A feed-forward UHRF1 read-write mechanism supports H3 multi- mono-ubiquitination and DNA methylation maintenance at CpG-sparse regions

The epigenetic inheritance of mammalian DNA methylation requires DNMT1 and its E3 ligase cofactor UHRF1. At newly replicated chromatin, UHRF1 recognition of hemi-methylated DNA and histone H3 N-terminal tails directs catalysis of H3K14, H3K18, and/or H3K23 mono-ubiquitination to recruit DNMT1. While it is appreciated that UHRF1 can deposit multiple mono-ubiquitin marks on a single H3 tail and that DNMT1 recognizes this state through tandem ubiquitin interacting motifs, the mechanism that promotes successive ubiquitination and the biological function of multi-mono-ubiquitination are unknown. Here, we show that UHRF1 directly binds its mono-ubiquitinated H3 products through a previously uncharacterized LGDDSL loop in Tudor 2 of its tandem Tudor domain (TTD) to promote further ubiquitin deposition. Disruption of this ubiquitin reading activity impairs H3 multi-mono-ubiquitination and accelerates DNA methylation loss within late-replicating, CpG-sparse genomic regions that are characteristic of partially methylated domains (PMDs) in cancer and aging cells. These methylation defects overlap those observed by disruption of UHRF1 ubiquitin ligase activity, providing convergent evidence that both writing and reading of H3 ubiquitination support CpG-sparse DNA methylation maintenance. Together, these findings establish a feed-forward ubiquitin read-write mechanism that generates multi-mono-ubiquitinated H3 and safeguards DNMT1-dependent DNA methylation maintenance at vulnerable genomic regions of the mammalian methylome.