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.     

SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing

This review summarizes genetic, molecular and biochemical studies of the SU(VAR)3-9 protein and the evidence for its key role in heterochromatin formation and heterochromatic gene silencing. The Su(var)3-9 locus was first identified as a dominant modifier of position-effect variegation (PEV) in Drosophila melanogaster. Together with Su(var)2-5 and Su(var)3-7, Su(var)3-9 belongs to the group of haplo-suppressor loci which show a triplo-dependent enhancer effect. All three genes encode heterochromatin-associated proteins. Su(var)3-9 is epistatic to the PEV modifier effects of Su(var)2-5 and Su(var)3-7, and it also dominates the effect of the Y chromosome on PEV. These genetic data support a central role of the SU(VAR)3-9 protein in heterochromatic gene silencing, one that is correlated with its activity as a histone H3-K9 methyltransferase (HMTase). In fact, SU(VAR)3-9 is the main chromocenter-specific HMTase of Drosophila. SU(VAR)3-9 and HP1, the product of Su(var)2-5, are main constituents of heterochromatin protein complexes and the interaction between these two proteins is interdependent. Functional analysis in fission yeast, Drosophila and mammals demonstrate that SU(VAR)3-9-dependent gene silencing processes are conserved in these organisms. This is also demonstrated by the rescue of Drosophila Su(var)3-9 mutant phenotypes with human SUV39H1 transgenes.     

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.     

Controlled expression of tagged proteins in Drosophila using a new modular P-element vector system

We have developed a new modular vector system that facilitates the combination of various DNA fragments as functional modules for P element-mediated transformation of Drosophila melanogaster. The basic vector pP?GS? contains unique sites for 17 restriction enzymes, including three 8-bp cutters, that allow one to combine various promoter elements, cDNAs and genomic DNA fragments, as well as protein tags and selectable marker genes, for a wide spectrum of transgene analyses. With this new vector system we analysed the chromosomal distribution of the Drosophila SU(VAR)3-9 protein tagged with EGFP, using hsp70-cDNA and genomic Su(var)3-9 constructs. We found preferential association of the tagged SU(VAR)3-9 with centric heterochromatin.     

Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis

Chromosoma - Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that...     

SU(VAR)3-7, a Drosophila heterochromatin-associated protein and companion of HP1 in the genomic silencing of position-effect variegation.

An increase in the dose of the Su(var)3-7 locus of Drosophila melanogaster enhances the genomic silencing of position-effect variegation caused by centromeric heterochromatin. Here we show that the product of Su(var)3-7 is a nuclear protein which associates with pericentromeric heterochromatin at interphase, whether on diploid chromosomes from embryonic nuclei or on polytene chromosomes from larval salivary glands. The protein also associates with the partially heterochromatic chromosome 4. As these phenotypes and localizations resemble those described by others for the Su(var)2-5 locus and its heterochromatin-associated protein HP1, the presumed co-operation of the two proteins was tested further. The effect of the dose of Su(var)3-7 on silencing of a number of variegating rearrangements and insertions is strikingly similar to the effect of the dose of Su(var)2-5 reported by others. In addition, the two loci interact genetically, and the two proteins co-immunoprecipitate from nuclear extracts. The results suggest that SU(VAR)3-7 and HP1 co-operate in building the genomic silencing associated with heterochromatin.     

Mechanisms of HP1-mediated gene silencing in Drosophila

Heterochromatin Protein 1 (HP1) is a structural component of silent chromatin at telomeres and centromeres. Euchromatic genes repositioned near heterochromatin by chromosomal rearrangements are typically silenced in an HP1-dependent manner. Silencing is thought to involve the spreading of heterochromatin proteins over the rearranged genes. HP1 associates with centric heterochromatin through an interaction with methylated lysine 9 of histone H3, a modification generated by SU(VAR)3-9. The current model for spreading of silent chromatin involves HP1-dependent recruitment of SU(VAR)3-9, resulting in the methylation of adjacent nucleosomes and association of HP1 along the chromatin fiber. To address mechanisms of silent chromatin formation and spreading, HP1 was fused to the DNA-binding domain of the E. coli lacI repressor and expressed in Drosophila melanogaster stocks carrying heat shock reporter genes positioned 1.9 and 3.7 kb downstream of lac operator repeats. Association of lacI-HP1 with the repeats resulted in silencing of both reporter genes and correlated with a closed chromatin structure consisting of regularly spaced nucleosomes, similar to that observed in centric heterochromatin. Chromatin immunoprecipitation experiments demonstrated that HP1 spread bi-directionally from the tethering site and associated with the silenced reporter transgenes. To examine mechanisms of spreading, the effects of a mutation in Su(var)3-9 were investigated. Silencing was minimally affected at 1.9 kb, but eliminated at 3.7 kb, suggesting that HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner.     

dSETDB1 and SU(VAR)3-9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster

Germline-stem cells (GSCs) produce gametes and are thus true "immortal stem cells". In Drosophila ovaries, GSCs divide asymmetrically to produce daughter GSCs and cystoblasts, and the latter differentiate into germline cysts. Here we show that the histone-lysine methyltransferase dSETDB1, located in pericentric heterochromatin, catalyzes H3-K9 trimethylation in GSCs and their immediate descendants. As germline cysts differentiate into egg chambers, the dSETDB1 function is gradually taken over by another H3-K9-specific methyltransferase, SU(VAR)3-9. Loss-of-function mutations in dsetdb1 or Su(var)3-9 abolish both H3K9me3 and heterochromatin protein-1 (HP1) signals from the anterior germarium and the developing egg chambers, respectively, and cause localization of H3K9me3 away from DNA-dense regions in most posterior germarium cells. These results indicate that dSETDB1 and SU(VAR)3-9 act together with distinct roles during oogenesis, with dsetdb1 being of particular importance due to its GSC-specific function and more severe mutant phenotype.     

