Cell, Vol. 107, 323–337, November 2, 2001, Copyright 2001 by Cell PressLossoftheSuv39h Histone MethyltransferasesImpairs Mammalian Heterochromatinand Genome Stabilitying centromeres and telomeres (Karpen and Allshire,1997) and to facilitate the extensive reorganization ofchromosomes during meiosis (Karpen et al., 1996; Dern-burg et al., 1996). Although alterations in chromatinstructure can be induced by sequence-specific recruit-Antoine H.F.M. Peters,1,5Do´nal O’Carroll,1,5,6Harry Scherthan,2,6Karl Mechtler,1Stephan Sauer,1Christian Scho¨fer,3Klara Weipoltshammer,3Michaela Pagani,1Monika Lachner,1Alexander Kohlmaier,1ment of transcription factors and chromatin remodelingSusanne Opravil,1Michael Doyle,1,6Maria Sibilia,1,6complexes (Kingston and Narlikar, 1999), or by the clus-and Thomas Jenuwein1,4tering of DNA repeats (Csink and Henikoff, 1998), DNA1Research Institute of Molecular Pathology (IMP)sequence per se appears not to be sufficient to mediateVienna Biocenterthe establishment of distinct chromosomal subdomainsDr. Bohrgasse 7(Murphy and Karpen, 1998; Henikoff et al., 2001). Fur-A-1030 Viennather, while changes in DNA methylation patterns corre-Austrialate with distinct epigenetic states (Bird and Wolffe,2Department of Human Biology1999), DNA methylation is virtually absent in S. pombeUniversity of Kaiserslauternand rare in Drosophila (Lyko, 2001), despite the epige-Erwin-Schro¨dingerstrasse, Geb. 14netic control operating in these organisms.D-67663 KaiserslauternRecently, a “histone code” hypothesis has been sug-Germanygested (Strahl and Allis, 2000), which predicts that differ-3Institute of Histology and Embryologyent modifications (e.g., acetylation, phosphorylation,University of Viennamethylation) of histone N termini are interdependent andSchwarzspanierstraße 17represent an evolutionarily conserved mechanism thatA-1090 Viennacan induce and stabilize functionally distinct chromo-Austriasomal subdomains (Jenuwein and Allis, 2001). For ex-ample, general underacetylation of histones (Jeppesenet al., 1992; Ekwall et al., 1997) and phosphorylation ofSummaryhistone H3 (Hendzel et al., 1997; Wei et al., 1999) arerequired for faithful chromosome segregation, presum-Histone H3 lysine 9 methylation has been proposedably by preserving the more compacted chromatinto provide a major “switch” for the functional organi-structure of pericentric heterochromatin. Furthermore,zation of chromosomal subdomains. Here, we showthe discovery of mammalian histone H3 lysine 9 specificthat the murine Suv39h histone methyltransferaseshistone methyltransferases (Suv39h HMTases) (Rea et(HMTases) govern H3-K9 methylation at pericentrical., 2000), which are heterochromatin-enriched enzymesheterochromatin and induce a specialized histonetransiently accumulating around centromeres during mi-methylation pattern that differs from the broad H3-K9tosis (Aagaard et al., 2000), revealed a regulatory mecha-methylation present at other chromosomal regions.nism in which the selective methylation of histone H3Suv39h-deficient mice display severely impaired via-at lysine 9 (H3-K9) creates a high-affinity binding sitebility and chromosomal instabilities that are associatedfor the heterochromatic HP1 proteins (Lachner et al.,with an increased tumor risk and perturbed chromo-2001; Bannister et al., 2001; Nakayama et al., 2001).some interactions during male meiosis. These in vivoGain- and loss-of-function studies for Suv39h enzymesdata assign a crucial role for pericentric H3-K9 methyl-in mammalian cell lines (Melcher et al., 2000; Rea etation in protecting genome stability, and define theal., 2000) suggest a major role for the SUV39H1-HP1Suv39h HMTases as important epigenetic regulatorsmethylation system in chromosome segregation. In ad-for mammalian development.dition, the SUV39H1 HMTase is also involved in localgene repression (Firestein et al., 2000), and is targetedto specific cell cycle genes through the tumor suppres-Introductionsor Rb (Nielsen et al., 2001).In S. pombe, the clr4 and swi6 genes, which encodeChromatin represents the physiological template of thethe fission yeast homologs of SUV39H1 and HP1, aregenetic information in all eukaryotic cells, and is func-required for heterochromatic gene silencing (Thon ettionally divided into euchromatic and heterochromatical., 1994; Allshire et al., 1995; Ivanova et al., 1998) andregions. This functional distinction has been proposedcentromere function (Ekwall et al., 1996). Disruption ofto be crucial for epigenetic control of gene expressionthe HMTase activity of Clr4 prevents pericentric associa-programs (Turner, 2000; Jenuwein and Allis, 2001), totion of Swi6 protein (Bannister et al., 2001) and impairsunderlie the specialized chromatin structure surround-H3-K9 methylation (Nakayama et al., 2001). Althoughclr4 and swi6 null mutants are viable, mutation of either4Correspondence: [email protected] leads to high rates of chromosome missegrega-5These authors contributed equally to this worktion (Ekwall et al., 1996). By contrast, HP1 null mutants6Present addresses: (D.O’C.) The Rockefeller University, New York;are lethal in Drosophila (Eissenberg et al., 1992) and(H.S.) Max Planck Institute for Molecular Genetics, Berlin; (M.D.)display a more severe phenotype, including aberrantInstitute of Botany, Vienna University; (M.S.) Institute of Dermatol-ogy, Vienna Universitychromosome segregation and telomeric fusions (KellumCell324Figure 1. Targeting and Genotyping of Suv39h1- and Suv39h2-Deficient Mice(A) Diagrammatic representation of the Suv39h1 and Suv39h2 genomic loci, the replacement vectors and the targeted alleles. Exons areindicated by black boxes with numbers referring to the starting amino acid positions of the respective exons (O’Carroll et al., 2000). Alsoshown are the diagnostic restriction sites and the external probes used for Southern blot analyses.(B) Southern blot analyses of PvuII- or HindIII-digested DNA isolated from offspring of Suv39h1⫹/⫺ or Suv39h2⫹/⫺ intercrosses.(C) Protein blot analyses of testis nuclear extracts from wild-type (wt), Suv39h1⫺/⫺ (Suv1⫺/⫺), and Suv39h2⫺/⫺ (Suv2⫺/⫺) mice with␣-Suv39h1 and ␣-Suv39h2 antibodies.(D) Suv39h double null (dn) mice are growth retarded at birth and during adulthood.and Alberts, 1995; Fanti et al., 1998). Surprisingly, Suv39h1 and Suv39h2 gene loci in the mouse. In addi-tion, we developed ␣-methH3-K9 antibodies which se-Su(var)3-9 mutants