Unconventional structure and function of PHD domains from Additional Sex Combs-like proteins

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 Additional Sex Combs-like 1–3 (ASXL1–3). A Plant Homeodomain (PHD) at the C-terminus of ASXL proteins is recurrently truncated in cancer, and was previously proposed to recognise epigenetic modifications on the N-terminal tail of histone H3. 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 to the two ions conventionally bound by PHD domains. Recently, the ASXL PHD was shown to bind an auxiliary set of PR-DUB interactors, named methyl CpG-binding domain proteins 5 (MBD5) and 6 (MBD6). We show that the ASXL PHD-MBD5 and -MBD6 complexes are stable in vitro, and surprisingly contain a composite Zinc-binding site at the interface between the two proteins. Overall, this data suggests an unconventional pairing of domains coordinate key functions of the PR-DUB — a non-canonical PHD domain from ASXL proteins obligately partners with MBD5 and 6, which were themselves misannotated because they cannot bind to methylated DNA.