., natural extracts or cell or tissue cultures), or biopharmaceuticals (e.g., proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzyme and DNAzyme) or sugars and lipids, etc. including, but not limited to.
본원에 따른 일 구현예에서는 저분자화합물이 시험물질로 사용될 수 있다. 상기 시험 물질은 합성 또는 천연 화합물의 라이브러리로부터 얻을 수 있으며 이러한 화합물의 라이브러리를 얻는 방법은 당업계에 공지되어 있다. 합성 화합물 라이브러리는 Maybridge Chemical Co.(UK), Comgenex(USA), Brandon Associates(USA), Microsource(USA) 및 Sigma-Aldrich(USA)에서 구입 가능하며, 천연 화합물의 라이브러리는 Pan Laboratories(USA) 및 MycoSearch(USA)에서 구입 가능하다. 시험 물질은 당업계에 공지된 다양한 조합 라이브러리 방법에 의해 얻을 수 있으며, 예를 들어, 생물학적 라이브러리, 공간 어드레서블 패러럴 고상 또는 액상 라이브러리 (spatially addressable parallel solid phase or solution phase libraries), 디컨볼루션이 요구되는 합성 라이브러리 방법, "1-비드 1-화합물" 라이브러리 방법, 그리고 친화성 크로마토그래피 선별을 이용하는 합성 라이브러리 방법에 의해 얻을 수 있다. 분자 라이브러리의 합성 방법은, DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 등에 개시되어 있다. 예를 들면 약물의 스크리닝 목적을 위해서는 화합물은 저분자량의 치료효과를 갖는 것이 사용될 수 있다. 예를 들면 중량이 400 Da, 600 Da 또는 800 Da과 같은 약 1000 Da 내외의 화합물이 사용될 수 있다. 목적에 따라 이러한 화합물은 화합물 라이브러리의 일부를 구성할 수 있으며, 라이브러리를 구성하는 화합물의 숫자도 수십개부터 수백만개까지 다양하다. 이러한 화합물 라이브러리는 펩타이드, 펩토이드 및 기타 환형 또는 선형의 올리고머성 화합물, 및 주형을 기본으로 하는 저분자 화합물, 예컨대 벤조디아제핀, 하이단토인, 바이아릴, 카보사이클 및 폴리사이클 화합물 (예컨대 나프탈렌, 페노티아진, 아크리딘, 스테로이드 등), 카보하이드레이트 및 아미노산 유도체, 디하이드로피리딘, 벤즈하이드릴 및 헤테로사이클 (예컨대 트리아진, 인돌, 티아졸리딘 등)을 포함하는 것일 수 있으나, 이는 단지 예시적인 것으로 이로 한정되는 것은 아니다.In one embodiment according to the present application, a low molecular weight compound may be used as a test substance. The test substance may be obtained from a library of synthetic or natural compounds, and methods for obtaining a library of such compounds are known in the art. Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and natural compound libraries are available from Pan Laboratories (USA) and It can be purchased from MycoSearch (USA). Test substances can be obtained by various combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution Required synthetic library methods, "1-bead 1-compound" library methods, and synthetic library methods using affinity chromatography selection can be obtained. Methods for synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, et al. For example, for drug screening purposes, compounds having a low molecular weight therapeutic effect may be used. For example, a compound having a weight of about 1000 Da, such as 400 Da, 600 Da, or 800 Da, may be used. Depending on the purpose, these compounds may form a part of a compound library, and the number of compounds constituting the library may also vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and template-based small molecule compounds such as benzodiazepines, hydantoins, biaryls, carbocycles and polycyclic compounds (such as naphthalene, phenothi azine, acridine, steroids, etc.), carbohydrates and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely exemplary. The present invention is not limited thereto.
또한 예를 들면 바이올로직스가 스크리닝에 사용될 수 있다. 바이올로직스는 세포 또는 바이오분자를 일컫는 것으로, 바이오분자란, 단백질, 핵산, 탄수화물, 지질 또는 생체내 및 생체외에서 세포 시스템 등을 이용하여 생산된 물질을 일컫는 것이다. 바이오분자를 단독으로 또는 다른 바이오분자 또는 세포와 조합으로 제공될 수 있다. 바이오분자는 예를 들면, 폴리뉴클레오타이드, 펩타이드, 항체, 또는 기타 혈장에서 발견되는 단백질 또는 생물학적 유기물질을 포함하는 것이다.Also, for example, biologics can be used for screening. Biologics refer to cells or biomolecules, and biomolecules refer to proteins, nucleic acids, carbohydrates, lipids, or substances produced using cell systems in vivo and in vitro. The biomolecule may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, polynucleotides, peptides, antibodies, or other proteins or biological organisms found in plasma.
일 구현예에서 본원에 따른 방법에서 후보물질을 선별할 수 있는 증가 또는 감소는 당업자라면 본원에 개시된 결과 및 당업자의 상식을 고려하여 적절하게 선택할 수 있으며, 예를 들면 이로 제한하는 것은 아니나 시험물질과 접촉되지 않은 대조군과 비교하여 시험물질의 존재하에서 약 10% 이상 증가 또는 감소, 약 20% 이상 증가 또는 감소, 약 30% 이상 증가 또는 감소, 약 40% 이상 증가 또는 감소, 약 50% 이상 증가 또는 감소, 약 60% 이상 증가 또는 감소, 약 70% 이상 증가 또는 감소, 약 80% 이상 증가 또는 감소, 약 80% 이상 증가 또는 감소, 약 100% 증가 또는 감소 또는 그 사이의 범위를 포함하는 시험물질을 후보물질로 선별할 수 있으나, 상기를 벗어나는 범위를 제외하는 것은 아니다. In one embodiment, an increase or decrease that can select a candidate substance in the method according to the present application can be appropriately selected by those skilled in the art in consideration of the results disclosed herein and common knowledge of those skilled in the art, for example, but not limited thereto, with the test substance and the test substance. increase or decrease by at least about 10%, increase or decrease by at least about 20%, increase or decrease by at least about 30%, increase or decrease by at least about 40%, increase or decrease by at least about 50%, or a test substance comprising a decrease, an increase or decrease of at least about 60%, an increase or decrease of at least about 70%, an increase or decrease of at least about 80%, an increase or decrease of at least about 80%, an increase or decrease of about 100%, or a range therebetween can be selected as a candidate material, but does not exclude a range outside the above.
이하, 본 발명의 이해를 돕기 위해서 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 하기의 실시예에 한정되는 것은 아니다.Hereinafter, examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the present invention is not limited to the following examples.
실시예 Example
실험재료 및 방법Experimental materials and methods
세포 배양 및 시약 조건부로 Oct4 결핍을 유도할 수 있는 (ZHBTc4) 마우스 배아 줄기 세포 라인은 기존에 알려져 있다. 세포 배양은 간략하게, 첫 1 ~ 2 계대에서 마우스 배아 섬유 아세포가 깔려 있는 상태에서 유지하였다. 이 상태로 안정화 후, 0.1% 젤라틴이 코팅 된 배양 접시를 사용하여 배아줄기세포를 배양 하였다. 배양액은 Dulbecco's modified Eagle medium (DMEM; Welgene)에 15% 소 태아 혈청 (FBS; Hyclone), 0.055 mM 베타-머캅토에탄올(b-mercaptoethanol), 2 mM L-글루타민산(L-glutamine), 0.1 mM 비필수 아미노산(non-essential amino acids), 5,000 units/ml 페니실린/스트렙토마이신 (penicillin/streptomycin) (GIBCO), 그리고 1,000 units/ml leukemia inhibitory factor (LIF) (Chemicon)가 첨가하여서 37℃의 가습 인큐베이터에서 배양하였다. 모든 세포주는 정기적으로 마이코 플라즈마 오염에 대해 테스트되었다. ZHBTc4의 배반포 분화 리 프로그래밍을 위해, 0.5 mg/ml 독시사이클린 (DOX) (# D3072, SIGMA)을 2일 동안 처리 하였다. ZHBTc4의 신경 외배엽 분화를 위해, 10 mg/ml 트랜스-레티노 산 (RA) (# R2625, SIGMA)을 4일 동안 LIF 없이 처리 하였다. 마우스 배아줄기세포에서의 DNA transfection은 Lipofectamine 3000 (Invitrogen)을 사용하여 수행되었다. CBP 및 p300의 녹다운에 대한 siRNA 서열은 http://gesteland.genetics.utah.edu/siRNA_scales에서 설계하였다. RNF20의 녹다운에 대한 siRNA 서열은 이전에 보고 된 바와 같이 사용되었다. 실험에 사용된 항체의 정보는 다음과 같다. : Novus 사의 항-PHF6 (# 68262); Abcam 사의 항-나노 그 (ab21624), 항 -H3 (ab1791), 항 -H2B (ab1790), 항 -H4 (ab10158), 항 -H3K4me3 (ab8580), 항 -H3K4me1 (ab8895), 항 -H3K27me3 (ab6002), 항 -H3K27Ac (ab4729), 항 -H3K9me3 (ab8898), 항 -H2BK12Ac (ab195494), 항 -RNF40 (ab191309) 및 항 -Cdx2 (ab157524); Santacruz 사의 항-USP44 (sc-377203), 항-p300 (sc-584), 항-CBP (sc-1211), 항-Oct4 (sc-5279), 항-GAPDH (sc-25778) 및 항-GST (sc-459); Bethly 사의 항-RNF20 (A300-714A); Active Motif 사의 항-H2BK120ub (# 39623); 시그마 사의 항 -Flag (# F3165) 및 항- β-actin (# A1978); Abm 사의 항-His (# G020). Cell Cultures and Reagents A mouse embryonic stem cell line (ZHBTc4) capable of conditionally inducing Oct4 deficiency is known. Cell cultures were briefly maintained in a litter of mouse embryonic fibroblasts in the first 1-2 passages. After stabilization in this state, embryonic stem cells were cultured using a culture dish coated with 0.1% gelatin. The culture medium was 15% fetal bovine serum (FBS; Hyclone), 0.055 mM beta-mercaptoethanol, 2 mM L-glutamine, 0.1 mM ratio in Dulbecco's modified Eagle medium (DMEM; Welgene). Non-essential amino acids, 5,000 units/ml penicillin/streptomycin (GIBCO), and 1,000 units/ml leukemia inhibitory factor (LIF) (Chemicon) were added in a humidified incubator at 37°C. cultured. All cell lines were routinely tested for mycoplasma contamination. For blastocyst differentiation reprogramming of ZHBTc4, 0.5 mg/ml doxycycline (DOX) (# D3072, SIGMA) was treated for 2 days. For neuroectoderm differentiation of ZHBTc4, 10 mg/ml trans-retinoic acid (RA) (# R2625, SIGMA) was treated without LIF for 4 days. DNA transfection in mouse embryonic stem cells was performed using Lipofectamine 3000 (Invitrogen). siRNA sequences for knockdown of CBP and p300 were designed at http://gesteland.genetics.utah.edu/siRNA_scales. The siRNA sequence for knockdown of RNF20 was used as previously reported. The information of the antibody used in the experiment is as follows. : Novus anti-PHF6 (#68262); Abcam anti-nanog (ab21624), anti-H3 (ab1791), anti-H2B (ab1790), anti-H4 (ab10158), anti-H3K4me3 (ab8580), anti-H3K4me1 (ab8895), anti-H3K27me3 (ab6002) ), anti-H3K27Ac (ab4729), anti-H3K9me3 (ab8898), anti-H2BK12Ac (ab195494), anti-RNF40 (ab191309) and anti-Cdx2 (ab157524); Anti-USP44 (sc-377203), anti-p300 (sc-584), anti-CBP (sc-1211), anti-Oct4 (sc-5279), anti-GAPDH (sc-25778) and anti-GST from Santacruz (sc-459); anti-RNF20 from Bethly (A300-714A); Anti-H2BK120ub from Active Motif (# 39623); anti-Flag (#F3165) and anti-β-actin (#A1978) from Sigma; Anti-His from Abm (# G020).
