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

Abstract

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.

Nucleolar Dominance of the Y Chromosome in Drosophila melanogaster

“…Drosophila rDNA genes also show moderate levels of H3K9me (Blattes et al 2006;Peng and Karpen 2007), arguing that heterochromatin-mediated repression is a general feature of rDNA regulation. While the Su(var)3-9 enzyme is one of the major H3K9 methyltransferases for Drosophila heterochromatin (Brower-Toland et al 2009), it is unknown how much histone methylation at the rDNA genes is due to the Su(var)3-9 enzyme. In any case, our genetic analysis shows that this enzyme only partially contributes to silencing of the X chromosome rDNA array, suggesting that rDNA silencing may be molecularly distinct from other forms of heterochromatic gene silencing.…”

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

“…Hairpin line construction was carried out as described (42) except that the piwi fragment was amplified from a cDNA clone, GM05853 (43), using the following primer pair: forward 5′-GCT CTA GAT CCG GTT GAG CTG GTA TCC AAG AA-3′ and reverse 5′-GCT CTA GAA GAT CGT CTC GGT GCG CAT AAC TT-3′. Seven transgenic lines with different insertion sites were recovered (SI Appendix, Table S4).…”

Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes

“…Previous work has demonstrated that Su(var)3-9 mutants display defects in H3K9 methylation patterns and reduced pericentric HP1a localization in both the ovary (Schotta et al 2002;Yoon et al 2008) and salivary glands (Brower-Toland et al 2009), and similar phenotypes have been observed in Eggless RNAi KD salivary glands (Brower-Toland et al 2009). When we use the mata driver to knock down either Su(var)3-9 or Eggless protein during meiotic prophase, we observe PHeMAAH defects at levels similar to those observed in HP1a and Piwi knockdown ovaries.…”

“…Unfortunately, all publically available Su(var)3-9 hairpins target sequences in this common exon, raising the possibility that the PHeMAAH defects that we observe in Su(var)3-9 KD oocytes result from disruption of eIF-g function rather than reduced H3K9 methyltransferase activity. To further investigate the requirement of the H3K9me2/3 mark for promoting centromere proximal association of achiasmate homologs, we knocked down Eggless (also known as dSETDB1), a second methyltransferase that is also required to maintain the H3K9me2/3 mark in the pericentric heterochromatin (Brower-Toland et al 2009). With two different hairpins targeting different sequences within the Eggless mRNA ( Figure S2), FM7a/X KD oocytes exhibited a significant increase in PHeMAAH defects compared to control oocytes (V20-1: 22.0% in KD vs. 13.6% in control, P = 0.005; V20-2: 28.4% in KD vs. 14.4% in control, P , 0.001; Figure 1F).…”