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H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins

Nature Structural & Molecular Biology volume 30, pages 800–811 (2023)Cite this article

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Abstract

The transmission and maintenance of genetic information in eukaryotic cells relies on the faithful duplication of the entire genome. In each round of division, excessive replication origins are licensed, with only a fraction activated to give rise to bi-directional replication forks in the context of chromatin. However, it remains elusive how eukaryotic replication origins are selectively activated. Here we demonstrate that O-GlcNAc transferase (OGT) enhances replication initiation by catalyzing H4S47 O-GlcNAcylation. Mutation of H4S47 impairs DBF4-dependent protein kinase (DDK) recruitment on chromatin, causing reduced phosphorylation of the replicative helicase mini-chromosome maintenance (MCM) complex and compromised DNA unwinding. Our short nascent-strand sequencing results further confirm the importance of H4S47 O-GlcNAcylation in origin activation. We propose that H4S47 O-GlcNAcylation directs origin activation through facilitating MCM phosphorylation, and this may shed light on the control of replication efficiency by chromatin environment.

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Fig. 1: H4S47 O-GlcNAcylation is required for efficient DNA replication.
Fig. 2: OGT regulates DNA replication through H4S47 O-GlcNAcylation.
Fig. 3: H4S47 O-GlcNAcylation facilitates origin activation by regulating MCMs.
Fig. 4: H4S47 O-GlcNAcylation recruits DDK through direct interaction with DBF4.
Fig. 5: S47 O-GlcNAcylation orients MCM association with H4 on chromatin.
Fig. 6: H4S47 O-GlcNAcylation fosters DNA unwinding for origin activation.
Fig. 7: Genome-wide investigation of H4S47 O-GlcNAcylation function at origins.

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Data availability

All the genome-wide sequencing datasets generated and analyzed during the current study are submitted to Gene Expression Omnibus (GEO) repository under the GEO accession number GSE205673. Human genome (hg38) was accessed from https://hgdownload.soe.ucsc.edu/goldenPath/hg38. Mouse genome (mm10) was accessed from https://hgdownload.soe.ucsc.edu/goldenPath/mm10. The mixed hg38-mm10 genome was made by concatenated fasta files of human genome (hg38) and mouse genome (mm10), with chromosome names of mouse genome modified to have the prefix ‘mm10_’. The concatenated fasta file was used to build bowtie2 reference using bowtie-build63. The fasta index file was generated using samtools faidx68 and can be found at https://github.com/maximewen/custom_ref. Source data are provided with this paper.

Code availability

The custom python script used for filtering pair-end reads can be found at https://github.com/maximewen/DNA_replication.

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Acknowledgements

We thank W.G. Dunphy (Division of Biology and Biological Engineering, California Institute of Technology) and Y. Gu (School of Medicine and Pharmacy, Ocean University of China) for providing helpful reagents. We acknowledge S. Lee (College of Pharmacy, Taipei Medical University), C. Xu, and L. Gong (School of Life Sciences, Northeast Normal University) for technical advice. This work was supported by grants from the National Natural Science Foundation of China (31600645 and 32070758 to Y.F., 31870896 to W.M., 32000412 to H.L., and 32000423 to Z.W). This work was also supported by the Ministry of Science and Technology of China (2022YFA1302801 to H.L.).

Author information

Author notes

  1. These authors contributed equally: Yingying Zou, Jiayao Pei, Haizhen Long.

Authors and Affiliations

  1. Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China

    Yingying Zou, Jiayao Pei, Kejian Dong, Tingting Wang, Ming Li, Zhexuan Zhao, Lirun Zhu, Gangxuan Zhang, Xin Jin, Yang Wang, Min Wei & Yunpeng Feng

  2. Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China

    Haizhen Long

  3. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Liting Lan

  4. University of Chinese Academy of Sciences, Beijing, China

    Liting Lan

  5. School of Medicine, Sun Yat-sen University, Shenzhen, China

    Zengqi Wen

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  1. Yingying Zou
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Contributions

