Functional Mechanisms of T1D Risk Variants and their Target Genes using 3D Epigenomics and Single Cell Approaches

使用 3D 表观基因组学和单细胞方法研究 T1D 风险变异及其靶基因的功能机制

基本信息

批准号：
10398021
负责人：
Struan F A Grant
金额：
$ 97.74万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10398021
关键词：
3-Dimensional ATAC-seq Alleles Antibodies Architecture Autoimmune Diseases B-Lymphocytes Bar Codes Bioinformatics CD19 gene CD8-Positive T-Lymphocytes CRISPR/Cas technology Cell Lineage Cells Cellular Indexing of Transcriptomes and Epitopes by Sequencing Chromatin Coupled DNA Dependence Disease susceptibility Enhancers Epitopes Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Genetic Risk Genetic Transcription Genetic Variation Genome Genotype Genotype-Tissue Expression Project Helper-Inducer T-Lymphocyte Human Human Genome Immune Immunophenotyping Individual Inflammatory Insulin Insulin-Dependent Diabetes Mellitus Life Link Maps Mediating Memory Meta-Analysis Morbidity - disease rate Natural Killer Cells Nature Nucleic Acid Regulatory Sequences Peripheral Blood Mononuclear Cell Phenotype Population Predisposition Regulator Genes Regulatory T-Lymphocyte Research Resolution Rest Role Sampling Single Nucleotide Polymorphism Site Specificity Surface Susceptibility Gene Symptoms T cell differentiation T-Lymphocyte Testing Therapeutic Intervention Tissues Tonsil Validation Variant base biobank cell type diabetes mellitus genetics diabetes risk epigenomics gene function gene interaction genome editing genome wide association study genome-wide immunoregulation indexing innovation molecular targeted therapies monocyte mortality novel prevent promoter risk variant single-cell RNA sequencing transcriptome transcriptome sequencing whole genome

项目摘要

ABSTRACT Type 1 diabetes (T1D) is an autoimmune disease, whose symptoms and complications result in increased morbidity, mortality, and life-long dependence on insulin. The Type 1 Diabetes Genetics Consortium (T1DGC, Stephen Rich, PI) conducted the largest T1D GWAS meta-analysis and identified over 40 T1D risk loci. The T1DGC refined the T1D risk variants by fine mapping with the ImmunoChip and constructing sets of 99% credible single nucleotide polymorphisms (SNPs) within each locus. Bioinformatic analyses of T1D-associated credible SNPs discovered enrichment in regions involved in gene regulation of immune-relevant cell types (CD4 and CD8 T cells, CD19 B cells). Despite these discoveries on the genetic basis of T1D, the mechanisms that define how T1D-associated SNPs contribute to disease susceptibility remain unclear. Here, we propose a powerful and innovative approach to define function of the T1D-associated SNPs and identify their target, causal effector genes through integrated analyses of T1D-relevant tissues. We will (Aim 1) generate an integrated 3D map of the gene regulatory architecture of T1D susceptibility by conducting ATAC-seq, RNA-seq and whole- genome promoter-focused Capture-C analysis on purified human cell populations; (Aim 2) perform single-cell immunophenotyping and eQTL analysis of T1DGC samples using CITE-seq, a single-cell RNA-seq approach guided by DNA-barcoded antibodies against lineage markers to characterize the immune cell subsets identified in Aim 1, allowing us to focus on SNPs that are regulatory and likely contribute to T1D risk; and (Aim 3) conduct functional validation of T1D SNP-gene regulatory effects using CRISPR/Cas9 genome editing in human immune cells to directly test whether these SNPs reside in regions that are required for T1D gene expression using Cas9-mediated editing. Our outstanding research team provides complementary and synergistic approaches to understanding the function and gene targets that modulate genetic risk of T1D. Together, we will establish the mechanisms underlying contribution of T1D-associated SNPs to inflammatory gene regulation and, by revealing the SNP-enhancer-gene link in 3D, are poised to discover novel molecular targets for therapeutic intervention to treat or prevent T1D.

抽象的 1型糖尿病（T1D）是一种自身免疫性疾病，其症状和并发症导致增加发病率，死亡率和终身对胰岛素的依赖。 1型糖尿病遗传联盟（T1DGC，史蒂芬·里奇（Stephen Rich），PI）进行了最大的T1D GWAS荟萃分析，并确定了40多个T1D风险基因座。这 T1DGC通过使用免疫芯片和99％可信的构造组进行精细映射来完善T1D风险变体每个基因座中的单核苷酸多态性（SNP）。 T1D相关可信的生物信息学分析 SNP在与免疫相关细胞类型的基因调节的区域中发现了富集（CD4和CD8 T细胞，CD19 B细胞）。尽管在T1D的遗传基础上发现了这些发现，但定义的机制 T1D相关的SNP如何促进疾病易感性尚不清楚。在这里，我们建议强大而创新的方法来定义与T1D相关SNP的功能并确定其目标通过对T1D相关组织的综合分析进行效应基因。我们将（AIM 1）产生一个集成的3D 通过进行ATAC-SEQ，RNA-SEQ和全部 - 以基因组启动子为中心的捕获-C分析对纯化的人类细胞群体；（AIM 2）执行单细胞使用Cite-Seq（单细胞RNA-Seq）对T1DGC样品的免疫表型和EQTL分析通过针对谱系标记的DNA钢筋抗体引导的方法，以表征免疫细胞亚群在AIM 1中确定，使我们能够专注于监管的SNP，并可能导致T1D风险；和（目标 3）使用CRISPR/CAS9基因组编辑对T1D SNP基因调节效应进行功能验证在人类免疫细胞中，直接测试这些SNP是否驻留在T1D基因所需的区域使用CAS9介导的编辑表达。我们杰出的研究团队提供互补的和了解调节T1D遗传风险的功能和基因靶标的协同方法。我们将共同建立与T1D相关SNP对炎症的贡献的基础机制基因调节并通过揭示3D中的SNP-增强剂 - 基因链接，可以发现新的分子治疗或预防T1D的治疗干预靶标。