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. 2014 Sep 24;9(9):e108267.
doi: 10.1371/journal.pone.0108267. eCollection 2014.

tBRD-1 selectively controls gene activity in the Drosophila testis and interacts with two new members of the bromodomain and extra-terminal (BET) family

Ina Theofel  1 , Marek Bartkuhn  2 , Tim Hundertmark  1 , Thomas Boettger  3 , Stefanie M K Gärtner  1 , Katja Leser  1 , Stephan Awe  1 , Michael Schipper  1 , Renate Renkawitz-Pohl  1 , Christina Rathke  1
Affiliations

tBRD-1 selectively controls gene activity in the Drosophila testis and interacts with two new members of the bromodomain and extra-terminal (BET) family

Ina Theofel et al. PLoS One. .

Abstract

Multicellular organisms have evolved specialized mechanisms to control transcription in a spatial and temporal manner. Gene activation is tightly linked to histone acetylation on lysine residues that can be recognized by bromodomains. Previously, the testis-specifically expressed bromodomain protein tBRD-1 was identified in Drosophila. Expression of tBRD-1 is restricted to highly transcriptionally active primary spermatocytes. tBRD-1 is essential for male fertility and proposed to act as a co-factor of testis-specific TATA box binding protein-associated factors (tTAFs) for testis-specific transcription. Here, we performed microarray analyses to compare the transcriptomes of tbrd-1 mutant testes and wild-type testes. Our data confirmed that tBRD-1 controls gene activity in male germ cells. Additionally, comparing the transcriptomes of tbrd-1 and tTAF mutant testes revealed a subset of common target genes. We also characterized two new members of the bromodomain and extra-terminal (BET) family, tBRD-2 and tBRD-3. In contrast to other members of the BET family in animals, both possess only a single bromodomain, a characteristic feature of plant BET family members. Immunohistology techniques not only revealed that tBRD-2 and tBRD-3 partially co-localize with tBRD-1 and tTAFs in primary spermatocytes, but also that their proper subcellular distribution was impaired in tbrd-1 and tTAF mutant testes. Treating cultured male germ cells with inhibitors showed that localization of tBRD-2 and tBRD-3 depends on the acetylation status within primary spermatocytes. Yeast two-hybrid assays and co-immunoprecipitations using fly testes protein extracts demonstrated that tBRD-1 is able to form homodimers as well as heterodimers with tBRD-2, tBRD-3, and tTAFs. These data reveal for the first time the existence of single bromodomain BET proteins in animals, as well as evidence for a complex containing tBRDs and tTAFs that regulates transcription of a subset of genes with relevance for spermiogenesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. tBRD-1 is required for gene activity in the testis.
(A) Distribution of gene expression changes observed when comparing wild-type and tbrd-1 mutant testis. The volcano plot shows the logarithm of the probability of the t-test as a function of the logarithm of fold change for each reporter on the microarray. Reporter with an absolute log2-fold change ≥1 and a corrected p-value ≤0.05 are plotted in red (up-regulated) and green (down-regulated). (B) Quantitative real-time PCR (qPCR) using cDNA of wild-type, tbrd-11 and tbrd-1-eGFP; tbrd-11 testes. Transcript levels of CG13946, CG18673, CG32457, CG42827, CG42828, CG17917, Yp3 and TfIIA-S were significantly reduced in tbrd-1 mutant testes compared to wild-type and tbrd-1-eGFP; tbrd-11 testes. P-values for significance: * p≤0.05, ** p≤0.01 and *** p≤0.001. NS: not significant. (C,D) Significantly changed genes in tbrd-1 mutants and sa mutants. (C) 144 genes were significantly down-regulated in both tbrd-1 and sa mutants. (D) 56 genes were significantly up-regulated in tbrd-1 and sa mutants. (E) Genes down-regulated (left boxplot) and up-regulated (right boxplot) in tbrd-1 mutant tested were analyzed for the associated transcriptional changes observed in sa mutant testes (all: all genes).
Figure 2
Figure 2. tBRD-2 and tBRD-3 represent two new types of BET proteins.
Scheme of full-length tBRD-1 (A), tBRD-2 (B) and tBRD-3 (C) proteins. Bromodomains are indicated in yellow, NET domains in green and the SEED domain in blue.
Figure 3
Figure 3. In primary spermatocytes tBRD-2 co-localizes with tBRD-1 and tBRD-3.
