Molecular characterization of substrate-induced ubiquitin transfer by UBR7-PHD finger, a newly identified histone H2BK120 ubiquitin ligase.

Monoubiquitination of histone H2B at lysine 120 plays a vital role in active transcription and DNA damage response pathways. UBR7 has been recently identified as an H2BK120 monoubiquitin ligase. However, the molecular details of its ubiquitin transfer mechanism are not well understood. Here, we report that PHD finger of UBR7 is essential for its association with E2 UbcH6 and consequent ubiquitin transfer to its substrate histone H2B. We have also identified the critical region of UbcH6 involved in this function and shown that the residues stretching from 114 to 125 of histone H2B C-terminal tail are sufficient for UBR7/UbcH6-mediated ubiquitin transfer. We also employed antibody-independent mass spectrometry to confirm UBR7 mediated ubiquitination of H2B C-terminal tail. We have demonstrated that the PHD finger of UBR7 forms a dimer and this dimerization is essential for ubiquitination of histone H2B. We have mapped the critical residues involved in dimerization and mutation of these residues abrogates E3 ligase activity and is associated with cancer. Furthermore, we have compared the mode of ubiquitin discharge from UbcH6 mediated by UBR7 and RNF20 through thioester hydrolysis assay. Interestingly, binding of substrate H2B to UBR7 induces conformational change in the PHD finger, which triggers ubiquitin transfer from UbcH6. However RNF20 RING finger alone is sufficient to promote the release of ubiquitin from UbcH6. Overall, the mechanism of ubiquitin transfer by the newly identified E3 ubiquitin ligase UBR7 is markedly different from that of RNF20.