AGO1 regulates pericentromeric regions in mouse embryonic stem cells

Abstract

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.