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

基本信息

批准号：
10634553
负责人：
Jennifer Elizabeth Phillips-Cremins
金额：
$ 43.39万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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/10634553
关键词：
3-Dimensional Affect Amyotrophic Lateral Sclerosis Anxiety Architecture B-Lymphocytes Binding Binding Proteins Binding Sites Biological Assay CCCTC-binding factor CGG repeat 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 Link Lysine Maps Methyltransferase Modeling Modification Molecular Mutate Mutation Neurodegenerative Disorders Neurodevelopmental Disorder Pathogenicity Pathologic Patients Pattern Proteins Publishing Quantitative Reverse Transcriptase PCR Regulatory Element Reporting Role Sampling Seizures Short Tandem Repeat Structure Symptoms Testing Translations Work YY1 Transcription Factor brain tissue chromatin modification chromosome conformation capture data integration demethylation density derepression epigenome genome-wide insight nervous system disorder neuropsychiatric symptom programs protein function recruit repaired respiratory social deficits stem transcriptome sequencing

项目摘要

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：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项目概要/摘要超过 30 种遗传性神经系统疾病，包括脆性 X 综合征 (FXS)、亨廷顿氏舞蹈病、肌萎缩侧索硬化症和弗里德赖希共济失调是由重复 DNA 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引起含有 STR 的基因的致病性沉默，导致严重的神经精神症状。一个加深对控制 STR 扩展如何促进的分子机制的了解转录沉默将有助于开发针对重复扩增障碍的治疗方法病理性基因表达变化。该提案的目的是了解高阶之间的联系染色质结构、抑制性染色质修饰、结构蛋白占据和基因 FXS 中的表达沉默。最近，我们发现几乎所有疾病相关 STR（daSTR）都是位于划分 3D 染色质域的边界处。 daSTR 专门定位于超高密度 CpG 岛屿边界，表明它们可能是 STR 上表观遗传不稳定或拓扑破坏的热点扩张。与这个想法一致，我们发现 FXS 患者表现出严重的边界破坏和丧失 CTCF 占用率与 FMR1 沉默程度相关。由于边界破坏， FMR1 基因从下游结构域经历了拓扑转换，该下游结构域包含许多假定的上游结构域的增强子缺乏调控元件。根据这些发现，我们假设 3D 基因组错误接线通过基因的拓扑开关与 FMR1 的病理性沉默存在因果关系从活跃的监管格局到沉默的监管格局。我们将通过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 沉默之间的因果关系。我们将化验突变 FMR1 边界处的 CTCF 和 YY1 结合位点后的结构和 FMR1 表达健康细胞中的 CRISPR-Cas9 和使用 dCas9-KRAB 阻遏物异位沉默 FMR1。其次，我们将创建 CTCF/YY1 结合、H3K9me3/H3K27me3 抑制域和 DNA 的全基因组图谱具有一系列 CGG daSTR 长度的 FXS 样品中的甲基化。通过计算整合这些数据，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 患者细胞中的基因组拓扑蛋白质和特定结构图案的主动去甲基化。我们将确定 3D 的程度单独的基因组工程或与线性表观基因组工程相结合将重新编程抑制性染色质标记并去抑制 FMR1。我们的工作意义重大，因为它将为 3D 技术提供新的视角表观遗传机制出了差错，可以在获得和进展过程中修复神经发育和神经退行性疾病状态。