GSE159192
Mechanism for the selective sequestration of a histone H3K9 methyltransferase at heterochromatin by the H3K9M mutation
Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild type histones, but the mechanisms by which these mutations function are highly controversial. We have previously shown that in fission yeast, H3K9M containing nucleosomes sequesters the H3K9 methyltransferase Clr4 to block H3K9 methylation{Shan, 2016 #119}. Using ChIP-sequencing, here we show that even though H3K9M containing nucleosomes are broadly distributed across the genome, Clr4 is mainly sequestered at pericentric repeats. The selective sequestration of Clr4 depends not only on H3K9M, but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4 associated E3 ubiquitin ligase complex. In vitro, H3K14ub enhances the interaction between H3K9M and Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Using bio-layer interferometry, we show that the H3K9M mutation reduced association rates (kon) of Clr4 with histone tails, suggesting that Clr4 intrinsically disfavors H3K9M. More importantly, H3K9M reduced the dissociation rate (koff) of Clr4 with histone tails, demonstrating that H3K9M slows down the turnover of Clr4 once it is bound. Finally, we generated mutations on Clr4 that specifically disrupt its interaction with H3K14ub and show that they reduce the inhibitory effects of H3K9MK14ub on Clr4 enzymatic activity in vitro and relieve the sequestration of Clr4 at pericentric repeats in vivo. Therefore, the selective sequestration of Clr4 at pericentric repeats by H3K9M is due to the presence of H3K14ub at these regions, which overcomes Clr4's aversion of H3K9M and reduces the dissociation of Clr4. Overall design: ChIP-seq of Flag-Clr4 with/without H3K9M, of H3-Flag and H3K9M-Flag and controls