Mammalian DNA maintenance methylation depends on histone but not PAF15 ubiquitination

In mammals, DNA methylation is an essential inheritable epigenetic modification maintained by the DNMT1-UHRF1 machinery in a UHRF1 E3 ubiquitin ligase-activity-dependent manner. UHRF1-catalyzed histone ubiquitination and PAF15 ubiquitination have been proposed to recruit DNMT1 to hemi-methylated CpGs. Here, we confirm that both PAF15 and histone H3 are robustly ubiquitinated by UHRF1 in a replication-dependent manner during S phase. However, loss of PAF15 or its ubiquitination, or overexpression of ubiquitination-deficient PAF15, does not alter DNMT1 recruitment to replicating DNA or global DNA methylation. Notably, the DNMT1 inhibitor GSK-3685032 strongly enhances histone ubiquitination, likely via UHRF1 activation driven by accumulated hemi-methylated CpGs, but markedly reduces PAF15 ubiquitination. We show that UHRF1-mediated PAF15 ubiquitination requires DNMT1, is strictly replication-coupled, and that PAF15 and histone ubiquitination are mediated by distinct UHRF1 pools. Taken together, our study demonstrates that hemi-methylated-DNA-stimulated local histone ubiquitination, rather than replication-coupled PAF15 ubiquitination, drives DNMT1 targeting for maintenance methylation.