짧은 헤어핀 RNA (shRNA) shRNA 녹다운 세포를 제작하기 위해, 렌티 바이러스 shRNA를 바이러스 패키징 플라스미드 (psPAX2 및 pMD2.G)와 함께 HEK293T 세포에 transfection 하였다. 세포가 60 ~ 70% 정도 자란 상태인 transfection 1일 후, 새로운 배지로 바꾸고 바이러스 수집을 위해 24시간 동안 유지하였다. 바이러스 수집 후, 배지를 여과하고 4X Lenti-X 수집기 (# 631232, TAKARA)와 혼합하고, 4℃에서 밤새 인큐베이션 하였다. 이후 수집한 바이러스를 폴리브렌과 섞어서 세포에 감염시켰다. shRNA 서열은 다음과 같다 :To construct short hairpin RNA (shRNA) shRNA knockdown cells, lentiviral shRNA was transfected into HEK293T cells together with viral packaging plasmids (psPAX2 and pMD2.G). One day after transfection, when the cells were in a state of 60 to 70% growth, the medium was changed and maintained for 24 hours for virus collection. After virus collection, the medium was filtered and mixed with a 4X Lenti-X collector (#631232, TAKARA) and incubated overnight at 4°C. Then, the collected virus was mixed with polybrene to infect the cells. The shRNA sequence is as follows:
shPhf6fwdshPhf6fwd
5'-CCGGGTTCAGCTCACAACAACATCACTCGAGTGATGTTGTTGTGTGAGCTGAACTTTTTG-3 ‘ 5'-CCGGGTTCAGCTCACAACAACATCACTCGAGTGATGTTGTTGTGTGAGCTGAACTTTTTG-3'
rev 5'-AATTCAAAAAGTTCAGCTCACAACAACATCACTCGAGTGATGTTGTTGTGAGCTGAAC-3'.rev 5'-AATTCAAAAAGTTCAGCTCACAACAACATCACTCGAGTGATGTTGTTGTGAGCTGAAC-3'.
shRnf20shRnf20
fwd 5'- CCGGCGCATCATCCTTAAACGTTATCTCGAGATAACGTTTAAGGATGATGCGTTTTTG-3 ‘fwd 5'-CCGGCGCATCATCCTTAAACGTTATCTCGAGATAACGTTTAAGGATGATGCGTTTTTG-3'
rev 5'- AATTCAAAAACGCATCATCCTTAAACGTTATCTCGAGATAACGTTTAAGGATGATGCG -3'.rev 5'- AATTCAAAACGCATCATCCTTAAACGTTATCTCGAGATAACGTTTAAGGATGATGCG -3'.
shRnf40shRnf40
fwd 5'- CCGGGACCACTCTAATCGAACCCATCTCGAGATGGGTTCGATTAGAGTGGTCTTTTTG-3 ‘fwd 5'-CCGGGACCACTCTAATCGAACCCATCTCGAGATGGGTTCGATTAGAGTGGTCTTTTTG-3'
rev 5' - AATTCAAAAAGACCACTCTAATCGAACCCATCTCGAGATGGGTTCGATTAGAGTGGTC - 3' .rev 5' - AATTCAAAAAGACCACTCTAATCGAACCCATCTCGAGATGGGTTCGATTAGAGTGGTC - 3' .
Phf6 Knock Out (KO) 마우스 ESC 생성 Phf6 KO 마우스 배아 줄기 세포 제작은 CRISPR-Cas9 시스템을 사용하였다. Phf6 표적화를 위해, 단일 가이드 RNA (sgRNA) 디자인을 GPP sgRNA Designer (CRISPRko)로부터 수행하였다. 선택한 sgRNA를 pRGEN-U6 벡터에 클로닝하고 ZHBTc4에 transfection 하였다. 퓨로마이신 처리에 의한 단일 콜로니 선택 후 Phf6 KO 콜로니를 수득하고, 면역 블롯팅으로 확인한 후 생거-시퀀싱에 의해 프레임-시프트 돌연변이를 확인하였다. Phf6 Knock Out (KO) mouse ESC generation Phf6 KO mouse embryonic stem cells were produced using the CRISPR-Cas9 system. For Phf6 targeting, a single guide RNA (sgRNA) design was performed from GPP sgRNA Designer (CRISPRko). The selected sgRNA was cloned into the pRGEN-U6 vector and transfected into ZHBTc4. Phf6 KO colonies were obtained after single colony selection by puromycin treatment, and after confirming by immunoblotting, frame-shift mutations were confirmed by Sanger-sequencing.
배아체 (EB) 형성 ZHBTc4 배아줄기세포를 행잉-드롭 방법에 의해 1000개의 세포/방울로 시작하였다. 방울 상태에서 2일 동안 유지한 후, 각 방울은 코팅되지 않은 멸균 커버 유리 및 둥근 바닥의 96웰 플레이트에 옮겨서 배양하였다. 배지는 매일 첨가를 혹은 바꾸어 주었고, 플레이트 이동은 EB에 대한 물리적 영향을 최소화하기 위해 최소로 고정되었다. 모든 EB는 LIF가 없는 배지를 사용하여 배양되었다. Embryo body (EB) formation ZHBTc4 embryonic stem cells were started at 1000 cells/drop by the hanging-drop method. After maintaining in the droplet state for 2 days, each droplet was transferred to an uncoated sterile cover glass and a round-bottom 96-well plate for incubation. Medium was added or changed daily, and plate movement was kept to a minimum to minimize physical impact on EBs. All EBs were cultured using LIF-free medium.
알칼리 포스파타제 (AP) 염색 AP 염색을 위해, 야생형 (WT) 및 Phf6 KO ZHBTc4를 12웰 플레이트에서 0.1% 젤라틴-코팅 된 커버 유리상에서 배양하였다. 웰당 104 개의 세포가 처음에 뿌려졌다. 2일 후, AP 염색은 알칼리성 포스파타제 검출 키트 (# SCR004, Millipore)를 사용하여 수행되었다. 제공된 프로토콜에 따라 염색을 수행 하였다. 간략하게, 각 세포를 1-2% 동안 4% 파라포름 알데하이드로 고정시키고, 실온에서 용액 (나프톨 / 패스트 레드 바이올렛 믹스)과 15-20분 동안 반응시켰다. 반응 후 용액을 씻어 내고 이미지 슬라이드를 만들었다. Alkaline Phosphatase (AP) Staining For AP staining, wild-type (WT) and Phf6 KO ZHBTc4 were cultured on 0.1% gelatin-coated cover glass in 12 well plates. 10 4 cells per well were initially seeded. After 2 days, AP staining was performed using an alkaline phosphatase detection kit (# SCR004, Millipore). Staining was performed according to the protocol provided. Briefly, each cell was fixed with 4% paraformaldehyde for 1-2% and reacted with the solution (naphthol/fast red violet mix) for 15-20 min at room temperature. After the reaction, the solution was washed and an image slide was made.
살아있는 세포 이미징 ZHBTc4 줄기세포 및 배아체의 성장을 추적하기 위해, JuLI Stage 실시간 세포 이력 기록기 (NanoEnTeK)가 사용되었다. 배아줄기세포 성장을 추적하기 위해, 104 세포를 0.1% 젤라틴-코팅 된 12웰 배양 접시에 뿌리고, 12시간 마다 세포가 자란 정도를 기록하였다. 기록이 완료된 후, 세포 성장 곡선은 JuLI에 의해 제공된 소프트웨어에 의해 분석되었다. 배아체 형성의 경우, 각각의 방울을 96웰 둥근 모양 플레이트의 각각의 웰로 이동시키고 12시간 마다 세포 형태를 기록 하였다. 그런 다음 제공된 소프트웨어로 이미지를 촬영하였다. 배아체 코어 외 세포 영역을 계산하기 위해, ImageJ 프로그램을 사용하여 7일에 촬영한 이미지에서 영역 크기를 계산하였다. Live Cell Imaging To track the growth of ZHBTc4 stem cells and embryoid bodies, a JuLI Stage real-time cell history recorder (NanoEnTeK) was used. To track embryonic stem cell growth, 10 4 cells were seeded in 0.1% gelatin-coated 12-well culture dishes, and the degree of cell growth was recorded every 12 hours. After the recording was complete, the cell growth curves were analyzed by the software provided by JuLI. For embryoid body formation, each drop was transferred to each well of a 96-well round plate and cell morphology was recorded every 12 h. Then, images were taken with the provided software. To calculate the cell area outside the embryonic core, the area size was calculated from the images taken on day 7 using the ImageJ program.
면역 세포 화학 염색법 면역 형광 촬영을 위한 배아체를 실험 전에 커버 슬립에서 성장시켰다 (커버 슬립 당 1개의 배아체). 배아체를 PBS에 섞은 2% 파라포름 알데히드로 10분 동안 고정시킨 다음, 실온에서 DPBS로 2회 세척 하였다. 고정 세포를 실온에서 5분 동안 PBS (PBS-T) 중 0.5% 트리톤 X-100으로 투과화시켰다. 0.1% PBS-T 중 10% FBS로 30분 동안 blocking을 수행하였다. 염색을 위해, 세포를 실온에서 4시간 동안 1차 항체와 함께 인큐베이션하고, 0.1% PBS-T로 4회 세척하고, 형광 표지 된 2차 항체 (Invitrogen)와 함께 1시간 동안 인큐베이션 하였다. 세포를 세척하고 DAPI (Sigma)와 함께 VECTASHIELD (H-1200, Vector Laboratories)에 의해 고정하였다. 형광은 Zeiss LSM700 공 초점 현미경 (Carl Zeiss)에서 촬영하였다. Immunocytochemical staining Embryo bodies for immunofluorescence imaging were grown on coverslips (1 embryo per coverslip) prior to experiments. Embryo bodies were fixed with 2% paraformaldehyde mixed in PBS for 10 minutes, and then washed twice with DPBS at room temperature. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS (PBS-T) for 5 min at room temperature. Blocking was performed with 10% FBS in 0.1% PBS-T for 30 minutes. For staining, cells were incubated with primary antibody for 4 h at room temperature, washed 4 times with 0.1% PBS-T, and incubated with fluorescently labeled secondary antibody (Invitrogen) for 1 h. Cells were washed and fixed by VECTASHIELD (H-1200, Vector Laboratories) with DAPI (Sigma). Fluorescence was captured on a Zeiss LSM700 confocal microscope (Carl Zeiss).
세포 융해 (Lysis) 융해 전 세포는 차가운 PBS로 간단히 헹구었다. 프로테아제 억제제가 첨가된 용해액 (50mM Tris-HCl pH 8.0, 200mM NaCl 및 0.5% NP-40)에 세포를 용해한 후, 브랜슨 소니파이어 450을 사용하여 초음파 처리로 세포를 용해하였다(출력 4, 10 ~ 12 펄스). 용해물은 브래드 포드 방법으로 정량하고 SDS-PAGE로 분석 하였다.Lysis Cells were briefly rinsed with cold PBS prior to lysis . Cells were lysed in a protease inhibitor-added lysate (50 mM Tris-HCl pH 8.0, 200 mM NaCl and 0.5% NP-40), and then the cells were lysed by sonication using a Branson Sonifier 450 ( outputs 4, 10 to 12 pulses). Lysates were quantified by the Bradford method and analyzed by SDS-PAGE.
Far western 블롯팅 대장균에서 GST 비드를 사용하여 GST-PHF6 단백질을 정제하였다. ZHBTc4 배아줄기세포 로부터 히스톤 추출물을 SDS-PAGE로 분리하였다. 크기에 따라 H3, H2B, H2A 및 H4 순서로 히스톤 옥타머를 분리 한 후, 정제된 GST-PHF6 단백질 0.1 mg/ml를 완충용액 (100 mM NaCl, 20 mM Tris-HCl [pH 7.6], 10% 글리세롤, 0.1% 트윈-20, 50mM ZnCl2, 2% 탈지 분유 및 1mM DTT) 에서 히스톤 추출물과 함께 반응시켰다. 히스톤이 분리된 멤브레인 상에 GST-PHF6의 결합 후, 항-GST 항체를 사용하여 면역 블롯 분석을 수행 하였다. Far western blotting E. coli GST-PHF6 protein was purified using GST beads. Histone extracts were separated from ZHBTc4 embryonic stem cells by SDS-PAGE. After separation of histone octamers in the order of H3, H2B, H2A and H4 according to size, 0.1 mg/ml of purified GST-PHF6 protein was added to a buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.6], 10% Glycerol, 0.1% Tween-20, 50 mM ZnCl 2 , 2% skim milk powder and 1 mM DTT) were reacted with histone extracts. After binding of GST-PHF6 to the histone-separated membrane, immunoblot analysis was performed using an anti-GST antibody.
시험관 내 히스톤 펩티드 풀다운 분석 분석 완충액 (250 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.05% NP-40, 및 50 mM ZnCl2)에서 0.3μg의 GST 정제한 단백질과 0.5μg의 바이오틴화 된 히스톤 펩티드를 밤새 인큐베이션 하였다. 이어서, 50% 슬러리의 스트렙타비딘 비드를 첨가하고 1시간 동안 추가로 인큐베이션 하였다. 이 후, 세척으로 비특이적 상호 작용을 제거한 후, 비드를 샘플링 완충액에서 비등시키고 펩티드-단백질 상호 작용을 면역 블롯팅에 의해 검출 하였다. 히스톤 H3 변형에 대한 바이오틴화 된 히스톤 펩티드는 Boston BioChems로부터 구입하였고, 히스톤 H2B 변형에 대한 것은 JPT peptide technology 로부터 구입 하였다. In vitro histone peptide pulldown assay 0.3 μg GST-purified protein and 0.5 μg biotinylated histone in assay buffer (250 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.05% NP-40, and 50 mM ZnCl2). Peptides were incubated overnight. Then, a 50% slurry of streptavidin beads was added and further incubated for 1 hour. Thereafter, after washing to remove non-specific interactions, the beads were boiled in sampling buffer and peptide-protein interactions were detected by immunoblotting. Biotinylated histone peptides for histone H3 modifications were purchased from Boston BioChems, and those for histone H2B modifications were purchased from JPT peptide technology.
