Single-cell dissection of chromatin architecture mechanisms connecting pathologic instability and transcriptional silencing

连接病理不稳定和转录沉默的染色质结构机制的单细胞解剖

• 批准号: 10684727
• 负责人: Rajan Jain
• 金额: $ 62.06万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684727
• 关键词: 3-Dimensional; Affect; Amyotrophic Lateral Sclerosis; Architecture; Biological Assay; Biological Models; Carbon; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; CpG Islands; DNMT3a; Data; Disease; Dissection; Engineering; Epigenetic Process; Etiology; Event; Exhibits; Exons; FMR1; Fibrosis; Fragile X Syndrome; Friedreich Ataxia; Functional disorder; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genome; Genome Mappings; Genome engineering; Genomic Instability; Genomics; Heterochromatin; Hi-C; Higher Order Chromatin Structure; Human; Human Genome; Hybrids; Hydrophobicity; Hypertrophy; Image; Individual; Intercistronic Region; Introns; Knowledge; Length; Link; Maps; Mediating; Mutation; Neurons; Nuclear; Onset of illness; Pathogenicity; Pathologic; Pathology; Patients; Pattern; Peptides; Peripheral; Physiological; Population; Positioning Attribute; RNA; Radial; Reporting; Repression; Resolution; Short Tandem Repeat; Somatic Cell; Technology; Testing; Tissues; Trinucleotide Repeat Expansion; Variant; Work; body system; cell type; cellular imaging; chromosome conformation capture; cohort; density; derepression; frataxin; gallium arsenide; genome-wide; genome-wide analysis; genomic data; genomic locus; human disease; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; insight; mortality; novel therapeutics; prototype; single molecule; stem

Short tandem repeat regions (STR) are distributed evenly across the human genome, and recent genome-wide studies have demonstrated that STRs are polymorphic across individuals and linked to gene expression levels. STR instability at key genomic loci has been causally linked to disease pathophysiology in a range of expansion disorders. We recently demonstrated that nearly all disease-associated STRs co-localize with boundaries demarcating topologically associated domains (TADs). Moreover, we have observed that pathologic STR instability and transcriptional silencing can destroy the associated boundary and shift genomic loci to the nuclear periphery. These results now open critical unanswered questions regarding whether and how STR expansion and pathologic alterations in gene expression are functionally linked to boundary integrity and radial positioning. Here, we focus on the prototypic repeat expansion disorder Friedreich’s ataxia (FRDA) in which expansion of a GAA STR in the first intron of the FRATAXIN (FXN) gene results in cardiac and neuronal pathology. The cardiac pathology, specifically hypertrophy, fibrosis, and occasional dilation of the ventricle, is the etiology of significant FRDA mortality. GAA expansion is associated with the silencing of FXN transcription and a repositioning of the locus to the nuclear periphery. However, it remains unclear if the change in genome folding, radial positioning, or reduced expression drives STR expansion or vice versa. A major technical barrier contributing to this knowledge gap is that STR instability and genome folding are classically evaluated in bulk populations, however they exhibit tremendous variation across individual somatic cells of the same subtype and among cell types within a pathologically affected tissue. Here, we seek to decipher the causal link among STR instability, transcription, radial positioning, and genome folding. Our central hypothesis is that disruption of long-range loops is the initial event triggered by STR expansion leading to a cascade of heterochromatin spreading, silencing, and loss of radial positioning. We will test our hypothesis by generating genome-wide, single-cell maps of chromatin accessibility, expression, and the repressive H3K9me3 heterochromatin mark in GAA-expanded and control iPS cells and iPS-derived cardiomyocytes. We will integrate genomics data with single-cell sequential Oligopaints/OligoSTORM imaging of TADs and local chromatin structure, as well as single molecule RNA FISH for FXN expression. We will implement multiple genome engineering strategies, including dCas9-VP64 FXN activation and dCas9-CTCF loop re-engineering in FRDA GAA-iPS cells, and dCas9-Krab-Dnmt3a FXN silencing and dCas9-Krab CTCF-mediated loop disruption in healthy iPS cells. We will assay the effect of genome engineering approaches on TADs, radial positioning, STR length, and FXN expression in single cells. Successful completion of the proposed work will shed light on the pathophysiological mechanisms underlying repeat expansion disorders by deciphering the cause-and-effect relationships among genome folding, radial positioning, transcription, and STR expansion.

短串联重复区域（STR）均匀分布在人类基因组中，最近在全基因组范围内 研究表明，STR 在个体之间具有多态性，并且与基因表达水平相关。 关键基因组位点的 STR 不稳定性与一系列扩展中的疾病病理生理学存在因果关系 失调。我们最近证明，几乎所有与疾病相关的 STR 都与边界共定位 划分拓扑关联域（TAD）。此外，我们观察到病理性 STR 不稳定性和转录沉默可以破坏相关边界并将基因组位点转移到核 周边。这些结果现在提出了关于 STR 是否以及如何扩展的关键未解答问题 基因表达的病理改变在功能上与边界完整性和径向定位相关。 在这里，我们重点关注原型重复扩张障碍弗里德赖希共济失调（FRDA），其中重复扩张 FRATAXIN (FXN) 基因第一个内含子中的 GAA STR 会导致心脏和神经元病理学。心脏的 病理学，特别是肥厚、纤维化和偶尔的心室扩张，是重要的病因。 FRDA 死亡率。 GAA 扩增与 FXN 转录的沉默和 FXN 转录的重新定位有关。 位于核外围。然而，目前尚不清楚基因组折叠、径向定位的变化是否 或减少的表达驱动 STR 扩展，反之亦然。造成这种情况的一个主要技术障碍 知识差距是 STR 不稳定性和基因组折叠通常在大量群体中进行评估，然而 它们在相同亚型的个体体细胞和细胞类型之间表现出巨大的差异 在受病理影响的组织内。在这里，我们试图破译 STR 不稳定性之间的因果关系， 转录、径向定位和基因组折叠。我们的中心假设是远程循环的破坏 是 STR 扩增触发的初始事件，导致异染色质扩散、沉默和级联反应 失去径向定位。我们将通过生成全基因组、单细胞染色质图来检验我们的假设 GAA 扩增和对照 iPS 中的可及性、表达和抑制性 H3K9me3 异染色质标记 细胞和 iPS 衍生的心肌细胞。我们将把基因组学数据与单细胞序列分析相结合 TAD 和局部染色质结构的 Oligopaints/OligoSTORM 成像，以及单分子 RNA FISH 用于 FXN 表达。我们将实施多种基因组工程策略，包括 dCas9-VP64 FXN FRDA GAA-iPS 细胞和 dCas9-Krab-Dnmt3a FXN 中的激活和 dCas9-CTCF 环重新设计 健康 iPS 细胞中的沉默和 dCas9-Krab CTCF 介导的环破坏。我们将测试其效果 单细胞中 TAD、径向定位、STR 长度和 FXN 表达的基因组工程方法。 成功完成拟议的工作将揭示潜在的病理生理机制 通过破译基因组折叠、放射状之间的因果关系来重复扩张障碍 定位、转录和 STR 扩展。