Fig 1: The Histone H3.3K27M mutant has an altered mitotic protein interaction network.(A) Venn diagram showing the distribution of FLAG-histone interacting proteins that are either specific for H3.3WT (n = 199) and mutant H3.3K27M (n = 102) cells, or common/enriched (n = 330) as identified by LC-MS/MS. (B) Graph depicting semi-quantitative spectral counts of H3.3WT and H3.3K27M common binding partners (black, n = 222) (differential spectral counts between dashed lines), and enriched binding partners (green: H3.3K27M - enriched, n = 76; grey: H3.3WT—enriched, n = 32). (C) H3.3K27M specific/enriched interaction network for Biological processes as analyzed using STRING. The illustrated network map was simplified for clarity by manual curation. Dashed circles represent a group of interacting proteins belonging to one Biological process. (D) GO-term analysis (for Biological processes) of interacting proteins using STRING reveals significant enrichment for chromosome organisation, mRNA splicing, DNA replication and translation in enriched H3.3K27M binding partners. GO = gene ontology, FDR = false discovery rate. (E) H3.3WT specific/enriched interaction network as analyzed using STRING. The illustrated network map was simplified for clarity by manual curation. Dashed circles represent a group of interacting proteins belonging to one biological process. (F) GO-term analysis (for Biological processes) of interacting proteins using STRING reveals significant enrichments for translation, regulation of apoptotic process and RNA processing in enriched H3.3WT binding partners. GO = gene ontology, FDR = false discovery rate. (G) DNA replication enriched network showing binding partners relation to the histone mutant H3.3K27M. (H) Western blot validation of FLAG-immunoprecipitations in H3.3WT, H3.3K27M and control mitotic lysates. (I) Quantification of the histone-bound fraction of H4, MCM2, 7 and 4 by densitometry. Signal was normalized to FLAG, and H4 was used as positive control. Data are presented as means ±SD (n = 4 experiments). *P = 0.0138 and 0.0107; **P = 0.0054 and 0.0057; ***P = 0.0003 (Multiple t-tests).