WO2020101880A1 - Compositions and methods of treating systemic lupus erythematosus - Google Patents

Abstract

The present invention includes compositions and methods for treating Systemic Lupus Erythematosus (SLE) comprising: identifying a subject in need of treatment for SLE; and providing the subject with an effective amount of an agent that blocks the binding of a DNA binding protein to a mutation in an epigenetic locus that affects transcription of RASGRP1 or RASGRP3.

Description

COMPOSITIONS AND METHODS OF TREATING SYSTEMIC LUPUS ERYTHEMATOSUS

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of novel compositions and methods of treating Systemic Lupus Erythematosus by targeting epigenetic targets.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under AR073941, AR060366, AI132532, MD007909, awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

The present application includes a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 24, 2019, is named OMRF2012WO SecLListing.txt and is 10.9 bytes in size.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is described in connection with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that disproportionately affects people of Asian, African, and Hispanic ethnicities and women, in particular, with higher incidence and disease severity. Yet, much of SLE etiology remains mysterious. It has been proposed that complex interactions amongst numerous genes and their products with pathogens and other environmental factors promotes dysregulation of both the innate and adaptive immune responses in SLE. Over 80 SLE susceptibility loci have been identified so far across multiple ethnic groups by genome-wide association studies (GWAS) and candidate gene studies. However, the precise underlying variants and functional mechanisms associated with disease are largely unidentified for the vast majority of these SLE-associated signals. Understanding SLE pathogenesis requires identification of true causal variants and the target genes and mechanisms by which they contribute to disease.

SLE is characterized by a wide range of autoantibody production, complement activation and immune complex deposition resulting in tissue and organ damage (i.e. kidney, lungs and central nervous system). New SLE cases are estimated at 2.0-7.6 cases per 100,000 persons per year, with a prevalence in the United States from 14.6-50 cases per 100,000 persons per year. SLE has a strong gender and ethnic bias. SLE primarily affects women (women: men = 9: 1) of childbearing age, and compared to Caucasians, SLE prevalence is 3-5 folds higher in African-Americans, Hispanics and Asians. SLE has a relatively strong genetic component (sibling risk ratio, /._s. ~30) compared to many other autoimmune diseases. Several candidate gene studies and genome wide association studies (GWAS) have identified over 90 SLE susceptibility genes with multiple risk loci.

An actual disease predisposing or causal variant from each locus as well as the associated molecular mechanisms involved in pathogenesis is not yet completely understood. Despite much progress, a need remains for novel compositions and methods for the treating Systemic Lupus Erythematosus. SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method of treating Systemic Lupus Erythematosus (SLE) comprising: identifying a subject in need of treatment for SLE; and providing the subject with an effective amount of an agent that blocks the binding of a DNA binding protein to a mutation in an epigenetic locus that affects transcription of RASGRP1 or RASGRP3. In one aspect, the method further comprises detecting the presence of a target locus selected from at least one of: rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, or rsl3725999, and providing a specific agent that blocks the binding of a DNA binding protein to the target locus if that mutation is present. In one aspect, the agent is 5-fluorouracil, olararib, meaparib, niraparib, talazoparib, veliparib, CEP 7922, E7016, ICG-001, C646, E1A, MI-3, or GSKI26. In another aspect, the epigenetic locus is at least one of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, or rsl3725999. In another aspect, the agent is a single stranded or double stranded oligonucleotide that is complementary to the sequence of SNP selected from at least one of: rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRP1 or RASGRP3 or mutations thereof that block or reduce binding of the DNA binding protein to the SNP. In another aspect, wherein the agent is a backbone-modified single stranded or double stranded oligonucleotide that is complementary to the sequence of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRP1 or RASGRP3. In another aspect, the agent is an anti-sense oligonucleotide that has a backbone modification that is selected from at least one of: a thio- or dithio-modified nucleic acids, methylphosphonate nucleic acids, 2’-O-methyl nucleic acids, 2’-0-methoxy nucleic acids, bridged nucleic acids, locked nucleic acids, arabinonucleic acids, anhydrohexitol nucleic acids, cyclohexenyl nucleic acids, threofuranosyl nucleic acids, or a peptide nucleic acid. In another aspect, the agent is delivered into a target cell via liposome, viral expression vector, CRISPR, particle(s), exosome(s), microvesicle(s), Zinc -finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), or a gene-gun. In another aspect, the agent is a peptide that blocks the DNA portion of the DNA binding protein specific for the epigenetic locus. In another aspect, the agent has a sequence selected from: 1 to 58. In another aspect, the agent is a small molecule inhibitor of RASGRPl or RASGRP3, or reduces the transcription of RASGRPl or RASGRP3. In another aspect, the agent blocks or reduces the binding of at least one of: lmRNP-K, H3K27Ac, H3K4Mel, H3K4Me3, P300, PARP1, or IRF1 to DNA.

In another embodiment, the present invention includes a method treating Systemic Lupus Erythematosus (SLE) comprising: identifying a subject in need of treatment for SLE; and providing the subject with an effective amount of an agent that blocks the binding of a DNA binding protein to intronic variants rs7170151, rs 11631591- rs9920715, that affect transcription of RASGRPl, or intronic variants rsl3385731 or rsl3725999 that affect transcription of RASGRP3. In one aspect, the agent is a single stranded or double stranded oligonucleotide that is complementary to the sequence of SNP selected from at least one of: rs7170151, rsl l631591-rs7173565, rsl3385731, rsl3725999, RASGRPl or RASGRP3 or mutations thereof that block or reduce binding of the DNA binding protein to the SNP. In another aspect, the agent is a backbone-modified single stranded or double stranded oligonucleotide that is complementary to the sequence of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rs 13725999, RASGRPl or RASGRP3. In another aspect, the agent is an anti-sense oligonucleotide that has a backbone modification that is selected from at least one of: a backbone modification is a thio- or dithio- modified nucleic acids, methylphosphonate nucleic acids, 2’-O-methyl nucleic acids, 2’-0-methoxy nucleic acids, bridged nucleic acids, locked nucleic acids, arabinonucleic acids, anhydrohexitol nucleic acids, cyclohexenyl nucleic acids, threofuranosyl nucleic acids, or a peptide nucleic acid. In another aspect, the agent is delivered into a target cell via liposome, viral expression vector, CRISPR, particle(s), exosome(s), microvesicle(s), Zinc -finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), or a gene-gun. In another aspect, the agent is a peptide that blocks the DNA portion of the DNA binding protein specific for the epigenetic locus. In another aspect, the agent has a sequence selected from: SEQ ID NOS: l - 58. In another aspect, the agent is 5- fluorouracil, olararib, rucaparib, niraparib, talazoparib, veliparib, CEP 7922, E7016, ICG-001, C646, E1A, MI-3, or GSK126. In another aspect, the agent is a small molecule inhibitor of RASGRPl or RASGRP3, or reduces the transcription of RASGRPl or RASGRP3. In another aspect, the agent blocks or reduces the binding of at least one of: lmRNP-K, H3K27Ac, H3K4Mel, H3K4Me3, P300, PARP1, or IRF1 to DNA.

In yet another embodiment, the present invention is a composition for suppressing proinflammatory gene expression in a subject with Systemic Lupus Erythematosus (SLE) comprising an effective amount of an agent that blocks the binding of a DNA binding protein to a mutation in an epigenetic locus that affects transcription. In another aspect, a target locus is selected from at least one of: rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, or rsl3725999, and a specific agent that blocks the binding of a DNA binding protein to the target locus is provided if that mutation is present. In another aspect, the agent inhibits or reduces the expression of RASGRPl or RASGRP3. In another aspect, the agent is 5-fluorouracil, olararib, rucaparib, niraparib, talazoparib, veliparib, CEP 7922, E7G16, ICG-001, C646, El A. MI-3, or GSK126. In another aspect, the agent is a small molecule inhibitor of RASGRP l or RASGRP3, or reduces the transcription of RASGRP l or RASGRP3. In another aspect, the epigenetic locus is at least one of rs7170151, rsl l631591-rs7173565, rs9920715, rs 13385731, or rsl3725999. In another aspect, the agent is a single stranded or double stranded oligonucleotide that is complementary to the sequence of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRPl or RASGRP3. In another aspect, the agent is a backbone-modified single stranded or double stranded oligonucleotide that is complementary to the sequence of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRPl or RASGRP3. In another aspect, the agent is an anti-sense oligonucleotide that has a backbone modification that is selected from at least one of: a backbone modification is a thio- or dithio-modified nucleic acids, methylphosphonate nucleic acids, 2’-0- methyl nucleic acids, 2’-0-methoxy nucleic acids, bridged nucleic acids, locked nucleic acids, arabinonucleic acids, anhydrohexitol nucleic acids, cyclohexenyl nucleic acids, threofuranosyl nucleic acids, or a peptide nucleic acid. In another aspect, the agent is delivered into a target cell via liposome, viral expression vector, CRISPR, particle(s), exosome(s), microvesicle(s), Zinc -finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), or a gene-gun. In another aspect, the agent is a peptide that blocks the DNA portion of the DNA binding protein specific for the epigenetic locus. In another aspect, the agent has a sequence selected from: 1 to 58. In another aspect, the agent is a small molecule inhibitor of RASGRPl or RASGRP3, or reduces the transcription of RASGRPl or RASGRP3. In another aspect, the agent blocks or reduces the binding of at least one of: lmRNP-K, H3K27AC, H3K4Mel, H3K4Me3, P300, PARP1, or IRF1 to DNA.

In yet another embodiment, the present invention is a method of identifying a subject in need of treatment for Systemic Lupus Erythematosus (SLE) and a therapy therefore, comprising: obtaining a DNA sample from the subject; determining the presence of one or more SNPs selected from at least one of rs7170151, rsl 1631591- rs7173565, rs9920715, rsl3385731, or rsl3725999; and determining if the subject has an increased expression of RASGRPl, or RASGRP3; and providing the subject with an agent that inhibits the binding of a DNA binding protein to the SNP. BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

FIG. 1A shows the framework of study design. The present study followed the inventors’ bioinformatics-prioritized potential functional SNPs with laboratory validation along many different dimensions.

FIG. IB shows the collocation of these selected potentially functional SNPs with H2K27ac, H3K4Mel, H3K4Me3 and DNAse hypersensitivity in lymphoblastoid GM12878, Naive CD4+, CD8+ T-cells and T-helper cells.

FIG. 2 shows a Meta-analysis in the RASGRP1 region. Blue diamond: lead SNP rs8032939 following initial meta analysis. Red circles: SNPs chosen for experimental validation. rsl l631591-rs7173565 are considered together due to their proximity; only rsl 1631591 is labeled. Purple diamond: the inventors previously reported (4) lead SNP rsl2900339. Linkage disequilibrium in the region (bottom) is notably different between European (EUR) and Asian (ASN).

FIG. 3. RASGRP1 expression levels in multiple tissues (skin, left; esophagus, right) correlates with rs7173565- rsl 1631591 genotypes. Reference allele T; alternate allele C (red).

FIGS. 4A and 4B show Chromatin interactions at the RASGRP1 region overlap with rsl 1631591 in GM12878 cells (4A). The three rings of orange marks show peaks for CD4+ T-cell H3K27Ac, H3K4Mel and H3K4Me3; the three rings of blue marks show peaks of CTCT, ETS1 and YY1 transcription factor binding. Colors in outer ring represent the 15-state classification of ChromHMM, notably: dark green for transcription, red for transcription start site/promoter, orange for enhancer. (4B). Promoter-capture Hi-C in Naive CD4+ T-cells. The blue side (Bait) represents the promoter of C15orf53 baited for the experiment. The red side (Target) represents the interaction side of the experiment. Intronic SNP rsl l631591 lies in the target (i.e. enhancer) side of the interaction with the baited promoter of C15orf53. Bait and Target colors under the gene show chromatin state in CD4+ T-cells using the chromHMM 25-state classification.

FIGS. 5A and 5B show allele-dependent binding to histones. (5A). H3K4Me3 (rsl l631591-rs7173565), and (5B). H3K4Mel (rs7170151) for lymphoblastoid cells as estimated with SNPhood (R-package). Top panel depicts the significant allelic fraction at each position relative to the SNP (position 0). Middle panel represents how significant the bias is at each position. Bottom panel shows the count of allele reads found in paternal (blue) and maternal (red) strands.

FIGS. 6A and 6B show Luciferase reporter assay for rs7170151, rsl l631591-rs7173565 and rs9920715 in 6A. Jurkat and 6B. HEK293 cells. Empty vector pGL4.26 was used as reference. NR: non-risk. P-values are for Student’s t-test.

FIGS. 7A and 7B show EMSA pulldown assay of rsl 1631591 (left) and rs7173565 (right) in Jurkat cells. EMSA was performed with biotin-labeled nucleotides containing the non-risk (TT) (41 bp) or risk (CC) (41 bp) polymorphisms along with the 5 bases-deleted SNP region (36 bp) for rsl 1631591 (FIG. 7A). Nuclear extracts were derived from Jurkat cell lines. The ~70 kDa apparent-MW protein/DNA complex shows allele-specific binding which is absent in the deleted nucleotides. Similar results were also observed for the SNP (rs7170151) (FIG. 7B). Lane 1 is molecular weight marker, lane 2 having the nucleotide with deleted SNP region, lane 3 is the Non risk nucleotides and lane 4 contains the risk genotype. Arrow representing the band of interest, which was cut for further analysis by mass spectrometry. FIG. 8 is a Western blot showing allele-specific binding of lmRNP-K against Jurkat cell lysate. The risk allele (C) of rsl 1631591 shows more binding than the non-risk allele (T). Densitometric measure of the bands T:C= 1 : 1.3.