시험관 내 히스톤 펩티드 결합 어레이 히스톤 펩티드 결합 어레이 키트는 ActiveMotif (# 13005)로부터 구입하였다. 제공된 프로토콜에 따라 다음 분석이 진행되었다. 간단하게, 4℃에서 밤새 5% 우유가 섞인 TTBS (10mM Tris-HCl [pH 7.5], 150mM NaCl 및 0.05% 트윈 -20)로 키트를 blocking 하였다. 이후, 키트를 실온에서 1시간 동안 결합 완충제 (100 mM KCl, 20 mM HEPES [pH 7.9], 1 mM EDTA, 10% 글리세롤 및 0.1 mM DTT) 중 0.3μg의 용리 된 GST-PHF6와 함께 배양 하였다. 이어서, GST 1차 항체 및 2차 항체를 키트에서 실온에서 1시간 동안 순차적으로 인큐베이션 하였다. 각 단계 사이에 TTBS로 세 번 세척 작업을 진행하였다. In Vitro Histone Peptide Binding Array Histone Peptide Binding Array Kit was purchased from ActiveMotif (#13005). The following assays were performed according to the provided protocol. Briefly, the kit was blocked with TTBS (10 mM Tris-HCl [pH 7.5], 150 mM NaCl and 0.05% Tween-20) mixed with 5% milk overnight at 4°C. The kit was then incubated with 0.3 μg of eluted GST-PHF6 in binding buffer (100 mM KCl, 20 mM HEPES [pH 7.9], 1 mM EDTA, 10% glycerol and 0.1 mM DTT) for 1 h at room temperature. Then, the GST primary antibody and secondary antibody were sequentially incubated for 1 hour at room temperature in the kit. Washing was performed three times with TTBS between each step.
단백질 발현 및 정제 PHF6 WT 및 E223S 돌연변이 체 (MT)는 로제타 (DE3) 대장균 세포에서 GST 융합 단백질로서 발현되었다. 0.25 mM 이소 프로필 1-티오-β-D-갈락토피라노시드 (IPTG)로 유도 한 후 20℃에서 밤새 성장시킨 후 세포를 수확하고, 500 mM NaCl 및 1mM 페닐 메탄 설 포닐 플루오 라이드 (PMSF)이 보충 된 포스페이트 완충 식염수 (PBS) 중 에멀시 플렉스 C3 (아베 스틴)을 사용하여 용해시켰다. 15분 동안 14,000 rpm에서 원심 분리한 후, 상청액을 글루타티온 아가 로스 컬럼 (Thermo Scientific)에 로딩 하였다. 평형 완충액 (20mM HEPES-NaOH [pH 7.5] 및 300mM NaCl)으로 컬럼 세척 한 후, 각 단백질을 용리 완충액 (100mM HEPES-NaOH [pH 7.5], 300mM NaCl 및 20mM 환원 글루타티온)으로 용리시켰다. 각각의 GST-PHF6 (WT 및 E223S)를 겔 여과 완충액 (20mM HEPES-NaOH [pH 7.5] 및 150mM NaCl)으로 사전 평형화 한 Superdex 200 크기 배제 컬럼인 HiTrap SP 양이온 교환 컬럼으로 정제하였다. 대조 실험을 위해, GST는 Rosetta (DE3) 대장균 세포에서 발현되었고 HiTrap SP 컬럼 대신 HiTrap Q 음이온 교환 컬럼을 사용하는 것을 제외하고는 전술 한 바와 유사하게 정제되었다. Protein expression and purification PHF6 WT and E223S mutants (MT) were expressed as GST fusion proteins in Rosetta (DE3) E. coli cells. Cells were harvested after induction with 0.25 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) followed by overnight growth at 20 °C, 500 mM NaCl and 1 mM phenylmethane sulfonyl fluoride (PMSF) This was dissolved using Emulciplex C3 (Avestin) in supplemented phosphate buffered saline (PBS). After centrifugation at 14,000 rpm for 15 minutes, the supernatant was loaded onto a glutathione agarose column (Thermo Scientific). After column washing with equilibration buffer (20 mM HEPES-NaOH [pH 7.5] and 300 mM NaCl), each protein was eluted with elution buffer (100 mM HEPES-NaOH [pH 7.5], 300 mM NaCl and 20 mM reduced glutathione). Each GST-PHF6 (WT and E223S) was purified on a HiTrap SP cation exchange column, a Superdex 200 size exclusion column, pre-equilibrated with gel filtration buffer (20 mM HEPES-NaOH [pH 7.5] and 150 mM NaCl). For control experiments, GST was expressed in Rosetta (DE3) E. coli cells and purified similarly as described above, except that a HiTrap Q anion exchange column was used instead of a HiTrap SP column.
MST (MicroScale Thermophoresis) 측정 결합성 측정은 Nanotemper Monolith NT.115pico로 수행되었다. H2B 펩타이드 (1-20) WT 및 K12Ac 변형을 갖는 펩티드는 Genscript로부터 구입하였고 각각의 GST-PHF6 (WT 및 E223S MT) 및 GST는 염료 NT-647 (Cy5) (Lumiprobe)로 표지되었다. 표지된 GST 및 GST-PHF6는 ~90nM의 농도로 사용되었다. 각각의 H2B 펩티드를 MST 완충액 (20mM HEPES-NaOH [pH 7.5], 150mM NaCl, 0.05% 트윈-20 및 0.5 mg / mL BSA)에서 ~ 880mM에서 26nM로 희석하고, 표지 된 단백질과 함께 10분 동안 실온에서 배양하였다. MST 측정은 40% MST 전력 및 25% LED 전력 (GST-PHF6 WT) 또는 12% LED 전력 (GST-PHF6 E223S 및 GST)으로 22℃에서 수행되었다. 각 데이터 세트는 MO를 사용하여 분석되었다 (Nanotemper Technology). MST (MicroScale Thermophoresis) Measurements Binding measurements were performed with a Nanotemper Monolith NT.115pico. H2B peptide (1-20) WT and peptides with K12Ac modifications were purchased from Genscript and GST-PHF6 (WT and E223S MT) and GST, respectively, were labeled with the dye NT-647 (Cy5) (Lumiprobe). Labeled GST and GST-PHF6 were used at a concentration of ˜90 nM. Dilute each H2B peptide from ~880 mM to 26 nM in MST buffer (20 mM HEPES-NaOH [pH 7.5], 150 mM NaCl, 0.05% Tween-20 and 0.5 mg/mL BSA), with labeled protein at room temperature for 10 min. cultured in MST measurements were performed at 22°C with either 40% MST power and 25% LED power (GST-PHF6 WT) or 12% LED power (GST-PHF6 E223S and GST). Each data set was analyzed using MO (Nanotemper Technology).
시험관 내 유비퀴틴화 분석 기질의 경우, H2B-Flag WT / K12R을 Phf6 KO 배아줄기세포에 형질 감염시키고, 모노-뉴 클레오솜을 함유하는 H2B-Flag를 MNase 소화에 의해 정제 하였다. 정제 후, 이들 뉴클레오솜을 E1, E2, E3 효소, 유비퀴틴, 50mM ZnCl2 및 10x 완충액 (500mM Tris-HCl [pH 7.5], 20mM ATP, 10mM MgCl2, 2mM DTT)과 50μl로 혼합 하였다. 이어서 37℃에서 1시간 동안 인큐베이션 하였다. 반응을 정지시키기 위해, 샘플링 완충액을 첨가하고 샘플을 100℃에서 10분 동안 비등시켰다. E1, E2 (UBCH3, UBCH6) 및 유비퀴틴은 Boston Biochems에서 구입했다. 정제된 GST-PHF6 WT/돌연변이는 E3 리가아제로 간주되었다.For the in vitro ubiquitination assay substrate, H2B-Flag WT/K12R was transfected into Phf6 KO embryonic stem cells, and H2B-Flag containing mono-nucleosomes was purified by MNase digestion. After purification, these nucleosomes were mixed with E1, E2, E3 enzymes, ubiquitin, 50 mM ZnCl2 and 10x buffer (500 mM Tris-HCl [pH 7.5], 20 mM ATP, 10 mM MgCl2, 2 mM DTT) in 50 μl. Then, it was incubated for 1 hour at 37°C. To stop the reaction, sampling buffer was added and the sample boiled at 100° C. for 10 minutes. E1, E2 (UBCH3, UBCH6) and ubiquitin were purchased from Boston Biochems. Purified GST-PHF6 WT/mutant was considered E3 ligase.
시험관 내 GST-풀다운 분석 E2 및 E3 관계로서 PHF6과 UCBH3 간의 상호 작용을 확인하기 위해, 시험관 내 유비퀴틴 화 분석에 의해 His-UBCH3을 유비퀴틴화 하였다. UBCH3-유비퀴틴 컨쥬게이션을 위해, 50ng의 E1, 0.5μg의 His-UBCH3 및 유비퀴틴을 유비퀴틴 화 분석 완충액과 함께 37℃에서 1시간 동안 배양 하였다. 다음으로, 1μg의 유비퀴틴-접합 된 His-UBCH3를 1ml 부피의 풀다운-검정 완충제 (20mM Tris-HCl [pH 7.8], 125mM NaCl, 10% 글리세롤, 0.1% NP-40 및 프로테아제 억제제)에서 비드 결합 된 GST 단독 또는 GST-PHF6 WT 또는 돌연변이와 함께 4℃에서 밤새 배양하였다. 반응 후, 동일한 완충액으로 비드를 4회 세척하고 샘플링 완충액으로 비드를 비등시켰다.In vitro GST-pulldown assay To confirm the interaction between PHF6 and UCBH3 as an E2 and E3 relationship, His-UBCH3 was ubiquitinated by an in vitro ubiquitination assay. For UBCH3-ubiquitin conjugation, 50 ng of E1, 0.5 μg of His-UBCH3 and ubiquitin were incubated with ubiquitination assay buffer at 37°C for 1 hour. Next, 1 μg of ubiquitin-conjugated His-UBCH3 was bead-bound in 1 ml volume of pull-down-assay buffer (20 mM Tris-HCl [pH 7.8], 125 mM NaCl, 10% glycerol, 0.1% NP-40 and protease inhibitor). GST alone or with GST-PHF6 WT or mutants were incubated overnight at 4°C. After the reaction, the beads were washed 4 times with the same buffer and the beads were boiled with the sampling buffer.
ChIP (Chromatin-Immunoprecipitation) 및 MNase-ChIP 분석 ChIP 분석은 기본적으로 보고된 바와 같이 수행되었다. ChIP 분석을 위해, 세포를 실온에서 15분 동안 1% 포름 알데히드에 의해 가교시켰다. 핵 펠렛을 RIPA 완충액에서 초음파 처리 하였다. MNase-ChIP의 경우, 핵 펠렛을 MNase 용해 완충액에 용해시키고 초음파 처리 대신에 37℃에서 10분 동안 MNase와 반응시켰다. 역가교는 65℃에서 밤새 수행되었다. MNase-ChIP 샘플의 역 가교 및 면역 블롯팅을 위해, 면역 침전 된 비드를 2X 샘플링 완충액과 혼합하고 100℃에서 45분 동안 비등시켰다. 정제 컬럼 (# 28105, QIAGEN)에 의해 DNA를 단리 하였다. 용리 된 DNA는 qRT-PCR에 의해 검출되었다. 모든 반응을 3회 수행 하였다. 이 연구에 사용 된 ChIP-qRT-PCR 프라이머는 다음 표 1과 같다. Chromatin-Immunoprecipitation (ChIP) and MNase-ChIP assays ChIP assays were performed essentially as reported. For ChIP assay, cells were crosslinked with 1% formaldehyde for 15 min at room temperature. Nuclear pellets were sonicated in RIPA buffer. For MNase-ChIP, nuclear pellets were dissolved in MNase lysis buffer and reacted with MNase at 37°C for 10 min instead of sonication. Backcrosslinking was performed overnight at 65°C. For reverse crosslinking and immunoblotting of MNase-ChIP samples, immunoprecipitated beads were mixed with 2X sampling buffer and boiled at 100 °C for 45 min. DNA was isolated by purification column (#28105, QIAGEN). Eluted DNA was detected by qRT-PCR. All reactions were performed in triplicate. The ChIP-qRT-PCR primers used in this study are shown in Table 1.
