An H3K14ub-H3K9me3 feedback circuit governs heterochromatin spreading and inheritance in fission yeast

Heterochromatin is a bistable chromatin state essential for genome stability and gene regulation. Its spreading and inheritance have long been explained by a "read-write" cycle in which histone methyltransferases bind pre-existing tri-methylation of histone H3 lysine 9 (H3K9me3) and propagate this mark to neighboring nucleosomes. However, the weak affinity and limited catalytic stimulation provided by H3K9me3 alone challenge this model. The fission yeast H3K9 methyltransferase Clr4 functions within the CLRC complex, which also catalyzes histone H3 lysine 14 ubiquitination (H3K14ub). Here we show that H3K14ub and H3K9me3 form a feedback loop: H3K14ub strongly stimulates Clr4 activity on nucleosomes, while both H3K14ub and H3K9me3 stabilize CLRC binding to chromatin. Even subtle perturbations that disrupt this feedback, such as mutating one of the three H3 genes to prevent ubiquitination or methylation, or impairing Clr3-mediated H3K14 deacetylation, compromises heterochromatin spreading and inheritance. Conversely, counteracting activities, such as H3K14 acetylation by Mst2 and H3K9 demethylation by Epe1, synergistically constrains heterochromatin expansion. Thus, rather than relying solely on the weak H3K9me3 "read-write" cycle, heterochromatin is maintained through an integrated circuit of ubiquitination, deacetylation, and methylation, which governs spreading and inheritance.