A functional mechanism for a non-coding variant near AGTR2 associated with risk for preterm birth

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Epigenetic and Transcriptional Dysregulation in T cells of Patients with Atopic Dermatitis

Rationale: Atopic dermatitis (AD) is linked to genetic and environmental risk factors. The effect of these factors on molecular and transcriptional events is not well understood. Immunologically, AD involves skin barrier defects and CD4+ T cells that produce inflammatory cytokines and amplify epidermal dysfunction Our objective was to investigate epigenetic mechanisms that may account for genetic susceptibility in CD4+ T cells. Methods: We measured chromatin accessibility (ATAC-seq), NFKB1 binding (ChIP-seq), and gene expression (RNA-seq) in anti-CD3/CD28 stimulated CD4+ T cells from 6 subjects with active moderate-to-severe AD and 6 age-matched non-allergic controls. Results: AD genetic risk loci were enriched for open chromatin regions in stimulated CD4+ T cells. The majority of ATAC-seq peaks were shared between matched AD-control pairs, consistent with those sections of chromatin being equally available. In contrast, NFKB DNA binding motifs were enriched in AD-dependent open chromatin. NFKB1 ChIP-seq identified genomic regions that were more strongly bound in AD cases, more strongly bound in controls, or shared between cases and controls. Chromatin that was strongly accessible and bound by NFKB1 in AD was enriched for AD genetic risk variants. Using whole genome sequencing data, we identified genotype-dependent accessible chromatin at AD risk loci corresponding to 32 genes with genotype-dependent expression in stimulated CD4+ T cells. Conclusions: The response of CD4+ T cells to stimulation is AD-specific and results in differential chromatin accessibility and transcription factor binding. These differences in transcriptional regulation result in epigenetic and transcriptional dysregulation in CD4+ T cells of patients with AD

Epigenetic and transcriptional dysregulation in CD4+ T cells in patients with atopic dermatitis

Atopic dermatitis (AD) is one of the most common skin disorders among children. Disease etiology involves genetic and environmental factors, with 29 independent AD risk loci enriched for risk allele-dependent gene expression in the skin and CD4+ T cell compartments. We investigated the potential epigenetic mechanisms responsible for the genetic susceptibility of CD4+ T cells. To understand the differences in gene regulatory activity in peripheral blood T cells in AD, we measured chromatin accessibility (an assay based on transposase-accessible chromatin sequencing, ATAC-seq), nuclear factor kappa B subunit 1 (NFKB1) binding (chromatin immunoprecipitation with sequencing, ChIP-seq), and gene expression levels (RNA-seq) in stimulated CD4+ T cells from subjects with active moderate-to-severe AD, as well as in age-matched non-allergic controls. Open chromatin regions in stimulated CD4+ T cells were highly enriched for AD genetic risk variants, with almost half of the AD risk loci overlapping AD-dependent ATAC-seq peaks. AD-specific open chromatin regions were strongly enriched for NF-κB DNA-binding motifs. ChIP-seq identified hundreds of NFKB1-occupied genomic loci that were AD- or control-specific. As expected, the AD-specific ChIP-seq peaks were strongly enriched for NF-κB DNA-binding motifs. Surprisingly, control-specific NFKB1 ChIP-seq peaks were not enriched for NFKB1 motifs, but instead contained motifs for other classes of human transcription factors, suggesting a mechanism involving altered indirect NFKB1 binding. Using DNA sequencing data, we identified 63 instances of altered genotype-dependent chromatin accessibility at 36 AD risk variant loci (30% of AD risk loci) that might lead to genotype-dependent gene expression. Based on these findings, we propose that CD4+ T cells respond to stimulation in an AD-specific manner, resulting in disease- and genotype-dependent chromatin accessibility alterations involving NFKB1 binding

Human cytomegalovirus infection coopts chromatin organization to diminish TEAD1 transcription factor activity

Human cytomegalovirus (HCMV) infects up to 80% of the world’s population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hr after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon 6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV’s established roles in these processes

