Fig. 1

Identification of PWWP2A as an H3K36me3 binding protein. a SILAC nucleosome affinity purifications were performed as previously described in Bartke et al.²². HeLa cells were labelled with heavy or light SILAC media (1) and nuclear proteins were extracted (2). Pairwise nucleosome affinity purifications were performed as follows (3): light and heavy nuclear extracts were mixed with unmodified and H3K36me3-modified recombinant nucleosomes respectively in the Forward experiment and vice-versa in the Reverse experiment. The paired nucleosome affinity purifications in the forward experiment were pooled for LC–MS/MS (4), and the same for the reverse experiment. b Unmodified and H3Kc36me3-modified nucleosomes were generated as described in Dyer et al.⁵¹. H3K36C purified histones were alkylated to completion to generate site-specific cysteine to generate a trimethyllysine analog, as described²³. c Identification of proteins enriched on unmodified vs. H3K36me3-modified nucleosomes. Proteins enriched on H3K36me3-modified nucleosomes are found in quadrant 3. d Domain structure of the PWWP2A protein. PWWP2A has a C-terminal PWWP domain (blue), the rest of the protein is highly disordered and contains two proline-rich (yellow) and one serine-rich (purple) region. PWWP2A was recently found to contain a H2A.Z-interacting region³³ (green). e ChIP-qPCR of FS2-PWWP2A and FS2-PWWP2A∆PWWP at the promoters and gene body regions of three genes expressed in mESCs, and a gene desert which does not harbour any active or repressive chromatin modifications. The error bar represents s.e.m. from four biological replicates, and significance was calculated by Student’s t-test, * indicates p-value <0.05