Fig 2: A double-positive mtp53 association seen for all MCM2-7 complex proteins in cytosol (x-axis) and nucleus (y-axis). Each dot (n = 1778) represents a protein with its position determined by its mPAI values in the cytosolic (x-axis) and the chromatin fractionations (y-axis). mPAI values were averaged if multiple peptides of the same protein were identified. Two side boxplots show the median, the lower and upper quartiles, and the range of mPAI values. The majority of points fall inside the x = -1, x = 1, y = -1, and y = 1 lines, indicating that abundance of most proteins are not significantly impacted by mtp53 knockdown in either fractionation. The mtp53 and the six members of the MCM2–7 complex fall in the top right quadrant, where protein levels are positively associated with mtp53 levels in both fractionations. PARP1 falls in the top left quadrant showing negative association with mtp53 in the cytosol but positive association in the nucleus, consistent with our previous experiment result.6 A searchable, zoomable, and clickable scatter plot of mPAI values for 4798 genes and 1330 associated pathways and gene sets is available at http://diverge.hunter.cuny.edu/~weigang/mpai-browser/

Fig 3: Distribution of mutant p53 association index (mPAI) scores in the cytosol (top) and chromatin (bottom) fractionations. Histograms of mPAI values (for equation see statistics in the methods) show close fit to normal curves (in dashed lines) obtained with the same mean (µ = 0.0060 for cytosol, µ= 0.085 for chromatin) and standard deviation (s = 0.92 for cytosol, s = 0.96 for chromatin). As expected (and as a negative control of the experiments and the mPAI statistic), most proteins are not significantly impacted by mtp53 depletion, showing -1 < mPAI < 1 (shaded in light gray). As a positive control, the mTP53 protein level (in red) shows significantly high positive mPAI values. As another positive control, the PARP1 levels (in blue) show contrasting mPAI values between the two fractionations, consistent with previous experimental results.6 The six components of the MCM2–7 complex (in green) show significant positive association with mtp53 in both cytosol and nucleus

Fig 4: 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).

Fig 5: Molecular basis for recognition of H3.3-H4 by DNAJC9(A) Multiple sequence alignment of DNAJC9 HBD: H. sapiens (NP_056005), M. musculus (NP_598842), G. gallus (NP_001186454), X. laevis (NP_001089275), D. rerio (NP_001002433), D. melanogaster (NP_001262473), and S. pombe (NP_594359). Under the alignment, red squares indicate residues of DNAJC9 interacting with H3.3-H4; “4A” highlights the multiple mutant disrupting interaction with H3.3-H4; black squares indicate residues of DNAJC9 HBD interacting with MCM2 HBD.(B–D) Enlarged views showing the interaction details between DNAJC9 HBD (magenta) and H3.3-H4 (blue and green, respectively).(E) Effects of DNAJC9 HBD mutants on histone binding using GST pull-downs. 4A1 and 4A corresponding to mutants “E195A E196A E199A A200E” and “Q224A R227A M238A Y242A,” respectively. Quantifications on H3.3 based on replicate experiments (n = 3), expressed as mean ± SEM percentage. The signal of H3.3 in WT lane from the same gel was set to 100%.(F) ITC results of DNAJC9 HBD WT and 4A mutant with histones (n = 3 independent experiments, error bars represent mean ± SD). DNAJC9 HBD WT binds H3.3-H4 with a Kd of 55.0 ± 19.7 nM and H3.1-H4 with a Kd of 39.5 ± 4.9 nM. Kd values represent the mean ± SD of independent measurements (n = 3). No binding was observed between DNAJC9 HBD 4A mutant and H3.3-H4 (n = 3).