H2A.Z facilitates licensing and activation of early replication origins

Abstract

DNA replication is a tightly regulated process that ensures the precise duplication of the genome during the cell cycle1. In eukaryotes, the licensing and activation of replication origins are regulated by both DNA sequence and chromatin features2. However, the chromatin-based regulatory mechanisms remain largely uncharacterized. Here we show that, in HeLa cells, nucleosomes containing the histone variant H2A.Z are enriched with histone H4 that is dimethylated on its lysine 20 residue (H4K20me2) and with bound origin-recognition complex (ORC). In vitro studies show that H2A.Z-containing nucleosomes bind directly to the histone lysine methyltransferase enzyme SUV420H1, promoting H4K20me2 deposition, which is in turn required for ORC1 binding. Genome-wide studies show that signals from H4K20me2, ORC1 and nascent DNA strands co-localize with H2A.Z, and that depletion of H2A.Z results in decreased H4K20me2, ORC1 and nascent-strand signals throughout the genome. H2A.Z-regulated replication origins have a higher firing efficiency and early replication timing compared with other origins. Our results suggest that the histone variant H2A.Z epigenetically regulates the licensing and activation of early replication origins and maintains replication timing through the SUV420H1–H4K20me2–ORC1 axis.

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Fig. 1: H2A.Z interacts with the pre-replication complex.
Fig. 2: H2A.Z binds SUV420H1 to promote H4K20me2 deposition and thereby recruit ORC1.
Fig. 3: H2A.Z regulates the recruitment of ORC1 to chromatin in order to license origins with higher firing efficiency.
Fig. 4: H2A.Z regulates early replication origins and replication timing.

Data availability

ChIP-seq, NS-seq, BrdU-seq and RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE134988. Further information and resources and reagents are available from G.L. upon reasonable request. Source data for Figs. 1, 2, 4, Extended Data Figs. 13, 58 and Supplementary Figures are provided with the paper.

Code availability

Custom codes used for data analysis in this paper can be found at https://github.com/Leavy-Zhang/RepliCode.

Change history

  • 14 January 2020

    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank M. Méchali for advice on nascent-strand sequencing; and B. Stillman and Z. Zhang for critical reading and discussion of the manuscript. This work was supported by grants to G.L. from the Ministry of Science and Technology of China (2017YFA0504202), the National Natural Science Foundation of China (31525013, 31630041 and 31521002), and the Chinese Academy of Sciences (CAS) Strategic Priority Research Program (XDB19040202). The work was also supported by the CAS Key Research Program on Frontier Science (QYZDY-SSW-SMC020) and a Howard Hughes Medical Institute (HHMI) international research scholar grant (55008737) to G.L. This work was also supported by a grant from the National Natural Science Foundation of China to L.Z. (31801062); the China Postdoctoral Science Foundation to Z.W. (2019M650871); the Ministry of Science and Technology of China (2018YFE0203302) and the National Natural Science Foundation of China (31871290) to P.C.; and the Ministry of Science and Technology of China to J.P. (2016YFA05023032). All fluorescence imaging data were collected at the Center for Bio-imaging, Core Facility for Protein Sciences (Institute of Biophysics, CAS). All radioactivity experiments were performed at the radioactive isotope laboratory (Institute of Biophysics, CAS), with guidance from H. J. Zhang in handling radioactive materials.