qRT-PCR primersqRT-PCR primers
Gene nameGene name Primer informationPrimer information Primer sequencePrimer sequence
Phf6 Phf6 Forward
ReverseForward
Reverse 		5'-TCTGTAGGGAGCACCGACAAT-3'
5'-TCTGTAGGGAGCACCGACAAT-3'5'-TCTGTAGGGAGCACCGACAAT-3'
5'-TCTGTAGGGAGCACCGACAAT-3'
Cdx2cdx2 Forward
ReverseForward
Reverse 		5'-CAAGGACGTGAGCATGTATCC-3'
5'-GTAACCACCGTAGTCCGGGTA-3'5'-CAAGGACGTGAGCATGTATCC-3'
5'-GTAACCACCGTAGTCCGGGTA-3'
Oct4Oct4 Forward
ReverseForward
Reverse 		5'-AGAGG ATCACCTTGGGGTACA-3'
5'-CGAAGCGACAGATGGTGGTC-3'5'-AGAGG ATCACCTTGGGGTACA-3'
5'-CGAAGCGACAGATGGTGGTC-3'
Tbx3Tbx3 Forward
ReverseForward
Reverse 		5'-GAACCTACCTGTTCCCGGAAA-3'
5'-CCATTGCCAGTGTCTCGAAAAC-3'5'-GAACCTACCTGTTCCCGGAAA-3'
5'-CCATTGCCAGTGTCTCGAAAAC-3'
Klf5Klf5 Forward
ReverseForward
Reverse 		5'-CCGGAGACGATCTGAAACAC-3'
5'-CAGATACTTCTCCATTTCACATCTTG-3'5'-CCGGAGACGATCTGAAACAC-3'
5'-CAGATACTTCTCCATTTCACATCTTG-3'
Plac1Plac1 Forward
ReverseForward
Reverse 		5'-CTTCAGCTACTCGGAGCAAAA-3'
5'-GTGAACATGATTGGGAGGGC-3'5'-CTTCAGCTACTCGGAGCAAAA-3'
5'-GTGAACATGATTGGGAGGGC-3'
Ascl2Ascl2 Forward
ReverseForward
Reverse 		5'-TCCTGGTGGACCTACCTGCTT-3'
5'-AGGTCAGTCAGCACTTGGCATT-3'5'-TCCTGGTGGACCTACCTGCTT-3'
5'-AGGTCAGTCAGCACTTGGCATT-3'
Gata2Gata2 Forward
ReverseForward
Reverse 		5'-CCTCCAGCTTCACCCCTAA-3'
5'-CAGAGAGGGGTGGCTGTG-3'5'-CCTCCAGCTTCACCCCTAA-3'
5'-CAGAGAGGGGTGGCTGTG-3'
Wnt7bWnt7b Forward
ReverseForward
Reverse 		5'-GGAGAAGCAAGGCTACTACAACCAGG-3'
5'-GCATCCACAAAGCGACGAGAAAA-3'5'-GGAGAAGCAAGGCTACTACAACCAGG-3'
5'-GCATCCACAAAGCGACGAGAAAA-3'
Fgfr2Fgfr2 Forward
ReverseForward
Reverse 		5'-CCACATTCAAGCAGTTGGTC-3'
5'-TTGTGTCGGGGTAACTAGGAG-3'5'-CCACATTCAAGCAGTTGGTC-3'
5'-TTGTGTCGGGGTAACTAGGAG-3'
Esx1Esx1 Forward
ReverseForward
Reverse 		5'-TTGGAGGGAGCAGACTACCAG-3
5'-CCAAAGTCGGAGTAGAAAGTTGT-3'5'-TTGGAGGGAGCAGACTACCAG-3
5'-CCAAAGTCGGAGTAGAAAGTTGT-3'
GapdhGapdh Forward
ReverseForward
Reverse 		5'-CATGGCCTTCCGTGTTCCTA-3'
5'-CCTGCTTCACCACCTTCTTGA-3'5'-CATGGCCTTCCGTGTTCCTA-3'
5'-CCTGCTTCACCACCTTCTTGA-3'
beta-actin beta-actin Forward
ReverseForward
Reverse 		5'-TAGCCATCCAGGCTGTGCTG-3'
5'-CAGGATCTTCATGAGGTAGTC-3'5'-TAGCCATCCAGGCTGTGCTG-3'
5'-CAGGATCTTCATGAGGTAGTC-3'
ChIP-qPCR primersChIP-qPCR primers
Gene nameGene name Primer informationPrimer information Primer sequencePrimer sequence
Cdx2 promoter Cdx2 promoter Forward
ReverseForward
Reverse 		5'-TCAACGTTTGTCCCCAGACA-3'
5'-ACGTCCTTGTCCAGAAGGTAGCT-3'5'-TCAACGTTTGTCCCCAGACA-3'
5'-ACGTCCTTGTCCAGAAGGTAGCT-3'
Gata2 promoter Gata2 promoter Forward
ReverseForward
Reverse 		5'-GTTGTCCAGCGGATCCTACC-3'
5'-ACGTGCACCTTCTGGGTATC-3'5'-GTTGTCCAGCGGATCCTACC-3'
5'-ACGTGCACCTTCTGGGTATC-3'
Msx2 promoter Msx2 promoter Forward
ReverseForward
Reverse 		5'-TGTTAACACCCGTTCCCCAG-3'
5'-ATACAGGAGCCCGGCAGATA-3'5'-TGTTAACACCCGTTCCCCAG-3'
5'-ATACAGGAGCCCGGCAGATA-3'
Klf6 promoter Klf6 promoter Forward
ReverseForward
Reverse 		5'-TGGCATAGTCTCCATGCTTCC-3'
5'-TTTTCGGGTACTGGGCTCTG-3'5'-TGGCATAGTCTCCATGCTTCC-3'
5'-TTTTCGGGTACTGGGCTCTG-3'
정량적 RT-PCR 총 RNA를 Trizol (Invitrogen)을 사용하여 추출하고 M-MLV cDNA Synthesis Kit (Enzynomics)를 사용하여 1 ~ 2μg의 총 RNA에서 역전사를 수행했다. mRNA의 양은 SYBR TOPreal qPCR 2X PreMix (Enzynomics)를 갖는 ABI 프리즘 7500 시스템 또는 BioRad CFX384에 의해 검출되었다. mRNA의 양은 ddCt 방법을 사용하여 계산하였고 Gapdh 및 β-actin을 대조군으로 사용 하였다. 모든 반응을 3회 수행 하였다. 이 연구에 사용 된 qRT-PCR 프라이머는 표 1과 같다. Quantitative RT-PCR total RNA was extracted using Trizol (Invitrogen) and reverse transcription was performed on 1-2 μg of total RNA using the M-MLV cDNA Synthesis Kit (Enzynomics). The amount of mRNA was detected by BioRad CFX384 or ABI Prism 7500 system with SYBR TOPreal qPCR 2X PreMix (Enzynomics). The amount of mRNA was calculated using the ddCt method and Gapdh and β-actin were used as controls. All reactions were performed in triplicate. Table 1 shows the qRT-PCR primers used in this study.
RNA 시퀀싱 분석 총 RNA는 DOX 처리 유무에 따라 각각 WT 및 Phf6 결핍 세포로부터 추출되었다. 그런 다음, Illumina의 TruSeq 프로토콜에 따라 가닥 mRNA-seq 라이브러리를 준비했다. HiSeq 플랫폼에서 원시 데이터를 생성 한 후 Trimmomatic (v0.36)에 의해 품질이 낮은 어댑터 및 베이스를 제거하기 위해 판독을 사전 처리하였다. 다음으로 STAR (v2.5.3)를 사용하여 판독 값을 정렬하고 TPM (transcripts per Million)을 RSEM (v1.3.0)으로 계산하였다. 유전자 당 판독 횟수를 사용하여, 정상 상태, DOX 처리 및 RA 처리에서 WT와 Phf6 KO의 모든 가능한 6가지 쌍별 비교를 위해 DESeq2 (v1.18.1)로 차별적으로 발현 된 유전자 (DEG)를 확인 하였다. R- (v 3.4.3)에서 4세트의 DEG의 결합에 대해 k- 평균 군집화를 수행 하였다. 유전자 세트 농축 분석 (GSEA)의 경우, 표현형 라벨은 2 : 2 : 50 : 10 = WT-DOX : KO-DOX : WT + DOX : KO + DOX로 정의되었고, 유전자 당 피어슨 상관 계수는 순위를 위해 사용되었다. 그런 다음 분자 시그니처 데이터베이스 (MSigDB) v6.2의 유전자 세트에 대해 농축 점수를 계산 하였다. 각 클러스터에 대한 WT와 Phf6 KO 세포 간의 DOX 반응성의 차이를 분석하기 위해, 먼저 각 샘플에 대해 각 클러스터에있는 유전자의 z- 점수 평균을 구하였다. 그 후, 각각의 WT 및 Phf6 KO 세포에서 DOX의 존재 또는 부재에 따른 평균값의 차이를 계산하였고, 이는 WT 및 Phf6 KO 세포의 DOX 반응성으로 간주되었다. 최종적으로, 클러스터에 의한 WT 및 Phf6 KO 세포 간의 DOX 반응성의 차이는 계산되었고 클러스터 당 PHF6의 존재 또는 부재에 따라 DOX 반응성 인 것으로 간주되었다. RNA sequencing analysis Total RNA was extracted from WT and Phf6-deficient cells with and without DOX treatment, respectively. Then, strand mRNA-seq library was prepared according to Illumina's TruSeq protocol. After generating raw data on the HiSeq platform, reads were preprocessed by Trimmomatic (v0.36) to remove poor quality adapters and bases. Next, reads were aligned using STAR (v2.5.3) and TPM (transcripts per million) was calculated as RSEM (v1.3.0). Using the number of reads per gene, differentially expressed genes (DEGs) were identified with DESeq2 (v1.18.1) for all possible pairwise comparisons of WT and Phf6 KOs in steady-state, DOX-treated and RA-treated. k-means clustering was performed for the binding of four sets of DEGs in R-(v 3.4.3). For gene set enrichment analysis (GSEA), phenotypic labels were defined as 2:2:50:10 = WT-DOX:KO-DOX:WT+DOX:KO+DOX, and Pearson correlation coefficient per gene used for ranking became An enrichment score was then calculated for a set of genes in the Molecular Signature Database (MSigDB) v6.2. To analyze the difference in DOX reactivity between WT and Phf6 KO cells for each cluster, we first averaged the z-scores of the genes in each cluster for each sample. Then, the difference of the mean value according to the presence or absence of DOX in each WT and Phf6 KO cells was calculated, which was considered as the DOX reactivity of WT and Phf6 KO cells. Finally, the difference in DOX reactivity between WT and Phf6 KO cells by cluster was calculated and considered to be DOX-responsive according to the presence or absence of PHF6 per cluster.
응답 차이 = | (∑ (+ DOX z- 점수) - WT (/ DOX z- 점수) WT) / n-(∑ (+ DOX z- 점수) - ∑ (-DOX z- 점수) KO) / n |Response difference = | (∑ (+ DOX z-score) - WT (/DOX z-score) WT) / n-(∑ (+ DOX z-score) - ∑ (-DOX z-score) KO) / n |
통계 분석 모든 실험은 3회 독립적으로 수행되었다. 값은 평균 ± SEM으로 표시되었다. ANOVA 테스트를 사용하여 유의성을 분석하였다. 0.05 미만의 P-값은 통계적으로 유의 한 것으로 간주되었다. Statistical Analysis All experiments were performed independently in triplicate. Values are expressed as mean ± SEM. Significance was analyzed using ANOVA tests. P-values less than 0.05 were considered statistically significant.
실시예 1. Phf6 결핍으로 인한 배반포 분화 및 리프로그래밍의 문제 규명Example 1. Identification of the problem of blastocyst differentiation and reprogramming due to Phf6 deficiency
포유 동물의 초기 발달 단계에서 PHF6의 잠재적인 기능을 확인하기 위해, 우리는 Phf6 결핍 배아줄기세포 (Phf6 KO ESCs)를 만들고, Phf6의 결핍이 배아줄기세포의 다능성 및 분화에 영향을 미치는지 여부를 조사하였다 (도 1A). 우리는 ZHBTc4 마우스 배아줄기세포 라인을 사용했는데, 이는 테트라사이클린(Tc) 또는 독시사이클린(DOX) 처리에 의해 Oct4가 결핍되도록 설계되어 있는 줄기세포이다. 배아줄기세포는 Oct4의 발현 억제 후 초기 배반포 계통으로 리프로그래밍된다는 것이 알려져 있다. 따라서, ZHBTc4 배아줄기세포 시스템은 시험 관 내에서 초기 단계 배아의 전체 계통을 확인할 수 있다는 이점이 있다. 면역 블롯 분석을 통해 우리는 Phf6 결핍 ZHBTc4 배아줄기세포 라인 (이하 Phf6 KO ESC)에서 PHF6의 발현이 완전히 사라졌음을 확인하였다 (도 1B). 또한, 짧은 헤어핀 RNA(short-hairpin RNA)로 Phf6의 발현을 억제하였을 때에도 ZHBTc4에서 PHF6 발현이 상당히 감소 된 것을 확인하였다 (도 1B).To determine the potential function of PHF6 in the early developmental stages of mammals, we generated Phf6-deficient embryonic stem cells (Phf6 KO ESCs) and investigated whether Phf6 deficiency affects the pluripotency and differentiation of embryonic stem cells. investigated ( FIG. 1A ). We used the ZHBTc4 mouse embryonic stem cell line, which is a stem cell designed to be depleted of Oct4 by tetracycline (Tc) or doxycycline (DOX) treatment. It is known that embryonic stem cells are reprogrammed to an early blastocyst lineage after inhibition of Oct4 expression. Therefore, the ZHBTc4 embryonic stem cell system has the advantage of being able to identify the entire lineage of early stage embryos in vitro. Through immunoblot analysis, we confirmed that the expression of PHF6 completely disappeared in the Phf6-deficient ZHBTc4 embryonic stem cell line (hereinafter, Phf6 KO ESCs) (Fig. 1B). In addition, it was confirmed that the expression of PHF6 in ZHBTc4 was significantly reduced even when the expression of Phf6 was suppressed with short-hairpin RNA ( FIG. 1B ).
