UHRF2 commissions the completion of DNA demethylation through allosteric activation by 5hmC and K33-linked ubiquitination of XRCC1

The transition of oxidized 5-methylcytosine (5mC) intermediates into the base excision repair (BER) pipeline to complete DNA demethylation remains enigmatic. We report here that UHRF2, the only paralog of UHRF1 in mammals that fails to rescue Uhrf1−/− phenotype, is physically and functionally associated with BER complex. We show that UHRF2 is allosterically activated by 5-hydroxymethylcytosine (5hmC) and acts as a ubiquitin E3 ligase to catalyze K33-linked polyubiquitination of XRCC1. This nonproteolytic action stimulates XRCC1’s interaction with the ubiquitin binding domain-bearing RAD23B, leading to the incorporation of TDG into BER complex. Integrative epigenomic analysis in mouse embryonic stem cells reveals that Uhrf2-fostered TDG-RAD23B-BER complex is functionally linked to the completion of DNA demethylation at active promoters and that Uhrf2 ablation impedes DNA demethylation on latent enhancers that undergo poised-to-active transition during neuronal commitment. Together, these observations highlight an essentiality of 5hmC-switched UHRF2 E3 ligase activity in commissioning the accomplishment of active DNA demethylation. [Display omitted] •UHRF2 is physically and functionally associated with the BER complex•UHRF2 is allosterically activated by 5hmC to catalyze K33-linked XRCC1 ubiquitination•XRCC1 ubiquitination aids its interaction with RAD23B and TDG incorporation into BER•UHRF2-fostered TDG-RAD23B-BER assembly promotes the completion of DNA demethylation Liu et al. discover that UHRF2 is physically and functionally associated with the BER complex. UHRF2 is allosterically activated by 5hmC to catalyze K33-linked ubiquitination of XRCC1. This nonproteolytic action fosters XRCC1’s interaction with RAD23B and facilitates TDG’s incorporation into the BER complex, thereby promoting the completion of DNA demethylation.