A previously-unrecognized motif of transcription factor RYBP, hotspot of cancer-related mutations, is essential for the integrity ofPolycombrepressive complex 1

Abstract

Polycomb repressive complex 1 (PRC1) catalyzes monoubiquitination of histone H2A on Lys119, promoting gene silencing. Cells at different developmental stages and in different tissues express different PRC1 isoforms. All isoforms share the same catalytic core (subunits RING1B and PCGF) and vary in the composition of regulatory subunits, clustering in two major classes. Canonical isoforms (cPRC1) are regulated by CBX-like subunits, while variant isoforms (vPRC1) are regulated by RYBP-like subunits. The molecular bases for how regulatory subunits affect the structural assembly of the complex and its catalytic activity are still largely unknown. To fill this knowledge gap, here we have specifically studied how RYBP regulates vPRC1 structure and function. Integrating the machine-learning algorithm AlphaFold2 and NMR, we have identified novel vPRC1 structural motifs in RING1B and RYBP. While the new RING1B motif is dispensable for vPRC1 assembly, the RYBP motif is essential for mediating inter-subunit interactions between RYBP and the catalytic RING1B-PCGF4 heterodimer. Importantly, the RYBP motif harbors cancer-related mutations systematically positioned on the same face of a putative transiently-forming α-helix. Biochemical, biophysical and enzymatic characterization of purified cancer-related mutants confirm that this region is crucial for the structural stability of the complex. Overall, our data offer novel insights into the molecular architecture of vPRC1 and the effects of its regulatory subunit on the biochemical, structural, enzymatic, and physio-pathological properties of the complex.

Unveiling the Binding between the Armadillo-Repeat Domain of Plakophilin 1 and the Intrinsically Disordered Transcriptional Repressor RYBP

“…The only ordered regions of this modeled protein are the zinc-finger domain (residues 23-47), the N-term helix (residue 59-69), and the β-hairpin motif (residue 165-175). Whereas the former and the latter are well-assessed binding spots of RYBP with some molecular partners [2], the N-term helix is a region with a high α-helical propensity predicted by AlphaFold to be fully folded and it has been recently shown that it is capable of interacting with a partner protein of RYBP, the RING1B-PCGF4 heterodimer [27]. All the other regions of the modeled RYBP are unfolded, and AlphaFold predicts that they have a low probability of adopting a structure (confidence score pLDDT < 60, where 0 is the minimum, and 100 is the maximum) [55].…”

“…From a structural point of view, RYBP is a 228-residue-long, highly basic intrinsically disordered protein (IDP) that binds to DNA [2,25]. As shown in Figure 1A, in the RYBP model, the primary structure includes a conserved zinc-finger motif at the N terminus, which is folded and contains the ubiquitin-binding domain [22,26] followed by a so-called 'N-term' helix [27] ending with the nuclear location signal (NLS) that allows for its nuclear translocation [28] and, further ahead, by a β-hairpin motif. The rest of the structure of the RYBP model is unfolded, including a polypeptide region rich in Lys and Arg residues and a C-terminal region with a high percentage of Ser and Thr residues.…”