Connecting 3D genome misfolding to transcriptional silencing in fragile X syndrome

将 3D 基因组错误折叠与脆性 X 综合征中的转录沉默联系起来

• 批准号: 10208688
• 负责人: Jennifer Elizabeth Phillips-Cremins
• 金额: $ 61.91万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10208688
• 关键词: 3-Dimensional; Affect; Amyotrophic Lateral Sclerosis; Anxiety; Architecture; B-Lymphocytes; Binding; Binding Proteins; Binding Sites; Biological Assay; Brain; CCCTC-binding factor; CRISPR/Cas technology; Carbon; Cataract; Cell Line; Cells; Chromatin; Chromatin Structure; Clinical; Complex; CpG Islands; DNA Methylation; DNA Sequence; Data; Defect; Dimensions; Disease; Distal; Engineering; Enhancers; Epigenetic Process; Exhibits; FMR1; Fragile X Syndrome; Friedreich Ataxia; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genome; Genome engineering; Goals; Histone H3; Huntington Disease; Hyperactivity; Inherited; Length; Light; Link; Lysine; Maps; Methylation; Methyltransferase; Modeling; Modification; Molecular; Mutate; Mutation; Neurodegenerative Disorders; Pathogenicity; Pathologic; Patients; Pattern; Proteins; Publishing; Quantitative Reverse Transcriptase PCR; Regulatory Element; Reporting; Role; Sampling; Short Tandem Repeat; Structure; Symptoms; Testing; Translations; Work; YY1 Transcription Factor; base; chromatin modification; chromosome conformation capture; demethylation; density; epigenome; genome-wide; induced pluripotent stem cell; insight; nervous system disorder; neuropsychiatric symptom; protein function; recruit; repaired; respiratory; social deficits; transcriptome sequencing

Title: Connecting 3D genome misfolding to transcriptional silencing in fragile X syndrome Project Summary/Abstract More than 30 inherited neurological disorders, including fragile X syndrome (FXS), Huntington's disease, amyotrophic lateral sclerosis, and Friedreich's ataxia, are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). In FXS, STR expansion above a critical length threshold causes pathogenic silencing of the STR-containing gene, resulting in severe neuropsychiatric symptoms. An increased understanding of the molecular mechanisms governing how STR expansion contributes to transcriptional silencing would facilitate efforts to develop treatments for repeat expansion disorders driven by pathologic gene expression changes. The objective of this proposal is to understand the link among higher-order chromatin architecture, repressive chromatin modifications, architectural protein occupancy, and gene expression silencing in FXS. Recently, we discovered that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic instability or topological disruption upon STR expansion. Consistent with this idea, we found that FXS patients exhibit severe boundary disruption and loss of CTCF occupancy in a manner that correlates with the degree of FMR1 silencing. Due to the boundary disruption, the FMR1 gene undergoes a topological switch from the downstream domain containing numerous putative enhancers to the upstream domain devoid of regulatory elements. Based on these findings, we hypothesized that 3D genome miswiring is causally linked to pathologic silencing of FMR1 via the gene's topological switch from an active to silenced regulatory landscape. We will test our hypothesis with three Specific Aims. First, we will dissect the cause-and-effect link between 3D genome misfolding and FMR1 silencing. We will assay architecture and FMR1 expression after mutating CTCF and YY1 binding sites at the FMR1 boundary with CRISPR-Cas9 and ectopically silencing FMR1 with the dCas9-KRAB repressor in healthy cells. Second, we will create genome-wide maps of CTCF/YY1 binding, H3K9me3/H3K27me3 repressive domains, and DNA methylation in FXS samples with a range of CGG daSTR lengths. By computationally integrating this data, we will elucidate how STR tract length affects distal epigenetic modifications to disrupt chromatin architecture and FMR1 expression. Third, we will re-engineer genome topology in FXS patient cells via synthetic architectural proteins and active de-methylation of specific architectural motifs. We will determine the degree to which 3D genome engineering alone or in combination with linear Epigenome engineering will reprogram repressive chromatin marks and de-repress FMR1. Our work is significant because it will shed new light into how 3-D epigenetic mechanisms go awry and can be repaired during the acquisition and progression of neurodevelopmental and neurodegenerative disease states.

标题：脆性X综合征患者3D基因组错误折叠与转录沉默之间的联系 项目摘要/摘要 30多种遗传性神经疾病，包括脆性X综合征(FXS)、亨廷顿病、 肌萎缩侧索硬化症和Friedreich共济失调是由重复DNA的不稳定扩张引起的 称为短串联重复序列(STR)的序列。在FXS中，STR扩展超过临界长度阈值 导致含有STR基因的病理性沉默，导致严重的神经精神症状。一个 增加了对STR扩展如何有助于 转录沉默将有助于开发针对由以下因素驱动的重复扩增障碍的治疗方法 病理基因表达发生变化。这项建议的目的是了解更高级别的 染色质结构、抑制性染色质修饰、结构蛋白占有率和基因 FXS中的表达沉默。最近，我们发现几乎所有与疾病相关的STR(DaSTR)都是 位于界定3D染色质结构域的边界上。DaSTR专门本地化到超高密度CpG 岛屿边界，表明它们可能是表观遗传不稳定或STR拓扑破坏的热点 扩张。与这一观点一致，我们发现FXS患者表现出严重的边界破坏和 CTCF占用的方式与FMR1沉默的程度相关。由于边界中断， FMR1基因从下游结构域经历了一次拓扑切换，该结构域包含许多可能的 上游领域缺乏监管要素的增强剂。基于这些发现，我们假设 3D基因组错误连接通过基因的拓扑开关与FMR1的病理性沉默有因果关系 从积极到沉默的监管格局。我们将用三个具体目标来检验我们的假设。首先，我们 将剖析3D基因组错误折叠和FMR1沉默之间的因果联系。我们将进行化验 FMR1边界CTCF和YY1结合位点突变后的构筑和FMR1表达 CRISPR-Cas9和用dCas9-KRAb抑制子异位沉默健康细胞中的FMR1。第二，我们将 创建CTCF/YY1结合、H3K9me3/H3K27me3抑制结构域和DNA的全基因组图谱 具有CGG daSTR长度范围的FXS样本中的甲基化。通过计算集成这些数据，我们 将阐明STR束长度如何影响远端表观遗传修饰以破坏染色质结构和 FMR1表达。第三，我们将通过合成架构重新设计FXS患者细胞的基因组拓扑 蛋白质和特定结构基序的活性去甲基化。我们将确定3D在多大程度上 基因组工程单独或与线性表观基因组工程结合将重新编程抑制 染色质标记和去抑制FMR1。我们的工作意义重大，因为它将为3D 表观遗传机制会出错，并可以在获得和进展期间修复 神经发育和神经退行性疾病状态。