Faeh, T. | Search bioRxiv

bioRxiv2021

ARGONAUTE proteins regulate a specific network of genes through KLF4 in mouse embryonic stem cells

The Argonaute proteins (AGO) are well-known for their essential role in post-transcriptional gene silencing in the microRNA (miRNA) biogenesis pathway. Only two AGOs (AGO1 and AGO2) are expressed in mouse embryonic stem cells (mESCs). The transcriptome of Ago mutant mESCs revealed a large and specific set of misregulated genes, compared to other miRNA biogenesis factor mutant cells, suggesting additional functions for the AGOs in stem cells. In this study, we endeavored to understand miRNA-independent roles of the AGOs in gene expression regulation through the integration of multiple datasets. Correlation of Ago mutant differential gene expression with ENCODE histone modification data of WT mESCs revealed that affected genes were regulated by the repressive histone modification H3K27me3. We validated this observation by performing chromatin immunoprecipitation followed by sequencing and observed a global loss of H3K27me3 in Ago mutant cells. Nevertheless, this reduction explains only a small part of the specific differential gene expression observed in Ago mutant mESCs. By integrating chromatin accessibility data in conjunction with prediction of transcription factor binding sites, we identified differential binding for five transcription factors, including KLF4 as a key modulator of more than half of the specific misregulation of gene expression 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. These data also highlight the importance of an integrative approach to unravel the variety of noncanonical functions of AGOs in mESCs.

molecular biology↗

bioRxiv2021

AGO1 regulates major satellite transcripts and H3K9me3 distribution at pericentromeric regions in mESCs

Argonaute proteins (AGOs), that play an essential role in cytosolic post-transcriptional gene silencing, have been also reported to function in nuclear processes like transcriptional activation or repression, alternative splicing and, chromatin organization. As most of these studies have been conducted in human cancer cell lines, the relevance of AGOs nuclear functions in the context of mouse early embryonic development remains uninvestigated. Here, we examined a possible role of the AGO1 protein on the distribution of constitutive heterochromatin in mouse Embryonic Stem Cells (mESCs). We observed a specific redistribution of the repressive histone mark H3K9me3 and the heterochromatin protein HP1, away from pericentromeric regions upon Ago1 depletion. Furthermore, we demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs and that their levels are partially restored upon AGO1 rescue. We also observed a similar redistribution of H3K9me3 and HP1 in Drosha_KO mESCs, suggesting a role for microRNAs (miRNAs) in the regulation of heterochromatin distribution in mESCs. Finally, we showed that specific miRNAs with complementarity to major satellites can partially regulate the expression of these transcripts. Summary blurbDepletion of AGO1 in mESCs leads to a redistribution of H3K9me3 and HP1 away from pericentromeric regions and is accompanied by an upregulation of major satellites transcripts.

cell biology↗