DNA hypomethylation promotes UHRF1-and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states

Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity toward H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes (TSGs). Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. By contrast, disrupting H3K18ub-dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy. • DNA hypomethylation leads to UHRF1-dependent increases in H3K18ub and H3K9me3 • Hemi-methylated DNA accumulation promotes H3K18ub that enhances SUV39H1/H2 activity • Disrupting UHRF1 activity prevents H3K9me3 accumulation but promotes PRC2 activity • Targeting H3K18ub stimulation of SUV39H1/H2 enhances DNMT1 inhibitor efficacy Liu et al. report that DNMT1 inhibition causes transient accumulation of hemi-methylated DNA at CpG islands, which stimulates UHRF1-mediated H3K18ub. This enhances SUV39H1/H2 activity, nucleates new H3K9me3 domains, and impedes PRC2 activity at DNA-methylation-silenced TSG promoters. Targeting this previously unrecognized H3K18ub-H3K9me3 crosstalk offers new strategies to enhance DNMT1 inhibitor therapy.