Correction: Stage-Dependent and Locus-Specific Role of Histone Demethylase Jumonji D3 (JMJD3) in the Embryonic Stages of Lung Development.

[This corrects the article DOI: 10.1371/journal.pgen.1004524.].

Epstein-Barr virus nuclear antigen 2 extensively rewires the human chromatin landscape at autoimmune risk loci.

The interplay between environmental and genetic factors plays a key role in the development of many autoimmune diseases. In particular, the Epstein-Barr virus (EBV) is an established contributor to multiple sclerosis, lupus, and other disorders. Previously, we showed that the EBV nuclear antigen 2 (EBNA2) transactivating protein occupies up to half of the risk loci for a set of seven autoimmune disorders. To further examine the mechanistic roles played by EBNA2 at these loci on a genome-wide scale, we globally examined gene expression, chromatin accessibility, chromatin looping, and EBNA2 binding in a B cell line that was (1) uninfected, (2) infected with a strain of EBV lacking EBNA2, or (3) infected with a strain that expresses EBNA2. We identified more than 400 EBNA2-dependent differentially expressed human genes and more than 5000 EBNA2 binding events in the human genome. ATAC-seq analysis revealed more than 2000 regions in the human genome with EBNA2-dependent chromatin accessibility, and HiChIP data revealed more than 1700 regions where EBNA2 altered chromatin looping interactions. Autoimmune genetic risk loci were highly enriched at the sites of these EBNA2-dependent chromatin-altering events. We present examples of autoimmune risk genotype-dependent EBNA2 events, nominating genetic risk mechanisms for autoimmune risk loci such as ZMIZ1 Taken together, our results reveal important interactions between host genetic variation and EBNA2-driven disease mechanisms. Further, our study highlights a critical role for EBNA2 in rewiring human gene regulatory programs through rearrangement of the chromatin landscape and nominates these interactions as components of genetic mechanisms that influence the risk of multiple autoimmune diseases.

Pervasive Sharing of Causal Genetic Risk Factors Contributes to Clinical and Molecular Overlap between Sjögren's Disease and Systemic Lupus Erythematosus.

SjD (Sjögren's Disease) and SLE (Systemic Lupus Erythematosus) are similar diseases. There is extensive overlap between the two in terms of both clinical features and pathobiologic mechanisms. Shared genetic risk is a potential explanation of this overlap. In this study, we evaluated whether these diseases share causal genetic risk factors. We compared the causal genetic risk for SLE and SjD using three complementary approaches. First, we examined the published GWAS results for these two diseases by analyzing the predicted causal gene protein-protein interaction networks of both diseases. Since this method does not account for overlapping risk intervals, we examined whether such intervals also overlap. Third, we used two-sample Mendelian randomization (two sample MR) using GWAS summary statistics to determine whether risk variants for SLE are causal for SjD and vice versa. We found that both the putative causal genes and the genomic risk intervals for SLE and SjD overlap 28- and 130-times more than expected by chance (p < 1.1 × 10-24 and p < 1.1 × 10-41, respectively). Further, two sample MR analysis confirmed that alone or in aggregate, SLE is likely causal for SjD and vice versa. [SjD variants predicting SLE: OR = 2.56; 95% CI (1.98-3.30); p < 1.4 × 10-13, inverse-variance weighted; SLE variants predicting SjD: OR = 1.36; 95% CI (1.26-1.47); p < 1.6 × 10-11, inverse-variance weighted]. Notably, some variants have disparate impact in terms of effect size across disease states. Overlapping causal genetic risk factors were found for both diseases using complementary approaches. These observations support the hypothesis that shared genetic factors drive the clinical and pathobiologic overlap between these diseases. Our study has implications for both differential diagnosis and future genetic studies of these two conditions.

Alteration of CTCF-associated chromatin neighborhood inhibits TAL1-driven oncogenic transcription program and leukemogenesis.

