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

Nature Structural & Molecular Biology volume 27, pages 1057–1068 (2020)Cite this article

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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.

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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.

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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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Authors and Affiliations

  1. Institut Curie, PSL Research University, CNRS UMR3664, Paris, France

    Júlia Torné, Dominique Ray-Gallet, Ekaterina Boyarchuk, Mickaël Garnier, Patricia Le Baccon, Antoine Coulon, Guillermo A. Orsi & Geneviève Almouzni

  2. Institut Curie, Sorbonne Université, CNRS UMR3664, Paris, France

    Júlia Torné, Dominique Ray-Gallet, Ekaterina Boyarchuk, Mickaël Garnier, Patricia Le Baccon, Antoine Coulon, Guillermo A. Orsi & Geneviève Almouzni

  3. Plateforme Imagerie PICT-IBiSA, Institut Curie, PSL Research University, Paris, France

    Mickaël Garnier & Patricia Le Baccon

  4. Institut Curie, PSL Research University, CNRS UMR168, Paris, France

    Antoine Coulon

  5. LBMC, Université de Lyon, ENS de Lyon, CNRS UMR5239, Université Claude Bernard Lyon 1, Lyon, France

    Guillermo A. Orsi

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  1. Júlia Torné
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Contributions

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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