Chromosoma (2004) 112: 308–315 DOI 10.1007/s00412-004-0275-7 RESEARCH ARTICLE James P. Jackson . Lianna Johnson . Zuzana Jasencakova . Xing Zhang . Laura PerezBurgos . Prim B. Singh . Xiaodong Cheng . Ingo Schubert . Thomas Jenuwein . Steven E. Jacobsen Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in Arabidopsis thaliana Received: 2 January 2004 / Accepted: 17 February 2004 / Published online: 10 March 2004 # Springer-Verlag 2004 Abstract The Arabidopsis KRYPTONITE gene encodes a member of the Su(var)3-9 family of histone methyltrans- ferases. Mutations of kryptonite cause a reduction of methylated histone H3 lysine 9, a loss of DNA methyl- ation, and reduced gene silencing. Lysine residues of histones can be either monomethylated, dimethylated or trimethylated and recent evidence suggests that different methylation states are found in different chromatin domains. Here we show that bulk Arabidopsis histones contain high levels of monomethylated and dimethylated, but not trimethylated histone H3 lysine 9. Using both immunostaining of nuclei and chromatin immunoprecip- itation assays, we show that monomethyl and dimethyl histone H3 lysine 9 are concentrated in heterochromatin. In kryptonite mutants, dimethyl histone H3 lysine 9 is nearly completely lost, but monomethyl histone H3 lysine 9 levels are only slightly reduced. Recombinant KRYP- TONITE can add one or two, but not three, methyl groups to the lysine 9 position of histone H3. Further, we identify a KRYPTONITE-related protein, SUVH6, which displays histone H3 lysine 9 methylation activity with a spectrum similar to that of KRYPTONITE. Our results suggest that multiple Su(var)3-9 family members are active in Arabidopsis and that dimethylation of histone H3 lysine 9 is the critical mark for gene silencing and DNA methylation. Introduction Epigenetic gene silencing in eukaryotic organisms is generally associated with the formation of heterochroma- tin, a complex of histone and non-histone proteins that combine to package the DNA tightly. The histones found in heterochromatin are characterized by specific post- translational modifications (Strahl and Allis 2000; Turner 2000). One of the best characterized modifications is methylation of lysine 9 of histone H3 (H3K9) (reviewed in Jenuwein and Allis 2001; Richards and Elgin 2002; Turner 2002). Additionally, in many eukaryotic organisms, including plants and mammals, cytosine DNA methylation is a necessary component of epigenetic gene silencing (reviewed in Martienssen and Colot 2001). Genetic screens in Arabidopsis thaliana have identified a number of components that are required for initiating and maintaining these epigenetic marks. The clark kent alleles of the SUPERMAN gene are silenced by DNA methylation, resulting in a superman like mutant pheno- type—flowers develop additional stamens and unfused carpels. KRYPTONITE (KYP), a histone methyltransfer- ase specific for H3K9, was identified in a screen for second site suppressors of the clark kent-stable allele (Jackson et al. 2002). KYP mutants were also uncovered independently in a screen for second site suppressors of Communicated by P. Shaw J. P. Jackson . L. Johnson . S. E. Jacobsen Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA Z. Jasencakova . I. Schubert Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), 06466 Gatersleben, Germany X. Zhang . X. Cheng Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA L. PerezBurgos . T. Jenuwein Research Institute of Molecular Pathology (IMP) at the Vienna Biocenter, Dr. Bohrgasse 7, 1030 Vienna, Austria P. B. Singh Nuclear Reprogramming Laboratory, Division of Gene Expression and Development, The Roslin Institute, Edinburgh, Midlothian, EH25 9PS, UK S. E. Jacobsen (*) Molecular Biology Institute, University of California, P.O. Box 951606, Los Angeles, CA 90095-1606, USA e-mail: jacobsen@ucla.edu