FIG. 9A shows ChIP-qPCR of sequences containing SNPs rsl l631591-rs7173565, rs7170151 or rs9920715 in Jurkat cells. SNP rs 11631591 showed 3-fold enrichment of lmRNP-K binding over IgG control. No significant enrichment at the other two SNPs was observed. P-values are for Student’s t-test.

FIG. 9B shows sequence chromatographs showing the difference in zygosity of rsl 1631591 between the input (equal binding to the two alleles, above) and the ChIP assay at the risk allele (2-3x more binding to the risk C allele, below).

FIGS. 10A and 10 B show the downregulation of lmRNP-K by 5-FU treatment. 5-FU treatment reduces linRNP-K expression levels in Jurkat cells. Jurkat cells were treated with DMSO vehicle or 5-FU (20 ng/mΐ) for 24 or 48 horns. lmRNP-K (10 A) and RASGRP1 (10B) were examined with GADPH as loading control.

FIG. 11 shows RASGRPl reduction influences the phosphorylation of ERK. 5-FU treatment reduces lmRNP-K and RASGRPl expression levels in Jurkat cells. Pretreatment with PMA increases levels of RASGRPl and phospho- ERK. Inhibition of linRNP-K with 5-FU decreases levels of RASGRPl and phospho-ERK, even after PMA stimulation.

FIG. 12 shows the RASGRP3 region for the three candidate SNPs. ChromHMM for GM12878, NHEK, NHLF and HSMM chromatin state tracks show a promoter prediction (red) for rsl3385731. rsl3385731 overlaps H3K27Ac, H3K4Mel, and DNAse I, whereas rsl2613020 and rsl3425999 overlap H3K27Ac and H3K4Me3. All three SNPs fall within active transcriptional elements (dreg).

FIG. 13 shows a meta-analysis of SNP rsl3385731 showing its association with SLE in Asian and non-Asian population.

FIG. 14 shows the PARP1 motif binding. FIMO prediction for the location of PARP1 binding motif on rs 13385731.

FIGS. 15A and 15B show the RASGRP3 expression differences in European samples. Allele-specific expression of FIG. 15A: RASGRP3 and FIG. 15B : IRF7 on European control cohorts (N=TT : 303/CC-CT: 42).

FIGS. 16A to 16D show functional analyses of intronic mutations. The two SNPs rsl3385731 and rsl2613020 were cloned into pGL4.26 for enhancer assay and in pGL4.14 for promoter assay. Difference combination is risk (R) and non-risk (NR) introduced by site-directed mutagenesis and tested for luciferase gene reporter assay in HEK-293T cells (16A) enhancer assay (16B) promoter assay. In another set of experiments, SNP rsl3425999 was cloned into pGL4.26 and pGL4.14 and a non-risk (NR) variant is introduced by site-directed mutagenesis (16C) enhancer assay (16D) promoter assay. The empty vector pGL4.26 and pGL4.14 were used as negative control. Results are expressed in a ratio of Firefly /Ranilla activity multiplied by 100. The p values are calculated from three replicates with unpaired student’s t test.

FIGS. 17A to 17C show, 17A, the protein DNA binding complexes formed with Jurkat nuclear extract showing the binding pattern. 17B, a Super-shift assay with PARP-1 antibody showed the mobility reduction of the DNA protein complex. 17C a Western blots analysis of DNA protein binding complex from the Toledo nuclear extract.

FIGS. 18 A to 18N show ChIP qPCR assay using different antibodies demonstrate binding affinity against risk and non-risk genotype of rsl3385731. (FIG. 18A) PARP1, (FIG. 18B) PARP1 identification through heterozygous sample. Motif scanning of IRF-1 region (FIG. 18C), IRF-8 (FIG. 18D). (FIG. 18E-F) chip qPCR analysis of IRF8 and IRF-1. (FIG. 18G) H3K27Ac (FIG. 18H) H3K4Me3 (FIG. 181) P300, (FIG. 18J) Brd4, (FIG. 18K) RNA Polymerases-II. Fig (FIG. 18L) overall binding profile of different proteins in SNP rsl3385731 region and (FIG. 18M) binding profile against SNP rsl3425999. (FIG. 18N) different protein binding profile in the intronic region containing all three SNPs. For ChIP qPCR analysis, first the non-parametric ANOVA (Bonferroni's multiple comparisons test) were checked between the IgG (control) vs the different SNPs as a group (risk and non-risk). If ANOVA shows significant difference, then Student’s t-test were performed within the group. Abbreviations used in the pictures 731, 3020 and 999 are the representation of the SNPs rsl3385731, rsl2613020 and rsl3425999 respectively.

FIGS. 19A to 19G show the effect of the risk genotypes on (FIG. 19A) RASGRP3 mRNA expression from LFRR cell line (FIG. 19B) Coriell cell lines (FIG. 19C) combined LFRR and Coriell cell line (999 FIG. 19D, 3020 Analysis FIG. 19E). (FIG. 19E) mRNA expression of RASGRP3 against SNP rsl2613020 and (FIG. 19F) mRNA expression of RASGRP3 against SNP rsl3425999. Fig (FIG. 19F-G) Protein expression difference between the non- Risk (CC) and Risk (TT) genotypes of rsl3385731. The three different genotypes are displayed on the X-axis. Y- axis represents the level of normalized expression for each assay. Each data point represents the expression level of RASGRP3 mRNA or RASGRP3 protein. Significant differences from the mean expression of the risk genotype were determined using an unpaired t-test.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as“a”,“an” and“the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

In one aspect, the present invention includes compositions and methods for identifying novel targets and inhibitors of epigenetic elements on intronic variants rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, or rsl3725999, that influence B-cell differentiation and Systemic Lupus Erythematosus disease progression.

The present invention includes two separate examples of novel targets for the treatment of Systemic lupus erythematosus (SLE), which is an autoimmune disease with a strong genetic component.

In the first example, the inventors recently identified a novel SLE susceptibility locus near RASGRP1, which governs the ERK/MAPK kinase cascade and B-/T-cell differentiation and development. However, precise causal RASGRP1 functional variant(s) and their mechanisms of action in SLE pathogenesis remain undefined. The goal was to fine-map this locus, prioritize genetic variants according to likely functionality, experimentally validate the contribution of three SNPs to SLE risk, and experimentally determine their biochemical mechanisms of action. The inventors performed a meta-analysis across six Asian and European cohorts (9,529 cases; 22,462 controls), followed by in silico bioinformatic and epigenetic analyses to prioritize potentially functional SNPs. The inventors experimentally validated the functional significance and mechanism of action of three SNPs in cultured T-cells. Meta-analysis identified 18 genome-wide significant (p<5xl0 ⁸) SNPs, mostly concentrated in two haplotype blocks, one intronic and the other intergenic. Epigenetic fine-mapping and allelic imbalance and eQTL analyses predicted three transcriptional regulatory regions with four SNPs (rs7170151, rsl l631591-rs7173565, and rs9920715) prioritized for functional validation. Lucif erase reporter assays indicated significant allele-specific enhancer activity for intronic rs7170151 and rsl l631591-rs7173565 in T lymphoid (Jurkat) cells, but not in HEK293 cells. Using EMSA, mass spectrometry and ChIP-qPCR analysis, the inventors detected allele-dependent interactions between heterogeneous nuclear ribonucleoprotein K (luiRNP-K) and rsl 1631591. Furthermore, inhibition of linRNP-K in Jurkat cells downregulated RASGRP1 and ERK/MAPK signaling. Comprehensive association, bioinformatics, and epigenetic analyses yielded functional variants of RASGRP1. Several SNPs were experimentally validated, with an intronic variant (rsl 1631591) yielding the strongest results. This SNP is located in a cell type-specific enhancer sequence, where its risk allele binds to the linRNP-K protein and modulates RASGRP1 expression in Jurkat cells. As risk allele dosage of rsl 1631591 correlates with increased R. ISGRP1 expression and ERK activity, the inventors suggest that this SNP may underlie SLE risk of this locus.

In the second example, the present inventions also determined the additional SLE association with RASGRP3 (RAS Guanyl Releasing Protein 3) as one of the most consistently replicated SLE signals. The inventors recently reported that multiple intronic variants explained RASGRP3-SLE association in Asians. The inventors hypothesized that these intronic variants influence RASGRP3 expression by modulating epigenetic regulation, which could be associated with SLE risk.

As used herein, the term“sample” refers to any biological sample that is isolated from a subject, which can include, without limitation, a single cell or multiple cells, fragments of cells, an aliquot of body fluid, whole blood, platelets, serum, plasma, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, synovial fluid, lymphatic fluid, ascites fluid, and interstitial or extracellular fluid. The term "sample" also encompasses the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, urine, or any other bodily fluids. As used herein, the term“blood sample” refers to whole blood or any fraction thereof, including blood cells, red blood cells, white blood cells or leucocytes, platelets, serum and plasma. Samples can be obtained from a subject by means including but not limited to venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art.

As used herein, the term“subject” or“patient” refers generally to a mammal, which includes, but is not limited to, a human, non-human primate, dog, cat, mouse, rat, cow, horse, and pig, without regard to gender or age. A subject can be one who has been previously diagnosed or identified as having an auto-immune and/or inflammatory disease, and which may have already undergone, or is undergoing, a therapeutic intervention for the auto-immune and/or inflammatory disease. However, a subject can also include a patient not previously diagnosed as having the auto immune and/or inflammatory disease, for example, a subject who exhibits one or more symptoms or risk factors for the auto-immune and/or inflammatory disease, or a subject who does not exhibit symptoms or risk factors for the auto-immune and/or inflammatory disease, or a subject who is asymptomatic for the auto-immune and/or inflammatory disease. As used herein, a“healthy control” refers to a healthy control that is not an SLE patient that has no clinical evidence of SLE.

Nucleic Acid Detection. In other embodiments for detecting protein expression, one may assay for gene transcription. For example, an indirect method for detecting protein expression is to detect mRNA transcripts from which the proteins are made.

Amplification of Nucleic Acids. Since many mRNAs are present in relatively low abundance, nucleic acid amplification greatly enhances the ability to assess expression. The general concept is that nucleic acids can be amplified using paired primers flanking the region of interest. As used herein, the term "primer," refers to any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single-stranded form, although the single- stranded form is often used.

Pairs of primers designed to selectively hybridize to nucleic acids corresponding to selected genes are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences. Once hybridized, the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.

The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual method. Alternatively, the detection may involve indirect identification of the product via chemilluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals.

A number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in their entirety.

A reverse transcriptase-PCR amplification procedure may be performed to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2001). Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Pat. No. 5,882,864. Standard PCR usually uses one pair of primers to amplify a specific sequence, while multiplex-PCR (MPCR) uses multiple pairs of primers to amplify many sequences simultaneously. The presence of many PCR primers in a single tube could cause many problems, such as the increased formation of misprimed PCR products and“primer dimers”, the amplification discrimination of longer DNA fragment and so on. Normally, MPCR buffers contain a Taq Polymerase additive, which decreases the competition among amplicons and the amplification discrimination of longer DNA fragment during MPCR. MPCR products can further be hybridized with gene-specific probe for verification. Theoretically, one should be able to use as many as primers as necessary. However, due to side effects (primer dimers, misprimed PCR products, etc.) caused during MPCR, there is a limit (less than 20) to the number of primers that can be used in a MPCR reaction. See also European Application No. 0 364 255, relevant portions incorporated herein by reference.

Another method for amplification is ligase chain reaction ("LCR"), disclosed in European Application No. 320 308, incorporated herein by reference in its entirety. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence. A method based on PCR and oligonucleotide ligase assay (OLA), disclosed in U.S. Pat. No. 5,912,148, may also be used. Alternative methods for amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB Application No. 2 202 328, and in PCT Application No. PCT/US1989/001025, from each relevant portions incorporated herein by reference.

Detection of Nucleic Acids. Following any amplification, it may be desirable to separate the amplification product from the template and/or the excess primer. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2001). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid. Separation of nucleic acids may also be effected by chromatographic techniques known in art. There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.

In certain embodiments, the amplification products are visualized. A typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.

In one embodiment, following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2001). One example of the foregoing is described in U.S. Pat. No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practice of the instant invention are disclosed in U.S. Pat. Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227, 5,932,413 and 5,935,791, each of which is incorporated herein by reference.

Nucleic Acid Arrays. Microarrays include a plurality of polymeric molecules spatially distributed over, and stably associated with, the surface of a substantially planar substrate, e.g., biochips. Microarrays of polynucleotides have been developed and find use in a variety of applications, such as screening and DNA sequencing. One area in particular in which microarrays find use is in gene expression analysis.

In gene expression analysis with microarrays, an array of "probe" oligonucleotides is contacted with a nucleic acid sample of interest, i.e., target, such as polyA mRNA from a particular tissue type. Contact is carried out under hybridization conditions and unbound nucleic acid is then removed. The resultant pattern of hybridized nucleic acid provides information regarding the genetic profile of the sample tested. Methodologies of gene expression analysis on microarrays are capable of providing both qualitative and quantitative information.

A variety of different arrays that may be used with the present invention are known in the art. The probe molecules of the arrays which are capable of sequence specific hybridization with target nucleic acid may be polynucleotides or hybridizing analogues or mimetics thereof, including: nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phophorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine and the like; nucleic acids in which the ribose subunit has been substituted, e.g., hexose phosphodiester; peptide nucleic acids; and the like. The length of the probes will generally range from 10 to 1,000 nucleotides, where in some embodiments the probes will be oligonucleotides and usually range from 15 to 150 nucleotides and more usually from 15 to 100 nucleotides in length, and in other embodiments the probes will be longer, usually ranging in length from 150 to 1,000 nucleotides, where the polynucleotide probes may be single- or double- stranded, usually single-stranded, and may be PCR fragments amplified from cDNA.

