DNA Elements Underlying Celiac and Crohn's Susceptibility

项目总结/摘要 针对乳糜泻和克罗恩病的强有力的全基因组关联研究（GWAS）已经确定， 许多非HLA易感基因座。遗传精细定位和表达数量性状基因座（eQTL）可以 缩小了基因可能解释GWAS信号的范围，但即使在这些方法之后， 导致核苷酸变化的原因往往是未知的。这种理解上的差距是实现目标的障碍 预防和治疗。正如我们先前的工作所开发的，绘制单倍型依赖的等位基因特异性CpG - 与给定疾病相关的细胞中的甲基化（hap-ASM）和甲基化数量性状基因座（mQTL），和 然后将这些表观遗传图谱与GWAS数据重叠，可以帮助我们深入研究DNA调控序列， GWAS信号背后的因果关系。我们的假设是，这种结合了遗传和表观遗传的图谱 策略，其次是功能测定，将能够确定调控DNA序列，有助于腹腔 疾病和克罗恩病的易感性和发病机制。首先，我们将进行CD 4+的甲基测序， CD 8 + T细胞和外周血单核细胞，以在全基因组范围内绘制hap-ASM。这些数据将精确定位- ASM差异甲基化区域（DMR）与GWAS峰在相同的单元型区块中， 克罗恩病由于hap-ASM通常反映等位基因特异性转录因子结合位点（TFBS）或绝缘子 因此，我们将使用一种独立的方法交叉验证我们的发现，转座酶测定- 可重复染色质测序（ATAC-Seq）。在我们的第二个目标中，我们将对hap-ASM进行排名和优先排序 DMR基于等位基因不对称性、ATAC-Seq重叠和连锁不平衡（LD）的强度 与GWAS峰值SNP，并进行高通量靶向亚硫酸氢盐测序，以精细映射排名靠前的 DMR，无论是在血液来源的细胞和粘膜T细胞从腹腔和克罗恩病患者。我们的第三个目标， 为了明确测试特定TFBS在DMR中的功能作用，我们将使用CRISPR删除这些TFBS。 Jurkat细胞和正常T细胞中的DNA序列。我们将评分缺失对局部甲基化的影响， 模式和mRNA水平。这些数据将澄清我们的基本认识 乳糜泻和克罗恩病的易感性和发病机制。

Project Summary/Abstract Well powered genome-wide association studies (GWAS) for celiac disease and Crohn's disease have identified numerous non-HLA susceptibility loci. Genetic fine-mapping and expression quantitative trait loci (eQTL) can narrow in on which genes probably account for the GWAS signals, but even after these approaches the locations of the causal nucleotide changes often remain unknown. This gap in understanding is a roadblock to targeted prevention and therapy. As developed in our prior work, mapping haplotype-dependent allele-specific CpG methylation (hap-ASM) and methylation quantitative trait loci (mQTLs) in cells relevant to a given disease, and then overlapping these epigenetic maps with GWAS data, can help to hone in on the DNA regulatory sequences that causally underlie the GWAS signals. Our hypothesis is that this combined genetic-epigenetic mapping strategy, followed by functional assays, will be able to identify regulatory DNA sequences that contribute to celiac disease and Crohn's disease susceptibility and pathogenesis. First, we will carry out Methyl-Seq of CD4+ and CD8+ T cells and peripheral blood monocytes, to map hap-ASM genome-wide. These data will pinpoint hap- ASM differentially methylated regions (DMRs) in the same haplotype blocks as GWAS peaks for celiac and Crohn's. Since hap-ASM often reflects allele-specific transcription factor binding site (TFBS) or insulator occupancies, we will cross-validate our findings using an independent method, Assay for Transposase- Accessible Chromatin Sequencing (ATAC-Seq). In our second aim, we will rank and prioritize the hap-ASM DMRs based on the strength of the allelic asymmetries, ATAC-Seq overlaps, and linkage disequilibrium (LD) with GWAS peak SNPs, and perform high-throughput targeted bisulfite sequencing to fine-map the top-ranked DMRs, both in the blood-derived cells and in mucosal T cells from celiac and Crohn's patients. In our third aim, to definitively test the functional roles of specific TFBS in the DMRs, we will use CRISPR to delete these sequences in Jurkat cells and in normal T cells. We will score effects of the deletions on local methylation patterns and mRNA levels of each of the nearby genes. These data will clarify our fundamental understanding of susceptibility and pathogenesis of celiac disease and Crohn's disease.