The histone variant H3.3 is enriched at enhancers and active genes, as well as repeat regions such as telomeres and retroelements, in mouse embryonic stem cells (mESCs)1,2,3. Although recent studies demonstrate a role for H3.3 and its chaperones in establishing heterochromatin at repeat regions4,5,6,7,8, the function of H3.3 in transcription regulation has been less clear9,10,11,12,13,14,15,16. Here, we find that H3.3-specific phosphorylation17,18,19 stimulates activity of the acetyltransferase p300 in trans, suggesting that H3.3 acts as a nucleosomal cofactor for p300. Depletion of H3.3 from mESCs reduces acetylation on histone H3 at lysine 27 (H3K27ac) at enhancers. Compared with wild-type cells, those lacking H3.3 demonstrate reduced capacity to acetylate enhancers that are activated upon differentiation, along with reduced ability to reprogram cell fate. Our study demonstrates that a single amino acid in a histone variant can integrate signaling information and impact genome regulation globally, which may help to better understand how mutations in these proteins contribute to human cancers20,21.
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We thank members of the Banaszynski and Hon laboratories for helpful discussions; E. Duncan, H. Yu, L. Kraus, H. Zhu, and S. Morrison for critical comments on this manuscript; A. Shoshnev for the illustrations used as templates in this manuscript; UTSW BioHPC for computational infrastructure; UTSW McDermott Center and the Children’s Research Institute for providing next-generation sequencing services; UTSW Flow Cytometry Core for single cell isolation; UTSW Proteomics Core for mass spectrometry analysis. L.A.B. is a Virginia Murchison Linthicum Scholar in Medical Research (UTSW Endowed Scholars Program) and a Peterson Investigator of the Neuroendocrine Research Foundation (NETRF). This work was supported in part by CPRIT RR140042, The Welch Foundation I-1892, DoD KCRP KC170230, and NIH R35 GM124958 (L.A.B.), the American-Italian Cancer Foundation (S.M.), the Taiwan Postdoctoral Research Abroad Fellowship (Y.-C.T), and the Green Center for Reproductive Biology Sciences.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Related to Fig. 1. a, Average profile showing enrichment of H3 and H3.3 at promoter-distal enhancers (± 3 kb from TSS) in mESC, related to Fig. 1a. b, Immunoblot of whole cell lysates from WT and H3.3 KO2 (independent clone) mESCs. Blot is representative of three independent experiments. See also Supplementary Fig. 10 for all the blots in this figure. c, Immunoblot with the indicated antibodies of whole cell lysates from WT and H3.3 KO mESCs. Blot is representative of three independent experiments. d, Dot blot array showing H3K27ac antibodies reactivity for either H3.1K27ac or H3.3K27ac peptides. Blot is representative of two independent experiments. e, Quantification of histone acetylation and methylation levels in WT and H3.3 KO mESCs by mass spectrometry (Mod SpecTM, Active Motif). Relative abundance across samples is reported. Data represented as mean ± s.d (n = 3). f, Bar graph showing the number of H3K27ac peaks that show changes in H3K27ac levels between WT and H3.3 KO mESC at enhancers (± 3 kb from TSS). g, Boxplots showing H3K27ac enrichment levels for distal enhancers that show reduced (n = 13,135) and increased (n = 2,454) H3K27ac enrichment in H3.3 KO mESCs compared to WT. The bottom and top of the boxes correspond to the 25th and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers correspond to 1.5× interquartile range and outliers are excluded. h, Average profile of H3K27ac enrichment at enhancers in WT and H3.3 KO mESCs using a monoclonal antibody. i,j, ChIP-qPCR validation from independent experiments of H3K27ac enrichment at 16 enhancers in WT and 2 clonal H3.3 KO mESCs. k, Ratio (log2) of H3K27ac enrichment in WT versus H3.3 KO mESCs at promoters and enhancers showing reduced acetylation in the absence of H3.3. x axis values > 0 indicate reduced H3K27ac enrichment in the absence of H3.3. l,m,n, Average profile of H3K18ac (l) H3K64ac (m) and H3K122ac (n) enrichment at enhancers in WT and H3.3 KO mESCs. o,p, Average profile (o) and ChIP-qPCR validation (p) of H3K4me1 enrichment at enhancers in WT and H3.3 KO mESCs.
