Using Ubiquitin Fusion Proteins Study The Molecular Mechanism Of DNMT1 Recruitment

In mammalian cells,DNMT1 is the primary enzyme responsible for maintenance of DNA methylation during DNA replication.UHRF1 recognizes hemimethylated DNA via the SRA domain and catalyzes H3 ubiquitination.DNMT1 is recruited to DNA replication forks to maintain DNA methylation by its interaction with UHRF1 and binding of ubiquitinated H3.Besides H3,ubiquitination on other proteins including UHRF1 and PCNA also occurs at replication fork.Whether ubiquitination on these proteins also contributes to DNMT1 recruitment remains to be investigated.The answer to this question will help us to better understand the molecular mechanisms of DNMT1 mediated maintainence of DNA methylation.In this study,we constructed UHRF1-UB,UHRF1 mutant-UB,SRA-UB,PCNAUB fusion proteins.Through Co-IP assay,we found that the UB fusion proteins did not show increased interaction with DNMT1 in comparison to the congnate proteins.We also tested whether these UB fusion proteins could rescue DNA methylation defect in Uhrf1-/-mouse embryonic stem(ES)cells.We found that expressing these fusion proteins could not rescue DNA methylation defect,whereas the wildtype UHRF1 fully rescued DNA methylation.As DNMT1 was shown to bind ubiquitinated H3,we also constructed a UB-H3 fusion protein.Using pull-down assay,we observed that DNMT1 indeed bound ubiquitinated H3 prepared from cells but not UB-H3 fusion.In addition,we found that expression of UB-H3 fusion in Uhrf1-/-ES cells failed to rescue DNA methylation.Collectively,our results indicate that DNMT1 selectively binds ubiquitinated H3 but not all ubiquitinated proteins,suggesting a profound substrate-and site-specificity in binding of ubiquitinated proteins by DNMT1.