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. 2018 Oct 8;9(1):4155.
doi: 10.1038/s41467-018-06697-x.

The H3K9 Methyltransferase SETDB1 Maintains Female Identity in Drosophila Germ Cells

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Free PMC article

The H3K9 Methyltransferase SETDB1 Maintains Female Identity in Drosophila Germ Cells

Anne E Smolko et al. Nat Commun. .
Free PMC article

Abstract

The preservation of germ cell sexual identity is essential for gametogenesis. Here we show that H3K9me3-mediated gene silencing is integral to female fate maintenance in Drosophila germ cells. Germ cell specific loss of the H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, WDE, and HP1a, leads to ectopic expression of genes, many of which are normally expressed in testis. SETDB1 controls the accumulation of H3K9me3 over a subset of these genes without spreading into neighboring loci. At phf7, a regulator of male germ cell sexual fate, the H3K9me3 peak falls over the silenced testis-specific transcription start site. Furthermore, H3K9me3 recruitment to phf7 and repression of testis-specific transcription is dependent on the female sex determination gene Sxl. Thus, female identity is secured by an H3K9me3 epigenetic pathway in which Sxl is the upstream female-specific regulator, SETDB1 is the required chromatin writer, and phf7 is one of the critical SETDB1 target genes.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
H3K9me3 is required for germ cell differentiation. ac Reduced H3K9me3 staining in germ cells upon SETDB1 and WDE depletion (GLKD, see text for details). Representative confocal images of a wild-type (WT), setdb1 GLKD, and wde GLKD germarium stained for H3K9me3 (green, white in a”–c”). Germ cells were identified by α-VASA staining (magenta). Scale bar, 25 μm. Insets show a higher magnification of a single germ cell, in which the nucleus is outlined by a white dashed line in a”–c”. dg Undifferentiated germ cells accumulate in setdb1 GLKD, wde GLKD, and hp1a GLKD mutant germaria. Representative confocal images of wild-type and mutant germaria stained for DNA (magenta) and α-Spectrin (cyan) to visualize spectrosomes (sp), fusomes (fu), and somatic cell membranes. Scale bar, 25 μm. h Quantification of mutant germaria with 0, 1–5, and >5 round spectrosome-containing germ cells. The number of scored germaria (n) is indicated
Fig. 2
Fig. 2
SETDB1, WDE, and HP1a depletion leads to female-to-male reprogramming at phf7. a Depletion of H3K9me3 pathway members leads to ectopic expression of the testis-specific phf7-RC isoform. RT-qPCR analysis of the testis phf7-RC transcript in wild-type testis, wild-type and mutant ovaries. Expression, normalized to the total level of phf7, is shown as fold change relative to testis. Primers are shown in panel F. Error bars indicate standard deviation (s.d.) of three biological replicates. be Depletion of H3K9me3 pathway members leads to ectopic expression of the testis-specific PHF7 protein. Ovaries from animals carrying an HA-PHF7 transgene stained for HA (green, white in B’-E’). Germ cells were identified by α-VASA staining (magenta). Scale bar, 25 μm. f RNA-seq data confirms ectopic expression of the testis-specific phf7-RC isoform. Genome browser view of the phf7 locus. Tracks show RNA-seq reads aligned to the Drosophila genome (UCSC dm6). All tracks are viewed at the same scale. The screen shot is reversed so that the 5’ end of the gene is on the left. The reads that are unique to the mutant ovaries are highlighted in gray. The two phf7 transcripts, phf7-RA and phf7-RC, are indicated. phf7-RC is normally only expressed in testis (blue arrow). phf7-RA is normally expressed in ovaries (pink arrow). Primers for RT-qPCR are indicated by arrowheads: gray for phf7-RC, red for total phf7
Fig. 3
Fig. 3