The probe molecules on the surface of the substrates will correspond to selected genes being analyzed and be positioned on the array at a known location so that positive hybridization events may be correlated to expression of a particular gene in the physiological source from which the target nucleic acid sample is derived. The substrates with which the probe molecules are stably associated may be fabricated from a variety of materials, including plastics, ceramics, metals, gels, membranes, glasses, and the like. The arrays may be produced according to any convenient methodology, such as preforming the probes and then stably associating them with the surface of the support or growing the probes directly on the support. A number of different array configurations and methods for their production are known to those of skill in the art and disclosed in U.S. Pat. Nos. 5,445,934, 5,532,128, 5,556,752, 5,242,974, 5,384,261, 5,405,783, 5,412,087, 5,424,186, 5,429,807, 5,436,327, 5,472,672, 5,527,681, 5,529,756, 5,545,531, 5,554,501, 5,561,071, 5,571,639, 5,593,839, 5,599,695, 5,624,711, 5,658,734, 5,700,637, and 6,004,755, relevant portions incorporated herein by reference.

Following hybridization, where non-hybridized labeled nucleic acid is capable of emitting a signal during the detection step, a washing step is employed where unhybridized labeled nucleic acid is removed from the support surface, generating a pattern of hybridized nucleic acid on the substrate surface. A variety of wash solutions and protocols for their use are known to those of skill in the art and may be used. Where the label on the target nucleic acid is not directly detectable, one then contacts the array, now comprising bound target, with the other member(s) of the signal producing system that is being employed. For example, where the label on the target is biotin, one then contacts the array with streptavidin-fluorescent conjugate under conditions sufficient for binding between the specific binding member pairs to occur. Following contact, any unbound members of the signal producing system will then be removed, e.g., by washing. The specific wash conditions employed will necessarily depend on the specific nature of the signal producing system that is employed, and will be known to those of skill in the art familiar with the particular signal producing system employed. The resultant hybridization pattem(s) of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid, where representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement and the like.

Prior to detection or visualization, where one desires to reduce the potential for a mismatch hybridization event to generate a false positive signal on the pattern, the array of hybridized target/probe complexes may be treated with an endonuclease under conditions sufficient such that the endonuclease degrades single stranded, but not double stranded DNA. A variety of different endonucleases are known and may be used, where such nucleases include: mung bean nuclease, SI nuclease, and the like. Where such treatment is employed in an assay in which the target nucleic acids are not labeled with a directly detectable label, e.g., in an assay with biotinylated target nucleic acids, the endonuclease treatment will generally be performed prior to contact of the array with the other member(s) of the signal producing system, e.g., fluorescent-streptavidin conjugate. Endonuclease treatment, as described above, ensures that only end-labeled target/probe complexes having a substantially complete hybridization at the 3' end of the probe are detected in the hybridization pattern. Following hybridization and any washing step(s) and/or subsequent treatments, as described above, the resultant hybridization pattern is detected. In detecting or visualizing the hybridization pattern, the intensity or signal value of the label will be not only be detected but quantified, by which is meant that the signal from each spot of the hybridization will be measured and compared to a unit value corresponding the signal emitted by known number of end-labeled target nucleic acids to obtain a count or absolute value of the copy number of each end-labeled target that is hybridized to a particular spot on the array in the hybridization pattern.

RNA Sequencing. RNA-seq (RNA Sequencing), also called Whole Transcriptome Shotgun Sequencing (WTSS), is a technology that utilizes the capabilities of Next-Generation Sequencing (NGS) to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time. The transcriptome of a cell is dynamic; it continually changes as opposed to a static genome. The recent developments of next-generation sequencing allow for increased base coverage of a DNA sequence, as well as higher sample throughput. This facilitates sequencing of the RNA transcripts in a cell, providing the ability to look at alternative gene spliced transcripts, post-transcriptional changes, gene fusion, mutations/SNPs and changes in gene expression. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries, Ongoing RNA-Seq research includes observing cellular pathway alterations during infection, and gene expression level changes in cancer studies. Prior to NGS, transcriptomics and gene expression studies were previously done with expression microarrays, which contain thousands of DNA sequences that probe for a match in the target sequence, making available a profile of all transcripts being expressed. This was later done with Serial Analysis of Gene Expression (SAGE).

The present invention also includes small molecule inhibitors of proteins that interact with DNA, including, e.g., agents that block binding of lmRNP-K, H3K27Ac, H3K4Mel, H3K4Me3, P300, PARP1, or IRF1 to DNA. Non limiting examples of agents that block PARPl binding to DNA include, e.g., olararib, rucaparib, niraparib, talazoparib, veliparib, CEP 7922, E7016 and the like. Non-limiting examples of agents that are IRF1 inhibitors include IRF2. Non-limiting examples of agents that are P300 inhibitors include ICG-001, C646, or E1A. Non limiting examples of agents that are H3K4me3 inhibitors include MI-3 and GSK126.

Treatments for SLE. Thus, the present invention contemplates the detection of certain biomarkers followed by a change in the treatment of SLE, which may include using standard therapeutic approaches where indicated. In general, the treatment of SLE involves treating elevated disease activity and trying to minimize the organ damage that can be associated with increased inflammation and increased immune complex formation/deposition/complement activation. Foundational treatment can include corticosteroids and/or anti- malarial drags. EXAMPLE 1. Mechanistic characterization of RASGRP1 variants identifies an hnRNP-K-regulated transcriptional enhancer contributing to SLE susceptibility.

Previously, the inventors reported a novel SLE susceptibility signal near the RAS guanyl-releasing protein 1 ( RASGRP1 ) in Asians (4). The inventors identified several associated variants, the most significant being an intergenic variant (rsl2900339) between RASGRRI and C15orfi3 (4). However, the actual predisposing variants, target genes, and underlying mechanisms of action for this region are largely unknown. RASGRP1 belongs to a family of RAS guanyl nucleotide-releasing proteins (RASGRPs) comprising four members (RASGRP1 through RASGRP4). They all have a diacylglycerol (DAG)-binding Cl catalytic domain. Upon antigen stimulation, DAG binding and PLC signaling drive RASGRPs to the membrane, where they play important roles in RAS activation (5, 6). RASGRP1, originally cloned from the brain (7), was later found highly expressed in T lymphocytes (8); small amounts of RASGRP1 expression can also occur in B lymphocytes , neutrophils, mast cells, and natural killer cells (9-11). RASGRP1 inhibition impairs T-cell expansion and increases susceptibility to Epsfein-Barr virus infection, as well as suppressing proliferation of activated T-cells occurring in autoimmune conditions ( 12). A heterozygous mutation in RASGRP1 correlates with autoimmune lymphoproliferative syndrome (ALPS)-like disease (13). The ratio of normal RASGRP1 isoforms to isofoims missing exon- 11 may be linked to defective poly [ADP-iibose] polymerase 1 (PARP1) expression and reduced lymphocyte survival in SLE patients (14, 15). Aberrant splice variants accumulate in SLE patients and adversely affect T-cell fmsction(16). R/iSGRPJ overexpression in T-cells increases RAS-ERK signaling (17. 18) and enhances disease progression in a mouse SLE model ( 19) and hitman lupus (20). These observations suggest that fOiSGRPI dysfunction is mechanistically associated with autoimmune phenotypes, including SLE

In this example, the inventors fine-mapped an SLE locus near R. iSGRRI that the inventors previously identified (4). Using trans-ethnic meta-analysis across six Asian and European cohorts followed by bioinformatic analyses and experimental validation, the inventors identified SLE predisposing variants and defined mechanisms by which these functional variants contribute to SLE pathogenicity.

Patients and Data. The inventors used all associated SNP data at this locus from six cohorts reported previously (Table 1). The inventors began with their published Asian cohort report (see supplementary table 5 in Reference (4)) and augmented this with two publicly available sets of GWAS summary statistics (21, 22) and a partially published Japanese cohort (23). The inventors’ original report contained three Asian cohorts (Korean, Han Chinese, and Malaya Chinese). Japanese samples included samples (456 cases and 1,102 controls) collected under support of the Autoimmune Disease Study Group of Research in Intractable Diseases, Japanese Ministry of Health, Labor and Welfare, and the BioBank Japan Project (23), and added samples obtained at Kyoto University, Japan. SLE classification followed the American College of Rheumatology criteria (24). All sample collections were approved by the Institutional Review Board of the Oklahoma Medical Research Foundation as well as by the collaborating institutions.

Quality Control. SNP quality control for the inventors’ initial Asian cohort has been described elsewhere (4). Quality control for European, Han Chinese 2, and Japanese samples was described in the original publications (21- 23). All SNPs in the study were in Hardy -Weinberg equilibrium (P>lxl0 ⁶), and had minor allele frequency >0.5%. Genotypic missingness was <10%. In order to match risk alleles between cohorts, the inventors compared their allele frequencies to the parent populations from the 1000 Genomes Project. The inventors used the SNP reference dbSNP142 as the SNP-naming convention in common for all variants. SNP imputation for all cohorts was described in their original publications. For this study, SNPs with r² and imputation quality information <0.7 were dropped. Study design. In order to identify RASGRP1 (Entrez Gene ID: 10125, incorporated herein by reference) functional variants and their mechanisms of action, the analysis followed the workflow presented in FIG. 1A. The inventors first extracted all summary GWAS information in and around RASGRP1 (118 SNPs) from Supplementary Table 5 in the inventors’ previous study of Asian SLE (4). The inventors combined these results with a European (21), an Asian (22), and a partially published Japanese cohort (23), to perform meta-analysis. SNPs that passed the genome wide significant association threshold (p = 5xl0 ⁸) were further annotated with functional information. A series of bioinformatics and epigenomic analyses was conducted for each of the candidate SNPs including their effects on gene expression (expression quantitative trait loci, eQTLs), transcription factor binding, promoter/enhancer activities and chromatin interaction sites. Together, the inventors prioritized and nominated SNPs with stronger association signals and with higher annotated likelihood of being functional. Finally, the inventors experimentally validated predicted functions of the nominated SNPs in Jurkat and HEK293 cell lines. Following SNP prioritization, the inventors performed electrophoretic mobility shift assays (EMSAs), followed by mass spectrometry, chromatin immuno -precipitation quantitative PCR (ChIP-qPCR), and inhibition-based expression assays.

Association analysis and trans-ethnic meta-analysis. Association analysis for all cohorts was performed using PLINK (25) and SNPTEST. Meta-analysis for all cohorts was performed in METAL (26) using cohort sample size correction and standard error correction to estimate the 95% confidence interval for odds ratios. Heterogeneity of odds ratios was estimated and informed the use of P_meta values in the study. Variants with P_meta<5xl0 ³ were selected for further study.

Bioinformatic analysis. Given that candidate SNPs were located in non-coding regions of the genome, the inventors performed a thorough epigenetic annotation of the variants. Initial annotation of epigenetic features was performed in Haploreg (27). Each SNP in the region was collocated with active and regulatory histone marks including H3K27Ac, H3K4Mel and H3K4Me3, and DNase hypersensitivity sites (DHS) in GM12878, and CD4+ and CD8⁺ T cells (FIG. IB). Histone modifications and DHS data were obtained from the ENCODE project (28) and the BLUEPRINT epigenome project (29).

SNP prioritization. The inventors used a prioritization algorithm to narrow down the large list of SNPs for further validation. The strategy consisted of two Bayesian algorithms to score each SNP (3dSNP (30) and RegulomeDB (31)), as well as additional expression, epigenetic, and preferential allele-specific information about each SNP. First the inventors used the 3dSNP (30) tool to assign functional weights based on the presence of enhancers, promoters, experimentally determined (ChIP-seq) transcription factor binding sites (TFBSs), TFBS motif matching, evolutionary conservation, and presence of 3D chromatin interactions. The inventors assigned a 3dSNP weight of 2 to SNPs greater than two standard deviations above the mean, a weight of 1 for scores above the mean, and a weight of 0 for the rest. RegulomeDB (31) scores were also assigned for each candidate SNP, and converted to an associated weight. Each functional category, i.e. eQTL, enhancer/super-enhancer, rSNP (32), capture Hi-C, TFBS, and allele-specific expression/binding was assigned a weight of 1 if the SNP had this feature. Finally, the inventors summed all weights for each SNP and nominated the top SNPs for further experimental validation.

Expression quantitative trait loci (eQTLs). All the candidate SNPs were annotated for the presence of eQTLs changing expression of RASGRP1 and its surrounding genes in multiple tissues. The inventors used expression databases for whole blood (33, 34), immune cell lines (35), and multiple tissues (36) (GTEx Analysis Release V6p). In order to identify quantitative changes in methylation in blood cell lines, the inventors used the WP10 database from the Blueprint epigenome project (37).

Transcription factor binding sites (TFBSs). In order to identify allele-specific effects on transcription factor binding (TFBSs), the inventors used the motifBreakr (38) algorithm implemented in R, as well as the PERFECTOS-APE algorithm that identifies fold-changes in binding affinity of SNPs against HOCOMOCOIO, HOMER, JASPER, Swiss Regulon, and HT-Selex motif databases. The inventors selected only TFBSs that had at least 5-fold change in affinity.

Assessing SNP effects on Enhancer/Promoter sequences. The inventors assessed whether each SNP was located within regulatory (enhancer/promoter) regions across multiple cell lines using active histone marks (H3K27Ac, H3K4Mel and H3K4Me3) collocation implemented in the 3dSNP application (30). Super-enhancers were annotated using the dbSuper (39), Prestige (40) and EnhancerAtlas (41) databases.

