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: 10709509
• 负责人: Thomas Ernst Malachowski
• 金额: $ 4.77万
• 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/10709509
• 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; Disease Progression; 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; Phenotype; Plasmids; Printing; Proteins; RNA; RNA Binding; RNA Sequences; RNA-Binding Proteins; Reader; Reporting; Role; Seminal; Short Tandem Repeat; Structure; Synapses; Techniques; Testing; Transcript; Transcriptional Regulation; Transfection; Transgenes; Tremor/Ataxia Syndrome; Triplet Multiple Birth; Work; X Chromosome; autism spectrum disorder; autosome; chromatin modification; clinical phenotype; derepression; epigenetic silencing; experimental study; genome editing; genome-wide; histone modification; human disease; induced pluripotent stem cell; nerve stem cell; nervous system disorder; overexpression; pharmacologic; 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 脆弱的X综合征（FXS）和脆弱的X相关/震颤共济失调综合征（FXTA）是两个胸膜病变 其特征是位于5'中的CGG短串联重复（Str）的不稳定膨胀 脆弱X智力低下1（FMR1）基因的未翻译区域。从野外扩展CGG道 类型长度（wt，<55 CGG str）到预先享用（PM，55-200 CGGS）导致FMR1急剧增加 它编码的蛋白质水平（FMRP）没有明显升高的转录。扩展到满 突变（FM，> 200 CGGS），通过DNA甲基化将FMR1沉默，因此，FMRP也会减少到 基线水平。在启动子和CGG道上也没有去除DNA甲基化或转基因 救援不能完全恢复健康的表型，表明FMR1和FMRP失调并不是唯一的 疾病驱动因素。我建议的目的是研究RNA介导的机制驱动 FXS中与疾病相关的H3K9ME3沉积，反式相互作用和全基因组的StR不稳定性。我的 中心假设是FMR1或FMR1-AS1 RNA会影响病理异染色质沉积 CGG长度依赖性方式通过在隔离的关键染色质读取器，作家和橡皮擦之间切换 在包含体和形成有毒的DNA：RNA结构局部和远端基因座。我已经制定了我的 假设基于我们最近令人惊讶的观察结果，即（1）巨型尺度异染色质结构域 在常染色体和X染色体上获取，并在异位间染色体中连接 相互作用FM FXS的方式取决于CGG str的长度和（2）FM的缩短 CGG至PM或PM长度CGG RNA的过表达可以逆转FXS中的病理H3K9me3沉积。 此外，在已建立的文献中，PM长度CGG RNA形成核包容物，而FM长度 CGG可以形成有毒的DNA-RNA R环，但在FX发作和进展和机械方面的相互作用 与异染色质的连接未知。我将通过采用最先进的技术来检验我的假设 在诱导的多能干细胞中，例如切割和运行，HI-C，MAPR，RADICL-SEQ和CHLRP-MS， 横跨一系列CGG STR扩展和工程剪切的神经祖细胞（IPSC-NPC） 区域。成功完成实验后，我将阐明核CGG的蛋白质成分 RNA包含体，RNA的位置和序列：DNA杂交和R环，异染色质 放置和基因组折叠具有全基因组作为CGG膨胀和收缩的函数。我的 工作很重要，因为II将阐明FXS可能通过RNA介导的机制 亚核体中的异染色质，并建立了有关相互作用的基本知识 基于RNA的包含体和RNA-DNA杂交基因组在重复扩张障碍中。我们的模型 FXS中全基因组的异染色和基因沉默也将揭示可能的新 H3K9me3在其他人类条件下的机制，例如癌症，神经退行性和衰老。