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

The Argonaute proteins (AGOs) are well known for their role in post-transcriptional gene silencing in the microRNA (miRNA) pathway. Here we show that in mouse embryonic stem cells, AGO1&2 serve additional functions that go beyond the miRNA pathway. Through the combined deletion of both Agos, we identified a specific set of genes that are uniquely regulated by AGOs but not by the other miRNA biogenesis factors. Deletion of Ago2&1 caused a global reduction of the repressive histone mark H3K27me3 due to downregulation at protein levels of Polycomb repressive complex 2 components. By integrating chromatin accessibility, prediction of transcription factor binding sites, and chromatin immunoprecipitation sequencing data, we identified the pluripotency factor KLF4 as a key modulator of AGO1&2-regulated genes. Our findings revealed a novel axis of gene regulation that is mediated by noncanonical functions of AGO proteins that affect chromatin states and gene expression using mechanisms outside the miRNA pathway. [Display omitted] •AGO1&2 regulate a specific set of genes in mESCs, independently of the miRNA pathway•PRC2 proteins are downregulated in Ago2&1_KO mESCs, leading to H3K27me3 global loss•AGO1&2 regulate gene expression through the pluripotency factor KLF4 Müller et al. report that AGO1&2 are required for the regulation of a specific set of genes in mESCs, independently of their roles in the miRNA pathway. Integration of multiple omics datasets revealed the pluripotency factor KLF4 as a key modulator of AGO1&2-regulated genes.