Y.Z. and J.P. conducted the majority of the experiments and analyzed the data. H.L., L.L., and Z.W. carried out SNS-seq, Repli-seq and associated data analysis. K.D. performed immunofluorescence. J.P. and G.Z. assisted with chromatin immunoprecipitation. T.W. and L.Z. performed the DNA fiber assay. M.L. constructed Flag-H4WT, Flag-H4S47A, Flag-OGT, and GFP-OGT plasmids. Z.Z. analyzed mass spectrometry data. X.J. and Y.W. provided support in flow cytometry. Y.Z., M.W., and Y.F. wrote the paper. Y.F. supervised the project. All authors discussed the results and commented on the paper.

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Correspondence to Yunpeng Feng.

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Nature Structural & Molecular Biology thanks John Hanover and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Beth Moorefield, Carolina Perdigoto and Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 H4S47 O-GlcNAcylation regulates DNA replication.

a, Detection of H4S47 O-GlcNAcylation by mass spectrometry. Histone H4 extracted from HEK293T cells were analyzed by mass spectrometry. Tandem mass spectrum of peptides with O-GlcNAcylation on S47 of H4 is shown. The y and b fragmentations were used to map the O-GlcNAcylation site. b, Quantification of H4 O-GlcNAcylation. WB from Fig. 1a were quantified. n = 4 biologically independent experiments. P values were calculated by unpaired, two-tailed t-tests, means ± s.d., n.s., non-significant. a.u., arbitrary unit. c, Functional assessment of mutations at H4S47 in DNA replication. HEK293T cells were transfected with Flag-H4WT, Flag-H4S47A for 24 h and cultured with or without PUG (100 μM) for another 24 h prior to EdU staining and FACS analysis. d, e, Evaluation of replication efficiency in HT1080 and A549 cells. HT1080 (d) or A549 (e) cells were transfected and treated as in Fig. 1a before the analyse of replication efficiency by immunofluorescence. Representative images (left) and associated quantifications (right) are demonstrated. Cells positive for both PCNA and Flag were marked with dashed lines. From left, n = 1,981, 2,232, 1,553, 1,181 (d) and n = 821, 963, 827, 1,306 (e) cells. P values were calculated by unpaired, two-tailed t-tests. For all box plots, the bottom, middle line and top of the box and whiskers indicate the 25th, 50th, 75th and 10–90th percentiles, respectively and means were shown as red ‘+’ signs. n.s., non-significant. Scale bar, 10 μm. a.u., arbitrary unit.

Source data

Extended Data Fig. 2 H4S47 O-GlcNAcylation influences cell proliferation.

a-c, Impact from H4S47 O-GlcNAcylation on cell proliferation. HEK293T (a), HT1080 (b) and A549 (c) cells were transfected with Flag-H4WT or -H4S47A and incubated with or without PUG (100 μM). Cell numbers were counted every 24 h. n = 3 biologically independent experiments. Two-way ANOVA Tukey’s multiple comparisons test, means ± s.d., n.s., non-significant. d, Functional assessment of mutations at H4S47 in DNA replication. HEK293T cells were transfected with Flag-H4WT, Flag-H4S47A, Flag-H4S47D or Flag-H4S47E for 48 h prior to the detection of replication efficiency by FACS analysis. e, DNA fiber assay for replication fork function. Frequency distribution of CIdU track lengths (left) and inter-origin distances (right) for experiments in Fig. 1d were shown. f, Assessment of DNA damage response. HEK293T cells transfected and treated as in Fig. 1a were analyzed by WB with indicated antibodies. HU (3 mM) treatment for 3 h was included as a positive control.

Source data

Extended Data Fig. 3 H4S47 O-GlcNAcylation does not affect cell viability.

a, b, Analysis of cell survival and apoptosis. HEK293T cells were transfected and treated as in Extended Data Fig. 2a. Etoposide (10 μM) treatment for 24 h was used as a positive control. Following staining with Annexin V and PI, cells were analyzed by flow cytometry. Representative FACS profiles (a) and quantifications of apoptotic cells (b, left) and viable cells (b, right) are demonstrated. n = 3 biologically independent experiments. P values were calculated by Tukey’s multiple comparison test (two-way ANOVA), means ± s.d., n.s., non-significant.

Source data

Extended Data Fig. 4 OGT and OGA distribution during the cell cycle progression.

a, b, Examination of proteins in cells synchronized at different stages of the cell cycle. HEK293T cells synchronized at G1/S were released and collected at indicated time points. Experimental setup and FACS profiles depicting the cell cycle progression were demonstrated (top). Both total proteins (a) and chromatin pellets (b) were analyzed by WB (bottom). Coomassie-stained total histones, H4 and β-actin served as loading controls. Asyn, asynchronization. H4S47ph, H4 phosphorylated at S47.

Source data

Extended Data Fig. 5 OGT promotes DNA replication through H4S47 O-GlcNAcylation.

a, Quantification of H4 O-GlcNAcylation levels. WB from Fig. 2d were quantified. n = 4 biologically independent experiments. P values measured by unpaired, two-tailed t-tests, means ± s.d., n.s., non-significant. a.u., arbitrary unit. b, Evaluation of replication efficiency by EdU incorporation. HEK293T cells were transfected with p3 × Flag-CMV-10 (empty vector) or Flag-OGT for 48 h prior to the detection of replication efficiency with immunofluorescence analysis. HU (3 mM) treatment for 3 h was used as a positive control. Representative images (left) and quantifications of EdU intensities of PCNA-positive cells (right) were demonstrated. From left, n = 1,551, 1,063, 1,234 cells. P values were calculated by unpaired, two-tailed t-tests. For all box plots, the bottom, middle line and top of the box and whiskers indicate the 25th, 50th, 75th and 10–90th percentiles, respectively and means were shown as red ‘+’ signs. Scale bar, 10 μm. a.u., arbitrary unit. c, Analysis of protein O-GlcNAcylation. HEK293T cells were incubated with or without OSMI-1 (50 μM) for 24 h and analyzed by WB with CTD110.6 antibody. d, Examination of EdU intensity by FACS. HEK293T cells were treated as described in (c) and pulsed with EdU for 30 min. HU treatment (3 mM) for 3 h was included as a positive control. e, Representative images from Fig. 2e. Cells positive for Flag (H4) were selected for the quantification of EdU intensities. Scale bar, 10 μm. f, g, DNA fiber assay. Experimental setup (f, top), representative images (f, bottom) as well as frequency distribution of CIdU track lengths (g, left) and inter-origin distances (g, right) for Fig. 2f were shown. Scale bar, 10 μm.

Source data

Extended Data Fig. 6 Functional evaluation of H4S47 O-GlcNAcylation in cells challenged with replication stresses.

a, b, Impact from Glc or Gln deficiency on DNA replication. From representative experiments included in Fig. 2g (a) and Fig. 2h (b), mean intensities of EdU of Flag- and PCNA-positive cells were shown. From left, n = 4,896, 3,249, 4,968, 3,316, 4,488, 3,083, 5,131, 2,413 (a) and n = 8,404, 2,180, 4,397, 2,514, 8,862, 3,090, 7,592, 1,599, 4,413, 1,801 (b) cells. c-f, Influences from HU or CPT on H4 O-GlcNAcylation and DNA replication. HEK293T cells were transfected as in Fig. 1a for 24 h, and then cultured in medium containing HU (c, d) or CPT (e, f) at different concentrations as indicated for another 24 h. Subsequently, cells were subjected to Flag IP for the detection of H4 O-GlcNAcylation (c, e, top) and immunofluorescence as in Fig. 1c for replication efficiency analysis (c, e, bottom). Different samples were loaded on the same gel, but not in adjacent lanes (e, top). Curves (c, e, bottom) based on means of EdU intensities from cells positive for both Flag and PCNA were shown. n = 3 (c, e, bottom) biologically independent experiments. For curves, P values calculated by Sidak’s multiple comparison test (two-way ANOVA) (c, e, bottom). From left, n = 2,074, 2,878, 1,707, 2,467, 1,185, 2,987, 2488, 2,819 (d) and n = 1,714, 2,244, 3,790, 1,214, 1,968, 432, 476, 670 (f) cells. For all box plots, P values were calculated by Tukey’s multiple comparison test (one-way ANOVA of unpaired t-tests); The bottom, middle line and top of the box and whiskers indicate the 25th, 50th, 75th and 10–90th percentiles, respectively and means were shown as ‘+’ signs (a, b, d and f). n.s., non-significant, a.u., arbitrary unit.

