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Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription

Abstract

Nucleosomes represent a challenge in regard to transcription. Histone eviction enables RNA polymerase II (RNAPII) progression through DNA, but compromises chromatin integrity. Here, we used the SNAP-tag system to distinguish new and old histones and monitor chromatin reassembly coupled to transcription in human cells. We uncovered a transcription-dependent loss of old histone variants H3.1 and H3.3. At transcriptionally active domains, H3.3 enrichment reflected both old H3.3 retention and new deposition. Mechanistically, we found that the histone regulator A (HIRA) chaperone is critical to processing both new and old H3.3 via different pathways. De novo H3.3 deposition is totally dependent on HIRA trimerization as well as on its partner ubinuclein 1 (UBN1), while antisilencing function 1 (ASF1) interaction with HIRA can be bypassed. By contrast, recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows absolute dependency on ASF1–HIRA interaction. We propose a model whereby HIRA coordinates these distinct pathways during transcription to fine-tune chromatin states.

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Fig. 1: Short-term old H3.3 loss is dependent on transcriptional activity.
Fig. 2: H3.3 is enriched and dynamically exchanged at transcriptionally active domains.
Fig. 3: HIRA is required for both deposition of new H3.3 and retention of old H3.3 at transcriptionally active domains.
Fig. 4: Transcription is required for HIRA-dependent H3.3 recycling.
Fig. 5: Different partnerships for HIRA in new H3.3 deposition versus recycling.
Fig. 6: HIRA–ASF1 interaction is required for old H3.3 recycling.
Fig. 7: The HIRA complex coordinates deposition of new H3.3 via UBN1 and recycling of old H3.3 via ASF1.

Data availability

The raw image dataset on which this paper is based is too large to be rendered available via public repositories, but is fully available upon request. Associated analysis code is also available upon request. All measurements reported in the figures are available as Source data files.

References

  1. Luger, K. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251–260 (1997).

    CAS  PubMed  Google Scholar 

  2. Knezetic, J. A. & Luse, D. S. The presence of nucleosomes on a DNA template prevents initiation by RNA polymerase II in vitro. Cell 45, 95–104 (1986).

    CAS  PubMed  Google Scholar 

  3. Lorch, Y., LaPointe, J. W. & Kornberg, R. D. Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones. Cell 49, 203–210 (1987).

    CAS  PubMed  Google Scholar 

  4. Janicki, S. M. et al. From silencing to gene expression: real-time analysis in single cells. Cell 116, 683–698 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Thiriet, C. & Hayes, J. J. Replication-independent core histone dynamics at transcriptionally active loci in vivo. Genes Dev. 19, 677–682 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Yadav, T., Quivy, J.-P. & Almouzni, G. Chromatin plasticity: a versatile landscape that underlies cell fate and identity. Science 361, 1332–1336 (2018).

    CAS  PubMed  Google Scholar 

  7. Teves, S. S. & Henikoff, S. Transcription-generated torsional stress destabilizes nucleosomes. Nat. Struct. Mol. Biol. 21, 88–94 (2014).

    CAS  PubMed  Google Scholar 

  8. Belotserkovskaya, R. FACT facilitates transcription-dependent nucleosome alteration. Science 301, 1090–1093 (2003).

    CAS  PubMed  Google Scholar 

  9. Natsume, R. et al. Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4. Nature 446, 338–341 (2007).

    CAS  PubMed  Google Scholar 

  10. Williams, S. K., Truong, D. & Tyler, J. K. Acetylation in the globular core of histone H3 on lysine-56 promotes chromatin disassembly during transcriptional activation. Proc. Natl Acad. Sci. USA 105, 9000–9005 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Lee, C.-K., Shibata, Y., Rao, B., Strahl, B. D. & Lieb, J. D. Evidence for nucleosome depletion at active regulatory regions genome-wide. Nat. Genet. 36, 900–905 (2004).

    CAS  PubMed  Google Scholar 

  12. Dion, M. F. et al. Dynamics of replication-independent histone turnover in budding yeast. Science 315, 1405–1408 (2007).

    CAS  PubMed  Google Scholar 

  13. Deal, R. B., Henikoff, J. G. & Henikoff, S. Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328, 1161–1164 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Deaton, A. M. et al. Enhancer regions show high histone H3.3 turnover that changes during differentiation. Elife 5, e15316 (2016).

    PubMed  PubMed Central  Google Scholar 

  15. Kireeva, M. L. et al. Nucleosome remodeling induced by RNA polymerase II. Mol. Cell 9, 541–552 (2002).

    CAS  PubMed  Google Scholar 

  16. Bondarenko, V. A. et al. Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II. Mol. Cell 24, 469–479 (2006).

    CAS  PubMed  Google Scholar 

  17. Kulaeva, O. I., Hsieh, F.-K. & Studitsky, V. M. RNA polymerase complexes cooperate to relieve the nucleosomal barrier and evict histones. Proc. Natl Acad. Sci. USA 107, 11325–11330 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Kujirai, T. et al. Structural basis of the nucleosome transition during RNA polymerase II passage. Science 362, 595–598 (2018).

