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

<div><p>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 <i>Drosophila</i>. 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 <i>tbrd-1</i> mutant testes and wild-type testes. Our data confirmed that tBRD-1 controls gene activity in male germ cells. Additionally, comparing the transcriptomes of <i>tbrd-1</i> 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 <i>tbrd-1</i> 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.</p></div

In primary spermatocytes tBRD-2 co-localizes with tBRD-1 and tBRD-3.

<p>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.</p

tBRD-1 interacts with tBRD-2 and the tTAF Sa in yeast two-hybrid experiments.

<p>(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.</p

tBRD-1 is not essential for co-localization of tBRD-2 and tBRD-3.

<p>Single primary spermatocytes from heterozygous (A panels) and homozygous <i>tbrd-1¹</i> (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 <i>tbrd-1¹</i> mutant spermatocytes. (A′″,B′″) Hoechst DNA staining. (A″″,B″″) Phase-contrast images. Scale bars: 5 µm.</p

Model for tBRD-1, tBRD-2, tBRD-3 and tTAF function in primary spermatocytes.

<p>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.</p

Recruitment of tBRD-1 and tBRD-3 to chromatin is independent of tTAF Sa.

<p>Single primary spermatocytes from heterozygous (A panels) and homozygous <i>sa²</i> (B panels) mutants that express tBRD-1-eGFP stained with anti-tBRD-3 antibody. (A″,B″) In heterozygous and homozygous <i>sa²</i> 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.</p

tBRD-2 and tBRD-3 represent two new types of BET proteins.

<p>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.</p

Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways