tBRD-1 Selectively Controls Gene Activity in the Drosophila Testis and Interacts with Two New Members of the Bromodomain and Extra-Terminal (BET) Family

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.

Control of Drosophila Growth and Survival by the Lipid Droplet-Associated Protein CG9186/Sturkopf

“…Question marks symbolize uncertainties. (D) Whole-fly extracts or extracts of dissected testes or accessory glands of 7-day-old virgin or mated escargot-Gal4 GFP flies (here, the promotor of the escargot gene drives expression of GFP in the stem-cell like secondary cells of the accessory gland) were probed with antisera specific for CG9186, the testis marker tBRD-1 (Theofel et al, 2014), anti-GFP to detect stem cells, and a-tubulin as a general loading control. Note that the GFP antibody detects a second, unspecific band in the accessory gland sample (*).…”

“…Western Blotting SDS-PAGE and western blotting were carried out according to standard procedures as reported previously (Thiel et al, 2013). We used the following antibodies: rat anti CG9186 (Thiel et al, 2013): 1:3000; mouse anti alpha-Tubulin (Sigma Aldrich, St. Louis, USA): 1:3000, rabbit anti-GFP (Torrey Pines Biolabs, Secaucus, USA): 1:2000, rabbit anti tBRD-1 (Theofel et al, 2014; kind gift from C. Rathke and R. Renkawitz-Pohl): 1:1000. All peroxidase conjugated secondary antibodies were ordered from Pierce (Thermo Scientific, Dreieich, Germany).…”

tBRD-1 and tBRD-2 regulate expression of genes necessary for spermatid differentiation

“…tBRD-1 and tBRD-2 full-length yeast constructs are described in Theofel et al (2014). Mutated tbrd-1 and tbrd2 ORFs were PCR amplified using specific primers with linked restriction sites () and ligated into pGADT7 (bait vector) and pGBKT7 (prey vector).…”

Prtl99C Acts Together with Protamines and Safeguards Male Fertility in Drosophila

“…In in situ hybridization of testes using a Prtl99C-specific probe, transcripts were richly distributed from early spermatocytes to late spermatids, but not in the hub region or seminal vesicles ( Figure 1C), while tbrd-2 transcripts, which encode a spermatocyte-specific protein, were limited to the spermatocyte stage ( Figure S1D; Theofel et al, 2014). As the 942-Prtl99C-eGFP fusion protein was first detected in late canoe stage nuclei ( Figure 1D), shortly before protamines appeared ( Figure S1A), we conclude that Prtl99C mRNAs were translationally repressed.…”

“…To generate the 386-Prtl99C-eGFP construct, the Prtl99C gene together with 386 bp upstream of the translational start codon was PCR amplified using genomic DNA and primers with linked EcoRI and SpeI restriction sites. The PCR fragment was inserted into the Drosophila P element vector pChabDsalDlacZ-eGFP (Theofel et al, 2014) modified according to Thummel et al (1988). Transgenic fly strains were established in a w 1118 background.…”