Aberrant activation of the TAL1 is associated with up to 60% of T-ALL cases and is involved in CTCF-mediated genome organization within the TAL1 locus, suggesting that CTCF boundary plays a pathogenic role in T-ALL. Here, we show that -31-Kb CTCF binding site (-31CBS) serves as chromatin boundary that defines topologically associating domain (TAD) and enhancer/promoter interaction required for TAL1 activation. Deleted or inverted -31CBS impairs TAL1 expression in a context-dependent manner. Deletion of -31CBS reduces chromatin accessibility and blocks long-range interaction between the +51 erythroid enhancer and TAL1 promoter-1 leading to inhibition of TAL1 expression in erythroid cells, but not T-ALL cells. However, in TAL1-expressing T-ALL cells, the leukemia-prone TAL1 promoter-IV specifically interacts with the +19 stem cell enhancer located 19 Kb downstream of TAL1 and this interaction is disrupted by the -31CBS inversion in T-ALL cells. Inversion of -31CBS in Jurkat cells alters chromatin accessibility, histone modifications and CTCF-mediated TAD leading to inhibition of TAL1 expression and TAL1-driven leukemogenesis. Thus, our data reveal that -31CBS acts as critical regulator to define +19-enhancer and the leukemic prone promoter IV interaction for TAL1 activation in T-ALL. Manipulation of CTCF boundary can alter TAL1 TAD and oncogenic transcription networks in leukemogenesis.

Validation of low-coverage whole-genome sequencing for mitochondrial DNA variants suggests mitochondrial DNA as a genetic cause of preterm birth.

Preterm birth (PTB), or birth that occurs earlier than 37 weeks of gestational age, is a major contributor to infant mortality and neonatal hospitalization. Mutations in the mitochondrial genome (mtDNA) have been linked to various rare mitochondrial disorders and may be a contributing factor in PTB given that maternal genetic factors have been strongly linked to PTB. However, to date, no study has found a conclusive connection between a particular mtDNA variant and PTB. Given the high mtDNA copy number per cell, an automated pipeline was developed for detecting mtDNA variants using low-coverage whole-genome sequencing (lcWGS) data. The pipeline was first validated against samples of known heteroplasmy, and then applied to 929 samples from a PTB cohort from diverse ethnic backgrounds with an average gestational age of 27.18 weeks (range: 21-30). Our new pipeline successfully identified haplogroups and a large number of mtDNA variants in this large PTB cohort, including 8 samples carrying known pathogenic variants and 47 samples carrying rare mtDNA variants. These results confirm that lcWGS can be utilized to reliably identify mtDNA variants. These mtDNA variants may make a contribution toward preterm birth in a small proportion of live births.

Genome-wide chromatin occupancy of BRDT and gene expression analysis suggest transcriptional partners and specific epigenetic landscapes that regulate gene expression during spermatogenesis.

BRDT, a member of the BET family of double bromodomain-containing proteins, is essential for spermatogenesis in the mouse and has been postulated to be a key regulator of transcription in meiotic and post-meiotic cells. To understand the function of BRDT in these processes, we first characterized the genome-wide distribution of the BRDT binding sites, in particular within gene units, by ChIP-Seq analysis of enriched fractions of pachytene spermatocytes and round spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exons, and introns of genes. BRDT binding sites in promoters overlapped with several histone modifications and histone variants associated with active transcription, and were enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS, and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE, and RFX in round spermatids. Subsequent integration of the ChIP-seq data with available transcriptome data revealed that stage-specific gene expression programs are associated with BRDT binding to their gene promoters, with most of the BDRT-bound genes being upregulated. Gene Ontology analysis further identified unique sets of genes enriched in diverse biological processes essential for meiosis and spermiogenesis between the two cell types, suggesting distinct developmentally stage-specific functions for BRDT. Taken together, our data suggest that BRDT cooperates with different transcription factors at distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.

CTCF boundary remodels chromatin domain and drives aberrant HOX gene transcription in acute myeloid leukemia.

HOX gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant HOX gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal HOX gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the HOX gene loci. We discovered that the CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) is critical for establishing and maintaining aberrant HOXA9-HOXA13 gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active HOXA chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior HOXA genes. Consistent with the role of the CBS7/9 boundary in HOXA locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the HOXA locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.

Stage-dependent and locus-specific role of histone demethylase Jumonji D3 (JMJD3) in the embryonic stages of lung development.

