Abstract
The polycomb repressive‐deubiquitinase (PR‐DUB) complex removes ubiquitin from lysine residue 119 on histone H2A (H2AK119Ub) in humans. The PR‐DUB is composed of two central protein factors, the catalytic breast cancer type 1 susceptibility protein (BRCA1)‐activating protein 1 (BAP1) and one of three additional sex combs‐like 1–3 (ASXL1–3) proteins. A plant homeodomain (PHD) at the C terminus of ASXL proteins is recurrently truncated in cancer, was previously proposed to recognise epigenetic modifications on the N‐terminal tail of histone H3 and was recently shown to bind an auxiliary set of PR‐DUB interactors, named methyl CpG‐binding domain proteins 5 (MBD5) and 6 (MBD6). Here, we demonstrate that the ASXL PHD domain lacks features required for histone tail recognition and is unable to bind histone H3 epigenetic marks. Modelling the structure of the ASXL PHD using AlphaFold3 suggests that the domain has an atypical fold and that the isolated ASXL PHD can chelate a single zinc ion in vitro , compared with the two ions conventionally bound by PHD domains. Alternatively, we show that the ASXL PHD‐MBD5 and PHD‐MBD6 complexes are stable in vitro . A composite zinc‐binding site was shown to form at the interface between the ASXL2 PHD and MBD6 MBD domains, and is required for stable complex formation. Overall, these data suggest an unconventional pairing of domains coordinate key functions of the PR‐DUB—a noncanonical PHD domain from ASXL proteins partners with MBD5 or 6, which were themselves misannotated because they cannot bind to methylated DNA.