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

项目摘要 脆性X综合征（FXS）和脆性X相关/震颤性共济失调综合征（FXTAS）是两种脆性X病 其特征在于位于5'端的CGG短串联重复序列（STR）的不稳定扩增， 脆性X智力迟钝1（FMR 1）基因的非翻译区。CGG道从野生型的扩展 型长度（WT，<55 CGG STR）到前突变（PM，55-200 CGG）导致FMR 1显著增加 转录，其编码的蛋白质（FMRP）水平没有明显升高。在扩展到完全时 突变（FM，>200 CGG），FMR 1通过DNA甲基化沉默，因此，FMRP也减少到 基线水平。无论是启动子和CGG区的DNA甲基化的去除，还是转基因， 救援不能完全恢复健康的表型，表明FMR 1和FMRP失调不是唯一的 疾病司机我的建议的目的是研究RNA介导的机制， 疾病相关的H3 K9 me 3沉积，反式相互作用和FXS中全基因组STR不稳定性。我 中心假设是FMR 1或FMR 1-AS 1 RNA影响病理性异染色质沉积， CGG长度依赖的方式，通过在隔离关键染色质阅读器、写入器和擦除器之间切换 在包涵体中，并在局部和远端位点形成毒性DNA：RNA结构。我已经制定了我的 基于我们最近令人惊讶的观察，（1）Megabase规模的异染色质结构域 在常染色体和X染色体上获得，并在异位染色体间空间连接 以依赖于CGG STR的长度的方式与FM FXS相互作用，以及（2）FM FXS的削减， CGG到PM，或PM长度的CGG RNA的过表达，可以逆转FXS中的病理性H3 K9 me 3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可以形成毒性DNA-RNA R环，但它们在FXS发作和进展过程中的相互作用和机制可能与FXS的发生有关。 与异染色质的联系尚不清楚。我会用最先进的技术来验证我的假设 如CUT&RUN、Hi-C、MapR、RADICL-seq和ChIRP-MS在诱导多能干细胞分化为 神经祖细胞（iPSC-NPC）在一系列CGG STR扩增和工程化削减到更短 小册子。成功完成实验后，我将阐明核CGG的蛋白质组分 RNA包涵体，RNA的位置和序列：DNA杂合体和R环，异染色质 放置，和基因组折叠的特征基因组范围内的CGG的扩展和收缩的函数。我 这项工作是有意义的，因为它将阐明FXS可能通过RNA介导的 异染色质在亚核体和建立的基础知识之间的相互作用 重复扩增疾病中全基因组范围的RNA包涵体和RNA-DNA杂合体我们的模型 FXS中全基因组异染色质化和基因沉默的研究也将揭示可能的新的 H3 K9 me 3在其他人类疾病中的作用机制，如癌症，神经变性和衰老。

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.