Project 3

项目3

• 批准号: 10204936
• 负责人: MARK A. ATKINSON
• 金额: $ 26.82万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10204936
• 关键词: Address; Alleles; Alternative Splicing; B-Lymphocyte Subsets; Biological Assay; Biological Models; CRISPR/Cas technology; Candidate Disease Gene; Cell model; Cells; Cellular Assay; Chromatin; Clinical; DNA Methylation; Data; Development; Disease; Elements; Enhancers; Event; Exons; Gene Expression Profile; Genes; Genetic Enhancer Element; Genetic Polymorphism; Genetic Transcription; Genotype; Immune; Immunogenetics; Individual; Insulin-Dependent Diabetes Mellitus; Introns; Jurkat Cells; Knock-out; Knowledge; Lymphocyte; Lymphocyte Subset; Measures; Molecular Probes; Nucleic Acid Regulatory Sequences; Phenotype; Population; Process; Protein Isoforms; RNA; RNA Splicing; Regulation; Regulatory T-Lymphocyte; Risk; Sampling; Spliced Genes; Structure; T-Lymphocyte; Testing; Therapeutic Intervention; Tissues; Transcript; Untranslated RNA; case control; causal variant; cell type; diabetes pathogenesis; diabetes risk; differential expression; disorder prevention; exon skipping; genetic variant; genome wide association study; immune function; improved; innovation; insight; member; novel; novel strategies; overexpression; preventive intervention; protein function; risk variant; transcription factor; transcriptome; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Classical immunogenetics, linkage studies, and genome-wide association studies (GWAS) have identified a set of ~50 chromosomal regions that individually and significantly contribute to the risk for type 1 diabetes (T1D). Knowledge of the genes in these regions provides an opportunity to probe the molecular underpinnings of the disease, potentially facilitating improved disease prediction and/or novel strategies for prevention or therapeutic intervention. ImmunoChip fine mapping in T1D indicates that the majority of credible causative genetic variants for T1D overlap with tissue specific enhancer (but not promoter) elements, emphasizing the need for a context dependent understanding of the mode of action for genes contributing to T1D pathogenesis (Projects 1 and 2) as well as through the study of relevant cell types; an emerging notion for this P01 renewal (and the focus of Project 2). To address this need, Project 3 has, in preliminary studies, processed more than 800 RNA-Seq samples; transcriptionally profiling T and B lymphocyte subsets in both T1D cases and controls. These efforts have yielded three key observations: 1) Genes located in T1D-associated chromosomal regions are significantly enriched for cell-type specific alternative splicing events. These alternative splicing events include intron retention or exon skipping activities that are likely to have significant effects on the expression and function of the proteins encoded by these genes. 2) Identification of non-coding polymorphisms in regulatory regions that are recognized as credible causative variants for T1D and are significantly associated with these alternative splicing events in a highly tissue-specific manner. 3) Genes located in chromosomal regions associated with T1D that display correlated expression in lymphocytes, allowing them be clustered into co-expression modules that provide insights into regulation and function. Our preliminary findings suggest that transcript isoforms and, by implication, the protein isoforms that they encode, are altered in T1D through genetically regulated shifts in exon usage in specific cell types. To gain a comprehensive understanding of the impact of alternative splicing and alternative transcript usage on T1D risk, and to identify and characterize disease relevant transcripts and isoforms, we propose to apply long read sequencing to RNA from selected lymphocyte populations in T1D and jointly analyze short and long read data to obtain reliable, cell type specific, information on transcript structure and usage. We will then characterize the downstream effects of alternative splicing and transcript isoform usage on a key T1D gene, IKZF4, which anchors a co-expression module in regulatory T cells. Finally, we will characterize the impact of T1D causative genetic variants on chromatin accessibility and DNA methylation at the T1D candidate genes they flank. These studies are highly synergistic with Projects 1 and 2 where those efforts propose to utilize clinical samples and isogenic cellular models to examine key genotype:phenotype associations in specific cell subsets.

项目摘要/摘要 经典的免疫遗传学，连锁研究和全基因组关联研究（GWAS）已经确定了一个集合 在〜50个染色体区域中，它们单独且显着导致1型糖尿病的风险（T1D）。 这些区域中基因的知识提供了探测分子基础的机会 疾病，有可能促进改善疾病预测和/或预防或治疗的新策略 干涉。 T1D中的免疫光绘制映射表明大多数可靠的病因遗传变异 对于T1D与组织特异性增强子（但不是启动子）元素重叠，强调了上下文 依赖对导致T1D发病机理的基因作用方式的理解（项目1和2） 以及通过研究相关细胞类型的研究；此P01更新的新兴概念（以及 项目2）。为了满足这一需求，项目3在初步研究中已处理了800多个RNA-Seq 样品；在T1D病例和对照组中，转录分析T和B淋巴细胞亚群。这些努力 已经产生了三个关键观察：1）位于T1D相关的染色体区域中的基因显着 富含细胞类型的特定替代剪接事件。这些替代剪接事件包括内含子 保留或外显子跳过活动可能会对的表达和功能产生重大影响 这些基因编码的蛋白质。 2）在调节区域鉴定非编码多态性 被认为是T1D的可信因果变体，并且与这些替代方案显着相关 以高度组织特异性的方式进行剪接事件。 3）位于染色体区域的基因 T1D显示在淋巴细胞中相关表达的T1D，将它们聚集到共表达模块中 这提供了对调节和功能的见解。我们的初步发现表明，转录本同工型和， 暗示，它们编码的蛋白质同工型通过遗传调节的转移而改变了T1D 特定细胞类型中的外显子使用情况。要全面了解替代剪接的影响 以及关于T1D风险的替代笔录使用，并确定和表征与疾病相关的成绩单和 同工型，我们建议将长读测序应用于T1D中选定的淋巴细胞种群的RNA 共同分析简短和长读数据以获取可靠的细胞类型，特定于成绩单结构的信息 和用法。然后，我们将表征替代剪接和转录本的下游效应 在关键的T1D基因IKZF4上，该基因锚定调节T细胞中的共表达模块。最后，我们会的 表征T1D致病遗传变异对染色质可及性和DNA甲基化的影响 他们侧翼的T1D候选基因。这些研究与项目1和2相关的高度协同作用 建议利用临床样品和等源性细胞模型检查关键基因型：表型关联 在特定的细胞子集中。