Author information

H.L. carried out experiments and composed the figures. L.Z. performed bioinformatics analysis of next-generation sequencing data and generated figures. Z.W. analysed the phenotypic effects of the H2A.Z-knockdown on cell-cycle deficiency, and assisted with ChIP-seq experiments and the analysis of next-generation sequencing data. M.L. analysed the phenotype of H2A.Z CKO mice and provided T cells for biochemistry experiments. W.Z. and H.D. performed mass-spectrometry analysis of the H4K20 modification of histone methyltransferase products, mononucleosomes in vivo and cells treated with siNC or siHA.Z oligonucleotides. X.C. and F.Y. performed mass-spectrometry analysis of H2A.Z nucleosome binding partners. P.Z. and X.W. assisted with the quantification of the histone methyltransferase reaction, plasmid construction, and protein purification. T.L. and J.P. performed the in silico analysis of the interaction between H2A.Z and SUV420H1. L.C. assisted with the analysis of H2A.Z and H4K20me2 sequencing data. C.J. constructed the H2A.Z CKO mice and assisted with their phenotype analysis. G.W. assisted with plasmid construction and protein purification. P.C. and R.M. helped to discuss the project. M.Z. conceived the project on the phenotype of H2A.Z CKO mice. G.L. conceived and supervised the project, analysed the data and wrote the manuscript.

Correspondence to Mingzhao Zhu or Guohong Li.

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Extended data figures and tables

Extended Data Fig. 1 Analysis of phenotypes and gene expression after H2AFZ knockdown, and analysis of the interaction between H2A.Z and ORC1.

a, Statistical outcome of FACS analysis of apoptosis of cells treated with siNC or siH2A.Z oligonucleotides. Annexin is used as a marker of apoptotic cells; DAPI is 4′,6-diamidino-2-phenylindole, a nuclear marker. b, Representative images of cells undergoing senescence (within boxed regions), shown by β-galactosidase staining. c, Cell-cycle analysis of siNC or siH2A.Z HeLa cells. Cells were pulse labelled with BrdU and stained with propidium iodide (PI), and then analysed by FACS. d, Western blots showing the H2A.Z level when cells are released from arrest at G2/M phase after treatment with IAA. AID, auxin-inducible degron. e, Volcano plot showing genome-wide expression dynamics after H2AFZ knockdown; n = 33,835. Genes with a log2(fold change) of 1 or more and P-values of less than 0.01 were selected as differentially expressed genes. f, Real-time PCR analysis of gene expression in siNC or siH2A.Z cells. The expression level was normalized to that of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). g, Left, diagram showing the LacO/LacI targeting system in A03_1 cells. An interaction between a LacI–Cherry-tagged histone and an enhanced green fluorescent protein (EGFP)-tagged ORC protein would result in an overlap of green fluorescence with red fluorescence. Right, there is no interaction in this experiment between ORC1 and H2A or H2A.Z. Scale bar, 5 μm. h, Western blot analysis of the enrichment of H4K20me2, H3K36me3 (negative control), SUV420H1, ORC1, ORC2 and MCM2 on H2A or H2A.Z nucleosomes from G1-phase-synchronized cells. Data in panel a are means; n = 2 biological replicates, with dot plots overlaid. Data in panels c, f are means ± s.e.m.; n = 3 biological replicates; two-tailed unpaired t-test. The β-galactosidase staining in panel b, the FACS results in c, the western blots in d, h, and the fluorescence image in g were independently repeated three times with similar results. H4 was used as a loading control and sample processing control in d and h. For gel source data, see Supplementary Fig. 1. For imaging source data, see Supplementary Fig. 2. For the FACS gating strategy, see Supplementary Fig. 3. Source data

Extended Data Fig. 2 H2A.Z interacts with ORC1 in an H4K20me2-dependent manner.

a, b, High-performance liquid chromatography (HPLC) of unmodified H4 peptide and H4K20me2 peptide in H2A (a) and H2A.Z (b) nucleosomes. c, Western blot analysis showing the signal of SUV420H1 and H4K20me1/2/3 in shGFP cells or shSUV420H1 cells with stably expressed Flag-tagged H2A or H2A.Z. d, Real-time PCR analysis shows the level of expression of SUV420H1 and SUV420H2 in cells transfected with shRNA targeting GFP, SUV420H1 or SUV420H2. The expression level was normalized to that of GAPDH. e, Left, enrichment of ORC1 on H2A or H2A.Z mononucleosome from wild-type or SUV420H2-knockdown cells. Right, enrichment of ORC1 on H2A.Z mononucleosomes from wild-type or SUV420H1/2-knockdown cells. Data in panel d are means ± s.e.m.; n = 3 biological replicates; two-tailed unpaired t-test. The experiment in panel a was independently repeated four times with similar results. The experiments in panels c, e were independently repeated twice with similar results. H4 was used as a loading control and sample processing control in c and e, respectively. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 3 H2A.Z enhances the binding of SUV420H1 to promote its enzymatic activity.