Histone demethylases have emerged as important players in developmental processes. Jumonji domain containing-3 (Jmjd3) has been identified as a key histone demethylase that plays a critical role in the regulation of gene expression; however, the in vivo function of Jmjd3 in embryonic development remains largely unknown. To this end, we generated Jmjd3 global and conditional knockout mice. Global deletion of Jmjd3 induces perinatal lethality associated with defective lung development. Tissue and stage-specific deletion revealed that Jmjd3 is dispensable in the later stage of embryonic lung development. Jmjd3 ablation downregulates the expression of genes critical for lung development and function, including AQP-5 and SP-B. Jmjd3-mediated alterations in gene expression are associated with locus-specific changes in the methylation status of H3K27 and H3K4. Furthermore, Jmjd3 is recruited to the SP-B promoter through interactions with the transcription factor Nkx2.1 and the epigenetic protein Brg1. Taken together, these findings demonstrate that Jmjd3 plays a stage-dependent and locus-specific role in the mouse lung development. Our study provides molecular insights into the mechanisms by which Jmjd3 regulates target gene expression in the embryonic stages of lung development.

Attenuation of Forkhead signaling by the retinal determination factor DACH1.

The Drosophila Dachshund (Dac) gene, cloned as a dominant inhibitor of the hyperactive growth factor mutant ellipse, encodes a key component of the retinal determination gene network that governs cell fate. Herein, cyclic amplification and selection of targets identified a DACH1 DNA-binding sequence that resembles the FOX (Forkhead box-containing protein) binding site. Genome-wide in silico promoter analysis of DACH1 binding sites identified gene clusters populating cellular pathways associated with the cell cycle and growth factor signaling. ChIP coupled with high-throughput sequencing mapped DACH1 binding sites to corresponding gene clusters predicted in silico and identified as weight matrix resembling the cyclic amplification and selection of targets-defined sequence. DACH1 antagonized FOXM1 target gene expression, promoter occupancy in the context of local chromatin, and contact-independent growth. Attenuation of FOX function by the cell fate determination pathway has broad implications given the diverse role of FOX proteins in cellular biology and tumorigenesis.

Modeling of horizontal pleiotropy identifies possible causal gene expression in systemic lupus erythematosus.

Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune condition with complex causes involving genetic and environmental factors. While genome-wide association studies (GWASs) have identified genetic loci associated with SLE, the functional genomic elements responsible for disease development remain largely unknown. Mendelian Randomization (MR) is an instrumental variable approach to causal inference based on data from observational studies, where genetic variants are employed as instrumental variables (IVs). Methods: This study utilized a two-step strategy to identify causal genes for SLE. In the first step, the classical MR method was employed, assuming the absence of horizontal pleiotropy, to estimate the causal effect of gene expression on SLE. In the second step, advanced probabilistic MR methods (PMR-Egger, MRAID, and MR-MtRobin) were applied to the genes identified in the first step, considering horizontal pleiotropy, to filter out false positives. PMR-Egger and MRAID analyses utilized whole blood expression quantitative trait loci (eQTL) and SLE GWAS summary data, while MR-MtRobin analysis used an independent eQTL dataset from multiple immune cell types along with the same SLE GWAS data. Results: The initial MR analysis identified 142 genes, including 43 outside of chromosome 6. Subsequently, applying the advanced MR methods reduced the number of genes with significant causal effects on SLE to 66. PMR-Egger, MRAID, and MR-MtRobin, respectively, identified 13, 7, and 16 non-chromosome 6 genes with significant causal effects. All methods identified expression of PHRF1 gene as causal for SLE. A comprehensive literature review was conducted to enhance understanding of the functional roles and mechanisms of the identified genes in SLE development. Conclusions: The findings from the three MR methods exhibited overlapping genes with causal effects on SLE, demonstrating consistent results. However, each method also uncovered unique genes due to different modelling assumptions and technical factors, highlighting the complementary nature of the approaches. Importantly, MRAID demonstrated a reduced percentage of causal genes from the Major Histocompatibility complex (MHC) region on chromosome 6, indicating its potential in minimizing false positive findings. This study contributes to unraveling the mechanisms underlying SLE by employing advanced probabilistic MR methods to identify causal genes, thereby enhancing our understanding of SLE pathogenesis.