Chromatin interactions. Chromatin looping was identified using capture Hi-C assays obtained from 3D Genome (42), 3DSNP (30) and CHiCP (43); as well as from Promoter-capture Hi-C (44-47) experiments.

Allele-specific binding. Candidate SNPs within the association peaks were further targeted to assess allele-specific binding (ASB) of histone marks H3K4Mel and H3K4Me3 in and around them. ASB was calculated using seven heterozygous cell lines (GM10847, GM12890, GM18951, GM19239, GM19240, GM2610, and SNYDER). ASB was implemented in SNPhood (48).

Luciferase reporter assays. To test candidate SNP-containing regions for allele-specific enhancer activity; the inventors cloned all three SNPs (rsl l63159, rs7173565-rs7173565, and rs9920715) individually into the enhancer reporter plasmid 4.26 (Promega USA). In brief, genomic DNA from the Coriell cell line (obtained from NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research) was amplified using specific primers containing Kpnl and Hindlll sites. These amplified PCR products surrounding rsl 1631591 (481 bp), rs7170151 (579 bp) and rs9920715 (455 bp) were digested with Kpnl and Hindlll restriction enzymes and ligated to pGL4.26 plasmid (Promega USA). After cloning and transformation, the plasmids generated for each genotype were confirmed by direct Sanger DNA sequencing. To study cell type-dependence, the inventors used two different cell types, human embryonic kidney HEK293 and T lymphoid Jurkat cell lines. HEK293 cells were seeded in 24-well sterile plastic culture plates at a density of lxlO⁵ cells per well with complete growth medium. The cells were transfected with 500ng of pGL4.26 (with or without insert) along with 50 ng Renilla plasmid as control vector to control for differences in transfection efficiency. LipofectAMINE 3000 (Invitrogen, USA) was used for transfection into HEK293 cells, according to the manufacturer’s protocol. For Jurkat transfections, the inventors used the Neon Transfection System (Thermo Fisher Scientific). 5x10^s Jurkat cells were resuspended in 10 mΐ Buffer R (provided in the Neon Transfection Kit), and transfected with 2 pg of each plasntid containing the insert with risk or non-risk allele, along with 50 ng Renilla plasmid, using two pulses, 1050 V, 30 ms pulse width, according to the T-cell protocol provided for Neon. Firefly and Renilla luciferase activities were measured consecutively at 24h after transfection using Dual-luciferase assays (Promega) according to the manufacturer’s instructions. Luciferase activity was analyzed with Student’s t-test implemented in GraphPad Prism7. Differences between relative luciferase activity levels were considered significant if Student's t-test P-value <0.05.

Identification of DNA-binding proteins. Electrophoretic mobility shift assays (EMSAs) and DNA pulldown assays. Jurkat cell lines were obtained from ATCC and maintained in RPMI 1640 medium with 2 mm L-glutamine, 100 pg/ml each of streptomycin and penicillin, and 10% fetal bovine serum at 37 °C with 5% C0₂. Cells were harvested at a density of 8xl0⁵ cells/ml, and nuclear extracts were prepared using the NER nuclear extraction kit (Invitrogen) with complete protease inhibitor (Roche Diagnostics). Protein concentrations were measured using a BCA reagent. Biotinylated DNA sequence surrounding the candidate SNPs (rs7170151 and rsl 1631591) was prepared using a synthetic single-stranded DNA sequence (IDT USA). Biotinylated DNA sequence with a 5-bp deletion at the SNP region served as a control for the assay. Twenty-five pmol of each DNA product was bound to 1 mg Dynabeads® M-280 Streptavidin (Invitrogen, USA) as per the manufacturer’s recommendations. Dynabeads M-280 Streptavidin (Dynal, Inc., Lake Success, NY, USA) were prepared by washing three times in phosphate-buffered saline (pH 7.4) containing 0.1% bovine serum albumin and two times with Tris-EDTA containing 1M NaCl. Between each wash, beads were pulled down with a Dynal magnetic particle concentrator. Double-stranded, biotinylated oligonucleotides were added to the washed beads, and the mix was rotated for 20-30 min at 21 °C. Equal cpm of proteins translated in vitro were diluted to lx with binding buffer and mixed with— 100 pg of Dynabeads containing 10 pmol of the individual oligonucleotide probe in a final volume of 250 mΐ. The mixture was rotated at room temperature for 20 min. Proteins bound to the beads were separated from unbound proteins by successive washes, three times with 0.5x binding buffer and once with lx binding buffer. Higher stringency washes included two washes with 2x binding buffer. Beads and bound proteins were pulled down with a magnetic concentrator, suspended in lx sample buffer, boiled for 5 min, and resolved on SDS-PAGE gels followed by peptide mass fingerprint MALDI-MS analysis of single bands.

Mass spectrometry analysis. Mass spectrometry analysis was performed using a Thermo-Scientific LTQ-XL mass spectrometer coupled to an Eksigent splitless nanoflow HPLC system. Bands of interest were excised from the silver nitrate-stained Bis-Tris gel and de-stained with Farmer’s reducer (50 mM sodium thiosulfate, 15 mM potassium ferricyanide). The proteins were reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Samples were injected onto a 10 cm x 75 mm inner diameter capillary column packed with Phenomenex Jupiter C18 reverse phase resin. The peptides were eluted into the mass spectrometer at a flow rate of 175 nl/min. The mass spectrometer was operated in a data-dependent mode acquiring one mass spectrum and four CID spectra per cycle. Data were analyzed by searching all acquired spectra against the human RefSeq databases using Mascot (Matrix Science Inc., Boston, MA, USA). Minimum identification criteria required two peptides with ion scores greater than 50% and were verified by manual inspection. The inventors verified the identity of the assayed proteins by Western blot.

Confirmation of identified protein by Western blot. Mass spectrometry-identified proteins were confirmed by Western blot. Jurkat nuclear extracts after DNA pulldown assay were lysed in sample buffer [62.5 mM Tris HCl (pH 6.8 at 25°C), 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% wt/vol bromophenol blue]. Equal amounts of protein were loaded into a 10% SDS-PAGE gel (GTX gel BioRad USA). After it resolved, samples were blotted to Nitrocellulose paper using the Trans-blot Turbo Transfer System (BioRad, USA). Membranes were blocked using LI-COR blocking buffer for 2 horns and then incubated with primary antibody 1: 1000 dilution (lmRNP-K, Santa Cruz USA) at 4°C overnight, and with a donkey anti-mouse IR-Dye 800 (LI-COR, USA) secondary antibody for 1 h. The membrane was imaged with a LI-COR Odyssey using Auto-Scan. Background- subtracted signal intensity was quantified using Image Studio 4.0 software.

Chromatin immuno-precipitation (ChIP) assay followed by qPCR (ChiP-qPCR). Jurkat cells were cultured in DMEM. ChIP assays were performed using MAGnity Chip kit (Invitrogen, Carlsbad, CA, USA). Cells were fixed for 10 min with 1% formaldehyde to crosslink DNA-protein and protein-protein complexes. The crosslinking reaction was stopped using 1.25 M glycine for 5 min. The cells were lysed, sonicated to shear DNA and sedimented. Then, their diluted supernatants were incubated with 5pg lmRNP-K antibody. Ten percent of the diluted supernatants were saved as“input” for normalization. Several washing steps were followed by protein digestion using proteinase K. Reverse crosslinking was carried out at 65°C. DNA was subsequently purified and amplified by quantitative PCR on an SDS 7900 (Applied Biosystems) using specific primers. Because the Jurkat cell line is heterozygous for the SNPs rsl l631591 and rs7170151, the inventors performed Sanger DNA sequencing with the ChIP-eluted PCR product.

Inhibition of lmRNP-K and ERK phosphorylation. Inhibition of lmRNP-K was performed in Jurkat cell lines using 5-Fluorouracil (5-FU) (Sigma Aldrich, USA) as described previously (49). Jurkat cells were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (Invitrogen) and kept at 37°C in 5% C0₂ conditions. For 5-FU treatment, the drag was first dissolved in dimethyl sulfoxide (DMSO) and further diluted in medium before use. Cells were treated with 20ng/pl 5-FU unless otherwise stated. Next, to examine whether lmRNP-K and or RASGRP1 down-regulation by 5-FU led to inhibition of EKR to phosphorylation of ERK, cells were pretreated with PMA 5ug/pl for 30 minutes, prior to drag (5FU) treatment. Inhibition of hnRNP-K and RASGRP1 was detected using mRNA expression analysis with quantitative PCR and by Western blot.

Patients and samples. The inventors used five Asian cohorts and one cohort of European descent; sample sizes for the meta-analysis were 9,529 SLE cases and 22,462 controls (Table 1).

Table 1. Cohorts used in this study. We utilized samples from our previous report Sun et al. 2016 for RASGRPl SLE association. We added a Han Chinese (HC) and a European (EU) cohort from Morris et al. 2016 and a Japanese cohort containing the patients from Okada et al. 2012 and additional Japanese samples (JAP).

Fine-mapping, replication and meta-analysis of RASGRPl association. First, the inventors probed their previously reported SLE-associated region (chrl5: 38.4-39.2MB, hgl9) and extracted association results for six cohorts from the region containing the genes RASGRPl (RAS guanyl-releasing protein 1, a diacylglycerol-regulated guanine nucleotide exchange factor) and C15orf53 (encoding a protein of unknown function linked to alcohol dependence

Analysis of the association signals in the context of linkage disequilibrium (LD) of lOOOGenome populations (EUR, ASN; FIG. 2) identified two uncorrelated association signals (data not shown). The main signal occurred at rs8032939 in intron 2 (FIG. 2), while the second signal localized to the intergenic region between R. ISGIU’l and C15orfl3 , SNP rs9920715 (60 kb 5’ of RASGRP1 ; P_meta = 5.1xl0^-9; OR (95%CI) = 0.89(0.86-0.93)). Many (27 of 119 SNPs) of variants were intronic (FIG. 2). The inventors then examined the 18 GWS SNPs with bioinformatic and epigenomic analysis (Table 2). The inventors’ top SNP (rs8032939) was previously reported as a rheumatoid arthritis (RA)-associated SNP (51). Within the intronic signal, the inventors also identified rs8035957 (P_meta ⁼l-3xl0 ¹⁰), associated with Type I Diabetes (52).

Table 2. Meta-analysis of the RASGRP1 region. We identified 18 genome-wide significant SNPs in RASGRP1 (intron 2) and between R. ISGRPl and C15orf53 (intergenic). *Sun et al. 2016 supplementary table 5 was used as the discovery cohort and was replicated in Morris et al. 2016 (Han Chinese, HC; and European, EU) and in Okada et al. 2012 (Japanese, JAP) cohorts. OR: Odds ratio. 95% Cl: 95% confidence interval. HetPVal: P-value for heterogeneity meta-analysis test. Direction of OR is presented as + if OR >1 and - if OR<l. Note that all OR directions are consistent for all SNPs.

Table 2

Table 3. Relevant epigenetic features of genome-wide significant SNPs. We integrated scores from 3dSNP, RegulomeDB and rSNP with blood cell-specific information for eQTLs, enhancer/super-enhancer existence, promoter capture HI-C (PCHiC), transcription factor binding site (TFBS) dismption and allele-specific expression/binding (ASE/ASB) into a weighted score for SNP prioritization. The inventors chose the top three SNPs for further validation (rsl 1631591 and rs7173565 were used together because of the short distance between them).

Table 3

Evaluating functional SNPs. To identify functional SLE SNPs in and around RASGRP1, the inventors computed weighted scores for each SNP by integrating multiple sources of functional annotation, including allele-dependent gene expression, overlap with annotated enhancers and promoters, binding affinity to transcription factors and collocation with anchors in promoter-enhancer-capture Hi-C experiments (data not shown).

Gene expression. The inventors then identified allele-dependent changes in gene expression by annotating SNPs using eQTL databases for multiple tissues (Methods). All candidate LD SNPs were eQTLs in blood cell lines (3.2xlO ³>P>1.9xlO ⁴; as well as in skin, esophagus, and testis (Table 3). The intronic (main signal) SNPs affected expression of both RASGRP1 and C15orf53 , while the intergenic (secondary) SNPs (in LD with rs9920715) altered expression of only RASGRP1. RASGRP1 SNPs also affected expression of long non-coding RNAs (IncRNAs) RP11-102L12.2 and RPl 1-27514.2 in nonblood cell lines. All eQTL risk alleles increased expression of RASGRP1 in multiple cell lines (FIG. 3), but had opposing effects on the neighboring gene C15orf53. The inventors also found significant effects of two linked SNPs (rsl 1073344, rsl 1631591) on methylation of RASGRPl in T-cells and neutrophils, respectively.

Overlap with enhancers and super-enhancers. Then, the inventors investigated the potential of the candidate SNPs to act as enhancers of RASGRPl expression. Three GWS SNPs (rs6495979, rsl 1631591, and rs7173565) overlapped with ENCODE-annotated enhancers for RASGRPl in lymphoblastoid cells (GM12878, GM12892) and also in CD8⁺ T-cells. These three GWS SNPs (all intronic) localized to super-enhancers (i.e. collections of multiple contiguous enhancers (53)) for RASGRPl in CD4⁺ CD25 CD45RA⁺ naive cells, CD4⁺ CD25 CD45RO⁺ memory cells, CD8⁺ primary cells, CD4⁺ CD25 Ill 7⁺ phorbol myristate acetate (PMA)-stimulated Thl7 cells, and CD4⁺ CD25 1117 PMA-stimulated Thl7 cells. This suggests that these SNPs may regulate RASGRPl in T lymphocytes.

