FIG 2.

dLsd1 binds to the regulatory regions of the Pumilio complex genes and regulates the transcription of Nos and Brat. (A) dLsd1 binds to Nanos, Pumilio, and Brat regulatory regions. ChIP analysis of dLsd1 binding was performed using an anti-dLsd1-specific antibody in wild-type (w¹¹¹⁸) ovaries. Preimmune serum was used as a control for specificity. Int1 and Int2 are intergenic regions used as negative controls, and Bun is a previously identified dLSD1 target used as a positive control. ChIP data are the result of three independent immunoprecipitations. The y axis represents enrichment as percent input. (B) Depletion of dLsd1 results in downregulation of Nos and Brat expression in ovaries. RT-qPCR analysis of the expression of Pum, Nos, Brat, Vasa, Bam, and abd-A in ovaries depleted of dLsd1 using an Act5C driver. Tubulin was used as a control. The expression level was normalized against an Act5C wild-type control, and RP49 and RpL32 transcripts were used as a reference. (C) RT-qPCR analysis of the expression of Nanos, Pumilio, Brat, and Tubulin in w¹¹¹⁸ (wild-type) and dLsd1 ^ΔN homozygous mutant ovaries. The expression level was normalized to that of wild-type ovaries. (D) RT-qPCR analysis of the expression of Nanos, Pumilio, and Brat in ovariectomized carcasses depleted of dLsd1 using an Act5C-GAL4 driver. Act5C-GAL4 was used as a wild-type control. dLsd1 depletion was verified using dLsd1-specific primers, and the levels of the housekeeping gene Tubulin were monitored as a control. All experiments were performed at least in triplicate, and error bars indicate standard errors of the means. A Welch two-sample t test was performed to indicate significance (*, P < 0.05).