    CAS  PubMed  Google Scholar 

  19. Farnung, L., Vos, S. M. & Cramer, P. Structure of transcribing RNA polymerase II-nucleosome complex. Nat. Commun. 9, 5432 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Schwabish, M. A. & Struhl, K. Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 24, 10111–10117 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Schwartz, B. E. & Ahmad, K. Transcriptional activation triggers deposition and removal of the histone variant H3.3. Genes Dev. 19, 804–814 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ahmad, K. & Henikoff, S. The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 9, 1191–1200 (2002).

    CAS  PubMed  Google Scholar 

  23. Tagami, H., Ray-Gallet, D., Almouzni, G. & Nakatani, Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116, 51–61 (2004).

    CAS  PubMed  Google Scholar 

  24. Drane, P., Ouararhni, K., Depaux, A., Shuaib, M. & Hamiche, A. The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3. Genes Dev. 24, 1253–1265 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Lewis, P. W., Elsaesser, S. J., Noh, K.-M., Stadler, S. C. & Allis, C. D. DAXX is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proc. Natl Acad. Sci. USA 107, 14075–14080 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Goldberg, A. D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Ray-Gallet, D. et al. Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity. Mol. Cell 44, 928–941 (2011).

    CAS  PubMed  Google Scholar 

  28. Pchelintsev, N. A. et al. Placing the HIRA histone chaperone complex in the chromatin landscape. Cell Rep. 3, 1012–1019 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Clément, C. et al. High-resolution visualization of H3 variants during replication reveals their controlled recycling. Nat. Commun. 9, 3181 (2018).

    PubMed  PubMed Central  Google Scholar 

  30. Rai, T. S. et al. Human CABIN1 is a functional member of the human HIRA/UBN1/ASF1a histone H3.3 chaperone complex. Mol. Cell. Biol. 31, 4107–4118 (2011).

    PubMed  PubMed Central  Google Scholar 

  31. Banumathy, G. et al. Human UBN1 is an ortholog of yeast Hpc2p and has an essential role in the HIRA/ASF1a chromatin-remodeling pathway in senescent cells. Mol. Cell. Biol. 29, 758–770 (2009).

    CAS  PubMed  Google Scholar 

  32. Ricketts, M. D. et al. Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex. Nat. Commun. 6, 7711 (2015).

    PubMed  Google Scholar 

  33. Ricketts, M. D. et al. The HIRA histone chaperone complex subunit UBN1 harbors H3/H4- and DNA-binding activity. J. Biol. Chem. 294, 9239–9259 (2019).

  34. Ray-Gallet, D. et al. Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit. Nat. Commun. 9, 3103 (2018).

    PubMed  PubMed Central  Google Scholar 

  35. Tang, Y. et al. Structure of a human ASF1a–HIRA complex and insights into specificity of histone chaperone complex assembly. Nat. Struct. Mol. Biol. 13, 921–929 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Tyler, J. K. et al. The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555–560 (1999).

    CAS  PubMed  Google Scholar 

  37. Mello, J. A. Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway. EMBO Rep. 3, 329–334 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. English, C. M., Adkins, M. W., Carson, J. J., Churchill, M. E. A. & Tyler, J. K. Structural basis for the histone chaperone activity of Asf1. Cell 127, 495–508 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Loyola, B., Tiziana, R., Daniele & Almouzni, G. PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state. Mol. Cell 24, 306–316 (2006).

    Google Scholar 

  40. Nourani, A., Robert, F. & Winston, F. Evidence that Spt2/Sin1, an HMG-like factor, plays roles in transcription elongation, chromatin structure, and genome stability in Saccharomyces cerevisiae. Mol. Cell. Biol. 26, 1496–1509 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Svensson, J. P. et al. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin. Genome Res. 25, 872–883 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Jeronimo, C., Poitras, C. & Robert, F. Histone recycling by FACT and Spt6 during transcription prevents the scrambling of histone modifications. Cell Rep. 28, 1206–1218 (2019).

    CAS  PubMed  Google Scholar 

  43. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    CAS  PubMed  Google Scholar 

  44. Groth, A. et al. Human Asf1 regulates the flow of S phase histones during replicational stress. Mol. Cell 17, 301–311 (2005).

    CAS  PubMed  Google Scholar 

  45. Torné, J., Orsi, G. A., Ray-Gallet, D. & Almouzni, G. in Histone Variants Vol. 1832 (eds Orsi, G. A. & Almouzni, G.) 207–221 (Springer, 2018).

  46. Mayer, A. et al. Uniform transitions of the general RNA polymerase II transcription complex. Nat. Struct. Mol. Biol. 17, 1272–1278 (2010).

