Article Figures & Tables
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.
Article Navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Other journals from The Company of Biologists
Why should you publish your next paper in BiO?
Biology Open has strong credentials and publishing with us is easy and fast. BiO aims to provide rapid publication for scientifically sound observations and valid conclusions in cell biology, developmental biology and experimental biology. Submit your paper here; you’ll be in good company.
Recent developmental biology research highlights in Biology Open
Meningeal retinoic acid contributes to neocortical lamination and radial migration during mouse brain development
Carole Haushalter, Brigitte Schuhbaur, Pascal Dollé and Muriel Rhinn
Constitutively active Notch1 converts cranial neural crest-derived frontonasal mesenchyme to perivascular cells in vivo
Sophie R. Miller, Surangi N. Perera and Clare V. H. Baker
Cardiomyocyte-specific expression of the nuclear matrix protein, CIZ1, stimulates production of mono-nucleated cells with an extended window of proliferation in the postnatal mouse heart
Sumia A. Bageghni, Georgia A. Frentzou, Mark J. Drinkhill, William Mansfield, Dawn Coverley and Justin F. X. Ainscough
Cover article: Arabidopsis TOP1α is required for adaptive response to light and flower development
The June cover shows scanning electron microscopy of elongated and fragmented apical meristem of an Arabidopsis plant with fas5 mutation in the DNA topoisomerase TOP1α. In the related article, Kupriyanova et al. uncover a new function for TOP1α connected with response to light signal.
Featured article: Alteration of swing leg work and power during human accelerated sprinting
In our most-read article in May, Nagahara et al. report that the roles of respective swing leg joints intricately change with increases in running speed during accelerated sprinting.
Transfer to Biology Open
If your submission to one of our other journals, Development, Journal of Cell Science, Journal of Experimental Biology or Disease Models & Mechanism, is unsuccessful, did you know you can transfer your paper and any reviews directly to Biology Open? The majority of papers transferred with reviews are accepted for publication. Find out how here.