a, Liquid scintillation results from analysis of SUV420H1 histone methyltransferase activity, using H2A or H2A.Z mononucleosomes as substrates (n = 3 biological replicates). b, Mass-spectrometry analysis of H4K20me2 modification by SUV420H1, using H2A or H2A.Z mononucleosomes as substrates. c, Western blot analysis of products from histone methyltransferase assay of SUV420H1 using H2A or H2A.Z mononucleosomes as substrates. IB, immunoblot. d, Left, mass-spectrometry analysis of monomethylated and unmethylated H4 histones from chemical methylation reactions in vitro. Right, western blot analysis and 3H autography show that H2A.Z promotes the activity of SUV420H1 on an H4KC20me1 substrate. e, Upper panel, diagram showing four chimaeric mutants of H2A.Z, with the regions in red replaced with the corresponding regions of H2A. The sequences of the region containing D97 and S98 of H2A.Z and the corresponding region of H2A are shown below the diagram (in single-letter code). Lower panel, 3H autograph and liquid scintillation analysis of the methyltransferase activity of SUV420H1 on mononucleosomes containing H2A.Z chimaeric mutants. f, Western blot analysis following the pulldown of biotinylated mononucleosomes shows an interaction between SUV420H1 and mononucleosomes containing wild-type H2A.Z or the H2A.ZD97N/S98K mutant. g, The interaction between SUV420H1 and H2A, H2A.Z or H2A.ZD97N/S98K was analysed by LacO/LacI targeting. Scale bar, 5 μm. h, Statistical results from the LacO/LacI targeting assay of panel g, showing the percentage of cells in which EGFP–SUV420H1 co-localizes with the indicated histones. Data in panel a are means ± s.d.; n = 3 biological replicates. Data in panel b are means; n = 2 biological replicates, with dot plot overlaid. Data in panels e, h are means ± s.e.m.; n = 3 biological replicates; two-tailed unpaired t-test. The western blots in panels c, d, f, the 3H autography in panels d, e, the mass spectrometry in panel d and the fluorescence imaging in panel g were independently repeated three times with similar results. H4 was used as a loading control and sample processing control in panels cf. For gel source data, see Supplementary Fig. 1. For imaging source data, see Supplementary Fig. 2. Source data

Extended Data Fig. 4 H4K20me2 dosage dependent interaction between ORC1 and H4K20me2 nucleosomes.

a, b, Docking of the SUV420H1 and H2A.Z nucleosome structures shows the interaction between R257 of SUV420H1 and E64 of H4 (a), and the interaction between K333 of SUV420H1 and D97 of H2A.Z (b). c, Western blots and 3H autography analysis of the products of a histone methyltransferase assay using wild-type SUV420H1 or SUV420H1 mutants. d, Mass-spectrometry analysis of H4 histones with dimethylated KC20, produced through chemical methylation reactions in vitro; mass spectrometry was performed once to confirm the methylation state. e, Western blot analysis of the interaction between H2A.Z mononucleosomes with the H4KC20me2 modification and ORC1 or ORC1 BAH-domain mutants. f, Western blots show the interaction between ORC1 and H2A or H2A.Z mononucleosomes with different H4KC20me2 states. g, Western blots show the interaction between ORC1 and H2A.Z polynucleosomes with increasing ratios of a H4KC20me2 octamer. The western blots in panels c, eg and the 3H autography in panel c were independently repeated twice with similar results. H4 was used as a loading control in panel c. H3 was used as a loading control in panels eg. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 5 H2A.Z regulates H4K20me2 and ORC1 on a genome-wide level.