    CAS  PubMed  Google Scholar 

  47. Ghamari, A. et al. In vivo live imaging of RNA polymerase II transcription factories in primary cells. Genes Dev. 27, 767–777 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhang, G. & Wang, W. RPA interacts with HIRA and regulates H3.3 deposition at gene regulatory elements in mammalian cells. Mol. Cell 65, 272–284 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Yoh, S. M., Lucas, J. S. & Jones, K. A. The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation. Genes Dev. 22, 3422–3434 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Kimura, H., Sugaya, K. & Cook, P. R. The transcription cycle of RNA polymerase II in living cells. J. Cell Biol. 159, 777–782 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Delbarre, E., Ivanauskiene, K., Kuntziger, T. & Collas, P. DAXX-dependent supply of soluble (H3.3-H4) dimers to PML bodies pending deposition into chromatin. Genome Res. 23, 440–451 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Ricci, M. A., Manzo, C., García-Parajo, M. F., Lakadamyali, M. & Cosma, M. P. Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo. Cell 160, 1145–1158 (2015).

    CAS  PubMed  Google Scholar 

  53. Lu, H. et al. Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature 558, 318–323 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Boehning, M. et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 25, 833–840 (2018).

    CAS  PubMed  Google Scholar 

  55. Vos, S. M. et al. Structure of activated transcription complex Pol II–DSIF–PAF–SPT6. Nature 560, 607–612 (2018).

    CAS  PubMed  Google Scholar 

  56. Groth, A., Rocha, W., Verreault, A. & Almouzni, G. Chromatin challenges during DNA replication and repair. Cell 128, 721–733 (2007).

    CAS  PubMed  Google Scholar 

  57. Horard, B., Sapey-Triomphe, L., Bonnefoy, E. & Loppin, B. ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization. Epigenetics Chromatin 11, https://doi.org/10.1186/s13072-018-0189-x (2018).

  58. Venkatesh, S. et al. Set2 methylation of histone H3 lysine 36 suppresses histone exchange on transcribed genes. Nature 489, 452–455 (2012).

    CAS  PubMed  Google Scholar 

  59. Gan, H. et al. The Mcm2–Ctf4–Polα axis facilitates parental histone H3-H4 transfer to lagging strands. Mol. Cell 72, 140–151 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Adam, S., Polo, S. E. & Almouzni, G. Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA. Cell 155, 94–106 (2013).

    CAS  PubMed  Google Scholar 

  61. Gregersen, L. H. & Svejstrup, J. Q. The cellular response to transcription-blocking DNA damage. Trends Biochem. Sci. 43, 327–341 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Hall, C. et al. HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel Cyclin-cdk2 substrate whose expression blocks S-phase progression. Mol. Cell. Biol. 21, 1854–1865 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Corpet, A. et al. Asf1b, the necessary Asf1 isoform for proliferation, is predictive of outcome in breast cancer: specific importance of Asf1b in proliferation. EMBO J. 30, 480–493 (2011).

    CAS  PubMed  Google Scholar 

  64. Green, E. M. et al. Replication-independent histone deposition by the HIR complex and Asf1. Curr. Biol. 15, 2044 (2005).

  65. Helmuth, J. A., Paul, G. & Sbalzarini, I. F. Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images. BMC Bioinformatics 11, 372 (2010).

    PubMed  PubMed Central  Google Scholar 

  66. Lagache, T., Lang, G., Sauvonnet, N. & Olivo-Marin, J.-C. Analysis of the spatial organization of molecules with robust statistics. PLoS ONE 8, e80914 (2013).

    PubMed  PubMed Central  Google Scholar 

  67. Martini, E., Roche, D. M. J., Marheineke, K., Verreault, A. & Almouzni, G. Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells. J. Cell Biol. 143, 563–575 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. Domrane for generation of preliminary data with transcription inhibitors. We thank J.-P. Quivy and D. Jeffery for critical reading of the manuscript, and the PICT-IBiSA@Pasteur Imaging Facility of the Institut Curie, a member of the France Bioimaging National Infrastructure (no. ANR-10-INBS-04). This work was supported by la Ligue Nationale contre le Cancer (Equipe labellisée Ligue): ANR-11-LABX-0044_DEEP and ANR-10-IDEX-0001-02 PSL, ANR-12-BSV5-0022-02 ‘CHAPINHIB’, ANR-14-CE16-0009 ‘Epicure’, ANR-14-CE10-0013 ‘CELLECTCHIP’, EU project 678563 ‘EPOCH28’, ERC-2015-ADG-694694 ‘ChromADICT’, ANR-16-CE15-0018 ‘CHRODYT’, ANR-16-CE12-0024 ‘CHIFT’, ANR-16-CE11-0028 ‘REPLICAF’ and PSL-AFdL ‘TRACK’.

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G.A. and G.A.O. supervised the work. G.A., G.A.O. and J.T. conceived the strategy and wrote the paper. J.T. performed epifluorescence and cell biology experiments. J.T and G.A.O analyzed the data. D.R.-G. designed and performed biochemistry experiments. E.B. carried out transcription experiments with inhibitor drugs. M.G. designed imaging analysis methods. P.L.B. performed live cell imaging experiments. A.C. performed fits and interpretation of kinetic data. Critical reading and discussion of all data involved all authors.

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Correspondence to Guillermo A. Orsi or Geneviève Almouzni.

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Torné, J., Ray-Gallet, D., Boyarchuk, E. et al. Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription. Nat Struct Mol Biol 27, 1057–1068 (2020). https://doi.org/10.1038/s41594-020-0492-7

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