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Extended Data Fig. 7 Confirmation of the regulation of MCM complex by H4S47 O-GlcNAcylation.

a, b, Assay for subunits in MCM complex associating with H4. HT1080 (a) or A549 (b) cells were transfected and treated as in Fig. 1a. IgG served as a negative control. Representative experiment (left) and quantification of MCM2/6 levels were shown (right). n = 3 (a), n = 4 (b) biologically independent experiments. P values were calculated by Tukey’s multiple comparison test (two-way ANOVA), means ± s.d., n.s., non-significant. c, d, Examination of MCM2 pS53 in chromatin fraction. HT1080 (c) or A549 (d) cells were transfected and treated as in Fig. 1a. Chromatin pellets were assayed with indicated antibodies. Representative experiment (left) and quantification of MCM2 pS53 levels were shown (right). n = 3 biologically independent experiments. P values were measured by paired, two-tailed t-tests, means ± s.d., n.s., non-significant. a.u., arbitrary unit.

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Extended Data Fig. 8 Confirmation of DDK function in the regulation of DNA replication by H4S47 O-GlcNAcylation.

a, Analysis of CDK2 and Cyclin E association with H4. HEK293T cells were transfected and treated as in Fig. 1a. IgG served as a negative control. b, c, Quantification of DBF4 and CDC7. WB from Fig. 4b (b) and Fig. 4c (c) were quantified, respectively. n = 3 biologically independent experiments. d, In vitro assay of H4 O-GlcNAcylation. His-H4WT and -H4S47A isolated from BL21 competent E. coli, which co-expressed OGT, were analyzed by WB with O-GlcNAcylation antibody CTD110.6. e, Quantification of DBF4, CDC7 and MCM2 pS53 levels for experiments in Fig. 4e. n = 3 biologically independent experiments. f, Overexpression of DBF4 enhances DNA replication. GFP-DBF4 or pEGFP-N1 (empty vector) was transfected along with Flag-H4WT or -H4S47A into HEK293T cells for 48 h prior to the detection of replication efficiency. From left, n = 4,183, 4,882, 3,133, 6,626 cells. P values were calculated by unpaired, two-tailed t-tests. For all box plots, the bottom, middle line and top of the box and whiskers indicate the 25th, 50th, 75th and 10–90th percentiles, respectively and means were shown as red ‘+’ signs. g, Quantification of MCM2/7 and DBF4 for experiments in Fig. 5c. n = 3 biologically independent experiments. h, DNA fiber assay. Experimental setup (left) and frequency distribution of inter-origin distances (right) for Fig. 6a were shown. i, Detection of H4S47 O-GlcNAcylation by mass spectrometry. Tandem mass spectrum of peptides containing S47 O-GlcNAcylation from experiments described in Fig. 6d was shown. The y, a and b fragmentations were used to map the O-GlcNAcylation site. P values calculated by Tukey’s multiple comparison test (two-way ANOVA), means ± s.d., n.s., non-significant (b, c, e and g). a.u., arbitrary unit.

