hnRNPA2/B1协同Tet2在脆性X相关的震颤/共济失调综合征中异常调控5-羟甲基化胞嘧啶水平引起浦肯野细胞变性的机制

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a heritable neurodegenerative disorder in fragile X premutation carriers with FMR1 alleles containing 55-200 CGG repeats. An RNA toxic gain-of-function model was known as the primary disease mechanism. Overexpression of riboRNA containing ninety CGG repeats (rCGG) alone in Drosophila retina is sufficient to cause neurodegeneration. Murine PC-specific expression of a transgene carrying ninety CGG repeats is sufficient to induce FXTAS phenotypes outside the context of the FMR1 gene. Our previous studies identified hnRNP A2/B1 and Pur α as rCGG specific RNA binding proteins, which could be sequestered from their normal function by rCGG binding. Although impressive progress has been made, how the sequestration of RNA-binding proteins (RBPs) affects normal PC functions, thereby contributing to FXTAS pathogenesis, particularly on transcription levels, remains unknown. Cytosine covalent modifications epigenetically influence transcriptional states and ultimately cellular identity, function and fate. 5-hydroxymethylcytosine (5hmC) converted from 5-methylcytosine by the ten-eleven translocation (TET) family of proteins has been found recently to play key roles in neuronal functions. 5hmC levels are approximately ten times higher in CNS such as PCs than in other somatic tissues. A global reduction of 5hmC was found in cerebella of rCGG mice. Although sequestration of RBPs serves as an attractive model for the FXTAS onset, the link to transcriptome alteration is unclear. Our preliminary data found that PC-specific hnRNP A2/B1 overexpression ameliorated rCGG-mediated PC apoptosis. IP-western showed an explicit interaction between hnRNP A2/B1 with C-terminal enzymatic domain of Tet2. Electrophoretic mobility shift assay demonstrated a dose-dependent interaction of hnRNP A2/B1 with single strand DNA. By performing FLAG ChIP-Seq from cerebella of mice specifically expressing FLAG-hnRNP A2/B1 in PCs, we generated hnRNP A2/B1 genome-wide binding sites specifically in PCs and confirmed its DNA binding ability in vivo. Genome-wide hnRNP A2/B1 ChIP-Seq in mouse embryonic stem cells revealed strong overlap with published Tet2 binding sites. Utilizing PC-specific hnRNP A2/B1 and Tet2 knockout mice combined with various technologies especially high-throughput sequencing, we will systematically investigate the epigenetic roles of hnRNP A2/B1 coordinating with Tet2 to regulate genome-wide 5hmC level and transcriptome in PC and reveal its contribution to PC degeneration in FXTAS. Our study will unveil the novel function of hnRNP A2/B1 in 5hmC regulation. We will clarify the pathogenic mechanism that hnRNP A2/B1 sequestration by rCGG repeats blocks its interaction with Tet2 then alters cytosine modification dynamics and the transcriptome in PCs, which could contribute to the PC degeneration and FXTAS onset. Our study will also facilitate the understanding of ectopic changes at the transcription level in normal PC function and PC-related diseases featuring ataxia.

FXTAS是脆性X致病基因FMR1前突变rCGG重复序列的RNA毒性导致的疾病。rCGG小鼠PC中全基因组范围的5hmC显著减少。我们鉴定hnRNPA2/B1是rCGG特异结合蛋白。前期工作中，我们发现过表达hnRNPA2/B1改善rCGG小鼠PC变性,hnRNPA2/B1与5hmC通路关键酶Tet2特异相互作用,体内外证实hnRNPA2/B1的DNA结合能力,得到hnRNPA2/B1在PC中的靶DNA，发现hnRNPA2/B1与Tet2共享大量靶基因。本课题将利用PC特异性敲除hnRNPA2/B1与Tet2小鼠结合多种高通量测序等方法，解析hnRNPA2/B1调控5hmC的新功能，阐明hnRNPA2/B1被rCGG过度捕获阻碍其与Tet2相互作用，从而改变PC中基因组5hmC动态修饰和转录组水平，引起PC变性的FXTAS致病机制。该研究还有助于揭示正常PC功能和PC变性的分子调控网络。

脆性X相关的震颤共济失调综合征(Fragile X associated Tremor/Ataxia Syndrome，FXTAS)是脆性X前突变导致的一类以目的性的震颤和共济失调，伴随着外周神经病变，植物神经功能紊乱，以及记忆和执行功能缺欠的渐进性认知水平下降等一系列表型的神经系统疾病。我们鉴定出FXTAS患者中rCGG的靶RNA结合蛋白hnRNPA2/B1，还发现hnRNPA2/B1与Tet2之间存在相互作用，但它们在FXTAS中的致病机制尚未明确。我们首先明确了hnRNPA2/B1对bubble structured DNA和5mC修饰DNA的强烈结合偏好性，明确其结合靶DNA的模式偏好和表观遗传作用。在hnRNPA2/B1特异性浦肯野细胞(Purkinje cell, PC)敲除小鼠中，获得hnRNPA2/B1在转录水平和转录后水平具体调控的基因，发现hnRNPA2/B1通过表观遗传学调控作用于其靶基因的表达。我们还利用PC条件性敲除Tet2小鼠研究在PC中hnRNPA2/B1与Tet2的相互作用，证明hnRNPA2/B1与靶DNA体内结合依赖于Tet2。我们筛选出5hmC与rCGG小鼠PC中转录水平显著变化且受hnRNPA2/B1和Tet2共同调控的基因共34个，并在FXTAS果蝇模型中验证了7个靶基因对神经变性的有效性。本研究阐明了hnRNPA2/B1协同Tet2在FXTAS的PC中5hmC水平降低最终导致PC变性的致病机制，对明确FXTAS的致病机制和后续治疗具有重大意义。