Related to Fig. 1. a, Immunoblot with the indicated antibodies of whole cell lysates from WT and HIRA, ATRX and DAXX KO mESCs. Blot is representative of three independent experiments. See also Supplementary Fig. 11 for all the blots in this figure. b, Boxplot showing H3.3 enrichment at enhancers in WT and HIRA, ATRX and DAXX KO mESCs (n = 17,589). WT & HIRA KO P < 2.2 × 10−16, WT & ATRX KO P = 1.664 × 10−14, WT & DAXX KO P = 0.0007246 by Wilcoxon rank sum two-side test. The bottom and top of the boxes correspond to the 25th and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers correspond to 1.5× interquartile range and outliers are excluded. c, Immunoblot with the indicated antibodies from WT and HIRA KO mESCs whole cell lysates loaded with increased protein concentration (1x, 2x). Blot is representative blot of two independent experiments. d, Average profile of H3K27ac enrichment at enhancers in WT and HIRA KO mESCs. e, Venn diagram showing overlap between H3.3 KO and HIRA KO reduced H3K27ac peaks. f, Average profile of H3K27ac enrichment at enhancers in WT, ATRX KO, and DAXX KO mESCs. g, Schematic of CRISPR/Cas9 strategy for generation of HIRA KO mESCs and immunoblot showing complete loss of HIRA. Blot is representative of four independent experiments. h, Average profile of H3K27ac enrichment in WT and HIRA KO2 mESCs at distal enhancers. i, Venn diagram showing overlap between HIRA KO and HIRA KO2 reduced H3K27ac peaks. j, ChIP-qPCR validation from independent experiments of H3K27ac enrichment at enhancers in all four chaperone KO mESCs line compared to the corresponding WT mESCs.
Related to Fig. 2. a, Quantification of Acetyl-CoA levels from 5×106 cells (Sigma MAK039) in WT and H3.3 KO mESC. Data represented as mean ± s.d. (n = 3), two-tailed t-test, n.s. = 0.9. b, HDAC activity (BioVision K331-100) in WT and H3.3 KO mESC nuclear extracts in the presence or absence of the HDAC inhibitor Sodium Butyrate (NaBut). Data represented as mean ± s.d. (n = 5), two-tailed t-test, n.s. = 0.45. c, Immunoblot of cytoplasmic (C) and nuclear extracts (NE) from WT and H3.3 KO mESCs. Blot is representative of three independent experiments. See also Supplementary Fig. 11b. d, Immunoblot of whole cell lysates from WT mESCs transfected with p300 and CBP shRNA. Blot is representative of four independent experiments. See also Supplementary Fig. 11b. e, Heatmaps and average profiles of H3K27ac (left), ATAC-seq (middle), and p300 (right) enrichment in WT and H3.3 KO mESCs at enhancers. 3kb around the center of enhancers are displayed for each analysis. Each row represents a single enhancer (n =17,589). f, Heatmap and average profile of ATAC-seq in WT and HIRA KO mESCs at enhancers. 3kb around the center of enhancers are displayed for each analysis. Each row represents a single enhancer (n = 17,589).
Related to Fig. 2. a, Boxplots showing H3-Flag-HA (left) and H3K27ac (right) enrichment (ChIP-qPCR) at 10 enhancer regions in H3.3 KO mESCs expressing exogenous histones (H3.2 and H3.3) compared to WT mESCs. (n = 10). The bottom and top of the boxes correspond to the 25th and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers correspond to 1.5× interquartile range and outliers are excluded. b, Immunoblot of whole cell lysates from WT and H3.3 KO mESCs exogenously expressing histones and indicated histone mutants. Blot is representative of two independent experiments. See also Supplementary Fig. 12a for all the blots in this figure. c, Venn diagrams showing drastically reduced H3.3S31ph peaks in WT mESC treated with Chek1inhibitor (Chk1i). p = 6.419 x 10-14 by hypergeometric test. Ctrl peaks (n = 11,259), Cheki peaks (n = 533). d, Violin plot showing H3.3S31ph in WT and mESC treated with Chek1 inhibitor (Chk1i). ***P < 2.2 × 10−16 by Wilcoxon rank sum two-side test (n = 11,259). Violin plots displayed as boxplot in panel a. e, Genome-wide distribution of H3.3S31ph binding sites (percent of total number of sites. H3.3S31ph ChIP-seq identified elements that fell within 300 kb from coding regions were analyzed based on their distance from nearest genes using CEAS. The pie chart represents percent distribution. f, ChIP-qPCR of H3.3S31ph enrichment at enhancers in WT and HIRA2 KO mESCs. Data are expressed as percentage of input and represent mean ± s.d. (n = 8), two-tailed t-test **P = 0.0016. g, Immunoblot of whole cell lysates from WT mESCs treated with indicated concentration of Chk1 kinase inhibitor (SB218078, ‘SB’) or Aurora B kinase inhibitor (ZM447439, ‘ZM’) for 4 hrs. Blot is representative of three independent experiments. h, Immunoblot of whole cell lysates from wild-type mESCs treated with Chk1 inhibitor for the indicated times. Blot is representative of three independent experiments.