The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster

In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, we demonstrate that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, we show that distal heterochromatin (blocks h26-h29) is the only one within the chromocenter to form a big "puff"-like structure. The "puffed" heterochromatin has not only unique morphology but also very special protein composition as well: (i) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (ii) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, we show that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization.     

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 evolution of the histone methyltransferase gene Su(var)3-9 in metazoans includes a fusion with and a re-fission from a functionally unrelated gene

Background  

In eukaryotes, histone H3 lysine 9 (H3K9) methylation is a common mechanism involved in gene silencing and the establishment of heterochromatin. The loci of the major heterochromatic H3K9 methyltransferase Su(var)3-9 and the functionally unrelated γ subunit of the translation initiation factor eIF2 are fused in Drosophila melanogaster. Here we examined the phylogenetic distribution of this unusual gene fusion and the molecular evolution of the H3K9 HMTase Su(var)3-9.     

Position-effect variegation and the genetic dissection of chromatin regulation in Drosophila

In position-effect variegation (PEV) genes become silenced by heterochromatisation. Genetic dissection of this process has been performed by means of dominant suppressor [Su(var)] and enhancer [E(var)] mutations. Selective genetic screens allowed mass isolation of more than 380 PEV modifier mutations identifying about 150 genes. Genetic fine structure studies revealed unique dosage dependent effects. Most of the haplo-dependent Su(var) and E(var) genes do not display triplo-dependent effects. Several Su(var) loci with triplo-dependent opposite enhancer effects have been identified and shown to encode heterochromatin-associated proteins. From these the evolutionary conserved histone H3 lysine 9 methyltransferase SU(VAR)3-9 plays a central role in heterochromatic gene silencing. Molecular function of most PEV modifier genes is still unknown also including genes identified with mutations displaying lethal interaction to heterochromatin. Their analysis should contribute to further understanding of processes connected with regulation of higher order chromatin structure and epigenetic programming.     

SET-domain proteins of the Su(var)3-9, E(z) and trithorax families

SET-domain (SET: Su(var)3-9, E(z) and Trithorax)-containing proteins were collected through sequence searches of the available databases. After removing redundancies, the proteins belonging to three families, SU(VAR)3-9, E(Z) and Trithorax, were selected. Analysis of the relationship between the different members is based on pairwise alignment, compilation, and comparison of their SET-domains. The level of homology of the SET-domains defined the distribution of the proteins into families and into clades within the families. The architecture of the entire protein supported the distribution pattern built upon SET-domain similarity. Parallel cladistic and protein-architecture analyses outlined two plausible criteria for predicting function.     

Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31

The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, we have isolated human (SUV39H1) and mouse (Suv39h1) homologues of the dominant Drosophila modifier of position-effect-variegation (PEV) Su(var)3-9. Mammalian homologues contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31, currently the only other characterized mammalian SU(VAR) homologue. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin.     

Molecular landscape of modified histones in Drosophila heterochromatic genes and euchromatin-heterochromatin transition zones

Constitutive heterochromatin is enriched in repetitive sequences and histone H3-methylated-at-lysine 9. Both components contribute to heterochromatin's ability to silence euchromatic genes. However, heterochromatin also harbors hundreds of expressed genes in organisms such as Drosophila. Recent studies have provided a detailed picture of sequence organization of D. melanogaster heterochromatin, but how histone modifications are associated with heterochromatic sequences at high resolution has not been described. Here, distributions of modified histones in the vicinity of heterochromatic genes of normal embryos and embryos homozygous for a chromosome rearrangement were characterized using chromatin immunoprecipitation and genome tiling arrays. We found that H3-di-methylated-at-lysine 9 (H3K9me2) was depleted at the 5' ends but enriched throughout transcribed regions of heterochromatic genes. The profile was distinct from that of euchromatic genes and suggests that heterochromatic genes are integrated into, rather than insulated from, the H3K9me2-enriched domain. Moreover, the profile was only subtly affected by a Su(var)3-9 null mutation, implicating a histone methyltransferase other than SU(VAR)3-9 as responsible for most H3K9me2 associated with heterochromatic genes in embryos. On a chromosomal scale, we observed a sharp transition to the H3K9me2 domain, which coincided with increased retrotransposon density in the euchromatin-heterochromatin (eu-het) transition zones on the long chromosome arms. Thus, a certain density of retrotransposons, rather than specific boundary elements, may demarcate Drosophila pericentric heterochromatin. We also demonstrate that a chromosome rearrangement that created a new eu-het junction altered H3K9me2 distribution and induced new euchromatic sites of enrichment as far as several megabases away from the breakpoint. Taken together, the findings argue against simple classification of H3K9me as the definitive signature of silenced genes, and clarify roles of histone modifications and repetitive DNAs in heterochromatin. The results are also relevant for understanding the effects of chromosome aberrations and the megabase scale over which epigenetic position effects can operate in multicellular organisms.