Chromatin interactions. Since all candidate SNPs reside outside of the RASGRPl promoter, the inventors investigated if the SNPs overlapped with anchors in promoter-enhancer connections though chromatin interactions. The inventors used promoter-capture Hi-C data on blood cell lines, in particular T-cells, to identify physical interactions between the intronic signal and the RASGRPl promoter (FIG. 4). The inventors also examined the physical interaction between the intergenic region (represented by rs9920715) and the promoters of RASGRPl and C15orf53. The inventors identified multiple significant promoter-enhancer interactions between the intronic signal and RASGRPl, C15orf53, FAM98B and SPRED1, and multiple interactions between the intergenic signal and the promoter of RASGRPl.

Effect on cytokine production. A critical feature in SLE pathogenicity is cytokine production (54) ; thus, the inventors investigated if these SNPs alter cytokine abundance. The candidate SNPs significantly increased expression of interleukins IL6 and IL22 and tumor necrosis factor (TNFa), while SNP rs9920715 exclusively increased IL22 expression. Allele-specific binding. The inventors found that 14 of the candidate GWS SNPs also had allele-specific binding (ASB) to H2K27ac in monocytes, neutrophils, and T-cells, while rs9920715 showed ASB with H3K4Mel in T-cells and neutrophils. To characterize the regulatory mechanisms involved, the inventors assessed ASB of histone marks H3K4Mel and H3K4Me3 at and around candidate SNPs (Table 3). The inventors identified a significant regulatory region associated with promoter mark H3K4Me3 with a higher binding affinity to the extended region (~1 kb) containing the risk alleles (both C) of intronic SNPs rsl l631591-rs7173565 (FIG. 5A). In addition, the inventors identified marginally significant ASB to enhancer mark H3K4Mel at SNPs rs6495979 and rs7170151, which tagged a regulatory region within ~500 bp (FIG. 5B). These data indicate that allele specific differences might affect chromatin interactions.

Validation of enhancer by luciferase assays. When testing in a luciferase reporter assay, rs7170151 and rs 11631591 showed marked (up to 10-fold over empty vector) enhancer activity in Jurkat cells (P=3.0xl0 ⁴, P=1.0xl0 ³, respectively) and less so (1.6-fold) in HEK293 cells (P=4.0xl0², P=3.0xl0³); however rs9920715 presented as a very weak enhancer only in HEK293 (P=4.1xl0 ²) (FIG. 6A, 6B). Furthermore, rs7170151 and rsl 1631591 showed dramatic allelic differences in enhancer function. Genomic regions containing homozygous risk alleles of rs7170151 (C) and rsl 1631591 (C) showed significantly higher enhancer activity (~50% increase; P=1.0xl0² and P=2.3xl0 ³, respectively; FIG. 6A) compared to non-risk alleles, but only in Jurkat cells. These results suggest allele-dependent enhancer activity is consistent with the allele-specific expression the inventors observed in the eQTL data. There were no significant differences in HEK293 cell lines (FIG. 6B), suggesting enhancer activity dependent on white blood cell-specific factors. The third intergenic SNP (rs9920715) did not show enhancer activity in any assayed cell type (FIG. 6A, 6B).

Transcription factor binding. The inventors next assessed allele-specific changes in transcription factor binding site (TFBS) affinity using five motif databases (see above). The inventors identified 256 TFBSs significantly affected by ten of these SNPs. Notably, the inventors found 43-fold higher affinity of promoter-specific TF YY1 to the non-risk allele (T) of rs7173565 and 42-fold higher affinity of TF GATA (GATA1..3.p2 motif) to the risk (T) allele of rs6495979. Interestingly, SLE-risk ETS1(55) binding had 10-fold higher affinity to the risk (C) allele of rs7173565, while SLE-risk IRF5 (56) bound 6- fold more tightly to the non-risk (C) allele of rs6495979.

Identification of DNA-binding proteins. The inventors detected DNA-binding protein complexes using electrophoretic mobility shift assays (EMSAs) and DNA pulldown assays using a 41 bp-long dsDNA containing the rsl l631591-rs7173565 (homozygous risk, CC; or homozygous non-risk, TT) alleles. The inventors prepared nuclear extracts from Jurkat (T-cells) and incubated them with biotin-labeled dsDNA (risk vs non-risk) bound to magnetic beads containing streptavidin. EMSA showed multiple bands of DNA-bound proteins (FIG. 7A, 7B). The inventors observed allele-specific binding of a protein complex at 75kDa. Although EMSA is not a quantitative assay, the inventors observed in multiple independent experiments that the intensity of the band with the risk (CC) oligo was darker than with the non-risk (TT) oligo, suggesting allele -specific differential binding (FIG. 7A, 7B). Using mass spectrometry analysis of bound proteins, the inventors identified heterogeneous nuclear ribonucleoprotein K (hnRNP-K) isoform b as the most abundant bound protein. hnRNP-K was also the protein whose binding was most diminished by substitution of the risk CC by non-risk TT nucleotides. The inventors also confirmed that the identified protein bound with the risk oligo for the region of rs 11631591 was hnRNP-K through EMSA followed by Western blot (FIG. 8).

SNPs bind to different transcription factors in an allele-specific manner. Using EMSA and mass spectrometry, the inventors showed that hnRNP-K protein has tighter binding affinity to the risk genotype (CC) of SNP rs 11631591. The inventors validated these findings using Jurkat (heterozygous (CT) at rs 11631591) to perform chromatin-immunoprecipitation (ChIP) followed by RT-qPCR (ChlP- qPCR). The inventors observed significant enrichment in binding of hnRNP-K antibody to the SNP region of rsl 1631591, but did not observe any binding of hnRNP-K to either rs7170151 or rs9920715 (FIG. 9A). To determine preferential or allele-specific binding, the inventors performed Sanger sequencing for the region containing rsl 1631591. Both alleles were present in the original input sample, however, only the risk allele (C) was detected significantly higher than the non-risk allele (T) in chromatograms of the ChIP-eluted PCR product (FIG. 9B). These data suggests a preferential allele- specific binding to hnRNP-K.

hnRNP-K plays an important role in RASGRP1 expression. To investigate the role of endogenous hnRNP-K in Jurkat cells, the inventors transiently inhibited hnRNP-K using 5-fluorouracil (5-FU). After 5-FU treatment (48 hours), the inventors observed significantly reduced mRNA expression for both hnRNP-K (P=1.4xl0 ³; FIG. 10A) and RASGRP1 (P=3.0xl0 ⁴; FIG. 10B). 5-FU-induced hnRNP-K downregulation correlated with reduced expression of RASGRP1. This result suggests that hnRNP-K plays an important role in RASGRP1 expression in Jurkat cells. Furthermore, the inventors observed the reduction of ERK phosphorylation with 5-FU after initial induction with PMA (FIG. 11).

In this example, the inventors fine-mapped their previously reported SLE locus near RAS guanyl- releasing protein 1 (RASGRP1), a lynchpin of T-cell development and the RAS/MAP kinase signaling cascade following antigen exposure. The inventors performed a trans-ethnic meta-analysis of the locus with cohorts of Asian and European descent, followed by multiple lines of bioinformatic analyses of its epigenetic context to prioritize SNPs as candidate causal variants. Experimental testing of the top candidates validated them as plausible variants underlying association of this locus with SLE (and perhaps other autoimmune phenotypes).

The inventors identified two independently associated regions correlated with RASGRP1 regulation and expression. The first signal lies in RASGRP1 intron 2, represented by SNPs rsl l631591-rs7173565 and rs7170151, which regulate RASGRP1 expression as eQTLs (esophageal mucosa and skin), enhancers (in CD8⁺ T-cells, and thymic and lymphoblastoid cell lines), and as interaction anchors with the nearby C15orf53 promoter. The SNPs in this region are within a robust enhancer, with the risk alleles (rs7170151-C and rsl l631591-C/rs7173565-C) greatly increasing RASGRP1 expression in multiple tissues (databases) and in Jurkat T-cells (these experiments). Furthermore, this enhancer is targeted by promoter interactions in CD8⁺ and CD4⁺ T-cells, B-cells, and monocytes (57) (FIG. 6A, 6B). The inventors also identified another intergenic signal around 60 kb 5’ of RASGRP1, at rs9920715, another SNP within promoter-interacting chromatin that acts as an eQTL for RASGRP1 in B- and T-cell lines (57). However, this SNP did not show enhancer activity in these assays.

Mammalian regulatory elements, especially those that are tissue-specific, show high in vivo nucleosome occupancy, which can effectively compete with TF binding (58, 59). This nucleosome-mediated restricted access to regulatory information is a key element for inducible or cell type-specific control of gene expression (60). In the current study, the inventors observed strong enhancer activity at rsl 1631591- rs7173565 or rs7170151 only in Jurkat but not HEK293 cells. Furthermore, these candidate SNPs show allele -specific RASGRP1 expression, with the risk alleles driving substantially more (~50%) expression than the non-risk alleles. Other studies on numerous complex diseases have demonstrated enrichment of disease-associated loci in cell type-specific regulatory regions of corresponding disease-relevant cell types (53, 61-64). Additional studies now document the direct effects of common variation in enhancer elements on enhancer states (65-68), gene expression (65, 69), and disease (70-74). Risk alleles of rsl 1631591 also showed significant binding to hnRNP-K protein in an allele-specific manner.

DNA/protein interaction assays demonstrated that hnRNP-K preferentially binds to sequences containing the rsl 1631591 risk (C) allele. The inventors confirmed this allele-specific binding by EMSA and ChIP DNA sequencing. The inventors only observed allele-specific binding of hnRNP-K at SNP rsl 16311591- rs7173565, but not at rs7170151 or rs9920715. The inventors also observed that inhibition of hnRNP-K correlates with RASGRP1 expression and ERK phosphorylation. These data suggest that SNP rsl 1631591 is a functional SNP and may directly contribute to modulating RASGRP1 expression. Abnormal expression of RASGRP1 isoforms play important roles in lymphocytes of SLE patients regardless of their clinical disease activity, and may contribute to impaired lymphocyte function and increased apoptosis in SLE patients (14). Abnormal RASGRP1 expression also induces ERK and JNK phosphorylation in the MAPK pathway, which in turn alters T-cell development, contributes to long-term organ damage and ultimately increases SLE susceptibility (19, 75, 76). In the present study the inventors also observed the role of RASGRP1 expression in the phosphorylation of ERK activity. Altogether, these results indicate increased RASGRP1 expression correlated with the risk alleles in these functional SLE loci and T-cell dysfunction. However, this study did not examine the differences in RASGRP1 isoform expression reportedly associated with SLE and correlated with low RASGRP1 expression (14).

In this study, the inventors characterized the genetic risk of SLE in RASGRP1. The inventors also propose a mechanism by which proposed functional SNPs could affect SLE pathogenesis. The inventors identified two functional regions affecting expression and regulation of RASGRP1 in an intronic region including two SNPs (rs 11631591 and rs7170151) and another in an intergenic region harboring SNP rs9920715. All identified SNPs are RASGRP1 eQTLs and exhibit regulatory potential through enhancer- promoter chromatin interactions. SNP rsl 1631591 showed T-cell-specific enhancer activity and an allele- specific interaction with hnRNP-K protein. Inhibition of hnRNP-K protein by 5-FU decreased expression of RASGRP1 in T-cells, suggesting that hnRNP-K plays an important role in RASGRP1 expression through interactions with the risk genotype of SNP rsl 1631591. These results are consistent with this SNP being an important factor contributing to SLE pathogenicity.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of nucleic acid-binding proteins implicated in various cellular processes including transcription and translation (75, 77). hnRNP- K is a highly multifunctional protein, with annotated roles in chromatin remodeling, transcription, splicing and translation (77). It is primarily referred to as an RNA-binding protein specific for“poly-C” repeats (78), but it actually prefers single-stranded DNA and can bind to double-stranded DNA (79). hnRNP-K can act as a transcriptional activator or repressor (80); notable examples include transcriptional repression of CD43 in leukocytes (81) and transcriptional activation of c-myc in B-cells (82). Its DNA- binding preference is found to be repeats of the CT motif, separated by several base pairs (79), confirmed by structure determination (83). There are several CT motifs in the immediate environment of rsl 1631591, whose hnRNP-K binding could be affected by the SNP. It should also be noted that several of the other abundant proteins pulled down by the double-stranded DNA EMSA are primarily annotated as RNA-binding proteins, including hnRNP-M and splicing factor U2AF. Other transcription factors were also abundant, including far upstream element-binding protein 3, supporting the notion that this locus is indeed transcriptionally active.

Taken together, the inventors have identified and mechanistically dissected, in this example, a lupus risk locus in the 2^nd intron of RASGRP1 , which regulates T- and B-cell development and the MAP kinase pathway. Single SNPs were found to control transcriptional activation and binding to several proteins, including the transcription factor hnRNP-K. Experiments confirmed that both the single base-pair risk-to- non-risk substitutions and pharmacological inhibition of hnRNP-K decreased MAPK signaling in T-cells. EXAMPLE 2. Intronic variants of the B-cell proliferator RASGRP3 affect expression and contribute lupus risk.

Systemic lupus erythematosus (SLE) is an inflammatory disease with complex genetic underpinnings. SLE association with RASGRP3 (RAS Guanyl Releasing Protein 3) is one of the most consistently replicated SLE signals. The inventors recently reported that multiple intronic variants explained RASGRP3-SLE association in Asians. The inventors hypothesized that these intronic variants influence RASGRP3 expression by modulating epigenetic regulation, which could be associated with SLE risk.

