Windei, the Drosophila Homolog of mAM/MCAF1, Is an Essential Cofactor of the H3K9 Methyl Transferase dSETDB1/Eggless in Germ Line Development

The epigenetic regulation of gene expression by the covalent modification of histones is a fundamental mechanism required for the proper differentiation of germ line cells during development. Trimethylation of histone 3 lysine 9 (H3K9me3) leads to chromatin silencing and the formation of heterochromatin by recruitment of heterochromatin protein 1 (HP1). dSETDB1/Eggless (Egg), the ortholog of the human methyltransferase SETDB1, is the only essential H3K9 methyltransferase in Drosophila and is required for H3K9 trimethylation in the female germ line. Here we show that Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF1, is an essential cofactor of Egg required for its nuclear localization and function in female germ line cells. By deletion analysis combined with coimmunoprecipitation, we have identified the protein regions in Wde and Egg that are necessary and sufficient for the interaction between the two proteins. We furthermore identified a region of Egg that gets covalently modified by SUMOylation, which may facilitate the formation of higher order chromatin-modifying complexes. Together with Egg, Wde localizes to euchromatin, is enriched on chromosome 4, and binds to the Painting of fourth (POF) protein. Our data provide the first genetic and phenotypic analysis of a mAM/MCAF1 homolog in a model organism and demonstrate its essential function in the survival of germ line cells

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

Differential Localization and Independent Acquisition of the H3K9me2 and H3K9me3 Chromatin Modifications in the Caenorhabditis elegans Adult Germ Line

Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells

Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms

SETDB1 Is Involved in Postembryonic DNA Methylation and Gene Silencing in Drosophila

DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3–9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian “Heterochromatin Protein 1”, to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development

Perturbation Analysis of Heterochromatin-Mediated Gene Silencing and Somatic Inheritance

Repetitive sequences in eukaryotic genomes induce chromatin-mediated gene-silencing of juxtaposed genes. Many components that promote or antagonize silencing have been identified, but how heterochromatin causes variegated and heritable changes in gene expression remains mysterious. We have used inducible mis-expression in the Drosophila eye to recover new factors that alter silencing caused by the bwD allele, an insertion of repetitive satellite DNA that silences a bw+ allele on the homologous chromosome. Inducible modifiers allow perturbation of silencing at different times in development, and distinguish factors that affect establishment or maintenance of silencing. We find that diverse chromatin and RNA processing factors can de-repress silencing. Most factors are effective even in differentiated cells, implying that silent chromatin remains plastic. However, over-expression of the bantam microRNA or the crooked-legs (crol) zinc-finger protein only de-repress silencing when expressed in cycling cells. Over-expression of crol accelerates the cell cycle, and this is required for de-repression of silencing. Strikingly, continual over-expression of crol converts the speckled variegation pattern of bwD into sectored variegation, where de-repression is stably inherited through mitotic divisions. Over-expression of crol establishes an open chromatin state, but the factor is not needed to maintain this state. Our analysis reveals that active chromatin states can be efficiently inherited through cell divisions, with implications for the stable maintenance of gene expression patterns through development

SU(VAR)3-7 Links Heterochromatin and Dosage Compensation in Drosophila

In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. We previously found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. In this study, we examine the impact of change in levels of SU(VAR)3-7 on dosage compensation. We first demonstrate that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, we show that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation

Buffering and the evolution of chromosome-wide gene regulation

Copy number variation (CNV) in terms of aneuploidies of both entire chromosomes and chromosomal segments is an important evolutionary driving force, but it is inevitably accompanied by potentially problematic variations in gene doses and genomic instability. Thus, a delicate balance must be maintained between mechanisms that compensate for variations in gene doses (and thus allow such genomic variability) and selection against destabilizing CNVs. In Drosophila, three known compensatory mechanisms have evolved: a general segmental aneuploidy-buffering system and two chromosome-specific systems. The two chromosome-specific systems are the male-specific lethal complex, which is important for dosage compensation of the male X chromosome, and Painting of fourth, which stimulates expression of the fourth chromosome. In this review, we discuss the origin and function of buffering and compensation using Drosophila as a model

Epigenetic Regulation of Learning and Memory by Drosophila EHMT/G9a

Behavioral phenotyping and genome-wide profiling of the histone modifier EHMT in Drosophila reveals a mechanism through which an epigenetic writer may control cognition