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.
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.
|Zamudio, Jesse R; Kelly, Timothy J; Sharp, Phillip A (2014) Argonaute-bound small RNAs from promoter-proximal RNA polymerase II. Cell 156:920-34|