RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells

项目摘要 10 - 11易位（泰特）蛋白通过调节DNA甲基化状态发挥关键作用。 氧化甲基胞嘧啶（5 mC），生成5-羟甲基胞嘧啶（5 hmC）， 表观遗传标记并参与主动去甲基化。与其他TET家族成员不同，TET 2不 含有一个DNA结合域，它是如何被募集到染色质中的仍不清楚。在此我们发现 TET 2由RNA结合蛋白Paraspeckle组分1（PSPC 1）通过转录方式募集。 活性基因座，包括内源性逆转录病毒（ERV），其长末端重复序列（LTR）已被增选 由哺乳动物基因组作为阶段和组织特异性转录调控模块。我们发现PSPC 1 TET 2和TET 2通过两种转录抑制， 组蛋白脱乙酰酶和ERV RNA通过5 hmC修饰的转录后不稳定。我们 这些发现为转录活性ERVs作为RNA的特异性对接位点的功能作用提供了证据 表观遗传调节和基因调控。该项目的目标是研究TET 2是否以及如何可能 通过RNA和RNA结合蛋白靶向染色质，导致RNA羟甲基化介导的 MERVL及其相关2C基因的调节对于ESC的多能性，而不是散发性 ESCs中的全能2C群体。我们假设TET 2的RNA依赖性染色质靶向是关键的， 直接RNA去甲基化和MERVL转录物的降解，这可能导致 是操纵干细胞和发育潜能的有效工具。以下三个目标将检验这一点 假设和探索干细胞潜能由TbPs、RBP和ERVs之间密切相互作用控制。第一章 建立TET 2在RNA修饰中的RBP依赖性功能; 2）探索TET 2在RNA修饰中的新功能。 通过调节用于ESC的多能性的RNA靶标的依赖性染色质靶向;以及3）操纵茎干细胞 RNA靶向CRISPR/RCas 9用于靶向MERVL RNA修饰的细胞和发育效力 胚胎干细胞和发育中的胚胎。第一个目标是建立TET 2在hm 5C修饰中的新功能， MERVL以PSPC 1依赖的方式转录。第二个目标将剖析hm 5C的后果 MERVL转录本的修饰及hm 5C介导MERVL的分子机制 降解第三个目标是探索通过直接MERVL RNA操纵干细胞和发育潜能 改性总的来说，这三个目标将提供相当多的新的见解RNA依赖性 TET 2的染色质靶向用于MERVL控制干细胞效力的转录后机制。的 该项目是非常重要的，因为它有望建立一个新的范式，在理解ERV法规，泰特 在RNA修饰和全能性中发挥作用。

PROJECT SUMMARY Ten-eleven translocation (TET) proteins play key roles in regulating the methylation status of DNA through oxidizing methylcytosines (5mC), generating 5-hydroxymethylcytosines (5hmC) that can both serve as stable epigenetic marks and participate in active demethylation. Unlike the other TET-family members, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We find that PSPC1 and TET2 contribute to ERV and ERV-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of ERV RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation. The goal of this project is to study whether and how TET2 may be targeted to chromatin via RNAs and RNA-binding proteins leading to RNA hydroxymethylation-mediated regulation of MERVL and their associated 2C genes for pluripotency of ESCs, as opposed to the sporadic totipotent 2C populations in ESCs. We hypothesize that RNA-dependent chromatin targeting of TET2 is critical for direct RNA demethylation and degradation of MERVL transcripts, which may lead to development of efficient tools in manipulating stem cell and developmental potency. The following three aims will test this hypothesis and explore stem cell potency control by an intimate interplay among TETs, RBPs, and ERVs. 1) Establish RBP-dependent functions of TET2 in RNA modification; 2) Explore novel TET2 functions in RNA- dependent chromatin targeting by regulating RNA targets for pluripotency of ESCs; and 3) Manipulate stem cell and developmental potency with RNA targeting CRISPR/RCas9 for targeted MERVL RNA modification in ESCs and developing embryos. The first aim will establish novel functions of TET2 in hm5C modification of MERVL transcripts in a PSPC1-dependent manner. The second aim will dissect the consequence of hm5C modification of MERVL transcripts and the molecular mechanism underlying hm5C-mediated MERVL degradation. The third aim will explore manipulating stem cell and development potency by direct MERVL RNA modification. Taken together, these three aims will provide considerable novel insight into RNA-dependent chromatin targeting of TET2 for the posttranscriptional mechanism of MERVL control in stem cell potency. The project is highly significant as it is expected to establish a new paradigm in understanding ERV regulation, TET functions in RNA modification, and totipotency.