우리는 이 야생형(WT)과 Phf6 KO 배아줄기세포를 비교하여 PHF6이 자가 재생 및 다능성과 같은 배아 줄기 세포의 주요 특성에 영향을 미치는지 여부를 확인했다. 그 결과, WT와 Phf6 KO 배아줄기세포의 세포 성장률은 서로 크게 다르지 않는 것을 확인하였다 (도 1C). 또한, 배아줄기세포의 다능성에 대한 PHF6의 영향을 조사하기 위해 알칼리성 포스파타아제 (AP) 활성을 염색해서 측정한 결과, 둘 간의 AP 염색이 차이가 없음을 확인했다 (도 1D). 이러한 결과는 Phf6를 shRNA 녹다운으로도 Phf6 KO과 동일한 결과가 나오는 것을 확인하였다 (도 1C 및 D). 다음으로, mRNA-시퀀싱을 통한 차등 발현 유전자 (DEG) 분석을 수행함으로써 WT 및 Phf6 KO 배아줄기세포 간의 유전체의 발현 정도를 비교 하였다 (도 1E). 그 결과, 단지 몇 개의 유전자의 발현이 PHF6에 의해 영향을 받았지만, 배아줄기세포에서 발현 정도가 상위 50%에 속하는 유전자들의 전체적인 발현에는 영향을 미치지 않음을 발견했다 (도 1F). 또한, Oct4, Nanog, Sox2 및 Klf4를 포함한 몇몇 배아줄기세포의 마커 유전자의 발현 역시 Phf6 결핍에 의해 영향을 받지 않았다 (도 1F). 이를 통해 우리는 PHF6이 배아 줄기 세포의 기능 유지에는 영향을 미치지 않는 것을 확인하였다.We compared these wild-type (WT) and Phf6 KO embryonic stem cells to determine whether PHF6 affects key characteristics of embryonic stem cells, such as self-renewal and pluripotency. As a result, it was confirmed that the cell growth rates of WT and Phf6 KO embryonic stem cells were not significantly different from each other (FIG. 1C). In addition, as a result of staining and measuring alkaline phosphatase (AP) activity to investigate the effect of PHF6 on the pluripotency of embryonic stem cells, it was confirmed that there was no difference in AP staining between the two ( FIG. 1D ). These results confirmed that the same results as Phf6 KO were obtained even with shRNA knockdown of Phf6 ( FIGS. 1C and D). Next, by performing differential expression gene (DEG) analysis through mRNA-sequencing, the expression levels of the genomes between WT and Phf6 KO embryonic stem cells were compared (Fig. 1E). As a result, it was found that the expression of only a few genes was affected by PHF6, but did not affect the overall expression of genes whose expression level was in the top 50% in embryonic stem cells (FIG. 1F). In addition, the expression of several embryonic stem cell marker genes, including Oct4, Nanog, Sox2 and Klf4, was also not affected by Phf6 deficiency (FIG. 1F). Through this, we confirmed that PHF6 did not affect the maintenance of embryonic stem cell function.
다음으로, PHF6이 분화 단계에서 계통-특이적인 유전자 발현에 영향을 미치는지 확인하기 위해, 우리는 배아줄기세포를 초기 배반포 단계로 리프로그램하는 DOX를 처리하거나 신경 외배엽으로 분화시키는 레티노산 (RA)을 처리한 후, WT와 Phf6 KO ESC 간의 유전자 발현 프로파일을 비교하였다 (도 1E). 우선, DOX 또는 RA 처리에 의해 상당히 상향 조절 된 유전자에 대한 유전자 온톨로지 (GO) 분석을 수행 하였다. GO 분석의 결과, ZHBTc4 배아줄기세포는 DOX 처리에 의해 태반 발달로 나아가는 배반포 계통으로 리프로그래밍하고, RA 처리에 의한 신경계 발달로 나아가는 신경 외배엽 계통으로의 ESC의 분화가 잘 유도되었음을 확인하였다 (도 1G). 다음으로, DOX 또는 RA 처리 하에서 WT와 Phf6 KO를 비교하여 DEG를 분석한 결과, 예상 한 바와 같이 상이한 분화 상태들 사이에서는 단지 몇 개의 유전자만 공유하는 것을 확인하였다. GO 분석 결과, 흥미롭게도 DOX 처리 하에서 Phf6 결핍에 의해 발현이 낮아진 유전자군에는 태반 발달에 관련된 유전자들이 많이 속한 반면, RA 처리 하에서 Phf6 결핍에 의해 발현에 영향을 받은 유전자군에는 신경 분화에 알려진 유전자들이 거의 속하지 않는 것을 확인하였다 (도 1H). 이를 통해, PHF6이 계통-특이적 방식으로 기능하고 특히 배반포 계통의 분화 단계에 중요한 유전자들의 발현에 역할을 한다는 것을 확인할 수 있었다.Next, to determine whether PHF6 affects lineage-specific gene expression at the differentiation stage, we treated DOX to reprogram embryonic stem cells to an early blastocyst stage or retinoic acid (RA) to differentiate into neuroectoderm. After treatment, gene expression profiles between WT and Phf6 KO ESCs were compared ( FIG. 1E ). First, gene ontology (GO) analysis was performed for genes significantly upregulated by DOX or RA treatment. As a result of the GO analysis, it was confirmed that ZHBTc4 embryonic stem cells were reprogrammed into a blastocyst lineage leading to placental development by DOX treatment, and differentiation of ESCs into a neuroectoderm lineage leading to nervous system development by RA treatment was well induced (Fig. 1G). ). Next, as a result of analyzing DEG by comparing WT and Phf6 KO under DOX or RA treatment, it was confirmed that only a few genes were shared between different differentiation states as expected. As a result of GO analysis, interestingly, genes related to placental development belonged to the gene group whose expression was lowered by Phf6 deficiency under DOX treatment, whereas genes known for neural differentiation were found in the gene group whose expression was affected by Phf6 deficiency under RA treatment. It was confirmed that it hardly belongs to (Fig. 1H). Through this, it was confirmed that PHF6 functions in a lineage-specific manner and plays a role in the expression of genes that are particularly important in the differentiation stage of the blastocyst lineage.
실시예 2. PHF6는 배반포 분화 마커 유전자들의 전사 활성 인자임을 규명Example 2. Identifying that PHF6 is a transcriptional activator of blastocyst differentiation marker genes
배아 줄기 세포의 배반포 세포로의 리프로그래밍 동안 PHF6의 기능을 자세히 식별하기 위해, 우리는 DOX 처리 유무에 따른 WT와 Phf6 KO ESCs 사이 DEG에 대한 k- 평균 클러스터링 (k = 6)을 수행하였다 (도 2A). 그 후, 각 클러스터에서 WT와 Phf6 KO 배아줄기세포 사이의 DOX 반응의 차이를 측정한 결과, 클러스터 1이 WT와 Phf6 KO 간의 DOX 반응의 차이가 가장 큰 것을 확인할 수 있었다 (도 2B). 이 결과는 클러스터 1 내에서 유전자 발현의 조절이 PHF6에 크게 의존한다는 것을 시사한다. 클러스터 1 내의 유전자 발현 양상을 확인한 결과, 배아 줄기 세포 상태 (-DOX)에서 WT와 Phf6 KO 사이에 거의 또는 전혀 차이를 보이지 않았지만, 배반포 세포로의 리프로그래밍 상태 (+ DOX)에서 발현이 상당히 증가하고, 이 증가된 발현이 Phf6 KO에서 상당히 감소한 유전자들이 모인 클러스터임을 확인할 수 있었다 (도 2C). 또한, DOX 처리를 통해 발현이 억제되는 유전자들의 클러스터 (클러스터 2 및 4)에 대한 WT와 Phf6 KO의 차이를 확인해 본 결과, 이들 클러스터의 유전자 발현의 차이는 클러스터 1에 비해 높지 않음을 확인할 수 있었다 (도 2B). 클러스터 1에 속한 유전자들의 생물학적 기능을 GO 분석을 통해 확인한 결과, 예상한 대로 배반포 분화로부터 파생되는 태반 발생이 가장 유의미한 생물학적 기능으로 확인되었다 (도 2D). 이 결과는 PHF6가 배반포 세포 리프로그래밍 동안 전사 활성 인자로서 기능한다는 것을 나타낸다.To further identify the function of PHF6 during reprogramming of embryonic stem cells into blastocyst cells, we performed k-means clustering (k = 6) for DEGs between WT and Phf6 KO ESCs with and without DOX treatment (Fig. 2A). Thereafter, as a result of measuring the difference in DOX response between WT and Phf6 KO embryonic stem cells in each cluster, it was confirmed that cluster 1 had the largest difference in DOX response between WT and Phf6 KO ( FIG. 2B ). These results suggest that the regulation of gene expression within cluster 1 is highly dependent on PHF6. As a result of confirming the gene expression pattern within cluster 1, there was little or no difference between WT and Phf6 KO in the embryonic stem cell state (-DOX), but the expression was significantly increased in the blastocyst cell reprogramming state (+ DOX), and , it could be confirmed that this increased expression is a cluster of genes significantly reduced in Phf6 KO (Fig. 2C). In addition, as a result of checking the difference between WT and Phf6 KO for clusters of genes whose expression is suppressed through DOX treatment (clusters 2 and 4), it was confirmed that the difference in gene expression in these clusters was not high compared to cluster 1. (Fig. 2B). As a result of confirming the biological functions of the genes belonging to cluster 1 through GO analysis, as expected, placental development derived from blastocyst differentiation was confirmed as the most significant biological function ( FIG. 2D ). These results indicate that PHF6 functions as a transcriptional activator during blastocyst cell reprogramming.
실시예 3. PHF6는 배반포 계통 결정을 위한 초기 배반포 계통 결정 유전자의 발현을 활성화시킴을 규명Example 3. It was identified that PHF6 activates the expression of early blastocyst lineage determining genes for blastocyst lineage determination
PHF6가 배반포 계통 결정에 대한 유전자들의 발현에 중요한 역할을 하는 것을 확인하였기 때문에, 우리는 실제로 PHF6가 전체 분화 상태에서 배반포 분화에 중요한 역할을 하는지를 확인해 보았다. 이를 위해 배아체 (EB) 형성 방법을 활용하고 Phf6 KO 및 WT 배아 줄기 세포에서 추출한 EB를 비교하여 분화 과정에서 PHF6의 역할을 확인해 보았다. 배아체 형성 실험은 원래 배아 줄기 세포를 3개의 계통(내배엽, 중배엽, 외배엽)으로 분화시키는 방법인데, 최근에는 이러한 배아체들을 부착시켜서 분화시키면서 3개의 배엽으로 분화되는 EB 코어뿐만 아니라 배아체 계통으로 분화되는 외부 세포층을 생성 할 수 있다는 것이 보고되었다. 이 방법을 사용해서 확인해 본 결과, Phf6 KO 배아 줄기세포로 형성한 배아체가 WT 배아체와는 다르게 외부 세포 층을 형성하는 데 실패함을 확인하였다 (도 3A). 또한, 이 배아체를 배아체 분화의 마커 유전자인 CDX2와 같이 면역 염색을 한 결과, WT 배아체의 외부 세포층에서 CDX2가 높게 발현됨을 확인함으로써, 이 외부 세포층이 배아체 계통으로 분화해 나가는 세포층임을 확인하였다 (도 3B). 흥미롭게도, PHF6는 외부 세포층에서 CDX2와 같이 발현되는 것을 확인할 수 있었다 (도 3C). 또한, Phf6 KO 배아체에서의 분화 동안 Cdx2, Plac1, Ascl2 및 Gata2를 포함한 배반포 마커 유전자의 mRNA 수준을 확인한 결과, WT 배반포에 비해 상당히 낮게 발현되는 것을 확인할 수 있었다 (도 3D). 이를 통해 PHF6이 배반포 분화로의 마커 유전자들의 전사 활성화에 결정적 역할을 하는 것을 확인할 수 있었다.Since we confirmed that PHF6 plays an important role in the expression of genes for blastocyst lineage determination, we checked whether PHF6 actually plays an important role in blastocyst differentiation in the state of total differentiation. To this end, we utilized the embryonic body (EB) formation method and compared EBs extracted from Phf6 KO and WT embryonic stem cells to confirm the role of PHF6 in the differentiation process. The embryonic body formation experiment was originally a method of differentiating embryonic stem cells into three lineages (endoderm, mesoderm, and ectoderm). It has been reported that it can generate a differentiated outer cell layer. As a result of checking using this method, it was confirmed that embryos formed with Phf6 KO embryonic stem cells failed to form an outer cell layer differently from WT embryos ( FIG. 3A ). In addition, as a result of immunostaining this embryoid body with CDX2, which is a marker gene for embryonic differentiation, it was confirmed that CDX2 was highly expressed in the outer cell layer of the WT embryo, indicating that this outer cell layer is a cell layer that differentiates into the embryonic lineage. was confirmed (FIG. 3B). Interestingly, it was confirmed that PHF6 was expressed like CDX2 in the outer cell layer (Fig. 3C). In addition, as a result of checking the mRNA levels of blastocyst marker genes including Cdx2, Plac1, Ascl2 and Gata2 during differentiation in Phf6 KO embryoid bodies, it was confirmed that they were expressed significantly lower than in WT blastocysts (FIG. 3D). Through this, it was confirmed that PHF6 plays a decisive role in the transcriptional activation of marker genes toward blastocyst differentiation.