Cell fate determination factor Dachshund reprograms breast cancer stem cell function.

The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here, endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo, reduced mammosphere formation, and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely, lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2, Nanog, and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog, KLF4, and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo.

Common inversion polymorphism at 17q21.31 affects expression of multiple genes in tissue-specific manner.

BACKGROUND: Chromosome 17q21.31 contains a common inversion polymorphism of approximately 900 kb in populations with European ancestry. Two divergent MAPT haplotypes, H1 and H2 are described with distinct linkage disequilibrium patterns across the region reflecting the inversion status at this locus. The MAPT H1 haplotype has been associated with progressive supranuclear palsy, corticobasal degeneration, Parkinson's disease and Alzheimer's disease, while the H2 is linked to recurrent deletion events associated with the 17q21.31 microdeletion syndrome, a disease characterized by developmental delay and learning disability. RESULTS: In this study, we investigate the effect of the inversion on the expression of genes in the 17q21.31 region. We find the expression of several genes in and at the borders of the inversion to be affected; specific either to whole blood or different regions of the human brain. The H1 haplotype was found to be associated with an increased expression of LRRC37A4, PLEKH1M and MAPT. In contrast, a decreased expression of MGC57346, LRRC37A and CRHR1 was associated with H1. CONCLUSIONS: Studies thus far have focused on the expression of MAPT in the inversion region. However, our results show that the inversion status affects expression of other genes in the 17q21.31 region as well. Given the link between the inversion status and different neurological diseases, these genes may also be involved in disease pathology, possibly in a tissue-specific manner.

Epigenome mapping in normal and disease States.

Epigenomes are comprised, in part, of all genome-wide chromatin modifications, including DNA methylation and histone modifications. Unlike the genome, epigenomes are dynamic during development and differentiation to establish and maintain cell type-specific gene expression states that underlie cellular identity and function. Chromatin modifications are particularly labile, providing a mechanism for organisms to respond and adapt to environmental cues. Results from studies in animal models clearly demonstrate that epigenomic variability leads to phenotypic variability, including susceptibility to disease that is not recognized at the DNA sequence level. Thus, capturing epigenomic information is invaluable for comprehensively understanding development, differentiation, and disease. Herein, we provide a brief overview of epigenetic processes, how they are relevant to human health, and review studies using technologies that enable epigenome mapping. We conclude by describing feasible applications of epigenome mapping, focusing on epigenome-wide association studies (eGWAS), which have the potential to revolutionize current studies of human diseases and will likely promote the discovery of novel diagnostic, preventative, and treatment strategies.

Haplotype-specific chromatin looping reveals genetic interactions of regulatory regions modulating gene expression in 8p23.1.

A major goal of genetics research is to elucidate mechanisms explaining how genetic variation contributes to phenotypic variation. The genetic variants identified in genome-wide association studies (GWASs) generally explain only a small proportion of heritability of phenotypic traits, the so-called missing heritability problem. Recent evidence suggests that additional common variants beyond lead GWAS variants contribute to phenotypic variation; however, their mechanistic underpinnings generally remain unexplored. Herein, we undertake a study of haplotype-specific mechanisms of gene regulation at 8p23.1 in the human genome, a region associated with a number of complex diseases. The FAM167A-BLK locus in this region has been consistently found in the genome-wide association studies (GWASs) of systemic lupus erythematosus (SLE) in all major ancestries. Our haplotype-specific chromatin interaction (Hi-C) experiments, allele-specific enhancer activity measurements, genetic analyses, and epigenome editing experiments revealed that: 1) haplotype-specific long-range chromatin interactions are prevalent in 8p23.1; 2) BLK promoter and cis-regulatory elements cooperatively interact with haplotype-specificity; 3) genetic variants at distal regulatory elements are allele-specific modifiers of the promoter variants at FAM167A-BLK; 4) the BLK promoter interacts with and, as an enhancer-like promoter, regulates FAM167A expression and 5) local allele-specific enhancer activities are influenced by global haplotype structure due to chromatin looping. Although systemic lupus erythematosus causal variants at the FAM167A-BLK locus are thought to reside in the BLK promoter region, our results reveal that genetic variants at distal regulatory elements modulate promoter activity, changing BLK and FAM167A gene expression and disease risk. Our results suggest that global haplotype-specific 3-dimensional chromatin looping architecture has a strong influence on local allelic BLK and FAM167A gene expression, providing mechanistic details for how regional variants controlling the BLK promoter may influence disease risk.

