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Figures
- Fig. 1.
The tBRD-1-eGFP fusion protein restores the localization of tBRD-2 to chromosomes in spermatocytes of tbrd-1 mutants. (A) Detection of tBRD-2 in heterozygous and homozygous tbrd-1 mutant testes using anti-tBRD-2 antibody. Actin detected with anti-Actin antibody served as the control. (B,C) Single primary spermatocyte nuclei of (B) heterozygous and (C) homozygous tbrd-1 mutants stained with an antibody against tBRD-2. (D–E′) Anti-tBRD-2 antibody staining of single primary spermatocyte nuclei of (D,D’) heterozygous and (E,E′) homozygous tbrd-1 mutants expressing a tBRD-1-eGFP fusion protein. Arrows, chromosomal regions; arrowheads, nucleolus. Scale bars: 5 µm.
- Fig. 2.
The region between the two bromodomains is required for tBRD-1 homodimerization, and the C-terminus interacts with the extra-terminal domain of tBRD-2. (A,B) Schematic overview of the full-length and mutated (A) tBRD-1 and (B) tBRD-2 proteins analyzed in yeast two-hybrid experiments for interaction with (A,B) full-length tBRD-1 and (A) tBRD-2 proteins. +, interaction observed; −, no interaction observed. (C) Schematic representation of the observed interaction between tBRD-1 and tBRD-1 via the region between the two bromodomains. (D) Schematic representation of the observed interaction between the C-terminus of tBRD-1 with the extra-terminal domain of tBRD-2. BD, bromodomain; NET, NET domain; SEED, SEED domain.
- Fig. 3.
tBRD-1 associates with acetylated histones. (A) Association of recombinant tBRD-1-Flag and tBRD-2-Flag proteins with non-acetylated and acetylated histone H3 and H4 peptides [H3(1–22), H3(14–33), H3(23–42), H4(1–20)] was examined in peptide pull-down assays. Bound proteins were analyzed in western blots using tBRD-1- or tBRD-2-specific antibodies. Acetylated proteins are indicated, e.g. H3K9ac, K14ac: H3 acetylated on Lys9 and Lys14. (B-J) Single primary spermatocyte nuclei of w1118 flies stained with specific antibodies against histone H3 acetylated on (B) Lys9, (C) Lys14, (D) Lys18, (E) Lys23, (F) Lys27, and (G) Lys36 or against histone H4 acetylated on (H) Lys5, (I) Lys8, and (J) Lys12. Arrows, chromosomes; arrowheads, nucleoli. Scale bar: 5 µm.
- Fig. 4.
RNAi-mediated knockdown of tbrd-2 leads to defects in post-meiotic male germ cells. (A) qPCR using cDNA from tbrd-2 knockdown testes (bam≫tbrd-2RNAi) compared to control testes (bam-Gal4). Two different tbrd-2-specific primer pairs (tBRD-2-1 and tBRD-2-2) were used to detect tbrd-2 transcript levels. The values were normalized to the mRNA expression level of Rpl32. Three technical replicates were performed. One-way ANOVA was used to evaluate statistical significance. P-values for significance: ***P≤0.001 (post hoc Tukey's honest significant difference test). (B) Western blot analysis of tBRD-2 proteins in bam≫tbrd-2RNAi testes, and in control testes (bam-Gal4 and tbrd-2RNAi). The detection of Actin with anti-Actin antibodies served as a loading control. (C–C″) Single primary spermatocyte nuclei of (C) bam-Gal4, (C′) tbrd-2RNAi, and (C″) bam≫tbrd-2RNAi flies stained with anti-tBRD-2 antibody. Photos had the same exposure time. Scale bar: 5 µm. (D–D″) Phase-contrast images of (D) control bam-Gal4, (D′) control tbrd-2RNAi, and (D″) bam≫tbrd-2RNAi early round spermatids. Arrows, Nebenkerne. (E–E″) Replacement of histones by Mst77F in post-meiotic spermatid nuclei of (E) bam-Gal4, (E′) tbrd-2RNAi, and (E″) bam≫tbrd-2RNAi visualized by immunofluorescence staining using antibodies against histones (white) and Mst77F (green). Arrowheads: E,E′, needle-like structure of mature sperm nuclei; E″, unelongated, round sperm nuclei. Scale bars: 20 µm.
- Fig. 5.
tBRD-2 is required for gene expression. (A,B) Venn diagrams depicting the overlap of (A) 73 significantly down-regulated and (B) 104 significantly up-regulated probe sets in bam≫tbrd-2RNAi testes compared to the controls (bam-Gal4 and undriven tbrd-2RNAi). (C,D) qPCR using cDNA from 50 testes pairs of bam≫tbrd-2RNAi, tbrd-2RNAi, and bam-Gal4 testes. (C) Expression of genes CG13946, CG17917, CG18673, CG42827, CG42828 and Yp3. (D) Expression of TwdlV, CG1441, CG31750 and cutlet. The values were normalized to the expression of Rpl32. ANOVA with post hoc Tukey's honest significant difference test were used to evaluate statistical significance. P-values for significance: *P≤0.05; ***P≤0.001; NS, not significant.
- Fig. 6.
tBRD-1 and tBRD-2 share a subset of target genes with Aly, Med22, and Sa. (A–C) Scatter plots depicting transcript levels (log2-transformed gene expression values) in (A) aly, (B) Med22, and (C) sa mutant testes (y-axes) compared to wild-type control testes (x-axes). Green and blue dots represent significantly down-regulated genes in tbrd-1 mutant testes in comparison to control testes. Red and blue dots represent transcripts of genes in bam≫tbrd-2RNAi testes expressed significantly lower than in undriven tbrd-2RNAi. Blue dots represent transcripts that are affected by both tBRD-1 and tBRD-2.
- Fig. 7.
tBRD-1 and tBRD-2 require Aly function for sub-cellular localization. (A–D‴) Single primary spermatocyte nuclei of (A–A‴ and C–C‴) heterozygous and (B–B‴ and D–D‴) homozygous aly mutants stained with (A–A‴ and B–B‴) anti-tBRD-1 and (C–C‴ and D–D‴) anti-tBRD-2 antibodies. Arrows, chromosomal regions; arrowheads, nucleoli. A,B,C,D, immunofluorescent staining with anti-tBRD-1; A′,B′,C′,D′, Hoechst DNA staining; A″,B″,C″,D″, merged images; A‴,B‴,C‴,D‴, phase-contrast images. Scale bars: 5 µm.
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