실시예 4. PHF6은 H2BK12Ac 인식을 통해 H2BK120ub 레벨을 조절하는 에피지네틱 조절인자임을 규명Example 4. PHF6 was identified as an epigenetic regulator that regulates H2BK120ub levels through H2BK12Ac recognition
다음으로, 우리는 PHF6가 어떤 히스톤 변형을 인식하여 이러한 전사 조절 인자로써의 기능을 가지는지를 확인하였다. 우선 PHF6가 4종류의 히스톤 중 어떠한 히스톤의 변형을 인지하는지 확인하기 위해, ZHBTc4 ESC로부터 히스톤을 추출한 후 GST-PHF6 재조합 단백질과 함께 Far-western 실험 기법을 통해 PHF6가 직접 인지하는 히스톤을 확인하였다. 그 결과, GST-PHF6 재조합 단백질이 히스톤 H2B 및 H3을 선택적으로 인지한다는 것을 발견하였다 (도 4A). 다음으로, PHF6에 의해 인식되는 H2B 및 H3의 특정 변형을 확인하기 위해 히스톤 펩티드 배열 키트를 사용하여서 스크리닝을 진행하였다. 이전 Far-western 실험 결과와 일치하게, 스크리닝 결과 PHF6가 여러 종류의 히스톤 H2B 및 H3의 변형이 들어 있는 펩티드에 특이적으로 결합하는 것을 확인하였다. 이를 분석해 본 결과, 상위 5개의 히스톤 변형은 H2BK15Ac, H3K27Ac, H2BK12Ac, H3R26me2a 및 H3K27me2 였다 (도 4B). 이들을 독립적으로 확인하기 위해 각각의 H2B 또는 H3 변형에 대해 펩티드 결합 분석 실험을 수행하였고, 그 결과 PHF6이 시험관 내에서 H2BK12Ac에 특이적으로 결합한다는 것을 확인 하였다 (도 4C).Next, we confirmed that PHF6 has a function as a transcriptional regulator by recognizing which histone modifications. First, in order to confirm which histone modification among the four types of histones is recognized by PHF6, histones directly recognized by PHF6 were identified through the Far-western experiment technique together with GST-PHF6 recombinant protein after extracting histones from ZHBTc4 ESCs. As a result, it was found that the GST-PHF6 recombinant protein selectively recognized histones H2B and H3 ( FIG. 4A ). Next, screening was performed using a histone peptide array kit to identify specific modifications of H2B and H3 recognized by PHF6. Consistent with the results of the previous Far-western experiment, the screening results confirmed that PHF6 specifically binds to peptides containing several types of histones H2B and H3 modifications. As a result of this analysis, the top five histone modifications were H2BK15Ac, H3K27Ac, H2BK12Ac, H3R26me2a and H3K27me2 (FIG. 4B). To confirm them independently, peptide binding assays were performed for each H2B or H3 modification, and as a result, it was confirmed that PHF6 specifically binds to H2BK12Ac in vitro (Fig. 4C).
PHF6는 2개의 확장 PHD 도메인을 가지고 있기 때문에, 어느 확장 PHD에 의해 H2BK12Ac를 판독하는 PHF6의 능력이 부여되는지 여부를 명확히 하기 위해, PHF6의 두 확장 PHD의 아미노산 서열을 비교 하였다. 흥미롭게도, 2번째 확장 PHD 도메인만 특이적으로 음으로 하전된 아미노산이 모여있는 부위가 존재하는 것을 확인했다 (도 4D). 기존에 보고된 PHD 도메인에 의한 아세틸화 인식의 경우, 음으로 하전된 아미노산의 카르보닐 산소가 아세틸화의 아세틸아미드와 상호 작용함으로써 인지 기능에 중요하다고 보고된 바가 있다. 또한 보고되었던 PHF6의 확장 PHD2의 결정 구조를 확인해 본 결과, 음전하를 갖는 4개의 글루탐산 (E219, E220, E221 및 E223)이 아세틸 화 된 기질 인식 모티프를 형성하는 주요 잔기로의 가능성을 확인할 수 있었다. 이를 확인하기 위해 단백질의 구조에는 영향을 미치지 않으면서 전하를 없애는 글루탐산->세린 치환 돌연변이를 사용한 결과, PHF6의 223번 글루탐산 잔기를 세린으로 치환한 돌연변이(E223S)는 H2BK12 아세틸화 인식이 망가지는 것을 확인하였다 (도 4E). 또한, MST 분석을 사용한 결합 친화도 측정은 GST-PHF6 WT가 H2B WT 펩티드보다 H2BK12 아세틸 펩티드에 대해 ~ 5배 더 높은 친화력을 갖는 반면, GST-PHF6 E223S는 WT 및 K12Ac 펩티드 둘 다에 낮은 친화도로 결합하는 것을 확인하였다 (도 4F). 이들 데이터는 PHF6의 2번째 확장 PHD 도에인에서 글루탐산 모티프가 H2BK12Ac 인식에 결정적인 역할을 하는 것을 나타낸다.Since PHF6 has two extended PHD domains, to clarify whether the ability of PHF6 to read H2BK12Ac is conferred by which extended PHD, the amino acid sequences of the two extended PHDs of PHF6 were compared. Interestingly, only the second extended PHD domain specifically confirmed that there was a region where negatively charged amino acids were gathered ( FIG. 4D ). In the case of the previously reported acetylation recognition by the PHD domain, it has been reported that the carbonyl oxygen of a negatively charged amino acid interacts with the acetylamide of acetylation to be important for cognitive function. In addition, as a result of confirming the crystal structure of the reported PHF6 extended PHD2, four negatively charged glutamic acids (E219, E220, E221 and E223) were acetylated, confirming the possibility as a major residue forming a substrate recognition motif. To confirm this, a glutamic acid->serine substitution mutation that removes charge without affecting the protein structure was used. As a result, the mutation (E223S) in which glutamic acid residue 223 of PHF6 was replaced with serine (E223S) was found to impair H2BK12 acetylation recognition. was confirmed (FIG. 4E). In addition, binding affinity measurements using MST assay showed that GST-PHF6 WT had ~5-fold higher affinity for H2BK12 acetyl peptide than H2B WT peptide, whereas GST-PHF6 E223S had low affinity for both WT and K12Ac peptide. Binding was confirmed (FIG. 4F). These data indicate that the glutamic acid motif in the second extended PHD domain of PHF6 plays a critical role in H2BK12Ac recognition.
기존의 암 연구에서 H2BK12 아세틸화는 전사 활성화 마커로 알려져 있었다. 때문에 우리는 PHF6의 아세틸 인지 기능이 계통-특이적 유전자의 발현을 조절하는 데 중요하다는 가설을 세웠다. WT 및 Phf6 KO 배아줄기세포에서 H2BK120ub, H3K4me3, H3K27me3, H3K9me3 및 H2BK12Ac를 포함한 다양한 히스톤 변형을 조사 하였다. 그 중에서도, H2B 120번 잔기의 유비퀴틴화(H2BK120ub)의 레벨이 DOX 처리시 WT ESC의 것에 비해 Phf6 KO에서 상당히 감소되어 있는 것을 확인하였다 (도 4G). Phf6 KO 배아줄기세포에서 H2BK120 유비퀴틴화의 현저한 감소는 H2BK120 유비퀴틴화와 H2BK12 아세틸화 사이의 잠재적인 상호 작용을 짐작할 수 있게 해주었다. 이를 확인하기 위해, 우리는 히스톤 H2BK12 아세틸화를 비롯한 여러 아세틸화의 아세틸화 효소인 CBP/p300을 DOX를 처리한 WT 및 Phf6 KO 배아줄기세포에서 siRNA에 의해 녹다운하고, p300 또는 CBP의 녹다운이 H2BK120 유비퀴틴화에 영향을 미치는지 확인하였다. 흥미롭게도, p300 또는 CBP의 녹다운은 DOX 처리 상태에서 H2BK120 유비퀴틴화 및 H2BK12 아세틸화 둘 다 감소하는 결과를 유발하였고, 이는 H2BK120ub와 H2BK12Ac 사이의 상호 작용이 있다는 것을 암시한다 (도 4H). 보다 직접적으로 두 히스톤 변형 간의 상호 작용을 확인하기 위해, 우리는 H2B의 라이신 12번 잔기를 아르기닌으로 치환한 H2BK12R 돌연변이와, 라이신 120번 잔기를 아르기닌으로 치환한 H2BK120R 돌연변이를 사용하여서 각각의 히스톤 변형이 일어나지 않는 돌연변이에서 상대 히스톤 변형이 어떻게 변하는지를 확인하였다. 그 결과, 아세틸화 결핍 돌연변이(H2BK12R)는 H2B WT과 비교하여 H2BK120 유비퀴틴화가 현저한 감소한 것을 확인한 반면, H2BK120R 유비퀴틴 화 결핍 돌연변이는 H2BK12 아세틸화에 영향을 미치지 않는 것을 확인할 수 있었다 (도 4I). 이를 통해 우리는 PHF6이 선행 H2BK12 아세틸화 인식을 통해 H2BK120 유비퀴틴화를 조절한다는 것을 확인할 수 있었다.In previous cancer studies, H2BK12 acetylation was known as a marker of transcriptional activation. Therefore, we hypothesized that the acetyl recognition function of PHF6 is important for regulating the expression of lineage-specific genes. Various histone modifications including H2BK120ub, H3K4me3, H3K27me3, H3K9me3 and H2BK12Ac were investigated in WT and Phf6 KO embryonic stem cells. Among them, it was confirmed that the level of ubiquitination (H2BK120ub) of H2B residue 120 was significantly reduced in Phf6 KO compared to that of WT ESCs upon DOX treatment ( FIG. 4G ). The significant reduction of H2BK120 ubiquitination in Phf6 KO embryonic stem cells allowed us to speculate a potential interaction between H2BK120 ubiquitination and H2BK12 acetylation. To confirm this, we knocked down CBP/p300, an acetylation enzyme of several acetylations including histone H2BK12 acetylation, by siRNA in DOX-treated WT and Phf6 KO embryonic stem cells, and knockdown of p300 or CBP was H2BK120 It was confirmed whether it affects ubiquitination. Interestingly, knockdown of p300 or CBP resulted in decreased both H2BK120 ubiquitination and H2BK12 acetylation under DOX treatment, suggesting that there is an interaction between H2BK120ub and H2BK12Ac (Fig. 4H). To check the interaction between the two histone modifications more directly, we used the H2BK12R mutant in which the lysine 12th residue of H2B was substituted with arginine and the H2BK120R mutant in which the lysine 120th residue was substituted with arginine. It was confirmed how the relative histone modification changes in the mutation that does not occur. As a result, it was confirmed that the acetylation-deficient mutant (H2BK12R) showed a significant decrease in H2BK120 ubiquitination compared to H2B WT, whereas the H2BK120R ubiquitination-deficient mutant did not affect H2BK12 acetylation (Fig. 4I). Through this, we confirmed that PHF6 regulates H2BK120 ubiquitination through antecedent H2BK12 acetylation recognition.
