Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3

Epigenetic indexing of chromatin domains by histone lysine methylation requires the balanced coordination of methyltransferase and demethylase activities. Here, we show that SU(VAR)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. SU(VAR)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. Our data indicate an early developmental function for the SU(VAR)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which SU(VAR)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation.     

Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase

In Arabidopsis thaliana, heterochromatin formation is guided by double-stranded RNA (dsRNA), which triggers methylation of histone H3 at Lys-9 (H3 mK9) and CG plus non-CG methylation on identical DNA sequences. At heterochromatin targets including transposons and centromere repeats, H3 mK9 mediated by the Su(var)3-9 homologue 4 (SUVH4)/KYP histone methyltransferase (MTase) is required for the maintenance of non-CG methylation by the CMT3 DNA MTase. Here, we show that although SUVH4 is the major H3 K9 MTase, the SUVH5 protein also has histone MTase activity in vitro and contributes to the maintenance of H3 mK9 and CMT3-mediated non-CG methylation in vivo. Strikingly, the relative contributions of SUVH4, SUVH5, and a third related histone MTase, SUVH6, to non-CG methylation are locus-specific. For example, SUVH4 and SUVH5 together control transposon sequences with only a minor contribution from SUVH6, whereas SUVH4 and SUVH6 together control a transcribed inverted repeat source of dsRNA with only a minor contribution from SUVH5. This locus-specific variation suggests different mechanisms for recruiting or activating SUVH enzymes at different heterochromatic sequences. The suvh4 suvh5 suvh6 triple mutant loses both monomethyl and dimethyl H3 K9 at target loci. The suvh4 suvh5 suvh6 mutant also displays a loss of non-CG methylation similar to a cmt3 mutant, indicating that SUVH4, SUVH5, and SUVH6 together control CMT3 activity.     

The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9

Proteins containing the evolutionarily conserved SET domain are involved in regulation of eukaryotic gene expression and chromatin structure through their histone lysine methyltransferase (HMTase) activity. The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization. In Arabidopsis there are 10 SUVH and 5 SUVR genes encoding proteins similar to SU(VAR)3-9, and 4 SUVH proteins have been shown to control heterochromatic silencing by its HMTase activity and by directing DNA methylation. The SUVR proteins differ from the SUVH proteins in their domain structure, and we show that the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel domain at their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far. A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.     

Glimpses of evolution: heterochromatic histone H3K9 methyltransferases left its marks behind

In eukaryotes, histone methylation is an epigenetic mechanism associated with a variety of functions related to gene regulation or genomic stability. Recently analyzed H3K9 methyltransferases (HMTases) as SUV39H1, Clr4p, DIM-5, Su(var)3-9 or SUVH2 are responsible for the establishment of histone H3 lysine 9 methylation (H3K9me), which is intimately connected with heterochromatinization. In this review, available data will be evaluated concerning (1) the phylogenetic distribution of H3K9me as heterochromatin-specific histone modification and its evolutionary stability in relation to other epigenetic marks, (2) known families of H3K9 methyltransferases, (3) their responsibility for the formation of constitutive heterochromatin and (4) the evolution of Su(var)3-9-like and SUVH-like H3K9 methyltransferases. Compilation and parsimony analysis reveal that histone H3K9 methylation is, next to histone deacetylation, the evolutionary most stable heterochromatic mark, which is established by at least two subfamilies of specialized heterochromatic HMTases in almost all studied eukaryotes.     

Drosophila SETDB1 is required for chromosome 4 silencing

Histone H3 lysine 9 (H3K9) methylation is associated with gene repression and heterochromatin formation. In Drosophila, SU(VAR)3–9 is responsible for H3K9 methylation mainly at pericentric heterochromatin. However, the histone methyltransferases responsible for H3K9 methylation at euchromatic sites, telomeres, and at the peculiar Chromosome 4 have not yet been identified. Here, we show that DmSETDB1 is involved in nonpericentric H3K9 methylation. Analysis of two DmSetdb1 alleles generated by homologous recombination, a deletion, and an allele where the 3HA tag is fused to the endogenous DmSetdb1, reveals that this gene is essential for fly viability and that DmSETDB1 localizes mainly at Chromosome 4. It also shows that DmSETDB1 is responsible for some of the H3K9 mono- and dimethyl marks in euchromatin and for H3K9 dimethylation on Chromosome 4. Moreover, DmSETDB1 is required for variegated repression of transgenes inserted on Chromosome 4. This study defines DmSETDB1 as a H3K9 methyltransferase that specifically targets euchromatin and the autosomal Chromosome 4 and shows that it is an essential factor for Chromosome 4 silencing.     

Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

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.     

H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases

Transcribed inverted repeats are potent triggers for RNA interference and RNA-directed DNA methylation in plants through the production of double-stranded RNA (dsRNA). For example, a transcribed inverted repeat of endogenous genes in Arabidopsis thaliana, PAI1-PAI4, guides methylation of itself as well as two unlinked duplicated PAI genes, PAI2 and PAI3. In previous work, we found that mutations in the SUVH4/KYP histone H3 lysine 9 (H3 K9) methyltransferase cause a loss of DNA methylation on PAI2 and PAI3, but not on the inverted repeat. Here we use chromatin immunoprecipitation analysis to show that the transcribed inverted repeat carries H3 K9 methylation, which is maintained even in an suvh4 mutant. PAI1-PAI4 H3 K9 methylation and DNA methylation are also maintained in an suvh6 mutant, which is defective for a gene closely related to SUVH4. However, both epigenetic modifications are reduced at this locus in an suvh4 suvh6 double mutant. In contrast, SUVH6 does not play a significant role in maintenance of H3 K9 or DNA methylation on PAI2, transposon sequences, or centromere repeat sequences. Thus, SUVH6 is preferentially active at a dsRNA source locus versus targets for RNA-directed chromatin modifications.     

Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing

Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologues, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3-9 null mutants, H3-K9 methylation at chromocentre heterochromatin is strongly reduced, indicating that SU(VAR)3-9 is the major heterochromatin-specific HMTase in Drosophila. SU (VAR)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, SU(VAR)3-7. Notably, SU(VAR)3-9-HP1 interaction is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of HP1 at the chromocentre is nearly lost without affecting HP1 accumulation at the fourth chromosome. By contrast, in HP1 null mutants SU(VAR)3-9 is no longer restricted at heterochromatin but broadly dispersed across the chromosomes. Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of HP1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects. Together, these data indicate a central role for the SU(VAR)3-9 HMTase in heterochromatin-induced gene silencing in Drosophila.     

An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation

Cytosine methylation is critical for correct development and genome stability in mammals and plants. In order to elucidate the factors that control genomic DNA methylation patterning, a genetic screen for mutations that disrupt methylation-correlated silencing of the endogenous gene PAI2 was conducted in Arabidopsis: This screen yielded seven loss-of-function alleles in a SET domain protein with histone H3 Lys9 methyltransferase activity, SUVH4. The mutations conferred reduced cytosine methylation on PAI2, especially in non-CG sequence contexts, but did not affect methylation on another PAI locus carrying two genes arranged as an inverted repeat. Moreover, an unmethylated PAI2 gene could be methylated de novo in the suvh4 mutant background. These results suggest that SUVH4 is involved in maintenance but not establishment of methylation at particular genomic regions. In contrast, a heterochromatin protein 1 homolog, LHP1, had no effect on PAI methylation.     

Multiple SET Methyltransferases Are Required to Maintain Normal Heterochromatin Domains in the Genome of Drosophila melanogaster

Methylation of histone H3 lysine 9 (H3K9) is a key feature of silent chromatin and plays an important role in stabilizing the interaction of heterochromatin protein 1 (HP1) with chromatin. Genomes of metazoans such as the fruit fly Drosophila melanogaster generally encode three types of H3K9-specific SET domain methyltransferases that contribute to chromatin homeostasis during the life cycle of the organism. SU(VAR)3-9, dG9a, and dSETDB1 all function in the generation of wild-type H3K9 methylation levels in the Drosophila genome. Two of these enzymes, dSETDB1 and SU(VAR)3-9, govern heterochromatin formation in distinct but overlapping patterns across the genome. H3K9 methylation in the small, heterochromatic fourth chromosome of D. melanogaster is governed mainly by dSETDB1, whereas dSETDB1 and SU(VAR)3-9 function in concert to methylate H3K9 in the pericentric heterochromatin of all chromosomes, with dG9a having little impact in these domains, as shown by monitoring position effect variegation. To understand how these distinct heterochromatin compartments may be differentiated, we examined the developmental timing of dSETDB1 function using a knockdown strategy. dSETDB1 acts to maintain heterochromatin during metamorphosis, at a later stage in development than the reported action of SU(VAR)3-9. Surprisingly, depletion of both of these enzymes has less deleterious effect than depletion of one. These results imply that dSETDB1 acts as a heterochromatin maintenance factor that may be required for the persistence of earlier developmental events normally governed by SU(VAR)3-9. In addition, the genetic interactions between dSETDB1 and Su(var)3-9 mutations emphasize the importance of maintaining the activities of these histone methyltransferases in balance for normal genome function.     

The SRA methyl-cytosine-binding domain links DNA and histone methylation

Epigenetic gene silencing suppresses transposon activity and is critical for normal development . Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2). In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif.     

SET3p monomethylates histone H3 on lysine 9 and is required for the silencing of tandemly repeated transgenes in Chlamydomonas

SET domain-containing proteins of the SU(VAR)3-9 class are major regulators of heterochromatin in several eukaryotes, including mammals, insects, plants and fungi. The function of these polypeptides is mediated, at least in part, by their ability to methylate histone H3 on lysine 9 (H3K9). Indeed, mutants defective in SU(VAR)3-9 proteins have implicated di- and/or trimethyl H3K9 in the formation and/or maintenance of heterochromatin across the eukaryotic spectrum. Yet, the biological significance of monomethyl H3K9 has remained unclear because of the lack of mutants exclusively defective in this modification. Interestingly, a SU(VAR)3-9 homolog in the unicellular green alga Chlamydomonas reinhardtii, SET3p, functions in vitro as a specific H3K9 monomethyltransferase. RNAi-mediated suppression of SET3 reactivated the expression of repetitive transgenic arrays and reduced global monomethyl H3K9 levels. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrated that transgene reactivation correlated with the partial loss of monomethyl H3K9 from their chromatin. In contrast, the levels of trimethyl H3K9 or the repression of euchromatic sequences were not affected by SET3 downregulation; whereas dimethyl H3K9 was undetectable in Chlamydomonas. Thus, our observations are consistent with a role for monomethyl H3K9 as an epigenetic mark of repressed chromatin and raise questions as to the functional distinctiveness of different H3K9 methylation states.