First, the inventors used in-silico bioinformatics to define the potential regulatory effects of three candidate variants on gene expression using data from ENCODE, ROADMAP and GTEx databases. Next, the inventors used a combination of DNA pulldown, Electrophoretic Mobility Shift Assay (EMSA), Super-shift, Western blot, Mass Spectrometry and ChIP-qPCR to identify allele-specific DNA- bound proteins and differential histone marks. The inventors measured the RASGRP3 mRNA and protein expressions and also studied the enhancer/promoter activity of these variants. Bioinformatics predicted that these variants are located in active chromatin and have the potential to be dual enhancers/promoters. The inventors also predicted allele-specific binding to PARP1 and IRF1 at rs 13385731. The inventors observed significant (p<0.005) difference in RASGRP3 transcript and protein levels with increased expression in rs 13385731 risk genotype (TT). Luciferase assays demonstrated significant (p<0.005) allele-specific enhancer and promoter activities for rsl3385731. DNA pulldown and EMSA suggested allele-specific bound protein ~100 kDa, which was identified as PARP1 protein by Mass Spectrometry and later confirmed by Super-shift and Western blot. The inventors also verified differential allele-specific binding of PARP1 and IRF1 against rsl3385731 using ChIP-qPCR. Interestingly, while PARPl binding affinity is higher with risk (TT) genotype, IRF1 binding affinity is higher with non-risk (CC) genotype of rs 13385731.

This example shows that risk genotype of rsl3385731 & rsl3725999 are associated with increased RASGRP3 expression. Furthermore, these variants showed significant allele -specific binding to H3K27Ac, H3K4Mel, H3K4Me3, P300, PARPl and IRF1 proteins, which might alter expression of RASGRP3, contributing to SLE risk.

Among SLE susceptibility loci, RAS Guanyl Releasing Protein 3 (RASGRP3) [16] at 2p25.1-24.1 is a consistently replicated SLE signal across populations, although initially identified in Asians [10] Recently, in a large-scale association study, the inventors’ replicated genetic association at this locus in Asian populations, and also identified two genetic variants that explained RASGRP3-SLE association [17]. The strongest associated SNPs were rsl3425999 (R=3.60c1(T¹⁰) and rsl3385731 (R=5.91c1(T⁹); both SNPs are highly correlated (r²=0.95). The inventors also identified rsl2613020 as independently associated to SLE (P=2.72xl0 ⁶, r² with rsl3425999=0.104). These three SNPs are within 500 bp region of intronic region. SNP rsl3385731 (C/T) is a eQTL affecting RASGRP3 expression (P = 1.69E-03) [18, 19] Meta-analysis in Asian and non-Asian population showed that one of the SNP rsl3385731 RASGRP3 is consistently replicated. In spite of the evidence of genetic association at these SNPs, it is unclear whether these SNPs are functional or what are the underlying mechanisms by which these variants exert functional effects.

RASGRP3 protein (690 aa, 73.8 kDa, Entrez Gene ID: 25780, incorporated herein by reference) a member of the subfamily of GTPases, mostly expressed in B-cell, macrophages, and endothelial cells serves as an RAS activator by promoting acquisition of GTP to maintain the active GTP -bound state and is the key link between cell surface receptors and RAS activation [20, 21] RASGRP3 plays an important functional role in the formation and progression of a variety of human cancers [22-25] Many studies suggest that RASGRP3 lead to defective ERK/MAPK pathway signaling alter gene expression in T-cells [26] and also limiting pro-inflammatory cytokines against low doses of TLR4 [27] these changes in T cells might contribute to lupus pathogenesis. RASGRP3 also showed to induce Ras-MAPK activation via DAG and by phosphorylation by PKC in B-cells in melanoma [28] But there is far less information known about the potential role of RASGRP3 in B-cells for lupus pathogenesis. Bioinformatics and epigenetic analysis. In silico prediction of epigenetic regulation. Intronic SNPs are reportedly involved in gene regulation through the epigenetic changes that affect chromatin accessibility, as well as through differential allele-specific binding affinity to transcription factors [29] In order to investigate epigenetic regulation at these loci, the inventors thoroughly annotated these loci with data on the co-localization of DNAse-hypersensitive sites, chromatin interactions, and histone modifications, especially to H3K27Ac, H3K4Mel, and H3K4Me3 as predictors of enhancer and promoter activity [30]

SNPs characterization and prioritization. In order to understand how the candidate variants could be functional, the inventors used the integrated method 3DSNP[31], which assigned a weighted score to each variant based on the likelihood of it being a promoter, enhancer, eQTL, or in open chromatin. Prediction of the chromatin state at these variants was taken from ChromHMM [32] ChromHMM clusters chromatin modification patterns into 15 states using a hidden Markov model [32]

Transcription factors binding. In order to further understand allele-specific changes in gene regulation at these loci, the inventors assessed the binding probabilities to human transcription factors (TF) from four databases: (167 TFs) from Factorbook [33], (401 TFs) from HOCOMOCO [34], (664 TFs) from HOMER [35], and (181 TFs) from ENCODE-TF [36] For this approach, the inventors used a sum of log probabilities method over the set of position probability matrices implemented in the library motifbreakR [37] in R. This method identifies potential transcription factor binding affinity at each SNP assigning an increasing value between 0-1.

Recent studies suggest that transcription factors (TF) P300 [38] (a transcriptional co-activator) and PARP1 have a role in gene regulation [39] In order to test binding affinity of these TFs to the candidate SNPs, the inventors used a motif scanning algorithm implemented in FIMO [40] Sequences containing the candidate SNPs (±10bp) were extracted from NCBI.

Active transcriptional elements. In order to identify the active transcriptional regulatory elements around these target variants, the inventors annotated their genomic positions with discriminative regulatory elements (dreg) [41] These annotations were taken from publicly available global run on sequencing data (GRO-seq) for seven cell lines including GM12878 and K562 (GSE66031) [41]

In order to further assess the allelic-specific effect of these SNPs on regulation in a cell-specific manner, the inventors used the deltaSVM method [42] on DNAse I sensitivity data for the 50 base pair sequence surrounding each of these candidate SNPs on a set of 101 tissues. These results were validated using the pre-computed contextual analysis of transcription factor occupancy (CATO) statistic [43], which estimates the probability of TF binding disruption. SNPs were considered to have a significant regulatory effect if the ([deltaSVM])>5, or CATO>0.1.

In silico gene expression. In order to characterize gene expression between SLE cases and controls, the inventors used two publically available data sets of SLE case/control array experiments (GSE45291 [44], and GSE65391 [45]). Dataset GSE45291 assayed 292 SLE cases and 20 controls on the Afiymetrix HT HG-U133 array; while GSE65391 assayed 157 SLE cases and 46 controls on the Illumina HumanHT-12 V4.0 Expression Bead Chip. Both expression datasets assayed whole blood samples. Case/control differences in expression were estimated with Wald’s T-test implemented in R. In order to identify differences in co-expression among cases and controls, the inventors estimated Pearson’s correlation between pairs of expressed genes within cases and controls.

Given expression differences between SLE cases and controls, the inventors assessed the allelic effect (eQTL) of the three candidate SNPs on RNA expression levels of RASGRP3 (and flanking genes FAM98A and TTC27) using RNA-seq data for 422 (345 Europeans and 77 African Yoruba) controls from the GEUVADIS project [46] Allele-dependent expression differences were estimated with the Wald statistic implemented in PLINK [47], whereas pair-wise differences between genotypes were estimated using a t-test statistic implemented in R.

Cell lines and culture conditions. The Epstein-Barr virus-transformed lymphoblast cell lines were obtained from the Oklahoma Medical Research Foundation (OMRF), OK USA. At the time of the experiments, selected EBV-transformed B cell lines were thawed and, after overnight incubation at 37°C in a humid atmosphere with 5% C0₂, the culture media was transferred to 15 ml conical tubes and centrifuged for 10 minutes at 300xg. Cell pellets were re-suspended in fresh complete RPMI media in T- 75 flasks and expanded. One million sub-confluent cells were transferred to 50 ml conical tubes and further centrifuged as described above. Cells were washed twice with 5 ml IX PBS. Pellets were flash frozen in liquid nitrogen for 20-30 seconds and stored at -80°C until they were used for preparation of total RNA.

Luciferase reporter assay. Bioinformatics analysis suggests that the regions containing the three SNPs are located in active regulatory region and could play role as enhancer and or promoter. Furthermore, SNP rsl3385731 and rsl3425999 are in strong LD, so the inventors divide the whole region into two different segments, SNP rsl3385731 along with rsl2613020 in one 400 base segments and SNP rsl3425999 into another 300 base segment. To study allele-specific effects on expression, the inventors cloned the 400 base region of DNA containing two SNPs (rsl3385731 and rsl2613020) into pGL4.26 with minimal promoter (enhancer assay). The inventors created different combinations of risk and non risk alleles of rsl3385731 T>C and rsl2613020 T>G using the Quick-change II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). In a separate construct, the inventors cloned 300 base region containing SNP rs 13425999 into the pGL4.26. The SNP rs 13425999 allele T>C was changed from non-risk to risk using site directed mutagenesis. For the mutant strand synthesis reaction, a 100 ng double-stranded DNA template was used for each construct with an extension time of 11 min 30 s; DPN- I restriction enzyme digestion was extended to 2 h to remove all residual templates and to increase mutagenesis efficiency. Next, the different combinations of variants were sub-cloned into the promoter less pGL4.14 (promoter assay). After cloning and mutagenesis plasmids of all the combinations were verified by DNA sequencing. The inventors used the HEK293 cell line for luciferase experiments. HEK293 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in DMEM supplemented with 10% fetal bovine serum (Invitrogen). For both promoter and enhancer assays, the inventors used the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). HEK293 cells were co-transfected with a pGL4.26 or pGL containing risk vector containing a firefly luciferase and an internal control pGL4.74 vector expressing Ranilla luciferase (as a control for transfection normalization). The internal control provided the basal response, thus minimizing experimental variability caused by transfection with different lengths of DNA. Each experiment was performed in three replicates and was repeated at least three times. Luciferase activity was analyzed with the Student’s t-test implemented in GraphPad Prism 7. Differences between relative luciferase activity levels were considered significant if Student's t-test P value <0.05.

Quantification of protein-DNA interaction. Preparation of Nuclear Extract. Toledo and Jurkat cells were grown in RPMI medium and then harvested by centrifugation (10 minutes, 20°C, and 200 g). Nuclear extract was prepared using the NER nuclear extraction kit (Invitrogen) with complete protease inhibitor (Roche Diagnostics). Protein estimation of nuclear extract was measured using a BCA reagent.

[0100] DNA pull-down assays and EMSA. The biotinylated DNA sequence (41 base pair) of the candidate SNPs was prepared using a synthetic single stranded DNA sequence (IDT USA) (Table 4). Twenty-five pmol of each DNA product was bound to 1 mg Dynabeads® M-280 Streptavidin (Invitrogen) as per the manufacturer’s recommendations. Dynabeads M-280 Streptavidin (Dynal, Inc., Lake Success, NY, USA) were prepared by washing three times in phosphate-buffered saline (pH 7.4) containing 0.1% bovine serum albumin and two times with Tris-EDTA containing 1M NaCl. Between each wash, beads were pulled down with a Dynal magnetic particle concentrator. Double-stranded, biotinylated oligonucleotides were added to the washed beads, and the mix was rotated for 20-30 min at 21 °C. Equal cpm of proteins translated in-vitro were made to 1 x with binding buffer and mixed with ~100 pg of Dynabeads containing 10 pmol of the individual oligonucleotide probe in a final volume of 250 mΐ. The mixture was rotated at room temperature for 20 min. Proteins bound to the beads were separated from unbound proteins by successive washes, three times with 0.5 ^c binding buffer and once with 1 x binding buffer. Higher stringency wash included two washes with 2 ^c binding buffer. Beads and bound proteins were pulled down with a magnetic concentrator, suspended in 1 ^c sample buffer, boiled for 5 min, and resolved on SDS-polyacrylamide gels followed by peptide mass fingerprint MALDI-MS analysis of single bands. For competition experiments, a 100-fold molar excess or, as indicated, a competitor oligonucleotide was added to the mixture. After incubation at room temperature for 20 min, the mixture was separated in a 6% non-denaturing polyacrylamide gel in 0.5 ^c Tris borate-EDTA electrophoresis buffer at 4°C followed by gel drying and autoradiography. For Super-shift assays, 2 pg of antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated with nuclear extract for 20 min at room temperature before addition of Cy5 labeled DNA probes. Next, the identified proteins were confirmed by Western blot. Jurkat and Toledo cells were lysed in sample buffer [62.5 mM Tris HCl (pH 6.8 at 25°C) 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% wt/vol bromophenol blue]. Equal amounts of protein were loaded in a 10% SDS-polyacrylamide gel; as soon as it resolved, it was transferred to nitrocellulose, and then incubated with primary antibody (PARP1) at 4°C overnight, and with the secondary antibody for 1 h. Antigen-antibody complexes were detected with ECL reagent (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). These results were replicated experimentally three times.

Table 4. Synthetic single-stranded DNA sequences

Mass spectrometry analysis. Mass spectrometry analysis was performed using a Thermo- Scientific LTQ- XL mass spectrometer coupled to an Eksigent splitless nanoflow HPLC system. Bands of interest were excised from the silver nitrate stained Bis-tris gel and de-stained with Farmer’s reducer (50 mM sodium thiosulfate, 15 mM potassium ferricyanide). The proteins were reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Samples were injected onto a 10 cm x 75 mm inner diameter capillary column packed with Phenomenex Jupiter C18 reverse phase resin. The peptides were eluted into the mass spectrometer at a flow rate of 175 nL/min. The mass spectrometer was operated in a data- dependent mode acquiring one mass spectrum and four CID spectra per cycle. Data was analyzed by searching all acquired spectra against the human RefSeq databases using Mascot (Matrix Science Inc. Boston, MA, USA). Minimum identification criteria required two peptides with ion scores greater than 50 % and were verified by manual inspection. The inventors verified the identity of the assayed proteins by Western blot.