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Extended Data Fig. 9 Genome-wide investigation of H4S47 O-GlcNAcylation function in origin activation.

a, Heat maps of SNS signals. HEK293T cells were prepared as in Fig. 1a. Heat maps demonstrating SNS signals from RNase-treated and untreated samples. n = 37,590 (H4WT), n = 35,625 (H4WT + PUG), n = 29,103 (H4S47A), n = 27,034 (H4WT + PUG) peaks. b, Box plots of SNS signals among H4WT peaks. n = 37,590 peaks. RRPM, reference-adjusted reads per million. P values were calculated by unpaired, two-tailed t-tests using R. For box plots, the bottom, middle line and top of the box and whiskers indicate the 25th, 50th, 75th percentiles and minimum to maximum, respectively. c, Venn diagram showing the overlap among origins of H4WT and origins upregulated by PUG treatment. d, Enriched DNA motifs in replication origins. P values calculated by cumulative binomial distributions. e, Genome-wide distribution of SNS peaks. n = 37,590. UTR, untranslated region. TTS, transcriptional terminate sites. f, Enrichment of MCM6 at replication origins. HEK293T cells incubated with or without PUG (100 μM) for 24 h were assayed by ChIP with antibody against MCM6. The y axis is the ratio of DNA in immunoprecipitate to that in input. n = 4 (TOP1), n = 3 (MCM4), n = 3 (ChrX (ENm006)) biologically independent experiments. g, h, Association of Flag (H4) and MCM6 at replication origins. HEK293T cells transfected and treated as in Fig. 1a were analyzed by ChIP using antibody against Flag (H4) (g) or MCM6 (h). The y axis is the ratio of DNA in the immunoprecipitate to that in the input. n = 4 (g, TOP1), n = 3 (g, MCM4), n = 4 (g, ChrX (ENm006)), n = 3 (h) biologically independent experiments. Two-way ANOVA Tukey’s multiple comparisons test, means ± s.d., n.s., non-significant (f-h).

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Extended Data Fig. 10 Genome-wide evaluation of H4S47 O-GlcNAcylation function in replication timing.

a-i, Functional comparison between H4WT and H4S47A for replication timing. HEK293T and HT1080 cells were transfected as in Fig. 1a (b, d, e, f) and treated with PUG (100 μM) for 24 h (c, g, h, i). Following BrdU (100 μM) pulse-labelling for 2 h, S1, S2, S3 and S4 fractions (a) were isolated for genomic sequencing. b, c, Integrative Genomics Viewer (IGV) snapshots of the Log2 E/L (Early/Late) Repli-seq plots. Raw RT data (Log2 E/L ratio) is shown for a chromosomal region. d, g, Dot plot of replication timing values. RDs (replication timing domains) defined in wild type HEK293T (d, n = 2,493; g, n = 2,579) and HT1080 (d, n = 2,275; g, n = 2,245) were demonstrated. e, f, h, i, Analysis of replication timing values of early/late timing domains. Meta profiles show replication timing values of the length percentage aligned early (e, h) and late (f, i) timing domains. Replication timing domains in HEK293T (e, n = 1,129; h, n = 1,209) and HT1080 (e, n = 1,002; h, n = 963) with replication timing value > 0 are defined as early RDs (e, h, left). Replication timing domains in HEK293T (f, n = 1,356; i, n = 1,389) and HT1080 (f, n = 1,266; i, n = 1,237) with replication timing value < 0 are defined as late RDs (f, i, left). Heatmaps show replication timing values of both early (e, h, right) and late (f, i, right) timing domains in HEK293T and HT1080 cells. Replication timing value was counted 5 Mb upstream and downstream of the centre of each early or late timing domain. RT, replication timing; RD, replication timing domain; Mb, mega base pair; Chr1, chromosome 1.

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Zou, Y., Pei, J., Long, H. et al. H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins. Nat Struct Mol Biol 30, 800–811 (2023). https://doi.org/10.1038/s41594-023-00998-6

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