RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control

项目总结 Tet蛋白在DNA甲基化状态的调节中起关键作用 氧化甲基胞嘧啶(5mC)，生成5-羟甲基胞嘧啶(5hmC)，两者都可以作为稳定的 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，TET2不 含有一个DNA结合域，目前尚不清楚它是如何被招募到染色质的。在这里，我们展示了 TET2由RNA结合蛋白Paraspeckle Component 1(PSPC1)通过转录方式招募 活性基因座，包括其长末端重复序列(LTRs)已被增选的内源性逆转录病毒(ERV) 由哺乳动物基因组作为阶段和组织特定的转录调节模块。我们发现PSPC1 和TET2通过转录抑制参与ERV和ERV相关基因的调控 组蛋白脱乙酰酶与ERV RNA通过5hmC修饰的转录后失稳。我们的 研究结果为转录活性ERV作为RNA的特定对接位点的功能作用提供了证据 表观遗传调控和基因调控。这个项目的目标是研究TET2是否以及如何 通过RNA和RNA结合蛋白靶向染色质，导致RNA羟甲基化 MERVL及其相关2C基因对胚胎干细胞多能性的调控 胚胎干细胞中的全能2C群体。我们假设依赖于RNA的染色质靶向TET2是关键 直接RNA去甲基化和MERVL转录本的降解，这可能导致 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 假设并探索通过TETs、RBPs和ERV之间的密切相互作用来控制干细胞的效力。1) 建立RBP依赖的TET2在RNA修饰中的功能；2)探索TET2在RNA中的新功能 依赖染色质靶向调控RNA靶点实现ESCs的多能性；3)操纵茎 靶向CRISPR/RCas9的RNA靶向MERVL RNA修饰的细胞和发育潜能 胚胎干细胞和发育中的胚胎。第一个目标是建立TET2在hm5C修饰中的新功能 MERVL以PSPC1依赖的方式转录。第二个目标将剖析hm5c的后果。 MERVL转录本的修饰及hm5C介导MERVL的分子机制 退化。第三个目标将探索通过直接MERVL RNA操纵干细胞和发育潜力 修改。综合起来，这三个目标将为RNA依赖提供相当新的洞察力 TET2的染色质靶向在干细胞潜能中控制MERVL的转录后机制。这个 该项目具有重要意义，因为它有望在理解ERV监管方面建立一个新的范式 在RNA修饰中的功能，以及全能性。

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.