Chromatin immimo-precipitation (ChIP) assay. EBV transformed cells were cultured in DMEM, and ChIP assay was performed using MAGniiy Chip kit (Invitrogen, Carlsbad, CA, USA). Cells were fixed for 10 min with 1% formaldehyde to crosslink DNA protein and protein-protein complexes. Crosslinking reaction was stopped using 1.25 M glycine for 5 min. The cells were lysed, sonicated to shear DNA, sedimented and their diluted supernatants were incubated overnight with the respective antibodies. Ten percent of the diluted supernatants was saved as“input” for normalization. Several washing steps were followed by protein digestion using proteinase K. Reverse crosslinking was carried out at 65°C. DNA was subsequently purified and amplified by quantitative PCR on a SDS 7900 (Applied Biosystems) using specific primers. The inventors performed a non-parametric ANOVA (Bonferroni's multiple comparisons test) between the IgG (Control) vs the different SNPs as a group (risk & non-risk). For significant IgG and SNP (risk and or non-risk) comparisons, the inventors performed within-group (risk vs non-risk) Students t-test. All data shown represent the mean ± SD., n > 3, *p < 0.05, **p < 0.01, ***p < 0.001 (t- test).

RASGRP3 mRNA expression. Total mRNA was extracted using an RNeasy plus Mini kit (QIAGEN) following the manufacturer's instructions. Complementary DNA (cDNA) was prepared by using the iScript System (Bio-Rad) as per manufacturer's instructions. Quantitative real-time PCR reactions for gene expression analysis were performed using Power SYBR Green master mix (Applied Biosystems, Foster City, CA, USA) following parameters: initial denaturation for 10 min at 95°C, followed by 45 cycles for 10 sec at 95°C, for 30 sec at 60°C, and for 1 sec at 72°C, followed by a melting curve analysis and a final cooling step to 40°C. Quantitative PCR data was analyzed using the SDS7900 System Software vl.5 (Applied Biosystems). Relative expression levels of RASGRP3 normalized to expression of housekeeping gene Beta Actin were calculated by the comparative C_t method.

RASGRP3 protein expression. EBV-transformed B cells were harvested and lysed in Whole Cell Extraction Buffer (25mM Tris, 1% Triton X-100, 150mM NaCl, ImM EDTA and protease inhibitors). Protein concentration in each cell line was measured using Quick Start Bradford Protein Assay Kits and adjusted to a final protein concentration of 2mg/mL. RASGRP3 protein was detected on western blot using the antibody against RASGRP3 (Cell Signaling (C33A3), Beverly, MA, USA). Anti-beta actin antibodies were purchased from Cell Signaling Technology, Inc. and were used to detect protein expression of beta actin. Densitometry analysis of immunore active bands was performed using National Institutes of Health Image J (National Institutes of Health) applied to digital images of respective Western blots.

Crisper Cas9 deletion. For crisper indel analysis, sgRNA was generated by Synthego and mixed with the Cas9 protein to form the RNP complex which was transfected in the cell line using Nucleofection. In brief, Nucleofection was done with a Neon® Nucleofection and the 100 mΐ Neon® Transfection Kit (Thermo Fisher Scientific) with 1050V and 2 pulse of 20 ms. Typically, 5 ^c 10⁶ cells in exponential growth phase were spun down and resuspended in 100 mΐ resuspension buffer containing 10-30 mM of sgRNA along with 20uM of Cas9-2NLS protein in addition of GFP plasmid. Cells transfected with TE buffer (mock). Transfection efficiencies were determined 1 or 2 days post transfection by flow cytometry (Gallios, Beckman Coulter) measuring the percentage of GFP-expressing cells. A total of 1 ^c 10⁴ cells were analyzed using a 488 nm argon laser with a 525/50 filter. A minimum of 70% GFP positive cells was expected. Indel was later confirmed by sequencing.

Bioinformatics and epigenetic analysis. RASGRP3 produces three protein coding transcripts (NM_170672, NM_001139488 and NM_015376). SNPs rsl3385731, rsl2613020 and rsl3425999 are located in intron-1, the largest intron in the gene (~35Kb). More specifically, these three candidate SNPs are located close to the end of the promoter of NM 001139488 (FIG. 12) (58bp, 270 bp and 371bp from the promoter respectively).

Epigenetic regulation. SNP rsl3385731 overlaps peaks of DNAse I, H3K27Ac, H3K4Mel and H3K4Me3 in lymphoblastoid cell lines (GM12878), whereas rsl2613020 and rsl3425999 only overlap H3K27Ac and H3K4Me3 peaks. This suggests that rsl3385731 acts as both promoter as well as enhancer, while the other two SNPs (rsl2613020 and rsl3425999) act as enhancers only. This observation was further confirmed by the ChromHMM [32] core 15 state chromatin state prediction (FIG. 12), as well as the 3DSNP result (FIG. 13). rsl3385731 was assigned to the active promoter and enhancer region of the gene in lymphoblastoid cell lines (GM12878), NHEK, NHLF and HSMM; whereas, both rsl3425999 and 12613020 are only predicted as promoters in GM12878, and are predicted to act as strong enhancers in HUVEC and HSMM cell lines (FIG. 12).

Transcription factors binding affinity. In order to understand allelic-specific effects of these SNPs on gene regulation, the inventors explored their effect on binding affinity of transcription factors (TFs). The analysis showed a strong affinity of rsl3385731-T to TFs IRF1, IRF2, IRF5, IRF7, IRF9 and IRF8, as well as strong affinity of rsl2613020-T to REST, SIN3A and SOX15. Interestingly, rsl3425999-C strongly bound to SLE locus ETS1 and to FOXJ2 (Table 5). Motif scanning for the sequence surrounding rsl3385731 showed significant affinity for the PARP1 motif (AAATCAAA; P=2.65xl0 ⁵) (FIG. 14).

Table 5. Predicted allele-specific motif binding at our three SNPs. Transcription factors predicted to strongly bind to either reference (AlleleRel) or alternate allele (Allele Alt). MotifPos: Position within the sequence where the SNP is located. Table 5

Regulatory elements. Analysis of the discriminative regulatory elements in GM12878 and K562 cell lines, collocated these three SNPs within active transcriptional regulatory elements (FIG. 12). Furthermore, analysis of the predicted regulatory potential for these variants, identified only rsl3385731 as regulatory in K562 cells (delta=5.02).

Gene expression. SLE cases had significantly higher rate of gene expression than controls (PFDR=2.04X10^'3, GSE45291; 292 cases/20 controls). To understand the mode of regulation for these variants, the inventors assessed allelic-specific expression on RNA-seq expression data for European and African samples. The inventors identified a significant allelic effect of rsl3385731 and rsl3425999 on expression of RASGRP3 (P= 1.07x 1 O ². P=8.82xl0 ³) (FIGS. 15A and 15B) but not for rsl2613020. In a previous report[46], rsl3385731 (as well as correlated SNP rsl3425999) reportedly affected exon level expression of RASGRP3 in Europeans (Table 6).

Table 6. Exon-level expression difference. Exon-level expression assayed by GEUVADIS at rsl3385731 and rsl3425999.

Luciferase assay. The inventors performed reported assays with the fragment of the intron (400 bp) encompassing SNP rsl3385731 and SNP rsl2613020 ligated with pGL4.26 for the enhancer assay, and with pGL4.14 for the promoter assay. The alleles of both SNPs were later changed to risk or non-risk by site-directed mutagenesis. The inventors identified significant difference in enhancer activity (risk vs non-risk) for SNP rsl3385731 (P=0.0034) and for rsl2613020 (P=0.0013). Furthermore, the inventors observed a significant enhancer activity increase when both SNPs are risk (P=0.0062, FIG. 16A). In the promoter assay, the inventors observed a significantly larger expression at the risk allele of rsl3385731 (risk vs. non-risk genotype, P=0.0004). SNP rsl2613020 did not show a promoter effect (P=0.7884, FIG. 16C). In a separate construct, 300 bp long segment containing SNP rs 13425999 was cloned into an enhancer plasmid (pGL4.26) and a promoter plasmid (pGL4.14). Concordant with rsl2613020, rsl3425999 showed a significant difference in enhancer activity between risk and non-risk (P=0.0037), but no promoter activity differences (P=0.1624). There were significant differences between the risk allele of rsl3425999 and control plasmids for pGL4.26 and pGL4.14 (P=0.0002 and P=0.0011, respectively, FIGS. 16B & 16D). Overall, these results suggest that all three SNPs have enhancer activity, and rsl3385731 has allele-specific promoter activity.

Identification of DNA binding proteins. DNA binding protein complexes were detected by electrophoretic mobility shift assay (EMSA) and DNA pull down assay using a 41 base long dsDNA containing the candidate alleles. Nuclear extract was prepared from Jurkat (T-cells) and Toledo cell (B- cells) lines and further mixed with biotin labeled dsDNA (risk vs non-risk) bound to magnetic beads containing streptavidin. EMSA showed multiple bands of DNA bound proteins (FIG. 17A). The inventors observed differences in binding between the DNA/Protein complexes of risk and non-risk genotypes of SNP rs 13385731. The molecular weight of this complex was lOOkDa. The bound protein was identified as poly [ADP-ribose] polymerase 1 (PARP1) through mass-spectrometry. The inventors confirmed PARP1 binding with EMSA“supershift” experiments. This antibody super-shifted the DNA protein complex from 100 kDa to 200 kDa (FIG. 17B). Similar results for rsl3385731 were obtained using the nuclear extract of the Toledo cell line which confirmed PARP1 differential binding using western blot (FIG. 17C).

SNPs bind to different transcription factors in an allele-specific manner. The inventors’ bioinformatics analysis showed that these three intronic variants (rsl3385731, rsl2613020 and rsl345999) are located in a region of RASGRP3 gene that exhibits open chromatin, epigenetic marks of active enhancers, and are likely to interact with transcription factors including P300 and Pol II and NFkb (FIGS. 18A to 18N). Furthermore, these EMSA results showed that PARP1 protein has binding affinity to the risk genotype (TT) of SNP rs 13385731. To validate these findings, the inventors performed chromatin- immunoprecipitation (ChIP) followed by quantitative PCR using EBV-transformed cell lines carrying risk (TT) and non-risk (CC) genotypes for SNP rsl3385731.

The inventors observed significant enrichment in binding of PARP-1 antibody to the risk genotype of rsl3385731 (P= 1.20x10³). The inventors did not observe this result for SNPs rsl2613020 and rsl3425999. Control DNA region (GDF15), known to have high binding with PARP-1, was taken as positive control and demonstrated no allele-specific difference in enrichment as expected (FIG. 18A). These results were further confirmed on a heterogeneous (CT) genotype cell line through ChIP elution followed by PCR and DNA sequencing which suggested only the risk allele (T) was present (FIG. 18B).

[0101] Since the region having these SNPs is highly active, the inventors performed ChIP with P300, Pol-II, Brd4, H3k27Ac, H3K4Me3, H3K4Mel and IRF-1. The risk genotype of rsl3385731 showed significant allele-specific fold enrichment with P300 (P=2.0xl0⁴), Pol-II (P=3.50xl0²), Brd4 (P=6.0x 10 ⁴), H3K27Ac (P=2.92xl0 ¹), and H3K4Me3 (P=8.0xl0 ³) which suggests that the risk genotype for SNP rsl3385731 is in a highly active region for transcription factor binding. The inventors also observed significant allele-specific fold enrichment for rsl3425999 with Pol-II (P=4.0xl0 ⁴) and Brd4 (P=2.0x 10 ²), which again suggests active involvement of this SNP in transcription. Interestingly, IRF1 (Interferon- 1) antibody showed higher affinity to the non-risk genotype of rsl3385731 (P=3.0xl0 ³) and of rsl3425999 (P=1.9xl0²). (FIG. 18C-K). Motif scanning of IRF-1 region (FIG. 18C), IRF-8 (FIG. 18D). (FIG. 18E-F) chip qPCR analysis of IRF8 and IRF-1. (FIG. 18G) H3K27Ac (FIG. 18H) H3K4Me3 (FIG. 181) P300, (FIG. 18J) Brd4, (FIG. 18K) RNA Polymerases-II. Fig (FIG. 18F) overall binding profile of different proteins in SNP rsl3385731 region and (FIG. 18M) binding profile against SNP rsl3425999. (FIG. 18N) different protein binding profile in the intronic region containing all three SNPs. For ChIP qPCR analysis, first we check the non-parametric ANOVA (Bonferroni's multiple comparisons test) between the IgG (control) vs the different SNPs as a group (risk and non-risk). If ANOVA shows significant difference, then Student’s t-test were performed within the group. Abbreviations used in the pictures 731, 3020 and 999 are the representation of the SNPs rsl3385731, rsl2613020 and rsl3425999 respectively.

RASGRP3 gene expression analysis. Expression of RASGRP3 mRNA was assayed from Coriell lymphoblastoid cell lines. Genotypes for SNP rsl3385731 for the assayed samples were risk (TT=76 %), heterozygous (CT=17%), and non-risk (CC=7%). The inventors observed a significantly increased RASGRP3 mRNA expression for the risk genotype (TT) compared to non-risk (CC) + heterozygous (CT) genotypes (P=0.0023, FIG. 19A). The inventors also confirmed the increased expression of RASGRP3 protein by Western blot analysis; expression difference between risk genotype (TT) and non-risk (CC) genotypes for rsl3385731 was significant (P=0.0137, FIG. 19B-C). Together, these results suggest that genetic risk of SEE at this locus is positively correlated with gene expression of RASGRP3. (FIG. 19E) mRNA expression of RASGRP3 against SNP rsl2613020 and (FIG. 19F) mRNA expression of RASGRP3 against SNP rsl3425999. Fig (FIG. 19F-G) Protein expression difference between the non- Risk (CC) and Risk (TT) genotypes of rs 13385731. The three different genotypes are displayed on the X- axis. Y-axis represents the level of normalized expression for each assay. Each data point represents the expression level of RASGRP3 mRNA or RASGRP3 protein. Significant differences from the mean expression of the risk genotype were determined using an unpaired t-test.

