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: 10116703
• 负责人: Rajan Jain
• 金额: $ 64.32万
• 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/10116703
• 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 engineering; Genomic Instability; Genomics; Heterochromatin; Higher Order Chromatin Structure; Human; Human Genome; Hybrids; Hydrophobicity; Hypertrophy; Image; Individual; Intercistronic Region; Introns; Knowledge; Length; Light; 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; base; body system; cell type; cellular imaging; chromosome conformation capture; cohort; density; genome-wide; genome-wide analysis; genomic data; genomic locus; human disease; induced pluripotent stem cell; insight; mortality; novel therapeutics; single molecule; single-cell RNA sequencing; 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.

短串联重复序列（STR）在人类基因组中均匀分布，最近在全基因组范围内发现了STR基因座。 研究表明，STR在个体之间具有多态性，并与基因表达水平相关。 在一系列扩展中，关键基因组位点的STR不稳定性与疾病病理生理学有因果关系 紊乱我们最近证明，几乎所有疾病相关的STR与边界共定位 划分拓扑关联域（TADs）。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的边界，并将基因组位点转移到细胞核 外围。这些结果现在开启了关于STR扩增是否以及如何扩增的关键问题 基因表达的病理改变与边界完整性和径向定位功能相关。 在这里，我们专注于原型重复扩增障碍弗里德赖希共济失调（FRDA），其中扩增的一个 FRATAXIN（FXN）基因第一内含子中的GAA STR导致心脏和神经元病理学。心脏 病理学，特别是心室肥大、纤维化和偶尔的心室扩张，是显著的 FRDA死亡率。GAA扩增与FXN转录的沉默和GAA基因的重新定位有关。 到核外围。然而，目前还不清楚基因组折叠，径向定位， 或减少的表达驱动STR扩增，反之亦然。一个主要的技术障碍促成了这一点 知识差距是STR不稳定性和基因组折叠是在大量人群中进行经典评估的，然而， 它们在相同亚型的个体体细胞之间和细胞类型之间表现出巨大的差异 在一个受病理影响的组织中。在这里，我们试图破译STR不稳定性， 转录、径向定位和基因组折叠。我们的中心假设是， 是由STR扩增引发的初始事件，导致异染色质扩散、沉默和 失去径向定位。我们将通过生成全基因组的染色质单细胞图谱来验证我们的假设 可及性、表达和抑制性H3 K9 me 3异染色质标记在GAA扩增和对照iPS中 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， TADs和局部染色质结构的Oligopaints/OligoSTORM成像，以及单分子RNA FISH 对于FXN表达式。我们将实施多种基因组工程策略，包括dCas 9-VP 64 FXN 在FRDA GAA-iPS细胞中的激活和dCas 9-CTCF环再工程化，以及dCas 9-Krab-Dnmt 3a FXN 在健康iPS细胞中的dCas 9-Krab CTCF介导的环沉默和环破坏。我们将检验 关于单细胞中TAD、径向定位、STR长度和FXN表达的基因组工程方法。 成功地完成拟议的工作将阐明的病理生理机制， 通过破译基因组折叠、放射状和放射状之间的因果关系， 定位、转录和STR扩增。