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Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3

Nature Cell Biology volume 12, pages 853–862 (2010)Cite this article

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Abstract

In mammals, oocyte fertilization by sperm initiates development. This is followed by epigenetic reprogramming of both parental genomes, which involves the de novo establishment of chromatin domains. In the mouse embryo, methylation of histone H3 establishes an epigenetic asymmetry and is predominant in the maternal pronucleus. However, the roles of differential incorporation of histone H3 variants in the parental chromatin, and of modified residues within specific histone variants, have not been addressed. Here we show that the histone variant H3.3, and in particular lysine 27, is required for the establishment of heterochromatin in the mouse embryo. H3.3 localizes to paternal pericentromeric chromatin during S phase at the time of transcription of pericentromeric repeats. Mutation of H3.3 K27, but not of H3.1 K27, results in aberrant accumulation of pericentromeric transcripts, HP1 mislocalization, dysfunctional chromosome segregation and developmental arrest. This phenotype is rescued by injection of double-stranded RNA (dsRNA) derived from pericentromeric transcripts, indicating a functional link between H3.3K27 and the silencing of such regions by means of an RNA-interference (RNAi) pathway. Our work demonstrates a role for a modifiable residue within a histone-variant-specific context during reprogramming and identifies a novel function for mammalian H3.3 in the initial formation of dsRNA-dependent heterochromatin.

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Figure 1: Embryos expressing H3.3–GFP K27R are compromised in their development.
Figure 2: Developmental defects elicited on mutation of K27 are H3.3-specific.
Figure 3: H3.3 and H3.1 are incorporated at different times in the two pronuclei.
Figure 4: Expression of H3.3–GFP K27R leads to changes in some epigenetic marks.
Figure 5: H3.3 localizes to pericentromeric domains surrounding NLBs in the male pronucleus only.
Figure 6: Expression of H3.3–GFP K27R in the zygote leads to altered pericentromeric transcription, defects in chromosome segregation and mislocalization of HP1β.
Figure 7: Addition of dsRNA from major satellites in the zygote rescues the developmental phenotype of H3.3 K27R mutants.

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References

  1. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).

    Article  CAS  Google Scholar 

  2. Arney, K. L., Bao, S., Bannister, A. J., Kouzarides, T. & Surani, M. A. Histone methylation defines epigenetic asymmetry in the mouse zygote. Int. J. Dev. Biol. 46, 317–320 (2002).

    CAS  PubMed  Google Scholar 

  3. Lepikhov, K. & Walter, J. Differential dynamics of histone H3 methylation at positions K4 and K9 in the mouse zygote. BMC Dev. Biol. 4, 12 (2004).

    Article  Google Scholar 

  4. Santos, F., Peters, A. H., Otte, A. P., Reik, W. & Dean, W. Dynamic chromatin modifications characterise the first cell cycle in mouse embryos. Dev. Biol. 280, 225–236 (2005).

    Article  CAS  Google Scholar 

  5. Torres-Padilla, M. E., Bannister, A. J., Hurd, P. J., Kouzarides, T. & Zernicka-Goetz, M. Dynamic distribution of the replacement histone variant H3.3 in the mouse oocyte and preimplantation embryos. Int. J. Dev. Biol. 50, 455–461 (2006).

    Article  CAS  Google Scholar 

  6. Adenot, P. G., Mercier, Y., Renard, J. P. & Thompson, E. M. Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos. Development 124, 4615–4625 (1997).

    CAS  Google Scholar 

  7. Bouniol-Baly, C., Nguyen, E., Besombes, D. & Debey, P. Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation. Exp. Cell Res. 236, 201–211 (1997).

    Article  CAS  Google Scholar 

  8. Wouters-Tyrou, D., Martinage, A., Chevaillier, P. & Sautiere, P. Nuclear basic proteins in spermiogenesis. Biochimie 80, 117–128 (1998).

    Article  CAS  Google Scholar 

  9. Wright, S. J. Sperm nuclear activation during fertilization. Curr. Top. Dev. Biol. 46, 133–178 (1999).

    Article  CAS  Google Scholar 

  10. Wiekowski, M., Miranda, M., Nothias, J. Y. & DePamphilis, M. L. Changes in histone synthesis and modification at the beginning of mouse development correlate with the establishment of chromatin mediated repression of transcription. J. Cell Sci. 110, 1147–1158 (1997).

