Elucidating the RNA-mediated mechanisms governing H3K9me3 deposition in fragile X syndrome

阐明脆性 X 综合征中 H3K9me3 沉积的 RNA 介导机制

• 批准号: 10537509
• 负责人: Thomas Ernst Malachowski
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-12-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537509
• 关键词: 5' Untranslated Regions; Affect; Aging; Automobile Driving; Back; Binding; Binding Proteins; Biological Assay; CGG repeat; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Methylation; Data; Deposition; Disease; Distal; Engineering; Enzymes; Excision; FMR1; FXTAS; Fragile X Syndrome; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genome; Genomics; Heritability; Heterochromatin; Hi-C; Human; Hybrids; Inclusion Bodies; Intellectual functioning disability; Knowledge; Length; Light; Link; Literature; Location; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methylation; Modeling; Mutation; Nerve Degeneration; Normal Range; Nuclear; Nuclear Inclusion; Nuclear Pore Complex; Nucleotides; Pathologic; Pharmacology; Phenotype; Plasmids; Proteins; RNA; RNA Binding; RNA Sequences; RNA-Binding Proteins; Reader; Reporting; Repression; Role; Seminal; Short Tandem Repeat; Structure; Synapses; Techniques; Testing; Transcript; Transcriptional Regulation; Transfection; Transgenes; Triplet Multiple Birth; Work; X Chromosome; autism spectrum disorder; autosome; base; chromatin modification; clinical phenotype; epigenetic silencing; experimental study; genome editing; genome-wide; histone modification; human disease; induced pluripotent stem cell; nerve stem cell; nervous system disorder; overexpression; promoter; recruit; stem cell differentiation

Project Summary Fragile X Syndrome (FXS) and Fragile X-associated/Tremor Ataxia Syndrome (FXTAS) are two FRAXopathies which are characterized by the unstable expansion of a CGG short tandem repeat (STR) located in the 5' untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. Expansion of the CGG tract from wild type length (WT, <55 CGG STR) to premutation (PM, 55-200 CGGs) results in a dramatic increase in FMR1 transcription with no noticeable elevation in levels of the protein it encodes (FMRP). Upon expansion to full mutation (FM, >200 CGGs), FMR1 is silenced via DNA methylation and, consequently, FMRP also reduces to baseline levels. Neither the removal of DNA methylation over the promoter and CGG tract nor transgene rescue cannot fully restore healthy phenotypes suggesting FMR1 and FMRP dysregulation are not the only disease drivers. The objective of my proposal is to investigate the RNA-mediated mechanisms driving disease-associated H3K9me3 deposition, trans interactions, and genome-wide STR instability in FXS. My central hypothesis is that FMR1 or FMR1-AS1 RNAs influence pathological heterochromatin deposition in a CGG length-dependent manner by toggling between sequestering key chromatin readers, writers, and erasers in inclusion bodies and forming toxic DNA:RNA structures locally and at distal loci. I have formulated my hypothesis based on our recent surprising observations that (1) Megabase-scale heterochromatin domains are acquired on autosomes and the X chromosome and spatially connect in ectopic inter-chromosomal interactions FM FXS in a manner that is dependent on the length of the CGG STR and (2) cut-back of the FM CGG to PM, or overexpression of PM-length CGG RNA, can reverse pathologic H3K9me3 deposition in FXS. Moreover, in established literature, PM-length CGG RNA forms nuclear inclusion bodies, whereas FM-length CGG can form toxic DNA-RNA R loops, but their interplay during FXS onset and progression and mechanistic connection to heterochromatin is unknown. I will test my hypothesis by employing state-of-the-art techniques like CUT&RUN, Hi-C, MapR, RADICL-seq, and ChlRP-MS in induced pluripotent stem cells differentiated to neural progenitors (iPSC-NPCs) across a range of CGG STR expansions and engineered cut-backs to shorter tracts. Upon successful completion of my experiments, I will elucidate the protein components of nuclear CGG RNA inclusion bodies, the location and sequence of RNA:DNA hybrids and R loops, heterochromatin placement, and genome folding features genome-wide as a function of CGG expansion and contraction. My work is significant because ii will elucidate the mechanisms by which FXS might progress via RNA-mediated heterochromatin in subnuclear bodies and established fundamental knowledge about the interplay between RNA-based inclusion bodies and RNA-DNA hybrids genome-wide in repeat expansion disorders. Our models of genome-wide heterochromatinization and gene silencing in FXS will also shed light on possible new mechanisms for H3K9me3 in other human conditions such as cancer, neurodegeneration, and aging.

项目摘要 脆性X综合征(FXS)和脆性X相关/震颤共济失调综合征(FXTAS)是两种脆性X综合征 其特征是位于5‘端的CGG短串联重复序列(STR)的不稳定扩张 脆性X智力低下1(FMR1)基因的非翻译区。野生CGG束的扩增 类型长度(WT，&lt；55 CGG STR)到预突变(PM，55-200 CGG)导致FMR1显著增加 转录，其编码的蛋白质水平没有明显的升高(FMRP)。在扩展到完全时 突变(FM，&gt；200 CGGs)，FMR1通过DNA甲基化沉默，因此FMRP也减少到 基线水平。既不去除启动子和CGG区域上的DNA甲基化，也不去除转基因 救援不能完全恢复健康的表型，这表明FMR1和FMRP失调并不是唯一的 疾病驱动因素。我的建议的目的是研究RNA介导的驱动机制 FXS中与疾病相关的H3K9me3沉积、反式相互作用和全基因组STR不稳定性。我的 中心假说是FMR1或FMR1-AS1 RNA影响小鼠的病理性异染色质沉积 通过在隔离关键染色质读取器、写入器和擦除器之间切换而依赖于CGG长度的方式 在包涵体中形成有毒的DNA：局部和远端位置的RNA结构。我已经制定了我的 基于我们最近令人惊讶的观察到的假说：(1)百万碱基规模的异染色质结构域 是在常染色体和X染色体上获得的，并在异位染色体间空间连接 以依赖于CGG STR的长度和(2)FM的缩进的方式相互作用FM FXS CGG到PM，或PM长度的CGG RNA的过表达，可以逆转FXS中病理性的H3K9me3沉积。 此外，在已建立的文献中，PM长度的CGG RNA形成核包涵体，而FM长度的CGG RNA形成核包涵体 CGG可形成毒性DNA-RNA R环，但它们在FXS发生发展过程中的相互作用及其机制 与异染色质的联系尚不清楚。我将使用最先进的技术来检验我的假设 与Cut&Run、Hi-C、MAPR、RADICL-seq和ChlRP-MS一样，诱导多能干细胞分化为 一系列CGG STR扩展和工程缩减为较短的神经前体(IPSC-NPC) 大片大片。在我的实验成功完成后，我将阐明核CGG的蛋白质成分 RNA包涵体，RNA的位置和序列：DNA杂交体和R环，异染色质 放置，基因组折叠以全基因组为特征，作为CGG扩张和收缩的函数。我的 这项工作意义重大，因为II将阐明FXS可能通过RNA介导的进展机制 亚核体中的异染色质，并建立了关于相互作用的基础知识 重复扩张性疾病全基因组的基于RNA的包涵体和RNA-DNA杂交体。我们的模特 FXS全基因组异染色质和基因沉默的研究也将揭示可能的新的 H3K9me3在其他人类疾病中的作用机制，如癌症、神经变性和衰老。