a, Box plot showing the dynamics of H4K20me2 at H4K20me2 peak regions (n = 99,574) after knocking down both SUV420H1 and SUV420H2. b, Venn diagram showing the overlap between H4K20me1, H4K20me2 and H4K20me3 peaks genome wide. c, Heat maps and corresponding box plots showing the dynamics of H4K20me1, H4K20me2 and H4K20me3 at 10-kilobase regions around the centres of the H4K20me2 peaks (n = 99,574). d, e, Dot plot showing a positive correlation between H2A.Z and H4K20me2 (d) and H2A.Z and ORC1 (e) at H2A.Z peaks (n = 58,642). r, Pearson’s correlation coefficient. f, Mass-spectrometry analysis of chromatin H4K20me2 abundance after H2AFZ knockdown. g, Western blot analysis of ORC1, SUV420H1, MCM2, H4K20me1, H4K20me2 and H4K20me3 in the chromatin fraction from cells arrested at G1 phase. h, Box plot showing the nascent-strand (NS) signal of RNase-treated and untreated samples from the NS peaks (n = 41,850). i, j, Dot plot showing the dynamics of H4K20me2 (i; n = 99,574) and ORC1 (j; n = 100,917) after knocking down H2AFZ. k, Genome-wide distribution of plus-ZD-ORC1 peaks (n = 20,978). UTR, untranslated region. Data in panels a, c, h were analysed by two-tailed Wilcoxon test. Data in panel f are means (n = 2 biological replicates) with dot plots overlaid. Data in panels i, j were analysed by one-sided Fisher’s exact test without adjustments for multiple comparisons. The western blots in panel g were independently repeated twice with similar results, and H4 was used as a loading control and sample processing control. For the box plots in panels a, c, the centre lines represent the medians, the box limits are the 25th and 75th percentiles, and the whiskers are the minimum to maximum values. For the violin plots in panel h, the centres of the boxes represent the medians, the box limits are the 25th and 75th percentiles, and the upper and lower limits show the 95% confidence interval. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 6 H2A.Z regulates early-replication origins and replication timing.

a, Venn diagram showing the overlap between early-replication domains identified from our BrdU-IP-seq data (n = 3,362) and Repli-seq data sets in ref. 21 (n = 4,727). b, Distribution of ORC1 peaks and the 10-min BrdU signal (labelled in cells immediately after release from G1/S arrest) in length-normalized early-replication domains. c, Real-time PCR analysis of ChIP signals from H2A.Z, H4K20me2 and ORC1, and nascent-strand signals in siNC or siH2A.Z cells at target 3 (an early replication origin). d, Dot plot showing the correlation between the replication timing of replication domains (n = 3,362) identified by the 10-min BrdU signal and the dynamics of the 10-min BrdU signal after H2AFZ knockdown. r, Pearson’s correlation coefficient. e, Genome tracks of an early-replication domain show the decreased 10-min BrdU signal after H2AFZ knockdown. RT, replication timing. Data in panel c are means ± s.e.m.; n = 3 biological replicates; two-tailed unpaired t-test. The BrdU-seq experiments in panels b, e were independently repeated twice with similar results. Source data

Extended Data Fig. 7 H2A.Z regulates early-replication origins through SUV420H1.

a, FACS analysis of cell-cycle progression for siNC and siH2A.Z cells released from G1/S arrest. b, Statistical results from panel a. To quantify cell-cycle progression, we normalized the peak DAPI signal of all time points to the peak DAPI signal at 0 h (G1/S arrest). We defined the DAPI signal at the G1/S boundary as value 1; when cells entered G2/M phase, the value is near 2. c, Western blot analysis of H4K20me levels in SUV420H1-knockdown cells rescued by wild-type or mutant (R257A, K333A) SUV420H1. d, H4K20me2, NS and BrdU levels at early origins (targets 1, 3) and late origins (target 2) in SUV420H1-knockdown (‘Sh’) cells rescued by wild-type or mutant SUV420H1. Data in panels b, d are means ± s.e.m.; n = 4 biological replicates in b; n = 3 biological replicates in d; two-tailed unpaired t-test. The FACS experiment in panel a was independently repeated four times with similar results. The western blots in panel c were independently repeated three times with similar results. H4 was used as a loading control and sample processing control in panel c. For gel source data, see Supplementary Fig. 1. For the FACS gating strategy, see Supplementary Fig. 3. Source data