Depletion of H3K9me3 pathway members leads to ectopic expression of a similar gene set. a Comparisons of the deregulated gene expression profiles of setdb1, wde and hp1a GLKD ovaries reveals extensive similarities. Heat map comparing changes in gene expression in setdb1, wde, and hp1a GLKD ovaries compared to wild-type (WT) ovaries. Each row depicts a gene whose expression is deregulated at least 2-fold (FDR < 0.05) in all mutants when compared to wild-type. b Depletion of pathway members leads to ectopic expression of genes which are not normally expressed in ovaries. Scatter plots of significantly upregulated genes in setdb1, wde, and hp1a GLKD ovaries. The log2 fold change in gene expression is plotted against the log2 of the FPKM values in wild-type ovaries. Colored points indicate ectopically expressed genes (log2 FPKM < 0 in WT ovaries). c Venn diagram showing overlap of ectopically expressed genes in setdb1, wde, and hp1a GLKD ovaries. The amount of overlap is significantly higher than expected stochastically (P < 10–6). The significance of each two-way overlap was assessed using Fisher’s exact test performed in R, in each case yielding P < 10−6. The significance of the three-way overlap was assessed by a Monte Carlo simulation, yielding P < 10−6. For each genotype, the number (n) of ectopically expressed genes is indicated. d A majority of ectopically expressed genes are normally expressed in testis. Bar chart showing the percentage of ectopically expressed genes in setdb1, wde, and hp1a GLKD ovaries that are normally expressed in wild-type testis. Genes are assigned into groups based on expression in wild-type and bgcn mutant testis (see text for details): early stage spermatogonia genes (>2-fold increase in bgcn testis compared to wild-type testis, in blue), late-stage spermatocyte genes (>2-fold decrease in bgcn testis compared to wild-type testis, in green) and genes detected during both stages (FPKM > 1 in both samples, in red). Genes which are not expressed in either testis sample (FPKM < 1) are in gray. For each genotype, the number (n) of ectopically expressed genes is indicated
Fig. 4
Fig. 4
Loss of SETDB1 in female germ cells leads to H3K9me3 depletion on a select set of genes a Differential analysis of paired H3K9me3 ChIP-seq data sets identifies SETDB1-dependent H3K9me3 peaks. Scatter plot showing the significantly altered (FDR < 0.05) H3K9me3 peaks in setdb1 GLKD ovaries relative to wild-type (WT) ovaries. The x-axis is the log2 H3K9me3 enrichment of wild-type peaks subtracted from the log2 H3K9me3 enrichment of setdb1 GLKD peaks. Negative values indicate a reduction in H3K9me3 in mutant ovaries. b The H3K9me3 peak over the testis-specific phf7-RC TSS is decreased in setdb1 GLKD and snf148 mutant ovaries. Genome browser view of phf7 and neighboring genes CG9577 and rab35. c In setdb1 GLKD ovaries, ectopic expression of CG34434 is correlated with a decreased in H3K9me3 accumulation. H3K9me3 accumulation is also decreased in snf148 mutant ovaries. Genome browser view of CG34434 and its neighbors, rhoGAP5A and CG34435. RNA reads in wild-type (WT) and setdb1 GLKD are in blue and orange, respectively. The ChIP-seq reads are in black, green and red. CG34434 is shaded. d Average enrichment profile indicates that the H3K9me3 peaks at the 21 SETDB1-regulated genes are localized over the gene body. The average H3K9me3 enrichment profile on the average gene body (transcription start site to transcription end site) scaled to 1500 base pairs, ±3 kb. In red, the average enrichment profile of euchromatic genes which display an H3K9me3 peak in wild-type ovaries. In blue, the average H3K9me3 profile of the 21 genes which are both ectopically expressed and display a loss of H3K9me3 enrichment in setdb1 GLKD ovaries
Fig. 5
Fig. 5
Distribution of the 21 SETDB1/H3K9me3 regulated genes in the genome. The 21 genes are not clustered together in the genome. Gene positions are shown on the six major chromosome arms. Chromosome length is indicated in megabase pairs (Mb). See Supplementary Table 1 for the exact position of each gene. The normal expression pattern of these genes is indicated as follows: genes with testis-specific isoform (dark blue), genes expressed in testis and other tissues (turquoise) and genes not normally expressed in adult testis (red). See Table 1 for details
