Roles of histone H3K9 methyltransferases during Drosophila spermatogenesis.

Epigenetic regulation of gene expression by covalent modification of histones is important for germ line cell development. In mammals, histone H3 lysine 9 (H3K9)-specific histone methyltransferases (HMTases), such as G9a, SETDB1, and SUV39H, play critical roles, but the contribution of H3K9-specific HMTases in Drosophila remains to be clarified, especially in male sperm. Here, we performed immunocytochemical analyses with a specific antibody to dG9a, Drosophila G9a ortholog, and demonstrated localization in the cytoplasm from the growth to elongation stages of spermatogenesis. In the subsequent early canoe stage, strong dG9a signals were detected exclusively in nuclei, suggesting a regulatory role. However, mono-, di-, and trimethylated H3K9 signals were not extensively decreased in a homozygous dG9a null mutant throughout these stages. In contrast, mono- and trimethylated H3K9 signals were extensively decreased in a heterozygous DmSetdb1 mutant during spermatogenesis, and similar reduction in monomethylated H3K9 signals was observed in a homozygous Su(var)3-9 mutant. Therefore, DmSETDB1 is likely to be mainly responsible for mono- and trimethylation of H3K9 and SU(VAR)3-9 for monomethylation of H3K9 during spermatogenesis. However, the reduced methylation of H3K9 in premeiotic spermatocytes did not influence X-Y chromosome disjunction in male meiosis, suggesting that it may not be critical for spermatogenesis in Drosophila.

Identification of nuclear localization signals of Drosophila G9a histone H3 methyltransferase.

G9a is one of the well-characterized histone methyltransferases. G9a regulates H3K9 mono- and dimethylation at euchromatic region and consequently plays important roles in euchromatic gene regulation. Mammalian G9a contains several distinct domains, such as GHD (G9a homology domain), ANK, preSET, SET and PostSET. These domains are highly conserved between mammals and Drosophila. Although mammalian G9a has nuclear localization signal (NLS) in its N-terminal region, the amino acid sequences of this region are not conserved in Drosophila. Here we have examined the subcellular localization of a series of truncated forms of Drosophila G9a (dG9a). The identified region (aa337-aa470) responsible for nuclear localization of dG9a contains four short stretches of positively charged basic amino acids (NLS1, aa334-aa345; NLS2, aa366-aa378; NLS3, aa407-aa419; NLS4, aa461-aa472). Each of NLS1, NLS3 and NLS4 is sufficient for the nuclear localization when they are fused with the enhanced green fluorescent protein. These NLSs of dG9a are distinct from those of mammalian G9a in their positions and amino acid sequences.