Supplementary Figure 5 H3.3-specific phosphorylation stimulates p300 histone acetyltransferase activity.
Related to Fig. 3. a-b, Immunoblot (above) and densitometry (below) of p300 histone acetyltransferase assay using increasing concentrations of recombinant H3.3 (a) or H3.1 (b) nucleosomes that were preincubated with Chk1 with or without ATP. Blot is representative of three independent experiments. See also Supplementary Fig. 12b.
Related to Fig. 4. a,c,e, MA plot of gene expression in WT and H3.3 KO (a), HIRA KO1 (c), HIRA KO2 (e) mESCs. The x-axis indicates gene counts and the y-axis represents the log2 fold change in expression for KO versus WT mESCs. Genes in red and blue were differentially expressed (P < 0.05) with a fold change > 2. b, Venn diagrams showing the relationship between H3K27ac ChIP-seq and RNA-seq for regions that showed upregulation (top) or downregulation (bottom) in the absence of H3.3 in both data sets. Up regulated genes P = 6.419 × 10−14 and down regulated genes P = 3.581 × 10−12 by hypergeometric test. H3K27ac Up-regulated (n = 1,051), RNA-Seq Up-regulated (n = 824), H3K27ac Down-regulated (n = 2,796), RNA-Seq Down-regulated (n = 724). d,f, Venn diagrams showing the relationship between H3K27ac ChIP-seq and RNA-seq for regions that showed downregulation in the absence of H3.3 in HIRA KO cells. Down regulated genes P = 3.581 × 10-12 by hypergeometric test. (d) H3K27ac Down-regulated (n = 2,022), RNA-Seq Down-regulated (n = 704). (f) H3K27ac Down-regulated (n = 1736), RNA-Seq Down-regulated (n = 859). g, Alkaline phosphatase staining of WT and H3.3 KO mESCs in 2i/SL media. Scale bar = 200 μm. Image is representative of three independent experiments. h, Quantification of colony formation assay of WT and H3.3 KO mESCs. i, Growth curve of WT and H3.3 KO mESCs assessed using MTT assay at indicated times. Data represent mean ± s.d (n = 4).
Related to Fig. 4. a, Average profiles of H3.3, H3K4me1, H3K27ac and p300 enrichment in WT mESC at mESC and EBs enhancers. 3kb around the center of enhancers are displayed for each analysis. b, Brightfield images of embryoid body (EB) differentiation in WT and H3.3 KO cells at 4, 8 and 12 days. Scale bar = 1 mm. Image is representative of at least three independent experiments. c, Comparison of average areas of WT and H3.3 KO embryoid bodies at the indicated days of differentiation. Data represent the mean ± s.d., two-tailed t-test ***P = 7.52−53, **P = 0.0068, ***P = 0.00016. d, Heatmap representation of transcript levels (RT-qPCR) of markers of pluripotency (ESC) (Oct4, Nanog), mesoderm (Brachyury, Fgf5), endoderm (Gata4, Gata6) and ectoderm (Cdx2, Otx6) during EBs differentiation. e, Comparison of transcription changes at day 4 of EB differentiation for WT and H3.3 KO cells. Gene list represent genes whose expression decreases or increases in expression upon differentiation compared to WT ESCs. The x- and y-axis represent the fold change (log2) of RNA expression between D4 and D0 in WT and H3.3 KO, respectively. f, Immunoblot of H3.3 and Oct4 in WT and H3.3 KO embryoid bodies at the indicated days of differentiation. Blot is representative of two independent experiments. See also Supplementary Fig. 12c. g, Model figure representing differential requirement for H3.3 during ongoing transcription (left) and the initiation of transcription (right).
Related to Fig. 4. a, Average profiles of H3K27ac enrichment at EB enhancers in H3.3KO EBs expressing the indicated exogenous protein. 3kb around the center of enhancers are displayed for each analysis. b, Brightfield images of EB differentiation in WT and H3.3 KO cells and in H3.3 KO cells expressing exogenous histone mutants at day 4. Scale bar = 1 mm. Image is representative of three independent experiments. c, Average profiles of H3K27ac enrichment at ESC enhancers in H3.3KO EBs expressing the indicated exogenous protein. 3kb around the center of enhancers are displayed for each analysis. d, Schematic of H3K27ac levels in WT and in H3.3 KO cells expressing exogenous histone mutants at day 4 of differentiation at mESC (pluripotency-specific) and EB (differentiation-specific) enhancers.
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Martire, S., Gogate, A.A., Whitmill, A. et al. Phosphorylation of histone H3.3 at serine 31 promotes p300 activity and enhancer acetylation. Nat Genet 51, 941–946 (2019). https://doi.org/10.1038/s41588-019-0428-5
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