Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications

ARTICLE Received 29 May 2014 | Accepted 14 Nov 2014 | Published 18 Dec 2014 DOI: 10.1038/ncomms6868 OPEN Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications Christine van de Werken1, Godfried W. van der Heijden1,2, Cindy Eleveld1, Miriam Teeuwssen1, Mareike Albert3,w, Willy M. Baarends2, Joop S.E. Laven1, Antoine H.F.M. Peters3,4 & Esther B. Baart1 The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos. Our results show that histones carrying canonical cHC modifications are retained in cHC regions of sperm chromatin. These modified histones are transmitted to the oocyte and contribute to the formation of paternal embryonic cHC. Subsequently, the modifications are recognized by the H3K9/HP1 pathway maternal chromatin modifiers and propagated over the embryonic cleavage divisions. These results are in contrast to what has been described for mouse embryos, in which paternal cHC lacks canonical modifications and is initially established by Polycomb group proteins. Our results show intergenerational epigenetic inheritance of the cHC structure in human embryos. 1 Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Postbus 2040, 3000 CA Rotterdam, The Netherlands. 2 Department of Reproduction and Development, Erasmus MC, University Medical Center, Postbus 2040, 3000 CA Rotterdam, The Netherlands. 3 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland. 4 Faculty of Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland. w Present address: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. Correspondence and requests for materials should be addressed to E.B.B. (email: ). NATURE COMMUNICATIONS | 5:5868 | DOI: 10.1038/ncomms6868 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE F NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6868 ertilization marks the fusion of two specialized gametes— oocyte and sperm. In mammalian zygotes, the maternal and paternal genomes exist in an asymmetric chromatin configuration. Extensive reorganization of chromatin to the embryonic configuration is crucial for the developmental potency1. During this process, some information of parental origin needs to be retained to maintain imprinting2. Other chromatin domains, such as the constitutive heterochromatin (cHC), need to be reorganized to the somatic configuration to function properly3,4. Constitutive HC assembles mostly on telomeric, centromeric and pericentric regions, remains condensed throughout the cell cycle and is important for genome stability and chromosome segregation5. DNA sequences underlying cHC differ between species, but mainly consist of repeats and transposons. In mouse, most of the cHC is located pericentrically (pericentric heterochromatin (pHC)), a region with major satellite DNA repeats. In human, cHC is more dispersed across the genome6; classic satellite II and III DNA repeats localize to the pericentric region, but also to large blocks of cHC on chromosomes 1, 9, 16, the acrocentric chromosomes and Y7, also referred to as ‘knobs’5. The H3K9/HP1 pathway underlies the formation of cHC. A central event is the trimethylation of histone H3 at lysine 9 (H3K9me3) by histone methyltransferases (HMTs) Suv39h1 and Suv39h2 (refs 5,8,9). H3K9me3 serves as a docking place for the binding of heterochromatin protein 1 (HP1) isoforms, which results in chromatin compaction5. Subsequently, HP1 binds Suv4-20h1/2 HMTs, which trimethylate histone H4 at lysine 20 (H4K20me3) to further establish a compact chromatin structure5,10. Through an unidentified mechanism, H3K9me3 also facilitates the trimethylation of histone H3 at lysine 64 (H3K64me3), which has been suggested to stabilize cHC11,12. The H3K9/HP1 pathway is interwoven with the methylation of DNA, another mechanism for gene silencing prominent in cHC5,10. Together, all modifications eventually lead to the establishment of a condensed, transcriptionally repressed state that is epigenetically heritable through cell division. In mammalian oocytes, the maternal genome is marked by high levels of histone lysine methylation, whereas in spermatozoa the paternal genome is compacted with small proteins named protamines13. Current knowledge of resolution of this epigenetic asymmetry in early mammalian embryos is mainly based on mouse models1. Paternal pHC in mouse spermatozoa and zygotes is largely devoid of canonical cHC marks14. Re-establishment of the canonical pHC configuration is not performed by the H3K9/ HP1 pathway. Instead, during the earliest embryonic stages, maternally provided Polycomb repressive complex 1 (PRC1) localizes to paternal pHC, which subsequently becomes enriched for Polycomb repressive complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) (refs 3,15). The core PRC1 complex contains an E3 ligase Ring1a/b, which interacts with one of the orthologues of the Drosophila posterior sex combs (Mel18, Bmi1 or Nspc1), a Polyhomeiotic orthologue (Phc1, Phc2 or Phc3) and a Polycomb orthologue (Cbx2, Cbx4, Cbx6, Cbx7 or Cbx8) (ref. 16). The PRC2 core complex contains one of the HMTs, Ezh1 or Ezh2, together with the regulatory subunits Suz12 and Eed17. In somatic cells, Polycomb complexes are known to regulate the formation of facultative heterochromatin, a type of heterochromatin that is able to undergo changes in configuration in the context of regulation of gene expression. Thus, in mouse preimplantation embryos, the paternal pericentric DNA temporarily assumes a facultative heterochromatin packaging, to circumvent the inactivity of the H3K9/HP1 pathway. The PRC1/2 pathway thereby operates as a transient backup mechanism for pHC 2 formation3. During the eight-cell stage of mouse embryo development, the H3K9/HP1 pathway takes over again and the pHC of both parental origins gradually becomes equivalent for H3K9me3 (refs 3,18). Other pHC-associated marks, such as H3K64me3 and H4K20me3, remain undetected in the paternal chromatin until after compaction and implantation, respectively11,12,19. In this study, we addressed chromatin dynamics on cHC during human preimplantation embryo development. Our results identify striking differences with mouse: cHC in human embryos is not re-established by PRC1/2 action, but is transmitted and maintained by actors of the canonical H3K9/HP1 pathway. We show that human spermatozoa retain and transmit nucleosomes with cHC marks, such (...truncated)