Elucidating Nuclear Argonaute Function

描述（由申请人提供）：通过RNAi诱导的沉默复合物（RISC）介导microRNA对mRNA稳定性和翻译的细胞质转录后调节。越来越清楚的是，细胞质转录后的主要蛋白质效应物 沉默-Argonaute（Ago）蛋白质家族的成员-也位于细胞核中。来自酵母和线虫研究的多条证据表明Argonaute蛋白在细胞核中具有次要功能，但支持哺乳动物中这种功能的数据很少。Ago 2在miRNA和siRNA介导的mRNA调控中起核心作用，并被认为与染色质结合并调节转录，然而以前的报道在定义染色质结合位点和在全基因组范围内对细胞转录的结果方面受到限制。基于来自多个小组的报告，我们假设Argonaute蛋白家族在介导转录和转录后基因沉默中起作用。因此，我们建议阐明核Argonaute的功能，以便更好地定义完整的 Argonaute蛋白在生物学中功能谱。我们已经在先前描述的Argonaute敲除小鼠胚胎干（ES）细胞中产生了强力霉素诱导的FLAG-HA表位标记的Ago 2表达系统，允许Ago 2在Argonaute无效背景中以野生型水平表达。 具体地，我们提出（1）利用ChIP-Seq分析，采用表位-标签介导的Ago 2免疫沉淀，鉴定Ago 2的全基因组染色质结合位点，（2）利用PAR-CLIP确定Ago 2的核RNA结合配偶体，（3）使用ChIP-Seq来确定在存在或不存在Ago 2的情况下的全基因组染色质修饰，以及（4）询问野生型、Argonaute null和Dicer null ES细胞之间的染色质修饰和RNA表达差异。总之，该分析将允许我们产生由Ago 2通过染色质修饰和核RNA指导的转录沉默控制的转录调控的全基因组图谱。该分析将使我们能够进一步确定Ago 2在控制细胞转录网络中的作用。 公共卫生相关性：据报道，Argonaute表达在乳腺癌、结肠癌和卵巢癌中增加。此外，Argonaute的表达与培养的乳腺癌细胞的增殖和细胞运动性增加有关，这表明这些蛋白质可能 在癌症生长和转移中发挥作用。

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.