The Argonaute proteins (AGO) are well-known for their essential role in post-transcriptional gene silencing in the microRNA (miRNA) pathway. Only two AGOs are expressed in mouse embryonic stem cells (mESCs). The transcriptome of Ago mutant mESCs revealed a large and specific set of differentially expressed genes (DEGs), compared to other miRNA biogenesis factor mutant cells, suggesting additional functions for AGOs. Integration of Ago DEGs with ENCODE histone modification data of WT mESCs revealed a correlation with H3K27me3 chromatin mark. We validated experimentally this result and observed a global loss of H3K27me3, which was only partially explaining the DEGs observed in Ago mutant cells. By integrating chromatin accessibility data in conjunction with the prediction of transcription factor (TF) binding sites, we identified differential binding for five TFs, including KLF4 as a key modulator of more than half of the specific DEGs in the absence of AGO proteins. Our findings illustrate that in addition to chromatin state, information about transcription factor binding is more revelatory in understanding the multi-layered mechanism adopted by cells to regulate gene expression.

In the past years, several studies reported nuclear roles for the Argonaute (AGO) proteins associating them with transcriptional activation or repression, alternative splicing and, chromatin organization. However, as most of these experiments have been conducted in human cancer cell lines, the nuclear functions of the AGO proteins in mouse early embryonic development still remains elusive. In this study, we investigated possible nuclear functions of the AGO proteins in mouse Embryonic Stem Cells (mESCs). By biochemical assays, we observed that AGO1 and AGO2 are present in a small fraction in the nucleus and even less on chromatin in mESCs. To profile the nuclear interactome of the AGO proteins, we performed immunoprecipitation followed by Mass Spectrometry and identified three novel nuclear interactors for AGO1, namely DNMT3a, HP1a;, and ATRX. These interactors are well-known proteins involved in the establishment and maintenance of heterochromatin at pericentromeric regions. Indeed, upon depletion of Ago1, we observed a specific redistribution of the heterochromatin protein HP1a; and the repressive histone mark H3K9me3, away from pericentromeric regions. Furthermore, these regions are characterized by AT-rich tandem repeats known as major satellite sequences. We demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs. Interestingly, this phenotype was not caused by the loss of genome integrity at pericentromeres, as these could still form normally in Ago1_KO mESCs. Lastly, we showed that specific microRNAs loaded in AGO1, regulate the expression of the major satellite transcripts. Overall, our results demonstrate for the first time a novel role for AGO1 in regulating major satellite transcripts and localization of HP1a; and H3K9me3 at pericentromeres in mESCs.