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

Mammalian DNA methylation inheritance depends on DNMT1 and its E3 ligase cofactor UHRF1. At newly replicated chromatin, UHRF1 recognizes hemi-methylated DNA and histone H3 N-terminal tails, directing mono-ubiquitination of H3K14, H3K18, and/or H3K23 to recruit DNMT1. UHRF1 can deposit multiple mono-ubiquitin marks on a single H3 tail, and DNMT1 recognizes this multivalent state through tandem ubiquitin-interacting motifs, but how successive ubiquitination is promoted and what multi-mono-ubiquitination does biologically were unknown.

The study shows that UHRF1 directly binds its mono-ubiquitinated H3 products through a previously uncharacterized LGDDSL loop in Tudor 2 of its tandem Tudor domain (TTD), enabling further ubiquitin deposition.

Disrupting this ubiquitin-reading activity impairs H3 multi-mono-ubiquitination and accelerates DNA methylation loss specifically within late-replicating, CpG-sparse genomic regions characteristic of partially methylated domains (PMDs) in cancer and aging cells. These methylation defects overlap those caused by disrupting UHRF1 ubiquitin ligase activity, providing convergent evidence that both writing and reading H3 ubiquitination support CpG-sparse DNA methylation maintenance.

Together, the findings define a feed-forward ubiquitin read-write mechanism that generates multi-mono-ubiquitinated H3 and safeguards DNMT1-dependent DNA methylation maintenance at vulnerable regions of the mammalian methylome.