    CAS  PubMed  Google Scholar 

  11. van der Heijden, G. W. et al. Asymmetry in histone H3 variants and lysine methylation between paternal and maternal chromatin of the early mouse zygote. Mech. Dev. 122, 1008–1022 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Hake, S. B. et al. Expression patterns and post-translational modifications associated with mammalian histone H3 variants. J. Biol. Chem. 281, 559–568 (2006).

    Article  CAS  Google Scholar 

  15. McKittrick, E., Gafken, P. R., Ahmad, K. & Henikoff, S. Histone H3.3 is enriched in covalent modifications associated with active chromatin. Proc. Natl Acad. Sci. USA 101, 1525–1530 (2004).

    Article  CAS  Google Scholar 

  16. Polo, S. E., Roche, D. & Almouzni, G. New histone incorporation marks sites of UV repair in human cells. Cell 127, 481–493 (2006).

    Article  CAS  Google Scholar 

  17. Azuara, V. et al. Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532–538 (2006).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Probst, A. V., Santos, F., Reik, W., Almouzni, G. & Dean, W. Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote. Chromosoma 116, 403–415 (2007).

    Article  Google Scholar 

  20. Muchardt, C. et al. Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1α. EMBO Rep. 3, 975–981 (2002).

    Article  CAS  Google Scholar 

  21. Chen, E. S. et al. Cell cycle control of centromeric repeat transcription and heterochromatin assembly. Nature 451, 734–737 (2008).

    Article  CAS  Google Scholar 

  22. Djupedal, I. et al. RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing. Genes Dev. 19, 2301–2306 (2005).

    Article  CAS  Google Scholar 

  23. Verdel, A. et al. RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676 (2004).

    Article  CAS  Google Scholar 

  24. Grewal, S. I. & Elgin, S. C. Transcription and RNA interference in the formation of heterochromatin. Nature 447, 399–406 (2007).

    Article  CAS  Google Scholar 

  25. Lu, J. & Gilbert, D. M. Proliferation-dependent and cell cycle regulated transcription of mouse pericentric heterochromatin. J. Cell Biol. 179, 411–421 (2007).

    Article  CAS  Google Scholar 

  26. Puschendorf, M. et al. PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos. Nat. Genet. 40, 411–420 (2008).

    Article  CAS  Google Scholar 

  27. Motamedi, M. R. et al. HP1 proteins form distinct complexes and mediate heterochromatic gene silencing by nonoverlapping mechanisms. Mol. Cell 32, 778–790 (2008).

    Article  CAS  Google Scholar 

  28. Mellone, B. G. et al. Centromere silencing and function in fission yeast is governed by the amino terminus of histone H3. Curr. Biol. 13, 1748–1757 (2003).

    Article  CAS  Google Scholar 

  29. Wianny, F. & Zernicka-Goetz, M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat. Cell Biol. 2, 70–75 (2000).

    Article  CAS  Google Scholar 

  30. Lehnertz, B. et al. Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr. Biol. 13, 1192–1200 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Kami Ahmad for providing H3.3 constructs, Florence Cammas and Tony Kouzarides for HP1β cDNAs, Natacha Dreumont and James Stevenin for advice on RNA-shift, and Olivier Pourquie for critical reading of the manuscript. A.J.B. acknowledges funding from Cancer Research U.K.; M.E.T.-P. acknowledges funding from Programme National de Recherche Reproduction Endocrinologie (PNRRE) and the Avenir programme from INSERM, and ANR-09-Blanc-0114.

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Authors and Affiliations

  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre national de la recherche scientifique/Institut National de la Santé et de la Recherche Médicale U964, Université de Strasbourg, Illkirch, F-67404, France

    Angèle Santenard, Céline Ziegler-Birling, Marc Koch, Làszlò Tora & Maria-Elena Torres-Padilla

  2. The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK

    Andrew J. Bannister

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  1. Angèle Santenard
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Contributions

A.S. designed, performed and analysed most of the experiments in this study. C.Z.-B. and M.K. performed experiments. L.T. provided support during the initial phase of this work. A.J.B. contributed to the design of the project. M.E.T.-P. conceived the project, designed and supervised the study and performed experiments. A.S., A.J.B. and M.-E.T.-P wrote the manuscript.

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Correspondence to Maria-Elena Torres-Padilla.

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Santenard, A., Ziegler-Birling, C., Koch, M. et al. Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3. Nat Cell Biol 12, 853–862 (2010). https://doi.org/10.1038/ncb2089

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