Enrichment of Epstein Barr Virus in patients with Multiple Sclerosis

Abstract Multiple sclerosis (MS) is an autoimmune disease of the central nervous system affecting more than 2 million people worldwide and has been linked with Epstein-Barr Virus (EBV) infection. Previous studies by the Kottyan and Weirauch labs show that the EBNA2 protein of both EBV type 1 and 2 binds unique MS risk loci potentially causing changes at the transcriptional level contributing to MS disease progress. We hypothesize that type 1 and type 2 EBV will be detectable in the MS patient samples, and we aim to quantify EBV enrichment and identify EBV type in MS patient-derived blood samples. Analysis by qPCR showed that 30% of the control population (n=10) and 66% of the MS patient population (n=41) had detectable levels of EBV whereas the MS patients had a significantly higher frequency of EBV presence (X2 test p=0.0389). Of the samples that tested positive for EBV, 66% showed presence of type 1 EBV while 33% were unknown without a significant difference between MS patients and control group (X2 test p&amp;gt;0.9999). Due to issues with the limit of detection, droplet digital PCR was assessed for higher sensitivity compared to qPCR, but was found to have a comparable limit of detection to qPCR. To overcome the challenge of detecting low copy number viruses, we look toward targeted DNA capture and next generation sequencing to improve the sensitivity of EBV detection and specificity of EBV typing. Understanding the distribution of EBV types among MS patients would elucidate the precise role of EBV in MS development, potentially leading to improved treatment and prevention methods. Supported by grants from NIH grants R01 HG010730, R01 NS099068, R01 GM055479, and U01 AI130830 to MTW; R01 DK107502, R01 AI148276, U19 AI070235, U01 HG011172, and P30 AR070549 to LCK; R01 AR073228, R01 AI024717, and CCHMC ARC Award 53632 to MTW and LCK.</jats:p

A Lupus-Associated Variant in IRF7 amplifies IFN-α Production in response to TLR stimulation

Abstract Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by chronic immune activation, loss of self-tolerance and organ destruction. More than 90 genetic risk loci are implicated in the etiology of SLE, yet the mechanisms connecting these loci to disease risk are not well understood. We focus on a SLE-associated coding variant (rs1131665) at the IRF7 risk locus that results in an arginine (R, non-risk) to glutamine (Q, risk) amino acid change in the auto inhibitory domain of interferon regulatory factor-7 (IRF7), a key transcriptional regulator of the expression of and response to type I interferon (IFN). The majority of SLE patients exhibit elevated levels of circulating IFN, a feature that correlates with disease activity. We generated cell lines expressing the risk and non-risk variants of IRF7 and isolated plasmacytoid dendritic cells from mice with CRISPR-mediated introduction of a mouse homologue of the risk variant of IRF7. In both experimental systems, the rs1131665 lupus risk variant drives increased toll like receptor-7 (TLR-7) ligand-induced IFN-α expression at both the mRNA and protein levels compared to the non-risk variant of IRF7. Additionally, we performed a covariance analysis that predicts that amino acid residues at the 84, 140, 142 positions interact with the variant position in the three-dimensional structure of IRF7. Mutation of the amino acid at the 84 position from a glutamic acid (E) residue to a threonine (T) residue results in amplified IFN production in response to TLR-7 stimulation. Our findings suggest a genotype-dependent mechanism for lupus risk variants in IRF7 in promoting exaggerated IFN production in SLE. These experiments would not have been possible with out NIH NIAMS R01 AR073228. </jats:p

B-cell lymphoma 6 protein modulates function of human tonsillar innate lymphoid cells

Abstract Innate lymphoid cells (ILC) play a fundamental role in mucosal homeostasis and immunity via expression of cytokines such as IL-22, IL-17A and IFN-γ. However, the transcriptional network that controls ILC functional identity is incompletely defined. Previously, we demonstrated that BCL6 plays a key role in transcriptional regulation in mouse intestinal ILC1 and ILC3. Here, we performed in vitro cultures of ILCs isolated from human tonsil in the presence of cytokines that promote ILC1 or ILC3 in order to assess the role of BCL6 in functional plasticity of ILCs. ILCs were treated with IL-2 and IL-12/IL-1β or IL-23/IL-1β in the presence or absence of the BCL6 inhibitor FX-1. In the context of IL-12/IL-1β ILC1-promoting culture, FX-1 inhibition of BCL6 reduced expression of Tbet and IFN-γ. In contrast, BCL6 inhibition in the context of IL-23/IL-1β ILC3-inducing conditions had no effect on Tbet, IFN-γ, or RORγt. In either context, FX-1 resulted in reduced IL-22 but increased IL-17A expression in comparison to vehicle treated cultures. Thus, our data emphasizes the role of BCL6 as a regulator of human tonsil ILC functional identity.</jats:p