The US Department of Veterans Affairs Science and Health Initiative to Combat Infectious and Emerging Life-Threatening Diseases (VA SHIELD): A Biorepository Addressing National Health Threats.

Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has demonstrated the need to share data and biospecimens broadly to optimize clinical outcomes for US military Veterans. Methods: In response, the Veterans Health Administration established VA SHIELD (Science and Health Initiative to Combat Infectious and Emerging Life-threatening Diseases), a comprehensive biorepository of specimens and clinical data from affected Veterans to advance research and public health surveillance and to improve diagnostic and therapeutic capabilities. Results: VA SHIELD now comprises 12 sites collecting de-identified biospecimens from US Veterans affected by SARS-CoV-2. In addition, 2 biorepository sites, a data processing center, and a coordinating center have been established under the direction of the Veterans Affairs Office of Research and Development. Phase 1 of VA SHIELD comprises 34 157 samples. Of these, 83.8% had positive tests for SARS-CoV-2, with the remainder serving as contemporaneous controls. The samples include nasopharyngeal swabs (57.9%), plasma (27.9%), and sera (12.5%). The associated clinical and demographic information available permits the evaluation of biological data in the context of patient demographics, clinical experience and management, vaccinations, and comorbidities. Conclusions: VA SHIELD is representative of US national diversity with a significant potential to impact national healthcare. VA SHIELD will support future projects designed to better understand SARS-CoV-2 and other emergent healthcare crises. To the extent possible, VA SHIELD will facilitate the discovery of diagnostics and therapeutics intended to diminish COVID-19 morbidity and mortality and to reduce the impact of new emerging threats to the health of US Veterans and populations worldwide.

Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq.

Whole-genome resequencing is still a costly method to detect genetic mutations that lead to altered forms of proteins and may be associated with disease development. Since the majority of disease-related single nucleotide variations (SNVs) are found in protein-coding regions, we propose to identify SNVs in expressed exons of the human genome using the recently developed RNA-Seq technique. We identify 12 176 and 10 621 SNVs, respectively, in Jurkat T cells and CD4(+) T cells from a healthy donor. Interestingly, our data show that one copy of the TAL-1 proto-oncogene has a point mutation in 3' UTR and only the mutant allele is expressed in Jurkat cells. We provide a comprehensive dataset for further understanding the cancer biology of Jurkat cells. Our results indicate that this is a cost-effective and efficient strategy to systematically identify SNVs in the expressed regions of the human genome.

Cyclin D1 integrates G9a-mediated histone methylation.

Lysine methylation of histones and non-histone substrates by the SET domain containing protein lysine methyltransferase (KMT) G9a/EHMT2 governs transcription contributing to apoptosis, aberrant cell growth, and pluripotency. The positioning of chromosomes within the nuclear three-dimensional space involves interactions between nuclear lamina (NL) and the lamina-associated domains (LAD). Contact of individual LADs with the NL are dependent upon H3K9me2 introduced by G9a. The mechanisms governing the recruitment of G9a to distinct subcellular sites, into chromatin or to LAD, is not known. The cyclin D1 gene product encodes the regulatory subunit of the holoenzyme that phosphorylates pRB and NRF1 thereby governing cell-cycle progression and mitochondrial metabolism. Herein, we show that cyclin D1 enhanced H3K9 dimethylation though direct association with G9a. Endogenous cyclin D1 was required for the recruitment of G9a to target genes in chromatin, for G9a-induced H3K9me2 of histones, and for NL-LAD interaction. The finding that cyclin D1 is required for recruitment of G9a to target genes in chromatin and for H3K9 dimethylation, identifies a novel mechanism coordinating protein methylation.

