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: 10268225
• 负责人: Rajan Jain
• 金额: $ 63.66万
• 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/10268225
• 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; Hi-C; 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.

短串联重复序列区域(STR)在人类基因组中均匀分布，最近在全基因组范围内 研究表明，短串联重复序列在个体中是多态的，并与基因表达水平有关。 在一系列的扩展中，关键基因组座位上的STR不稳定性与疾病的病理生理相关 精神错乱。我们最近证明，几乎所有与疾病相关的STR都与边界共同定位 划分拓扑相关结构域(TADS)。此外，我们观察到病理性STR 不稳定和转录沉默可以破坏相关的边界，并将基因组基因座转移到核 外围设备。这些结果现在打开了关于STR是否以及如何扩展的关键悬而未决的问题 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，我们关注典型的重复扩张性疾病Friedreich‘s共济失调(FRDA)，在这种疾病中， Frataxin(FXN)基因第一内含子中的GAA STR导致心脏和神经元病理。心脏 病理，特别是肥大、纤维化和偶尔的脑室扩张，是显著的病因。 FRDA死亡率。GAA的扩展与FXN转录的沉默和FXN基因的重新定位有关 到核外围的轨迹。然而，目前尚不清楚基因组折叠、径向定位、 或减少表达推动STR扩展，反之亦然。导致这一点的一个主要技术障碍 然而，知识差距是，STR不稳定性和基因组折叠在大量群体中得到了经典的评估 它们在同一亚型的单个体细胞和不同细胞类型之间表现出巨大的差异 在病理性的组织内。在这里，我们试图破译STR不稳定性之间的因果联系， 转录、径向定位和基因组折叠。我们的中心假设是远程环路的中断 是由STR扩展触发的初始事件，导致异染色质扩散、沉默和 径向定位丢失。我们将通过生成全基因组的单细胞染色质图谱来检验我们的假设 GAA扩张型和对照iPS的可及性、表达和抑制性H3K9me3异染色质标记 细胞和iPS来源的心肌细胞。我们将把基因组数据与单细胞序列相结合 TADS和局部染色质结构以及单分子RNA FISH的寡核苷酸/寡核苷酸成像 用于FXN表达式。我们将实施多种基因组工程策略，包括dCas9-VP64 FXN FRDA GAA-iPS细胞中的激活和dCas9-CTCF环重组，以及dCas9-Krab-DNMT3A FXN 沉默和dCas9-Krab CTCF介导的健康iPS细胞环路中断。我们将分析其效果。 单细胞中TADS、径向定位、STR长度和FXN表达的基因组工程方法。 这项拟议工作的成功完成将有助于揭示 通过破译基因组折叠、放射之间的因果关系来实现重复扩张障碍 定位、转录和STR扩展。