Fig. 6
Fig. 6
H3K9me3 distribution is affected in snf148 mutant ovaries. a, b Reduced H3K9me3 staining in snf148 mutant germ cells. Confocal images of a wild-type (WT) and snf148 germaria stained for H3K9me3 (green, white in inset). Germ cells were identified by α-VASA staining (magenta). Scale bar, 25 μm. Insets show a higher magnification of a single germ cell, outlined by a dashed line. c, d SETDB1 protein localization is not altered in snf148 mutant germ cells. Confocal images of a germaria from wild-type and snf148 females carrying a copy an endogenously HA-tagged allele of setdb1 stained for HA (green, white in inset). Germ cells were identified by α-VASA staining (magenta). Scale bar, 12.5 μm. Insets show a higher magnification of a single germ cell, outlined by a dashed line. e Western blot of ovarian lysates from wild-type and snf148 females carrying a copy an endogenously HA-tagged allele of setdb1 probed with an antibody against the HA tag. α-tubulin the loading control. f Diagram of genetic pathway controlling H3K9me3 accumulation in female germ cells. In WT, SXL collaborates with SETDB1 to regulate H3K9me3 accumulation. snf148, by virtue of the fact that it interferes with Sxl splicing, leads to germ cells without SXL protein. This in turn leads to a defect in H3K9me3 accumulation without interfering with SETDB1 protein accumulation. g Differential analysis of paired H3K9me3 ChIP-seq data sets identifies peak changes in snf148 mutant ovaries. Scatter plot of significantly altered (FDR < 0.05) H3K9me3 peaks in snf148 ovaries relative to wild-type (WT) ovaries. The x-axis is the log2 H3K9me3 enrichment of wild type peaks subtracted from the log2 H3K9me3 enrichment of snf148 peaks. Negative values indicate a decreased H3K9me3 peak in mutant ovaries. h setdb1 GLKD and snf148 influence H3K9me3 accumulation on a similar set of genes, including the genes ectopically expressed in setdb1 GLKD ovaries. Plot comparing the significantly altered H3K9me3 peaks observed in snf148 ovaries to setdb1 GLKD ovaries. Genes that are not normally expressed in ovaries but are ectopically expressed in setdb1 GLKD are labeled in red or in blue. Blue indicates genes which are normally expressed in testis
Fig. 7
Fig. 7
Schematic summary of discrete facultative heterochromatin island assembly at phf7. In female germ cells, SETDB1, together with SXL, directs assembly of a highly localized H3K9me3 domain around the testis-specific TSS. In germ cells lacking SETDB1 or SXL protein, the dissolution of the H3K9me3 domain correlates with ectopic testis-specific phf7-RC transcription and PHF7 protein expression. Ectopic PHF7 protein activity leads to activation of downstream testis genes and a tumor phenotype

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References

    1. Lesch BJ, Page DC. Genetics of germ cell development. Nat. Rev. Genet. 2012;13:781–794. doi: 10.1038/nrg3294. - DOI - PubMed
    1. Salz HK, Dawson EP, Heaney JD. Germ cell tumors: Insights from the Drosophila ovary and the mouse testis. Mol. Reprod. Dev. 2017;84:200–211. doi: 10.1002/mrd.22779. - DOI - PMC - PubMed
    1. Murray SM, Yang SY, van Doren M. Germ cell sex determination: a collaboration between soma and germline. Curr. Opin. Cell Biol. 2010;22:722–729. doi: 10.1016/j.ceb.2010.09.006. - DOI - PMC - PubMed
    1. Shapiro-Kulnane L, Smolko AE, Salz HK. Maintenance of Drosophila germline stem cell sexual identity in oogenesis and tumorigenesis. Development. 2015;142:1073–1082. doi: 10.1242/dev.116590. - DOI - PMC - PubMed
    1. Yang SY, Baxter EM, van Doren M. Phf7 controls male sex determination in the Drosophila germline. Dev. Cell. 2012;22:1041–1051. doi: 10.1016/j.devcel.2012.04.013. - DOI - PMC - PubMed

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