Extended Data Fig. 8 H2A.Z is essential for DNA replication and cell proliferation during T-cell activation.

a, Diagram showing the construction of mice with H2A.Z conditionally knocked out in T cells. b, FACS analysis of T cells and B cells from the spleen of wild-type (H2A.Zf/f) or H2A.Z CKO (CD4creH2A.Zf/f) mice. c, Statistical analysis of T-cell numbers from panel b. d, Left, FACS analysis of BrdU incorporation in CD8+ T cells from the lymph node of wild-type and H2A.Z CKO mixed bone-marrow chimaeric mice. Right, statistical analysis of the percentage of BrdU-incorporating cells. FSC, forward scatter. e, FACS analysis of CFSE dilution in wild-type and H2A.Z CKO CD4+ T cells upon anti-CD3 and anti-CD28 stimulation for 72 h. f, Statistical analysis of the percentage of divided cells from panel e. g, Cell-cycle analysis of wild-type and H2A.Z CKO CD4+ T cells upon anti-CD3 and anti-CD28 stimulation for 48 h. PI, propidium iodide. h, Statistical analysis of cell-cycle distribution from panel g. i, Heat map and box plots showing the H4K20me2 and NS signals in 10-kb regions around H4K20me2 peaks (n = 53,788) and NS peaks (n = 26,283). j, Western blots showing H4K20me2 levels in active T cells from wild-type and H2A.Z CKO mice. k, Box plots showing H2A.Z, H4K20me2 and NS signals in ZD-NS (n = 18,382) and ZI-NS (n = 7,901) regions. Data in panels c, d, f and h are means ± s.e.m.; n = 4 biological replicates in panel c; n = 5 biological replicates in panel d; n = 3 biological replicates in panels f, h; two-tailed unpaired t-test. The data in panels i, k were analysed by two-tailed paired t-test and two-tailed unpaired t-test, respectively. FACS analyses in panels b, d, e, g were independently repeated four, five, three and three times, respectively, with similar results. The western blots in panel j were independently repeated three times with similar results, and H4 was used as a loading control and sample processing control. For the box plots in panels i, k, the centre lines represent medians, the box limits are the 25th and 75th percentiles, and the whiskers are the minimum to maximum values. For gel source data, see Supplementary Fig. 1. For FACS gating strategy, see Supplementary Fig. 3. Source data

Extended Data Table 1 Enriched DNA motifs in H2A.Z-regulated replication origins

Supplementary information

Supplementary Information

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Reporting Summary

Supplementary Figure

Supplementary Figure 1: Original gel source data. This figure contains original gel source data for the western bolts, commassie staining and 3H autograph results, with molecular weight markers and an indication of how the gels were cropped.

Supplementary Figure

Supplementary Figure 2: Original imaging data. This figure contains original fluorescence and imaging data with indication of how the regions of interests are selected.

Supplementary Figure

Supplementary Figure 3: FACS gating strategy. This figure shows the gating strategy for FACS data analysis, and the sorting gate strategy for naive T cells.

Supplementary Table

Supplementary Table 1: Mass spectrometry data of peptides immunoprecipitated by H2A or H2A.Z nucleosomes. Flag-H2A or Flag-H2A.Z nucleosomes were precipitated from HeLa cells, and the samples were analysed by mass spectrometry. The proteins identified for at least one time from three biological replicates of either H2A or H2A.Z nucleosome were included, and the number of unique peptide identified from each time were listed

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Long, H., Zhang, L., Lv, M. et al. H2A.Z facilitates licensing and activation of early replication origins. Nature 577, 576–581 (2020). https://doi.org/10.1038/s41586-019-1877-9

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