Gene silencing in eukaryotes is associated with the formation of heterochromatin, a complex of proteins and DNA that block transcription. Heterochromatin is characterized by the methylation of cytosine nucleotides of the DNA, the methylation of histone H3 at lysine 9 (H3 Lys 9), and the specific binding of heterochromatin protein 1 (HP1) to methylated H3 Lys 9 (refs 1-7). Although the relationship between these chromatin modifications is generally unknown, in the fungus Neurospora crassa, DNA methylation acts genetically downstream of H3 Lys 9 methylation. Here we report the isolation of KRYPTONITE, a methyltransferase gene specific to H3 Lys 9, identified in a mutant screen for suppressors of gene silencing at the Arabidopsis thaliana SUPERMAN (SUP) locus. Loss-of-function kryptonite alleles resemble mutants in the DNA methyltransferase gene CHROMOMETHYLASE3 (CMT3), showing loss of cytosine methylation at sites of CpNpG trinucleotides (where N is A, C, G or T) and reactivation of endogenous retrotransposon sequences. We show that CMT3 interacts with an Arabidopsis homologue of HP1, which in turn interacts with methylated histones. These data suggest that CpNpG DNA methylation is controlled by histone H3 Lys 9 methylation, through interaction of CMT3 with methylated chromatin.

Epigenetic silenced alleles of the Arabidopsis SUPERMAN locus (the clark kent alleles) are associated with dense hypermethylation at noncanonical cytosines (CpXpG and asymmetric sites, where X = A, T, C, or G). A genetic screen for suppressors of a hypermethylated clark kent mutant identified nine loss-of-function alleles of CHROMOMETHYLASE3 (CMT3), a novel cytosine methyltransferase homolog. These cmt3 mutants display a wild-type morphology but exhibit decreased CpXpG methylation of the SUP gene and of other sequences throughout the genome. They also show reactivated expression of endogenous retrotransposon sequences. These results show that a non-CpG DNA methyltransferase is responsible for maintaining epigenetic gene silencing.

The transition to flowering in Arabidopsis thaliana is delayed in fwa mutant plants. FWA was identified by loss-of-function mutations in normally flowering revertants of the fwa mutant, and it encodes a homeodomain-containing transcription factor. The DNA sequence of wild-type and fwa mutant alleles was identical in the genomic region of FWA. Furthermore, the FWA gene is ectopically expressed in fwa mutants and silenced in mature wild-type plants. This silencing is associated with extensive methylation of two direct repeats in the 5' region of the gene. The late flowering phenotype, ectopic FWA expression, and hypomethylation of the repeats were also induced in the ddm1 hypomethylated background. Mechanisms for establishment and maintenance of the epigenetic mark on FWA are discussed.

The Arabidopsis KRYPTONITE gene encodes a member of the Su(var)3-9 family of histone methyltransferases. Mutations of kryptonite cause a reduction of methylated histone H3 lysine 9, a loss of DNA methylation, 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 immunoprecipitation 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 KRYPTONITE 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.

P rimary effusion lymphoma (PEL) is an infrequent neoplasia of severe immunodeficiency and is usually observed in those with longstanding AIDS (1, 2). PEL exhibit features both in common with and distinct from other B cell neoplasms. Like myeloma, PEL arise from postgerminal center, developmentally mature B lymphocytes (1-3). Unlike myeloma, PEL grow in body cavities as liquid effusions, are almost always infected with human herpesvirus-8 (HHV-8) and lack expression of B-lineagespecific genes (reviewed in ref.2). The loss of B lineage gene expression in PEL could be caused by DNA methylation and epigenetic silencing. In accord with this idea, ␥-herpesvirus genomes such as HHV-8, herpesvirus saimiri, and Epstein-Barr virus, are commonly methylated (4, 5). The same mechanism(s) responsible for viral DNA methylation may also be involved in methylation and silencing of B lineage-specific genes in PEL.DNA methylation in mammalian cells largely occurs on cytosines in symmetric CpG dinucleotides and is associated with repressed gene transcription (6 -10). Methyl-binding domain proteins engage methylated CpG ( m CpG) and recruit histone deacetylase (HDAC) and transcriptional repressors to form stable repression complexes that induce local chromatin remodeling and gene silencing (11-16). Early mammalian embryos and germ cells, like plants and fungi, also methylate non-CpG cytosines (17-21). A recent report indicates that peripheral blood leukocytes may methylate the internal cytosine of symmetric CCTGG but not CCAGG motifs (22). However, little is known about the gene specificity, frequency, and functional significance of this latter type of symmetric non-CpG methylation.The B29 (Ig␤͞CD79b) component of the B cell surface receptor is encoded by the B29 gene and is absent in all PEL lines and some myelomas (1, 23). The B cell-specific B29 promoter is well characterized and provides an ideal model to analyze DNA methylation in B lineage gene silencing in PEL and myeloma. Early B cell factor (EBF), in concert with Octamer, Ets, Sp1, and Ikaros transcription factors, regulates B29 promoter activity in early B cell development, whereas non-EBF factors control B29 gene expression at later stages of B cell differentiation (24). The human B29 promoter contains 20 CpG dinucleotides and 6 CCAGG or CCTGG motifs within a 450-bp span (25). CpG dinucleotides are present in single Sp1 and EBF consensusbinding sites, whereas the CCAGG and CCTGG motifs are concentrated in a central promoter control region containing essential EBF sites.Here we report that the B29 promoter in PEL and nonexpressing myeloma cells is methylated at CpG and CC(A͞T)GG sites. Because the methylation pattern observed at these B29 promoter sites is similar to that reported in epigenetic retroviral silencing, we propose that B cell gene extinction occurs through a similar mechanism. We also find that CC(A͞T)GG methylation repressed B29 promoter activity and replaced transcription factors with new protein complexes. Materials and MethodsDNA Demethylation and HDAC In...

Plants with low levels of DNA methylation show a range of developmental abnormalities including homeotic transformation of floral organs. Two independent DNA METHYLTRANSFERASEI (METI) antisense transformants with low levels of DNA methylation had flowers with increased numbers of stamens which resembled flowers seen on the loss-of-function superman (sup) mutant plants and on transgenic plants that ectopically express APETALA3 (AP3). These METI antisense plants have both increased and decreased methylation in and around the sup gene, compared with untransformed controls. DNA from the antisense plants was demethylated at least 4 kb upstream of the sup gene, while there was dense methylation around the start of transcription and within the coding region of this gene; these regions were unmethylated in control DNA. Methylation within the sup gene was correlated with an absence of SUP transcripts. The pattern and density of methylation was heterogeneous among different DNA molecules from the same plant, with some molecules being completely unmethylated. Methylcytosine occurred in asymmetric sites and in symmetric CpA/TpG but rarely in CpG dinucleotides in the antisense plants. In contrast, segregants lacking the METI antisense construct and epimutants with a hypermethylated allele of sup (clark kent 3), both of which have active METI genes, showed a higher frequency of methylation of CpG dinucleotides and of asymmetric cytosines. We conclude that METI is the predominant CpG methyltransferase and directly or indirectly affects asymmetric methylation.