The DNA damage response, chromatin remodelling and transcriptional regulation are partially interdependent pathways that share a common feature, in that they are all regulated by posttranslational modifications of histones. Lysine-specific ubiquitination of histone H2A has been shown to be involved in these pathways. H2A can get ubiquitinated on three independent ubiquitination sites, by three different E3 ligases: BRCA1-BARD1, RNF2 — in a PRC1 complex — and RNF168. The ubiquitination status on these three sites has distinct signalling outcomes and represents a fine balance between the activities of ubiquitinating and deubiquitinating enzymes (DUBs).Yet, the lysine-specificity of H2A DUBs and the biological implications of specific H2A ubiquitination remained elusive.
Screening for H2A-specific DUBs, we found USP48 to be a DUB specific for the BRCA1-BARD1 induced H2A ubiquitination mark. Detailed biochemical analysis revealed a curious mode of action, where USP48 only efficiently cleaves BRCA1-ubiquitinated H2A when activated by an auxiliary ubiquitin on an adjacent lysine. Further analysis indicates that this auxiliary ubiquitin can be placed on the PRC1 target-site to activate cleavage of the BRCA1 target-site. These observation implies that USP48 antagonises BRCA1 in a PRC1-dependent manner and thus suggesting a potential crosstalk between these pathways.
Knockout-rescue experiments in cells established USP48 as a direct antagonist of BRCA1 signalling in the DNA damage response. Specifically, loss of USP48 disrupts the balance of repair pathway choice, promoting the use of error-prone single strand-annealing pathways. This is due to extended DNA-end resection, driven by BRCA1-BARD1 ubiquitin ligase activity and SMARCAD1-dependent chromatin remodelling, upon USP48 loss. DNA repair through single-strand annealing leads to large chromosomal deletions, which potentially implies a function of USP48-driven repair pathway choice in cancer development.
Collectively, our findings indicate that USP48 is a direct antagonist of BRCA1 ubiquitination activity that acts in concert with SMARCAD1-dependent chromatin remodelling and PRC1-catalysed histone ubiquitination.