Single primary spermatocyte nuclei of flies expressing tBRD-2-eGFP stained with anti-tBRD-1 (A panels) or anti-tBRD-3 (B panels). (A,B) tBRD-2-eGFP was visible over the chromosome territories (arrows). tBRD-2-eGFP partially co-localizes with tBRD-1 (A″) and tBRD-3 (B″) over the chromosomes (arrows). (A′″,B′″) Hoechst DNA staining. (A″″,B″″) Phase-contrast images. Scale bars: 5 µm.
Figure 4
Figure 4. Recruitment of tBRD-1 and tBRD-3 to chromatin is independent of tTAF Sa.
Single primary spermatocytes from heterozygous (A panels) and homozygous sa2 (B panels) mutants that express tBRD-1-eGFP stained with anti-tBRD-3 antibody. (A″,B″) In heterozygous and homozygous sa2 mutant spermatocytes tBRD-3 partially co-localizes with tBRD-1-eGFP over the chromosomes (arrows). (A′″,B′″) Hoechst DNA staining. (A″″,B″″) Phase-contrast images. Scale bars: 5 µm.
Figure 5
Figure 5. tBRD-1 is not essential for co-localization of tBRD-2 and tBRD-3.
Single primary spermatocytes from heterozygous (A panels) and homozygous tbrd-11 (B panels) mutants that express tBRD-2-eGFP stained with anti-tBRD-3 antibody. (B″) Partial co-localization of tBRD-2-eGFP and tBRD-3 over the chromosomes (arrows) was still detectable in homozygous tbrd-11 mutant spermatocytes. (A′″,B′″) Hoechst DNA staining. (A″″,B″″) Phase-contrast images. Scale bars: 5 µm.
Figure 6
Figure 6. Acetylation levels in primary spermatocytes influences tBRD-2-eGFP localization.
Pupal testis of tBRD-2-eGFP expressing flies were treated with anacardic acid (AA) (B panels) or TSA (D panels) for 24 hours in culture and afterwards spermatoyctes were stained with an antibody against tBRD-1. (A and C panels) Untreated control. (B) Incubation of testis with AA led to a spotted pattern of tBRD-2-eGFP at chromosome territories (arrow) compared to the control (A). (D) TSA treatment led to increased localization of tBRD-2-eGFP to the chromosomes (arrow) compared to the control (C). (B″,D″) Partial co-localization of tBRD-2-eGFP and tBRD-1 was not affected by AA or TSA treatment. (A′″,B′″,C′″,D′″) Hoechst DNA staining. (A″″,B″″,C″″,D″″) Phase-contrast images. Scale bars: 5 µm.
Figure 7
Figure 7. tBRD-1 interacts with tBRD-2 and the tTAF Sa in yeast two-hybrid experiments.
(A) Positive control (DBD-53+AD-T). (B) Negative control (DBD-Lam+AD-T). (C–F, J,K) tBRD-1, tBRD-2 and Sa fusion proteins showed no self-activity. (G) No homodimerization of tBRD-2 was detectable. (H,I) tBRD-1 and tBRD-2 heterodimer formation was visible. (L) tBRD-1 was able to homodimerize. (M,N) tBRD-1 and Sa could only interact when Sa was acting as the bait.
Figure 8
Figure 8. tBRD-1 co-precipitates with tBRD-1-eGFP and tBRD-2-eGFP from testes protein extracts.
(A) Proteins were analyzed by SDS-PAGE and immunoblotting using anti-tBRD-1 antibody. Lanes 1–3: testes extracts before immunoprecipitation (Input). The anti-tBRD-1 antibody detected a protein at about 56 kDa (tBRD-1 predicted molecular mass: 59.2 kDa) in protein extracts of tbrd-1-eGFP (lane 1) and wild-type (lane 2), but not in tbrd-1 mutant testis (lane 3). Additionally, a protein at about 90 kDa was detected in protein extracts of tbrd-1-eGFP testes (lane 1) that represents the tBRD-1-eGFP fusion protein. An unspecific protein at about 76 kDa was visible in all three extracts (asterisk). Lanes 4–6: eGFP-tagged tBRD-1 was immunoprecipitated (IP) with the GFP-Trap A Kit from testes protein extracts of tbrd-1-eGFP transgenic flies (lane 4), wild-type flies (lane 5) or tbrd-1 mutants (lane 6). tBRD-1 was detected in the IP from tbrd-1-eGFP testes (lane 4) but not from wild-type (lane 5) or tbrd-1 mutant testis. (B) Input and immunoprecipitates (IP performed as in A) from testes protein extracts of tbrd-2-eGFP transgenic flies were analyzed by SDS-PAGE and immunoblotting using anti-GFP and anti-tBRD-1 antibodies. tBRD-2-eGFP and tBRD-1 were detected in both immunoprecipitates (lanes 2 and 4).
Figure 9
Figure 9. Model for tBRD-1, tBRD-2, tBRD-3 and tTAF function in primary spermatocytes.
Scheme of a complex of tBRDs (green), tTAFs (orange) and so far unknown proteins (grey) that may activate transcription of a specific set of genes in primary spermatocytes. In our model tBRDs recognize and bind to acetylated histones (little black flags). This binding could in turn lead to the recruitment of tTAFs and additional transcription factors and subsequently gene activation.
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This work was supported by the German Research Foundation (DFG) (http://www.dfg.de/) within a research grant to CR (RA 2150/2-1) and within the TRR81 to MB and RR-P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.