Elucidating Nuclear Argonaute Function

描述(由申请人提供)：通过RNAi诱导的沉默复合体(RISC)，MicroRNAs对mRNA稳定性和翻译的细胞质转录后调节是介导的。越来越清楚的是，胞质转录后的主要蛋白质效应因子 沉默--ArgAerte(AGO)蛋白家族的成员--也定位于细胞核。在酵母和线虫中研究的多条证据表明，ArgAerte蛋白在细胞核中具有第二功能，但支持哺乳动物这种功能的数据很少。在哺乳动物编码的四种AGO蛋白中，Ago2在miRNA和siRNA介导的mRNA调控中发挥核心作用，并被认为与染色质结合并调节转录，但以往的报道仅限于确定染色质结合部位和在全基因组水平上对细胞转录的结果。根据多个小组的报告，我们假设ArgAerte蛋白家族在介导转录和转录后基因沉默方面发挥了作用。因此，我们建议阐明核宇航员的功能，以便更好地定义完整的 阿尔加索特蛋白在生物学中的功能谱。我们已经在前面描述的ArgAerte基因敲除小鼠胚胎干细胞(ES)中产生了一个多西环素可诱导的FLAG-HA表位标记的Ago2表达系统，允许在Argavite零背景下以野生型水平表达Ago2。具体地说，我们建议(1)利用表位标签介导的Ago2免疫沉淀，利用CHIP-Seq分析确定Ago2的全基因组染色质结合部位；(2)利用PAR-CLIP确定Ago2的核RNA结合伙伴；(3)使用CHIP-Seq来确定在有或没有Ago2的情况下全基因组的染色质修饰；(4)询问野生型、ArgAerte缺失和DICER缺失ES细胞之间的染色质修饰和RNA表达的差异。总而言之，这一分析将使我们能够生成由Ago2通过染色质修饰和核RNA指导的转录沉默控制的全基因组转录调控图。这一分析将使我们能够进一步确定Ago2在控制细胞转录网络中的作用。 公共卫生相关性：据报道，在乳腺癌、结肠癌和卵巢癌中Argonaute的表达增加。此外，在培养的乳腺癌细胞中，ArgAerte的表达与细胞增殖和细胞运动能力的增加有关，这表明这些蛋白可能 在癌症的生长和转移中起作用。

DESCRIPTION (provided by applicant): Cytoplasmic post-transcriptional regulation of mRNA stability and translation by microRNAs is mediated through the RNAi-induced silencing complex (RISC). It is becoming clear that the primary protein effectors of cytoplasmic post-transcriptional silencing - members of the Argonaute (Ago) family of proteins - are also localized in the nucleus. Multiple lines of evidence from work in yeast and nematodes have suggested that the Argonaute proteins have a secondary function in the nucleus, however data supporting this function in mammals is scant. Of the four mammalian-encoded Ago proteins, Ago2 plays a central role in both miRNA and siRNA mediated mRNA regulation and has been suggested to bind to chromatin and regulate transcription, however previous reports have been limited in defining chromatin binding sites and the result on cellular transcription on a genome-wide scale. Based on reports from multiple groups, we hypothesize that the Argonaute family of proteins play a role in mediating both transcriptional and post-transcriptional gene silencing. We therefore propose to elucidate the function of nuclear Argonaute in order to better define the full spectrum of Argonaute protein function in biology. We have generated a doxycycline-inducible FLAG-HA epitope-tagged Ago2 expression system in the previously described Argonaute knockout mouse embryonic stem (ES) cells, allowing for expression of Ago2 at wildtype levels in an Argonaute-null background. Specifically, we propose to (1) identify genome-wide chromatin binding sites of Ago2 utilizing ChIP-Seq analysis employing epitope-tag mediated Ago2 immunoprecipitation, (2) determine nuclear RNA binding partners of Ago2 utilizing PAR- CLIP, (3) use ChIP-Seq to determine genome-wide chromatin modifications in the presence or absence of Ago2 and (4) interrogate the chromatin modification and RNA expression differences between wildtype, Argonaute null and Dicer null ES cells. Together this analysis will allow us to generate a genome-wide map of transcriptional regulation as controlled by Ago2 through chromatin modifications and nuclear RNA- directed transcriptional silencing. This analysis will allow us to further define the role of Ago2 in controlling the cellular transcriptionl network. PUBLIC HEALTH RELEVANCE: Increased Argonaute expression has been reported in breast, colon and ovarian cancer. Furthermore, expression of Argonaute has been linked to increased proliferation and cell motility in cultured breast cancer cells, suggesting that these proteins may play a role in cancer growth and metastasis.