In the present example, the inventors characterized the functional consequences of three intronic genetic variants (rsl3385731, rsl2613020 and rsl3425999) that are associated with SEE in RASGRP3. Identification and functional characterization of causal variants responsible for disease predisposition is a fundamental goal for human genetics. Even though GWAS have identified thousands of variants reproducible associated with hundreds of complex genetic diseases [48], only a small fraction of these variants are presumed to be causal. This is due to the presence of linkage disequilibrium (LD) in the human genome, which streamlines GWAS discovery but renders causal variants statistically indistinguishable from non-causal variants in the same haplotype. Isolating causal from non-causal variants is a formidable task and most often involves a combination of genetic and bioinformatics’ approaches. In the present study, the inventors used a combination of these approaches to functionally characterize the genetic association of RASGRP3 genetic intronic variants with SLE. These data showed strong compelling evidence that these variants are causal variants, which showed strong enhancer/promoter activity as well as allele-specific binding of regulatory proteins.

Many large scale GWAS and high-density candidate gene studies, including the inventors’ recent study, showed a strong association of candidate SNPs with SLE [16, 17, 49, 50] Despite the strength of association, no single study has explained the functional/molecular aspect of these SNPs. RASGRP3 plays a significant role in many diseases by increasing the Ras activation in response to diacylglycerol (DAG) in rheumatoid arthritis [51], diabetes [52, 53], breast cancer [23] and prostate cancer [22-25], but its biological role in SLE is still unknown. Genome-wide association studies identified intronic SNPs rsl3385731, rsl2613020 and rsl3425999 associated with clinical features of SLE [16, 17, 50, 54] in Asian and non-Asian populations. The present study was designed to examine the functional aspect of these three candidate variants and their potential mechanistic role in SLE pathogenesis.

In these in-vitro studies, the inventors observed that the risk allele of rsl3385731 and rsl3425999 were correlated with higher expression of RASGRP3. This observation, in conjunction with the observed higher expression of RASGRP3 in some cases, shows that the role of this SNP is causal for SLE through its risk genotype associated promoter/enhancer activity with the concurrent higher levels of expression among RASGRP3 cases [18, 54]

These results show that a significant promoter/enhancer activity for the risk genotype of rsl3385731 and enhancer activity for rsl2613020 and rsl3425999. Together, these results demonstrate the risk alleles of rsl3385731, rsl2613020 and rsl345999 in gene expression modulation through the promoter/enhancer effects and through allele-specific binding to SLE -related IRF factors, and poly-ADP-(ribose) polymerase PARP1. Recent studies suggest that PARP1 is associated with SLE [55-57] Aberrant expression of PARP-1 impair survival of lymphocytes in SLE patients [55] But the nature of the relation between PARP1 and RASGRP3 is still unknown. These results show that, while PARP1 binding affinity is higher with risk (rsl3385731-TT) genotype, IRF1 binding is higher with non-risk (CC) genotype of rs 13385731. Together, these two proteins showed different allele-specific binding which shows that these proteins play an important role in the regulation of the gene expression of RASGRP3. Furthermore, the SNP rsl3425999 showed significant allele-specific binding to P300 and Pol-II, showing that the intronic region of RASGRP3 modulates gene expression with help from PARP1, IFR-1, P300 and Pol-II.

RASGRP3 is important in linking B cell receptor (BCR) activation to Ras signaling [21] Susceptibility locus RASGRP3 (ras-guanyl-releasing protein 3) is involved in SLE pathogenesis through B-cell signal transmission via Ras-ERK after B-cell receptor ligation and its potential impact on immunoglobulin production and B-cell proliferation [16] Lymphocyte activation is a key clinical feature of SLE which occurs in part through activation of Ras signaling pathway [58] [23, 25, 51] Here, the inventors show that risk alleles of these SNPs cause overproduction of RASGRP3 through their strong affinity to transcription binding proteins. This abnormal expression of RASGRP3 stimulates lymphocyte activation, which results in abnormal overstimulation of lymphocytes and contributes to SLE pathogenesis.

These results together present a picture of the functional role of three intronic RASGRP3 SNPs (rsl3385731, rsl2613020 and rsl3425999). Functional SNP rsl3385731 is an eQTL and the risk (TT) genotype associated with increased RASGRP3 expression. This SNP showed strong enhancer/promoter activity. Furthermore, this variant showed significant allele-specific binding to different transcription factors, which alters the expression of RASGRP3 and contributes to SLE risk. SNP rs 13425999, in strong LD with rsl3385731, also showed enhancer activity as well as allele-specific binding toward P300, Pol- II. Together, these results demonstrate that this intronic region regulates RASGRP3 expression and thus is directly involved in SLE pathogenicity. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.” The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as “comprise” and“comprises”),“having” (and any form of having, such as“have” and“has”),“including” (and any form of including, such as“includes” and“include”) or“containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein,“comprising” may be replaced with“consisting essentially of’ or“consisting of’. As used herein, the phrase“consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term“consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property (ies), method/process steps or limitation(s)) only.

The term“or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example,“A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (1), or equivalent, as it exists on the date of filing hereof unless the words“means for” or“step for” are explicitly used in the particular claim.

For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating Systemic Lupus Erythematosus (SLE) comprising:

identifying a subject in need of treatment for SLE; and

providing the subject with an effective amount of an agent that blocks the binding of a DNA binding protein to a mutation in an epigenetic locus that affects transcription of RASGRP1 or RASGRP3.

2. The method of claim 1, further comprising detecting the presence of a target locus selected from at least one of: rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, or rsl3725999, and providing a specific agent that blocks the binding of a DNA binding protein to the target locus if that mutation is present.

3. The method of claim 1, wherein the agent is 5-fiuorouracil, olararib, nicaparib, niraparib, talazoparib, veliparib, CEP 7922, E7016, ICG-001, C646, EI A, MI-3, or GSK 326.

4. The method of claim 1, wherein the epigenetic locus is at least one of rs7170151, rsl 1631591- rs7173565, rs9920715, rsl3385731, or rsl3725999.

5. The method of claim 1, wherein the agent is a single stranded or double stranded oligonucleotide that is complementary to the sequence of SNP selected from at least one of: rs7170151, rsl 1631591- rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRP1 or RASGRP3, or mutations thereof that block or reduce binding of the DNA binding protein to the SNP.

6. The method of claim 1, wherein the agent is a backbone -modified single stranded or double stranded oligonucleotide that is complementary to the sequence of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRP1 or RASGRP3.

7. The method of claim 6, wherein the agent is an anti-sense oligonucleotide that has a backbone modification that is selected from at least one of: a thio- or dithio-modified nucleic acids,

methylphosphonate nucleic acids, 2’-0-methyl nucleic acids, 2’-0-methoxy nucleic acids, bridged nucleic acids, locked nucleic acids, arabinonucleic acids, anhydrohexitol nucleic acids, cyclohexenyl nucleic acids, threofuranosyl nucleic acids, or a peptide nucleic acid.

8. The method of claim 1, wherein the agent is delivered into a target cell via liposome, viral expression vector, CRISPR, particle(s), exosome(s), microvesicle(s), Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), or a gene-gun.

9. The method of claim 1, wherein the agent is a peptide that blocks the DNA portion of the DNA binding protein specific for the epigenetic locus.

10. The method of claim 1, wherein the agent has a sequence selected from: 1 to 58.

11. The method of claim 1, wherein the agent is a small molecule inhibitor of RASGRP1 or RASGRP3, or reduces the transcription of RASGRP1 or RASGRP3.

12. The method of claim 1, wherein the agent blocks or reduces the binding of at least one of: hnRNP-K, H3K27Ac, H3K4Mel, H3K4Me3, P300, PARP1, or IRF1 to DNA.

13. A method treating Systemic Lupus Erythematosus (SLE) comprising:

identifying a subject in need of treatment for SLE; and

providing the subject with an effective amount of an agent that blocks the binding of a DNA binding protein to intronic variants rs7170151, rs 1163159 l-rs7173565, rs9920715, that affect transcription of RASGRPl, or intronic variants rsl3385731 or rsl3725999 that affect transcription of RASGRP3.

14. The method of claim 13, wherein the agent is a single stranded or double stranded

oligonucleotide that is complementary to the sequence of SNP selected from at least one of: rs7170151, rs 11631591 -rs7173565, rs9920715, rsl3385731, or rsl3725999, RASGRPl or RASGRP3, or mutations thereof that block or reduce binding of the DNA binding protein to the SNP.

15. The method of claim 13, wherein the agent is a backbone-modified single stranded or double stranded oligonucleotide that is complementary to the sequence of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRPl or RASGRP3.

16. The method of claim 15, wherein the agent is an anti-sense oligonucleotide that has a backbone modification that is selected from at least one of: a backbone modification is a thio- or dithio-modified nucleic acids, methylphosphonate nucleic acids, 2’ -O-methyl nucleic acids, 2’-0-methoxy nucleic acids, bridged nucleic acids, locked nucleic acids, arabinonucleic acids, anhydrohexitol nucleic acids, cyclohexenyl nucleic acids, threofuranosyl nucleic acids, or a peptide nucleic acid.

17. The method of claim 13, wherein the agent is delivered into a target cell via liposome, viral expression vector, CRISPR, particle(s), exosome(s), microvesicle(s), Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), or a gene-gun.

18. The method of claim 13, wherein the agent is a peptide that blocks the DNA portion of the DNA binding protein specific for the epigenetic locus.

19. The method of claim 13, wherein the agent has a sequence selected from: SEQ ID NOS: 1 - 58.

20. The method of claim 13, wherein the agent is 5-fluorouracil olararib, rucaparib, niraparib, talazoparib, veliparih, CEP 7922, E7016, ICG-001 , C646, El A, MI-3, or GSK126.

21. The method of claim 13, wherein the agent is a small molecule inhibitor of RASGRPl or RASGRP3, or reduces the transcription of RASGRPl or RASGRP3.

22. The method of claim 13, wherein the agent blocks or reduces the binding of at least one of: hnRNP-K, H3K27Ac, H3K4Mel, H3K4Me3, P300, PARP1, or IRF1 to DNA.

23. A composition for suppressing proinflammatory gene expression in a subject with Systemic Lupus Erythematosus (SLE) comprising an effective amount of an agent that blocks the binding of a DNA binding protein to a mutation in an epigenetic locus that affects transcription.

24. The composition of claim 23, wherein a target locus is selected from at least one of: rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, or rsl3725999, and a specific agent that blocks the binding of a DNA binding protein to the target locus is provided if that mutation is present.

25. The composition of claim 23, wherein the agent inhibits or reduces the expression of RASGRP1 or RASGRP3 .

26. The composition of claim 23, wherein the agent is 5-fluorouracil, oiararib, rucaparib niraparib, talazoparib, veliparih, CEP 7922, E7016, ICG-001 , C646, El A, MI-3, or GSK126.

27. The composition of claim 23, wherein the agent is a small molecule inhibitor of RASGRP1 or RASGRP3, or reduces the transcription of RASGRP1 or RASGRP3.

28. The composition of claim 23, wherein the epigenetic locus is at least one of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, or rsl3725999.

29. The composition of claim 23, wherein the agent is a single stranded or double stranded oligonucleotide that is complementary to the sequence of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRP1 or RASGRP3.

30. The composition of claim 23, wherein the agent is a backbone-modified single stranded or double stranded oligonucleotide that is complementary to the sequence of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, rsl3725999, RASGRP1 or RASGRP3.

31. The composition of claim 30, wherein the agent is an anti-sense oligonucleotide that has a backbone modification that is selected from at least one of: a backbone modification is a thio- or dithio- modified nucleic acids, methylphosphonate nucleic acids, 2’ -O-methyl nucleic acids, 2’-0-methoxy nucleic acids, bridged nucleic acids, locked nucleic acids, arabinonucleic acids, anhydrohexitol nucleic acids, cyclohexenyl nucleic acids, threofuranosyl nucleic acids, or a peptide nucleic acid.

32. The composition of claim 23, wherein the agent is delivered into a target cell via liposome, viral expression vector, CRISPR, particle(s), exosome(s), microvesicle(s), Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), or a gene-gun.

33. The composition of claim 23, wherein the agent is a peptide that blocks the DNA portion of the DNA binding protein specific for the epigenetic locus.

34. The composition of claim 23, wherein the agent has a sequence selected from: 1 to 58.

35. The composition of claim 23, wherein the agent is a small molecule inhibitor of RASGRP1 or RASGRP3, or reduces the transcription of RASGRP1 or RASGRP3.

36. The composition of claim 23, wherein the agent blocks or reduces the binding of at least one of: hnRNP-K, H3K27Ac, H3K4Mel, H3K4Me3, P300, PARP1, or IRF1 to DNA.

37. A method of identifying a subject in need of treatment for Systemic Lupus Erythematosus (SLE) and a therapy therefore, comprising:

obtaining a DNA sample from the subject;

determining the presence of one or more SNPs selected from at least one of rs7170151, rsl l631591-rs7173565, rs9920715, rsl3385731, or rsl3725999;

determining if the subject has an increased expression of RASGRP1, or RASGRP3; and providing the subject with an agent that inhibits the binding of a DNA binding protein to the

SNP.