Fig 2: DNAJC9 links heat shock biology to the histone chaperone networkDNAJC9 binds histone substrates that cannot engage other histone chaperones because of being monomeric, misfolded, or engaged in spurious interactions with RNA/DNA. DNAJC9 recruits HSP70-type enzymes through its J domain to fold and release of histones substrates with ATP-derived energy. DNAJC9-bound histones can enter the histone chaperone supply chain upstream of HAT1 for their eventual delivery to chromatin by ASF1. Alternatively, DNAJC9-bound histone dimers bypass ASF1 and engage with histone deposition chaperones during DNA replication and transcription.

Fig 3: J domain mutation traps DNAJC9 on chromatin genome-wide in a histone-dependent manner(A) DNAJC9 domain map with relevant mutations.(B) Western blots of soluble and chromatin extracts from cells expressing DNAJC9-MYC-FLAG WT, J, or 4AJ mutants compared with control cells. See also Figure S4.(C–E) Quantitative ChIP-seq of cells expressing DNAJC9-MYC-FLAG WT, J, or 4AJ mutants compared with control cells. ChIP-seq reads were quantitated in 10 kb windows with a 5 kb step. Plots represent data averaged from n = 2 biological replicates.(C) Visualization of spike-in normalized ChIP-seq signal in DNAJC9 WT, J, 4AJ, and control samples, quantitated with reference-adjusted reads per million (RRPM), and raw input reads over the region depicted.(D) Boxplots of spike-in normalized DNAJC9 ChIP-seq signal across the genome quantitated with reference-adjusted reads per million (RRPM), Log2(n + 1). Black line, median; whiskers, 1.5 × interquartile range.(E) Boxplots of input corrected signal for DNAJC9 J mutant over gene bodies and intergenic regions (left) or gene bodies parsed to active and inactive genes (right). Black line, median; whiskers, 1.5 × interquartile range.

Fig 4: DNAJC9 recruits the heat shock molecular chaperone machinery to fold histone H3-H4 substrates(A) DNAJC9 WT, 4A mutant, and control purifications subjected to triple SILAC-based mass spectrometry analysis. Ratios averaged from n = 2 biological replicates.(B) Histone purifications from soluble extracts of cells small interfering RNA (siRNA) depleted for DNAJC9, BAG2, or HSC7C compared with control (CTRL) siRNA and analyzed using label-free mass spectrometry (s0 = 0.5, false discovery rate [FDR] = 0.05, H3.1 n = 5 and H4 n = 4 biological replicates). Bubble plots colors represent Log2 ratios of median-normalized LFQ intensities (siRNA/siCTRL)M.N., and radii represent significance of changes (s0 = 0.5, FDR = 0.05); no imputed values shown.(C) Histone purifications from soluble extracts subjected to label-free mass spectrometry analysis (n = 3 biological replicates, s0 = 0.5, FDR = 0.05). Volcano plots represent differences in median-normalized LFQ intensities (LFQM.N.) with missing values imputed for factors observed three times in either replicate.(D) GST pull-down assays showing H3.3 WT- or H3.3 ED105AA-H4 binding to selected histone chaperones.In (A)–(C), proteins are referred to by human UniProt protein identification code.See also Figures S2 and S3 and Table S1.

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).