PHD를 보유하는 단백질은 전형적으로 히스톤 변형 상태의 변경을 위해 다른 효소를 동원하는 링커 단백질로서 작용한다. 따라서, 우리는 PHF6이 H2BK120 유비퀴틴화에 알려진 E3 유비퀴틴 리가아제 또는 탈 유비퀴티나제를 모집하여 H2BK120 유비퀴틴화를 조절할 가능성을 확인하였다. RNF20/40은 배아줄기세포가 신경 외배엽으로 분화하는 동안 H2BK120 유비퀴틴화에 대한 E3 리가아제로써 알려져 있었고, USP44는 이에 대한 탈 유비퀴티나제로서 기능하는 것으로 알려져 있었다. 우선 우리는 RNF20/40 및 USP44 발현 수준을 조사하여 이들 효소의 발현이 PHF6에 의해 조절되는지를 확인하였지만, 이들 중 어느 것도 WT ESC에 비해 Phf6 KO ESC에서 발현 수준이 유의미하게 변하지 않는 것을 확인하였다 (도 4J). 또한, 공동 면역 침전 분석을 통해 서로 물리적 결합이 있는지 확인한 결과, PHF6는 이들 중 어느 단백질과도 결합하지 않는 것을 확인할 수 있었다 (도 4K). 이 데이터는 PHF6이 RNF20/40 및 USP44와 독립적으로 H2BK120 유비퀴틴화를 조절한다는 것을 암시한다. 더 나아가 H2BK120 유비퀴틴화의 조절을 위한 PHF6의 기능이 RNF20/40의 E3 리가아제 기능과 독립적인지 여부를 추가로 증명하기 위해, DOX 처리의 유무에서 Phf6, Rnf20 및 Rnf40의 녹다운에 따른 H2BK120 유비퀴틴화를 비교 하였다. 그 결과, Phf6가 아닌 Rnf20/40의 녹다운은 DOX 처리가 없을 때에도 H2BK120 유비퀴틴화를 감소시키는 것을 확인할 수 있었다 (도 4L). 그러나, Phf6의 녹다운은 DOX 처리시에만 H2BK120 유비퀴틴화의 감소를 초래하였다 (도 4L). 더욱 중요하게는, Phf6의 녹다운은 Cdx2 및 Gata2를 포함하는 배반포 분화 마커 유전자의 발현을 특이적으로 감소시키는 반면, Rnf20 및 Rnf40의 녹다운은 DOX 처리시 Cdx2 및 Gata2의 발현에 영향을 미치지 않음을 확인하였다 (도 4M). 또한, 크로마틴-면역 침전 분석 결과, Phf6의 녹다운을 통해서는 Cdx2 및 Gata2의 프로모터에서 H2BK120 유비퀴틴화가 감소되지만, RNF20/40 녹다운에서는 영향을 받지 않는 것을 확인할 수 있었다 (도 4N). 이들 데이터는 PHF6이 배반포 분화 유전자의 전사 활성화를 위해 H2BK120ub 수준을 특이적으로 조절하고, 이는 기존에 알려진 E3 리가아제와는 무관하다는 것을 나타낸다.Proteins bearing PHD typically act as linker proteins that recruit other enzymes for alteration of histone modification state. Therefore, we confirmed the possibility that PHF6 modulates H2BK120 ubiquitination by recruiting E3 ubiquitin ligase or de-ubiquitinase known for H2BK120 ubiquitination. RNF20/40 was known as an E3 ligase for H2BK120 ubiquitination during the differentiation of embryonic stem cells into neuroectoderm, and USP44 was known to function as a de-ubiquitinase. First, we examined the expression levels of RNF20/40 and USP44 to determine whether the expression of these enzymes is regulated by PHF6, but none of these confirmed that the expression level was significantly altered in Phf6 KO ESCs compared to WT ESCs ( Figure 4J). In addition, as a result of confirming whether there is a physical bond to each other through co-immunoprecipitation analysis, it was confirmed that PHF6 did not bind to any of these proteins ( FIG. 4K ). These data suggest that PHF6 regulates H2BK120 ubiquitination independently of RNF20/40 and USP44. Furthermore, to further prove whether the function of PHF6 for the regulation of H2BK120 ubiquitination is independent of the E3 ligase function of RNF20/40, H2BK120 ubiquitination following knockdown of Phf6, Rnf20 and Rnf40 in the presence or absence of DOX treatment was investigated. compared. As a result, it was confirmed that knockdown of Rnf20/40 rather than Phf6 reduced H2BK120 ubiquitination even in the absence of DOX treatment ( FIG. 4L ). However, knockdown of Phf6 resulted in a decrease in H2BK120 ubiquitination only upon DOX treatment (Fig. 4L). More importantly, it was confirmed that knockdown of Phf6 specifically reduced the expression of blastocyst differentiation marker genes, including Cdx2 and Gata2, whereas knockdown of Rnf20 and Rnf40 did not affect the expression of Cdx2 and Gata2 upon DOX treatment. (Fig. 4M). In addition, as a result of chromatin-immunoprecipitation analysis, it was confirmed that although the knockdown of Phf6 reduced H2BK120 ubiquitination at the promoters of Cdx2 and Gata2, it was not affected by the knockdown of RNF20/40 ( FIG. 4N ). These data indicate that PHF6 specifically regulates H2BK120ub levels for transcriptional activation of blastocyst differentiation genes, independent of the previously known E3 ligase.
실시예 5. PHF6은 히스톤 H2B 단백질의 120번째 라이신 (H2BK120)에 대한 E3 유비퀴틴 리가아제로서 기능함을 규명Example 5. It was established that PHF6 functions as an E3 ubiquitin ligase for the 120th lysine (H2BK120) of the histone H2B protein.
이전 보고들에서 PHD 도메인 자체가 E3 리가아제 활성을 갖는 경우가 보고된 바가 있다. PHF6은 2번째 확장PHD 도메인를 통해 H2BK12Ac를 인식하고 H2B에서 K12Ac와 K120ub 사이에 PHF6 의존성이 존재하기 때문에, PHF6이 PHD 도메인를 통해 H2BK120ub에 대한 E3 유비퀴틴 리가아제 활성을 갖는지 여부를 조사하였다. 먼저, Micrococcal Nuclease를 사용하여 Phf6 KO 배아줄기세포에서 H2BK12R 아세틸화 결핍 돌연변이 및 H2B WT이 들어있는 모노-뉴클레오솜을 용리시켰다. 이어서, 정제된 GST-PHF6와 용리된 모노-뉴클레오솜을 혼합해 시험관 내 유비퀴틴화 분석 실험을 수행하였다 (도 5A). 그 결과, E2 리가아제 파트너로 UBCH6가 아닌 UBCH3가 작동했을 때 PHF6이 E3 유비퀴틴 리가아제로서 기능한다는 것을 발견하였다 (도 5B). 이 때, PHF6은 H2BK12R 아세틸화 돌연변이에는 H2BK120을 유비퀴틴화하지 못하는 것을 확인함으로써, PHF6이 선행 H2BK12Ac 인식을 통해 H2BK120ub에 대한 E3 유비퀴틴 리가아제로서 기능함을 추가로 확인하였다. 다음으로, 이 때 PHF6의 첫 번째 확장 PHD 도메인이 PHF6의 E3 유비퀴틴 리가아제 활성을 가지는지를 확인해 보았다. 시험 관내 유비퀴틴화 분석을 통해 PHF6 WT은 H2BK120을 유비퀴틴화하지만, C82A (아연 이온 포착을 방해함으로써 첫번째 확장 PHD 도메인의 기능적 활성이 망가진 돌연변이) 또는 E223S (두번째 확장 PHD 도메인의 H2BK12Ac을 인식 할 수 없는 돌연변이)에서 둘 다 H2BK120을 유비퀴틴화 하지 못하는 것을 확인하였다 (도 5C). 이 결과는 PHF6이 두번째 확장 PHD를 통해 H2BK12Ac를 인식하고 이어서 첫번째 확장 PHD 도메인을 통해 H2BK120 잔기를 유비퀴틴화 한다는 것을 암시한다.In previous reports, it has been reported that the PHD domain itself has E3 ligase activity. Since PHF6 recognizes H2BK12Ac through the second extended PHD domain and PHF6 dependence exists between K12Ac and K120ub in H2B, we investigated whether PHF6 has E3 ubiquitin ligase activity to H2BK120ub through the PHD domain. First, mononucleosomes containing H2BK12R acetylation-deficient mutants and H2B WT were eluted from Phf6 KO embryonic stem cells using Micrococcal Nuclease. Then, an in vitro ubiquitination assay was performed by mixing purified GST-PHF6 with the eluted mono-nucleosome (FIG. 5A). As a result, it was found that PHF6 functions as an E3 ubiquitin ligase when UBCH3 but not UBCH6 acted as an E2 ligase partner ( FIG. 5B ). At this time, by confirming that PHF6 could not ubiquitinate H2BK120 in the H2BK12R acetylation mutant, it was further confirmed that PHF6 functions as an E3 ubiquitin ligase for H2BK120ub through prior H2BK12Ac recognition. Next, it was checked whether the first extended PHD domain of PHF6 has E3 ubiquitin ligase activity of PHF6. An in vitro ubiquitination assay showed that PHF6 WT ubiquitinates H2BK120, but either C82A (a mutant that disrupts the functional activity of the first extended PHD domain by interfering with zinc ion capture) or E223S (a mutation that cannot recognize H2BK12Ac in the second extended PHD domain) ), it was confirmed that both did not ubiquitinate H2BK120 ( FIG. 5C ). These results suggest that PHF6 recognizes H2BK12Ac through the second extended PHD and then ubiquitinates the H2BK120 residue through the first extended PHD domain.
다음으로 배아줄기세포의 배반포 세포로의 재프로그래밍 단계에서 PHF6의 E3 유비퀴틴 리가아제로서의 기능을 확인하기 위해, 우리는 Phf6 KO 배아줄기세포에서 PHF6 WT, C82A 또는 E223S 돌연변이를 다시 발현시켜주었다. 그러나 예상치 못하게, C82A 돌연변이의 발현 수준이 세포 내에서는 현저하게 감소하는 것을 관찰하였다 (도 5D). 이전 BFl 증후군에서의 PHF6 돌연변이에 대한 연구에서, PHF6의 첫번째 코어 PHD 도메인의 돌연변이 (C45Y 및 C99F)는 PHF6의 단백질 안정성을 감소시키는 것이 보고되었다. 따라서 우리는 단백질 안정성에 영향을 주지 않으면서 PHF6의 E3 리가아제 활성이 망가지는 첫번째 확장PHD 도메인의 다른 주요 아미노산 잔기를 찾아보았다. PHD 도메인과 유사한 구조이자 E3 리가아제 활성을 갖는 것으로 알려진 RING 도메인에 대한 이전 연구들에서, 아연 양이온을 캡처하는 코어 잔기 (시스테인 혹은 히스티딘) 근처에 있는 소수성 아미노산 잔기가 E3 리가아제 활성에 중요한 것이 알려져 있었기 때문에, 우리는 PHF6의 첫번째 확장 PHD 도메인 내에서 이러한 인접 소수성 아미노산 잔기들이 PHF6의 E3 리가아제 활성을 발휘하는 데 중요한지 여부를 조사했다 (도 5E). 이를 위해, 첫번째 확장 PHD 도메인에서 종 간 보존된 소수성 잔기들을 찾고, 이들에 대한 돌연변이를 제작하여 시험관 내 유비퀴틴화 분석을 수행하였다. 흥미롭게도, 125번 메티오닌 잔기(M125) 및 129번 아르기닌 잔기(R129)에 대한 돌연변이는 PHF6의 E3 리가아제 활성이 사라지는 것을 확인하였다 (도 5F). 또한, PHF6 WT 및 E223S 돌연변이는 둘 다 UBCH3 E2 효소에 결합하는 능력을 유지하는 반면, M125A 및 R129A 돌연변이는 UBCH3에 결합하지 못하는 것을 확인하였고, 이러한 결과는 PHF6의 첫번째 PHD 도메인이 E3 리가아제 활성을 갖는 도메인임을 시사한다 (도 5G). 이러한 연구 결과를 바탕으로, 우리는 PHF6이 H2BK12Ac와 H2BK120ub를 연결하는 E3 유비퀴틴 리가아제 역할을 하는 이전에 인식 할 수 없었던 조절 분자 기반을 제시한다 (도 5H).Next, to confirm the function of PHF6 as an E3 ubiquitin ligase in the step of reprogramming embryonic stem cells into blastocyst cells, we re-expressed PHF6 WT, C82A or E223S mutants in Phf6 KO embryonic stem cells. However, unexpectedly, it was observed that the expression level of the C82A mutant was significantly decreased in cells ( FIG. 5D ). In a previous study of PHF6 mutations in BFl syndrome, it was reported that mutations in the first core PHD domain of PHF6 (C45Y and C99F) reduce the protein stability of PHF6. Therefore, we searched for other key amino acid residues in the first extended PHD domain in which the E3 ligase activity of PHF6 was disrupted without affecting protein stability. In previous studies on the RING domain, which has a structure similar to that of the PHD domain and known to have E3 ligase activity, it is known that hydrophobic amino acid residues near the core residue (cysteine or histidine) that capture zinc cations are important for E3 ligase activity. Therefore, we investigated whether these contiguous hydrophobic amino acid residues within the first extended PHD domain of PHF6 are important for exerting the E3 ligase activity of PHF6 (Fig. 5E). To this end, in vitro ubiquitination analysis was performed by finding cross-species conserved hydrophobic residues in the first extended PHD domain, and mutating them. Interestingly, mutations at methionine residue 125 (M125) and arginine residue 129 (R129) confirmed that the E3 ligase activity of PHF6 was lost ( FIG. 5F ). In addition, it was confirmed that the PHF6 WT and E223S mutants both retain the ability to bind to the UBCH3 E2 enzyme, whereas the M125A and R129A mutations do not bind to UBCH3. It suggests that the domain has (Fig. 5G). Based on these findings, we present a previously unrecognized regulatory molecular basis in which PHF6 acts as an E3 ubiquitin ligase linking H2BK12Ac and H2BK120ub (Fig. 5H).