Prediction of regulatory elements in mammalian genomes using chromatin signatures.

BACKGROUND: Recent genomic scale survey of epigenetic states in the mammalian genomes has shown that promoters and enhancers are correlated with distinct chromatin signatures, providing a pragmatic way for systematic mapping of these regulatory elements in the genome. With rapid accumulation of chromatin modification profiles in the genome of various organisms and cell types, this chromatin based approach promises to uncover many new regulatory elements, but computational methods to effectively extract information from these datasets are still limited. RESULTS: We present here a supervised learning method to predict promoters and enhancers based on their unique chromatin modification signatures. We trained Hidden Markov models (HMMs) on the histone modification data for known promoters and enhancers, and then used the trained HMMs to identify promoter or enhancer like sequences in the human genome. Using a simulated annealing (SA) procedure, we searched for the most informative combination and the optimal window size of histone marks. CONCLUSION: Compared with the previous methods, the HMM method can capture the complex patterns of histone modifications particularly from the weak signals. Cross validation and scanning the ENCODE regions showed that our method outperforms the previous profile-based method in mapping promoters and enhancers. We also showed that including more histone marks can further boost the performance of our method. This observation suggests that the HMM is robust and is capable of integrating information from multiple histone marks. To further demonstrate the usefulness of our method, we applied it to analyzing genome wide ChIP-Seq data in three mouse cell lines and correctly predicted active and inactive promoters with positive predictive values of more than 80%. The software is available at http://http:/nash.ucsd.edu/chromatin.tar.gz.

The effect of inversion at 8p23 on BLK association with lupus in Caucasian population.

UNLABELLED: To explore the potential influence of the polymorphic 8p23.1 inversion on known autoimmune susceptibility risk at or near BLK locus, we validated a new bioinformatics method that utilizes SNP data to enable accurate, high-throughput genotyping of the 8p23.1 inversion in a Caucasian population. METHODS: Principal components analysis (PCA) was performed using markers inside the inversion territory followed by k-means cluster analyses on 7416 European derived and 267 HapMaP CEU and TSI samples. A logistic regression conditional analysis was performed. RESULTS: Three subgroups have been identified; inversion homozygous, heterozygous and non-inversion homozygous. The status of inversion was further validated using HapMap samples that had previously undergone Fluorescence in situ hybridization (FISH) assays with a concordance rate of above 98%. Conditional analyses based on the status of inversion were performed. We found that overall association signals in the BLK region remain significant after controlling for inversion status. The proportion of lupus cases and controls (cases/controls) in each subgroup was determined to be 0.97 for the inverted homozygous group (1067 cases and 1095 controls), 1.12 for the inverted heterozygous group (1935 cases 1717 controls) and 1.36 for non-inverted subgroups (924 cases and 678 controls). After calculating the linkage disequilibrium between inversion status and lupus risk haplotype we found that the lupus risk haplotype tends to reside on non-inversion background. As a result, a new association effect between non-inversion status and lupus phenotype has been identified ((p = 8.18×10(-7), OR = 1.18, 95%CI = 1.10-1.26). CONCLUSION: Our results demonstrate that both known lupus risk haplotype and inversion status act additively in the pathogenesis of lupus. Since inversion regulates expression of many genes in its territory, altered expression of other genes might also be involved in the development of lupus.

Critical role of histone demethylase Jmjd3 in the regulation of CD4+ T-cell differentiation.

Epigenetic factors have been implicated in the regulation of CD4(+) T-cell differentiation. Jmjd3 plays a role in many biological processes, but its in vivo function in T-cell differentiation remains unknown. Here we report that Jmjd3 ablation promotes CD4(+) T-cell differentiation into Th2 and Th17 cells in the small intestine and colon, and inhibits T-cell differentiation into Th1 cells under different cytokine-polarizing conditions and in a Th1-dependent colitis model. Jmjd3 deficiency also restrains the plasticity of the conversion of Th2, Th17 or Treg cells to Th1 cells. The skewing of T-cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines. H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors. Our results identify Jmjd3 as an epigenetic factor in T-cell differentiation via changes in histone methylation and target gene expression.