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