Mechanistic insights into the stimulation of the histone H3K9 methyltransferase Clr4 by proximal H3K14 ubiquitination

H3K9 methylation, a conserved heterochromatin marker, is crucial for chromosome segregation and gene regulation. Clr4 is the sole known methyltransferase catalyzing H3K9 methylation in <i>Schizosaccharomyces pombe</i>. Clr4 K455/K472 automethylation and H3K14 ubiquitination (H3K14Ub) are vital activators of Clr4, ensuring appropriate heterochromatin deposition and preventing deleterious silencing. While automethylation's activation mechanism is uncovered, the mechanism of H3K14Ub's significantly stronger stimulation on Clr4 remains unclear. Here, we determined the crystal structures of Clr4 bound to ubiquitinated and unmodified H3 peptides at 2.60 and 2.39 angstrom, which revealed a synergistic mechanism underlying the pronounced stimulatory effect: H3K14Ub increases substrate affinity through multivalent interactions and facilitates the allosteric transition of Clr4 from an inactive apo conformation to a hyperactive "catalyzing state," including conformational changes in the αC-SET-insertion region, autoregulatory loop, and the β9/10 loop. We finally propose a multilevel structural model for the Clr4 catalytic-regulatory cycle. This work provides structural insights into the interplay between histone modifications and their collective impact on epigenetic regulation.