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.

短串联重复区域（STR）均匀地分布在人类基因组中，最近全基因组分布 研究表明，STR在个体之间是多态性的，并且与基因表达水平相关。 关键基因组基因局的StR不稳定性已随随便就与疾病的病理生理学联系在一起 疾病。我们最近证明，几乎所有与疾病相关的STR与边界共定位 划界拓扑相关的域（TAD）。此外，我们已经观察到病理学 不稳定性和转录沉默可以破坏相关的边界，并将基因组局部转移到核 周边。现在，这些结果对是否以及如何扩展开辟了关键的未解决的问题 基因表达的病理改变在功能上与边界完整性和径向定位有关。 在这里，我们专注于原型重复扩张障碍弗里德雷希（Friedreich）的共济失调（FRDA），其中膨胀 Frataxin（FXN）基因的第一个内含子中的GAA STR会导致心脏和神经元病理。心脏 病理学，特别是肥大，纤维化和心室偶尔扩张，是显着的病因 FRDA死亡率。 GAA扩展与FXN转录的沉默和一个存储库有关 核周围的基因座。但是，尚不清楚基因组折叠，径向定位的变化是否变化 或降低表达驱动器str膨胀，反之亦然。为此做出贡献的主要技术障碍 知识差距是，在批量种群中对Str不稳定性和基因组折叠进行了经典评估，但是 他们暴露了同一亚型和细胞类型之间各个体细胞的巨大变化 在病理影响的组织中。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 是由STR膨胀引起的初始事件，导致级联异染色质扩散，沉默和 径向定位的丧失。我们将通过生成染色质的全基因组的单细胞图来检验我们的假设 GAA扩展和控制IPS中的可访问性，表达和反射性H3K9me3异染色质标记 细胞和IPS衍生的心肌细胞。我们将将基因组数据与单细胞顺序集成 TAD和局部染色质结构以及单分子RNA FISH的寡聚体/寡聚型成像 用于FXN表达。我们将实施多种基因组工程策略，包括DCAS9-VP64 FXN 激活和DCAS9-CTCF循环在FRDA GAA-ips细胞中重新设计，DCAS9-KRAB-DNMT3A FXN 在健康的IPS细胞中，沉默和DCAS9-KRAB CTCF介导的循环破坏。我们将主张 基因组工程在单个细胞中的TAD，径向定位，STR长度和FXN表达的方法。 成功完成拟议的工作将阐明依据的病理生理机制 通过破译基因组折叠之间的因果关系来重复扩展障碍 定位，转录和STR扩展。