Cellular senescence is associated with the appearance of heterochromatin foci. These foci are presumed to contain proliferation-promoting genes that are transcriptionally repressed by the retinoblastoma (RB1)–E2F complex. Given that Argonaute (AGO) proteins, microRNAs (miRNAs) and other small noncoding RNAs have been implicated in heterochromatin formation and transcriptional gene silencing (TGS), Benhamed et al. analyzed the possible involvement of miRNAs and AGO proteins in senescence-associated repression of E2F target genes in human cells. Genome-wide identification of AGO-bound E2F target genes uncovered many genes involved in cell-cycle control, whereas AGO- and heterochromatin-bound miRNAs were positively correlated in senescent cells. AGO2 was found to accumulate in the nucleus of senescent cells, where it cooperates with RB1 to repress E2F target genes, as shown in promoter-reporter and AGO2 knockdown assays. Depletion of AGO2 delayed senescence onset in fibroblasts, whereas AGO2 overexpression induced an abrupt proliferative arrest. Members of the let-7 family of miRNAs were found to interact with both AGO2 and repressive heterochromatin. Promoter-reporter assays with an E2F target gene promoter containing a putative let-7f binding site showed that AGO2 and let-7f cooperate to induce TGS, but promoter binding of let-7f is AGO2 dependent. Together, these data suggest that cellular senescence can trigger miRNA-mediated TGS, which may contribute to tumor suppression by transcriptionally repressing proliferation-promoting genes. (Nat. Cell Biol. 14, 266–275, 2012)