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: 10447121
• 负责人: Jennifer Elizabeth Phillips-Cremins
• 金额: $ 47.14万
• 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/10447121
• 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综合征中3D基因组错误折叠与转录沉默的联系 项目总结/摘要 超过30种遗传性神经系统疾病，包括脆性X综合征（FXS），亨廷顿病， 肌萎缩侧索硬化症和弗里德赖希共济失调是由重复DNA的不稳定扩增引起的 短串联重复序列（STR）。在FXS中，STR扩增超过临界长度阈值 导致致病性STR基因沉默，导致严重的神经精神症状。一个 增加了对STR扩增如何有助于 转录沉默将有助于开发由以下因素驱动的重复扩增疾病的治疗方法： 病理性基因表达改变。本建议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、结构蛋白占有率和基因 FXS中的表达沉默。最近，我们发现几乎所有的疾病相关STR（daSTR）都是 位于划分3D染色质结构域的边界。daSTR特异性定位于超高密度CpG 岛边界，表明它们可能是表观遗传不稳定性或STR拓扑破坏的热点 扩张.与这一想法一致，我们发现FXS患者表现出严重的边界破坏和丧失， CTCF占有率与FMR 1沉默程度相关。由于边界中断， FMR 1基因经历了从下游结构域的拓扑开关， 增强子的上游结构域缺乏调控元件。基于这些发现，我们假设 3D基因组错误连接与FMR 1通过基因拓扑开关的病理沉默有因果关系， 从活跃的监管环境到沉默的监管环境。我们将用三个具体目标来检验我们的假设。一是 将剖析3D基因组错误折叠和FMR 1沉默之间的因果关系。我们将分析 突变FMR 1边界处的CTCF和YY 1结合位点后的结构和FMR 1表达， CRISPR-Cas9和在健康细胞中用dCas 9-KRAB阻遏物异位沉默FMR 1。二是 创建CTCF/YY 1结合、H3 K9 me 3/H3 K27 me 3抑制结构域和DNA的全基因组图谱 在具有一系列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序列长度如何影响远端表观遗传修饰以破坏染色质结构， FMR 1表达。第三，我们将通过合成的结构重建FXS患者细胞中的基因组拓扑结构， 蛋白质和特定结构基序的活性去甲基化。我们将决定3D技术 单独的基因组工程或与线性表观基因组工程组合将重新编程抑制性的 染色质标记和去抑制FMR 1。我们的工作意义重大，因为它将为3D技术的发展提供新的思路。 表观遗传机制出错，可以在获得和进展过程中修复。 神经发育和神经变性疾病状态。