PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos Mareike Puschendorf 1 , Re ´mi Terranova 1 , Erwin Boutsma 2 , Xiaohong Mao 3,6 , Kyo-ichi Isono 4 , Urszula Brykczynska 1 , Carolin Kolb 1 , Arie P Otte 5 , Haruhiko Koseki 4 , Stuart H Orkin 3 , Maarten van Lohuizen 2 & Antoine H F M Peters 1 In eukaryotes, Suv39h H3K9 trimethyltransferases are required for pericentric heterochromatin formation and function. In early mouse preimplantation embryos, however, paternal pericentric heterochromatin lacks Suv39h-mediated H3K9me3 and downstream marks. Here we demonstrate Ezh2-independent targeting of maternally provided polycomb repressive complex 1 (PRC1) components to paternal heterochromatin. In Suv39h2 maternally deficient zygotes, PRC1 also associates with maternal heterochromatin lacking H3K9me3, thereby revealing hierarchy between repressive pathways. In Rnf2 maternally deficient zygotes, the PRC1 complex is disrupted, and levels of pericentric major satellite transcripts are increased at the paternal but not the maternal genome. We conclude that in early embryos, Suv39h-mediated H3K9me3 constitutes the dominant maternal transgenerational signal for pericentric heterochromatin formation. In absence of this signal, PRC1 functions as the default repressive back-up mechanism. Parental epigenetic asymmetry, also observed along cleavage chromosomes, is resolved by the end of the 8-cell stage—concurrent with blastomere polarization—marking the end of the maternal- to-embryonic transition. In mammals, parental genomes are epigenetically distinct, despite their genetic resemblance 1 . During early mouse preimplantation develop- ment, parental genomes are highly asymmetric in epigenetic modifica- tions of DNA and associated chromatin 2–9 . At gamete fusion, the maternal genome exists in a nucleosomal configuration marked by distinct types of histone lysine methylation inherited from the oocyte. In contrast, following the histone-to-protamine exchange occurring during spermiogenesis, the paternal genome incorporates maternally provided histones and becomes de novo methylated at different lysine residues in a highly spatially and temporally coordinated manner. The function of parental epigenetic asymmetry for gene expression and genome reorganization 6,10 is largely enigmatic, as are the mechanisms of establishment, maintenance and resolution. A key question is whether parentally inherited epigenetic states affect de novo targeting and function of (maternally provided) epigenetic modifiers in cis and/or in trans in the early embryo, thereby directing gene expression over shorter or longer developmental time windows. Notably, trans- mission of the paternal genome in a nucleosomal state impairs DNA methylation reprogramming in early embryos 11 . Here, we study the transgenerational contribution of two distinct evolutionarily conserved classes of epigenetic modifiers in defining parental asymmetry at constitutive heterochromatin and euchromatin in preimplantation embryos. The first class consists of the Suv39h histone methyltransferases (HMTs), which are essential for constitutive heterochromatin forma- tion and function, gene repression and maintenance of genome integrity 12–15 . Suv39h-mediated H3K9me3 directs chromatin binding of the heterochromatic proteins HP1a, HPb and HPg (ref. 16), which target the two H4K20 di- and trimethylation-specific Suv4-20h HMTs and the Dnmt3a/3b DNA methyltransferases, to establish a transcrip- tionally repressed state 17–19 . The second class consists of Polycomb group (PcG) proteins, which are repressive chromatin factors required for maintaining cell iden- tity 14,20 . PcG proteins are classified into two groups of multimeric protein complexes termed polycomb repressive complexes (PRCs). In Drosophila melanogaster , PRC1 contains four core components for which multiple paralogs exist in mammals 21 . In vitro, PRC1 mediates repression by inhibiting chromatin remodeling, impairing the tran- scription machinery and by mediating chromatin compaction 22 . The mammalian and fly RING orthologs function as E3 ubiquitin ligases that monoubiquitinate H2A at lysine 119, a modification associated with gene repression 23,24 . PRC2 consists of Ezh2, Suz12 and Eed, which Received 14 November 2007; accepted 22 January 2008; published online 2 March 2008; corrected online 16 March 2008 (details online); doi:10.1038/ng.99 1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. 2 Division of Molecular Genetics and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. 3 Department of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Stem Cell Institute and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. 4 RIKEN Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku Yokohama City, Kanagawa 230-0045, Japan. 5 Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 406, 1098 SM Amsterdam, The Netherlands. 6 Present address: Novartis Institutes for BioMedical Research, Inc., 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. Correspondence should be addressed to A.H.F.M.P. (antoine.peters@fmi.ch). NATURE GENETICS VOLUME 40 [ NUMBER 4 [ APRIL 2008 411 ARTICLES © 2008 Nature Publishing Group http://www.nature.com/naturegenetics