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Monoubiquitylation of histone H2B (H2Bub1) is catalyzed mainly by the RNF20/RNF40 complex and erased by multiple deubiquitylating enzymes (DUBs). H2Bub1 influences many aspects of chromatin function, including transcription regulation and DNA repair. Cancer cells often display reduced levels of H2Bub1, and this reduction may contribute to cancer progression. The let-7 family of microRNAs comprises multiple members with reported tumor suppressive features, whose expression is frequently downregulated in cancer. We now report that let-7b and let-7c can positively regulate cellular H2Bub1 levels. Overexpression of let-7b and let-7c in a variety of non-transformed and cancer-derived cell lines results in H2Bub1 elevation. The positive effect of let-7b and let-7c on H2Bub1 levels is achieved through targeting of multiple mRNAs, coding for distinct components of the H2B deubiquitylation machinery. Specifically, let-7b and let-7c bind directly and inhibit the mRNAs encoding the DUBs USP42 and USP44, and also the mRNA encoding the adapter protein ATXN7L3, which is part of the DUB module of the SAGA complex. RNF20 knockdown strongly reduces H2Bub1 levels and increases the migration of non-transformed mammary epithelial cells and breast cancer-derived cells. Remarkably, overexpression of let-7b, which partly counteracts the effect of RNF20 knockdown on H2Bub1 levels, also reverses the pro-migratory effect of RNF20 knockdown. Likewise, ATXN7L3 knockdown also increases H2Bub1 levels and reduces cell migration, and this anti-migratory effect is abolished by simultaneous knockdown of RNF20. Together, our findings uncover a novel function of let-7 microRNAs as regulators of H2B ubiquitylation, suggesting an additional mechanism whereby these microRNAs can exert their tumor suppressive effects.

The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status.