실시예 6. PHF6은 H2BK120의 유비퀴틴화를 통해 배반포 계통 마커 유전자들의 발현에 미치는 영향 규명Example 6. Identification of the effect of PHF6 on the expression of blastocyst lineage marker genes through ubiquitination of H2BK120
PHF6의 두번째 확장 PHD 도메인은 H2BK12Ac의 인식을 담당하고, PHF6의 첫번째 확장 PHD 도메인은 E3 리가아제 활성을 발휘하기 때문에, DOX 처리를 한 배아 줄기 세포의 배반포 분화로의 분화 과정에서 PHF6의 이러한 활성을 추가로 확인하였다. 먼저, MNase-ChIP 분석을 수행하여 E223 잔기가 PHF6의 H2BK12Ac 인식에 중요하다는 것을 확인하였다. 실제로, PHF6 E223S 돌연변이는 H2BK12Ac가 포함된 모노-뉴클레오솜에 대한 결합이 상당히 감소한 반면, PHF6 WT 및 E3 리가아제 돌연변이 M125A는 H2BK12Ac이 포함된 모노-뉴클레오솜 결합 능력을 유지하는 것을 확인하였다 (도 6A). 또한, PHF6 WT가 인핸서의 마커인 H3K4me1이 포함된 모노-뉴클레오솜과는 결합하지 못함을 발견하였다. 이러한 결과는 PHF6이 H2BK12Ac를 인식함으로써 표적 유전자의 프로모터 영역에서 기능을 하는 것을 시사한다. 다음으로, PHF6 WT 또는 돌연변이를 Phf6 KO 배아줄기세포에 발현시켜준 후 H2BK120ub의 변화를 확인하였다. 그 결과, PHF6 WT을 발현시켜주었을 때는 H2BK120ub 수준이 다시 회복되는 것을 확인했지만, H2BK12Ac을 인식할 수 없는 돌연변이인 E223S 또는 E3 리가아제 활성이 없는 돌연변이인 M125A나 R129A의 발현은 H2BK120ub 수준이 회복되지 않는 것을 확인하였다 (도 6B). 다음으로, DOX 처리 유무에 따른 WT 및 Phf6 KO 배아줄기세포에서 Cdx2 및 Gata2의 프로모터에 대해 ChIP 분석을 수행하였다. 그 결과, DOX 처리는 WT 배아줄기세포에서 이러한 유전자들의 프로모터에서 H2BK120ub 수준과 함께 PHF6의 모집이 증가되는 것을 확인할 수 있었지만, Phf6 KO 배아줄기세포에서는 이 증가가 보이지 않는 것을 확인하였다 (도 6C). 또한, Phf6과 유무와 상관없이 발현되는 유전자인 Msx2의 프로모터에 대한 ChIP 분석에서는, WT와 Phf6 KO 배아줄기세포 사이에 PHF6 모집 및 H2BK120ub 수준의 유의미한 변화가 나타나지 않는 것을 확인하였다 (도 6D). 이에 더해, 프로모터 영역에서 H2BK120ub에 뒤이어 나타나는 H3K4me3의 수준도 확인하였다. 예상한 대로, Cdx2 및 Gata2 프로모터에서의 H3K4me3 수준은 DOX 처리에 의존하여 H2BK120ub 및 Phf6 증가에 따라 같이 증가하는 것을 확인하였다 (도 6C). 그러나 PHF6과 무관한 유전자인 Msx2의 프로모터에서는 H3K4me3 수준 역시 PHF6의 유무와 무관하게 증가하는 것을 확인하였다 (도 6D). 다음으로, 우리는 lenti-virus 방법을 통해 Phf6 KO 배아줄기세에서 PHF6 WT 또는 돌연변이들을 지속적으로 발현하는 세포주를 제작하고, 여기에서 PHF6 WT 또는 돌연변이가 발현하는 Phf6 KO 배아줄기세포에서 배반포 분화 마커 유전자들의 mRNA 수준을 비교하였다 (도 6E). qRT-PCR 분석을 통해 유전자들의 발현을 확인한 결과, PHF6 돌연변이들을 발현한 배아줄기세포들은 PHF6 WT이 발현하는 Phf6 KO 배아줄기세포와 비교했을 때 Cdx2, Ascl2, Wnt7b, Fgfr2 및 Plac1을 포함한 배반포 분화 마커 유전자들의 발현이 유의미하게 낮아져 있는 것을 확인하였다 (도 6F).Since the second extended PHD domain of PHF6 is responsible for the recognition of H2BK12Ac, and the first extended PHD domain of PHF6 exerts E3 ligase activity, this activity of PHF6 is inhibited during the differentiation of DOX-treated embryonic stem cells into blastocyst differentiation. further confirmed. First, MNase-ChIP analysis was performed to confirm that the E223 residue is important for H2BK12Ac recognition of PHF6. Indeed, it was confirmed that the PHF6 E223S mutant significantly reduced binding to H2BK12Ac-containing mononucleosomes, whereas PHF6 WT and E3 ligase mutant M125A retained the H2BK12Ac-containing mono-nucleosome binding ability ( 6A). In addition, it was found that PHF6 WT did not bind to the mono-nucleosome containing H3K4me1, which is an enhancer marker. These results suggest that PHF6 functions in the promoter region of the target gene by recognizing H2BK12Ac. Next, the change of H2BK120ub was confirmed after expressing PHF6 WT or mutant in Phf6 KO embryonic stem cells. As a result, it was confirmed that the H2BK120ub level was restored when PHF6 WT was expressed, but the expression of E223S, a mutation that does not recognize H2BK12Ac, or M125A or R129A, a mutant without E3 ligase activity, does not restore H2BK120ub levels. was confirmed (FIG. 6B). Next, ChIP analysis was performed on the promoters of Cdx2 and Gata2 in WT and Phf6 KO embryonic stem cells with or without DOX treatment. As a result, it was confirmed that DOX treatment increased the recruitment of PHF6 along with the H2BK120ub level in the promoter of these genes in WT embryonic stem cells, but this increase was not seen in Phf6 KO embryonic stem cells ( FIG. 6C ). In addition, in the ChIP analysis of the promoter of Msx2, a gene expressed with or without Phf6, it was confirmed that there was no significant change in PHF6 recruitment and H2BK120ub levels between WT and Phf6 KO embryonic stem cells (Fig. 6D). In addition, the level of H3K4me3 following H2BK120ub in the promoter region was also confirmed. As expected, it was confirmed that the H3K4me3 level in the Cdx2 and Gata2 promoters increased with the increase of H2BK120ub and Phf6 depending on DOX treatment ( FIG. 6C ). However, it was confirmed that the level of H3K4me3 in the promoter of Msx2, which is a gene independent of PHF6, also increased regardless of the presence or absence of PHF6 ( FIG. 6D ). Next, we constructed a cell line continuously expressing PHF6 WT or mutants in Phf6 KO embryonic stem cells using the lenti-virus method, and here, in PHF6 WT or mutant-expressing Phf6 KO embryonic stem cells, a blastocyst differentiation marker gene. mRNA levels were compared ( FIG. 6E ). As a result of confirming the expression of genes through qRT-PCR analysis, embryonic stem cells expressing PHF6 mutations showed blastocyst differentiation markers including Cdx2, Ascl2, Wnt7b, Fgfr2 and Plac1 when compared to PHF6 WT-expressing Phf6 KO embryonic stem cells. It was confirmed that the expression of the genes was significantly lowered (FIG. 6F).
이상에서 본원의 예시적인 실시예에 대하여 상세하게 설명하였지만 본원의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본원의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본원의 권리범위에 속하는 것이다.Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present application as defined in the following claims are also included in the scope of the present application. will belong to
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다.All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

Claims (9)

  1. PHF6 (PHD finger-containing protein 6) 단백질 및 12번째 라이신 잔기가 아세틸화로 변형된 히스톤 2B (H2BK12-Ac) 단백질을 제공하는 단계; providing a PHF6 (PHD finger-containing protein 6) protein and a histone 2B (H2BK12-Ac) protein in which the 12th lysine residue is modified by acetylation;
    상기 PHF6 단백질 및 H2BK12-Ac 단백질을 상기 PHF6의 상기 H2BK12-Ac의 인식을 조절할 것으로 기대되는 시험물질의 존재 중에서 접촉하는 단계;contacting the PHF6 protein and the H2BK12-Ac protein in the presence of a test substance expected to modulate the recognition of the H2BK12-Ac by the PHF6;
    상기 히스톤 단백질의 120번째 라이신 잔기의 유비퀴틴화 (H2BK120-ub)를 측정하는 단계; 및measuring ubiquitination (H2BK120-ub) of the 120th lysine residue of the histone protein; and
    상기 측정결과, 상기 시험물질로 처리되지 않은 대조군과 비교하여, 상기 시험물질로 처리된 세포에서 상기 히스톤 단백질의 120번째 라이신 잔기의 유비퀴틴화 수준에 변화가 있는 경우, 상기 시험물질을 상기 PHF6 활성 조절 후보물질로 선별하는 단계를 포함하는, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법.As a result of the measurement, when there is a change in the level of ubiquitination of the 120th lysine residue of the histone protein in the cells treated with the test substance compared to the control group not treated with the test substance, the test substance is adjusted to the PHF6 activity A method for screening a histone epigenetic modulator by PHF6, comprising the step of selecting a candidate substance.
  2. 제 1 항에 있어서, The method of claim 1,
    상기 PHF6 단백질 및 상기 H2BK12-Ac은 진핵세포 또는 인간을 제외한 동물모델로서 제공되는 것인, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법. The PHF6 protein and the H2BK12-Ac are provided as eukaryotic cells or animal models other than humans, Histone epigenetic modulator screening method by PHF6.
  3. 제 2 항에 있어서,3. The method of claim 2,
    상기 진핵세포는 인간을 제외한 동물의 배아줄기세포인, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법. The eukaryotic cells are embryonic stem cells of animals other than humans, histone epigenetic modulator screening method by PHF6.
  4. 제 1 항 내지 제 3 항 중 어느 한 항에 있어서, 4. The method according to any one of claims 1 to 3,
    상기 PHF6 단백질은 PHD1 및 PHD2 도메인을 포함하며, 상기 H2BK12-Ac는 상기 PHD2 도메인에 의해 인식되고, 상기 H2BK120의 유비퀴틴화는 상기 PHD1에 의한 것인, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법. The method for screening a histone epigenetic modulator by PHF6, wherein the PHF6 protein includes PHD1 and PHD2 domains, the H2BK12-Ac is recognized by the PHD2 domain, and the ubiquitination of the H2BK120 is by the PHD1.
  5. 제 1 항 내지 제 3 항 중 어느 한 항에 있어서, 상기 에피제네틱 조절제는 백혈병 치료제로 개발되는 것인, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법. The method according to any one of claims 1 to 3, wherein the epigenetic modulator is developed as a therapeutic agent for leukemia.
  6. PHF6의 PHD1 도메인; 또는 PHD2 도메인이 결실 또는 돌연변이된 PHF6 단백질 및 히스톤 2B 단백질을 제공하는 단계;PHD1 domain of PHF6; or providing a PHF6 protein and a histone 2B protein in which the PHD2 domain is deleted or mutated;
    상기 PHD1 도메인 또는 상기 PHD2 도메인이 결실 또는 돌연변이된 PHF6 단백질과 상기 히스톤 2B 단백질의 결합을 촉진시킬 것으로 기대되는 물질의 존재 중에서 상기 단백질을 접촉하는 단계; contacting the PHF6 protein in which the PHD1 domain or the PHD2 domain is deleted or mutated in the presence of a substance expected to promote binding of the histone 2B protein;
    상기 히스톤 2B 단백질의 120번째 라이신 잔기의 유비퀴틴화 수준을 측정하는 단계; 및 measuring the ubiquitination level of the 120th lysine residue of the histone 2B protein; and
    상기 측정 결과 시험물질과 처리되지 않은 대조군과 비교하여, 처리된 경우 히스톤 2B 단백질의 120번째 라이신 잔기의 유비퀴틴화가 증가한 경우, 상기 시험물질을 상기 PHF6 활성 조절 후보물질로 선별하는 단계를 포함하는, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법.As a result of the measurement, when the ubiquitination of the 120th lysine residue of the histone 2B protein is increased when treated, compared to the test substance and the untreated control, selecting the test substance as a candidate for regulating the PHF6 activity, PHF6 Histone epigenetic modulator screening method by
  7. 제 6 항에 있어서, 7. The method of claim 6,
    상기 PHD1 도메인; 또는 PHD2 도메인이 결실 또는 돌연변이된 PHF6 단백질 및 히스톤 2B 단백질은 진핵세포 또는 인간을 제외한 동물모델로서 제공되는 것인, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법. the PHD1 domain; Or PHF6 protein and histone 2B protein in which the PHD2 domain is deleted or mutated are provided as eukaryotic cells or animal models other than humans.
  8. 제 7 항에 있어서,8. The method of claim 7,
    상기 진핵세포는 인간을 제외한 동물의 배아줄기세포인, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법. The eukaryotic cells are embryonic stem cells of animals other than humans, histone epigenetic modulator screening method by PHF6.
  9. 제 6 항 내지 제 8 항 중 어느 한 항에 있어서, 상기 에피제네틱 조절제는 백혈병 치료제로 개발되는 것인, PHF6에 의한 히스톤 에피제네틱 조절제 스크리닝 방법.The method according to any one of claims 6 to 8, wherein the epigenetic modulator is developed as a therapeutic agent for leukemia.
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