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. 2015 Feb;5(2):140145.
doi: 10.1098/rsob.140145.

Two bromodomain proteins functionally interact to recapitulate an essential BRDT-like function in Drosophila spermatocytes

Shuhei Kimura  1 , Benjamin Loppin  2
Affiliations

Two bromodomain proteins functionally interact to recapitulate an essential BRDT-like function in Drosophila spermatocytes

Shuhei Kimura et al. Open Biol. 2015 Feb.

Abstract

In mammals, the testis-specific bromodomain and extra terminal (BET) protein BRDT is essential for spermatogenesis. In Drosophila, it was recently reported that the tBRD-1 protein is similarly required for male fertility. Interestingly, however, tBRD-1 has two conserved bromodomains in its N-terminus but it lacks an extra terminal (ET) domain characteristic of BET proteins. Here, using proteomics approaches to search for tBRD-1 interactors, we identified tBRD-2 as a novel testis-specific bromodomain protein. In contrast to tBRD-1, tBRD-2 contains a single bromodomain, but which is associated with an ET domain in its C-terminus. Strikingly, we show that tbrd-2 knock-out males are sterile and display aberrant meiosis in a way highly similar to tbrd-1 mutants. Furthermore, these two factors co-localize and are interdependent in spermatocytes. We propose that Drosophila tBRD-1 and tBRD-2 associate into a functional BET complex in spermatocytes, which recapitulates the activity of the single mammalian BRDT-like protein.

Keywords: Drosophila; bromodomain and extra terminal family; spermatocyte; tBRD-1; tBRD-2.

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Figures

Figure 1.
Figure 1.
Generation of tbrd-1 null mutant. (a) Representations of Fs(1)hS (short isoform of Fs(1)h) and tBRD-1 proteins. Bromodomain 1 (BD1) of tBRD-1 showed 44% identity with BD1 of Fs(1)h and 40% identity with bromodomain 2 (BD2) of Fs(1)h. BD2 of tBRD-1 showed 23% identity with BD2 of Fs(1)h, but no significant homology (−) with BD1 of Fs(1)h. Sequence identity was analysed using ClustalW. BD, bromodomain; ET, ET domain; SEED, seed motif. Blue boxes showed the peptides used as antigens for anti-tBRD-1 antibody generation. (b) A map of the tbrd-1 gene locus. The deletion range in tbrd-12 and two rescue constructs (tbrd-1 genomic rescue and tbrd-1::mRFP1-1×Flag rescue) are shown. Triangle indicates the position of the P(GSV6)GS13976 insertion mobilized to generate the tbrd-12 allele. (c) Western blotting analysis of tBRD-1 protein. Anti-α-tubulin antibody was used as control. Wild-type and tbrd-1 genomic rescue lanes showed endogenous tBRD-1 protein at about 65 kDa (black arrowhead), which is not detected in tbrd-12 extracts. In the tbrd-1::mRFP1-1×Flag rescue lane, a higher molecular weight band (white arrowhead) was observed, corresponding to mRFP1-1×Flag fused tBRD-1. (d) Phase contrast view of spermatids at onion stage. In wild-type (left panel), haploid nuclei (white circles) and mitochondrial derivatives, so-called Nebenkern (black circles), are all at the same size. By contrast, spermatids from tbrd-12 testes (right panel) contain nuclei of various sizes and irregularly shaped mitochondrial derivatives. Note that in these preparations, spermatids occasionally fuse during the course of live observation. Scale bar, 10 μm.
Figure 2.
Figure 2.
tBRD-1 interacts with a novel ET domain-containing protein, tBRD-2. (a) A scheme for the identification of the tBRD-1 interacting proteins using immunoprecipitation (IP) of exogenous tBRD-1. In the result table, coverage indicates the percentage coverage of identified polypeptides in full-length protein, no. peptides indicates the number of identified distinct peptides and peptide spectrum matches (no. PSMs) indicates the total number of identified peptides. (b) A schematic view of the identification of the tBRD-1 interacting proteins using IP of endogenous tBRD-1. SDS-gel was stained with the silver method. IgG lane was used as control. Proteins identified by LC-MS after in-gel digestion are indicated. MHC, myosin heavy chain; ZIP, zipper; PRM, paramyosin; PSI, P-element somatic inhibitor; PABP, poly(A) binding protein; HSP68, heat shock protein 68; Rb97D, ribonuclear protein at 97D. (c) Representation of CG7229/tBRD2 protein in Drosophila. BD of tBRD-2 showed 40% identity with BD1 of Fs(1)h and 36% identity with BD2 of Fs(1)h. It also showed 36% identity with BD1 of tBRD-1; however, it showed no significant homology (−) with BD2 of tBRD-1. Sequence identity was analysed using ClustalW. BD, bromodomain; ET, ET domain; SEED, seed motif. (d) Sequence alignment of ET domain using ClustalW. Black boxes and white lettering, identical amino acids; grey boxes and black lettering, same group of amino acids.
Figure 3.
Figure 3.
Meiotic defects in the tbrd-2 mutant. (a) Schematic of the tbrd-2 gene locus, the pW25-tbrd-2 construct used for homologous recombination and the tbrd-2::eGFP-6×His rescue construct. (b) Genomic PCR analysis of the tbrd-2 locus using the primers represented as ‘for’ and ‘rev’ in (a). rp49 primers were used for control amplification. (c–e) Confocal images showing the localization of tBRD-2::eGFP in testes. (c) Apical tip of the testis. tBRD-2::eGFP gradually appeared during primary spermatocyte growth. The testis is outlined with a white line. Scale bar, 10 μm. (d) Mature primary spermatocytes. tBRD-1 and tBRD-2::eGFP signals are almost completely overlapping. Scale bar, 5 μm. (e) Mature primary spermatocytes and prophase I meiotic cells (outlined cells, characterized by the presence of two prominent asters and condensed chromatin). tBRD-2::eGFP is not detected in spermatocytes in prophase I. Scale bar, 10 μm. (f) Phase contrast view of post-meiotic spermatids at onion stage. tbrd-2HR1(i) and tbrd-2HR1; tbrd-12 double mutant (iii) present similar defects. Spermatids in the tbrd-2::eGFP-6×His rescue (ii) appear normal. Scale bar, 10 μm. (g) Phase contrast view of mature primary spermatocytes. Primary spermatocytes appear normal for all genotypes. Scale bar, 10 μm. (h–m) Confocal images of spermatocytes in meiosis I. Scale bar, 5 μm. (h,i) Wild-type: (h) G2 stage, (i) prophase I. (j–m) tbrd-2HR1: (j) G2 stage, (k–m) prophase I-like. (k) Although the chromatin appeared condensed, asters are not present. (l) Spermatocytes with asters in aberrant disposition. (m) Meiosis I with aberrant number of asters.
Figure 4.
Figure 4.
tBRD-1 and tBRD-2 interdependency in primary spermatocytes. (a,b) Confocal images. Scale bar, 5 μm. (a) tBRD-1::mRFP1 nuclear localization is dependent on tBRD-2. In tbrd-2HR1 heterozygous (i), tBRD-1::mRFP1 is localized in nucleolus and chromatin. In tbrd-2HR1 homozygous (ii), the tBRD-1::mRFP1 signal is strongly decreased. (b) tBRD-2::eGFP is dependent on tBRD-1. In tbrd-12 heterozygous (i), tBRD-2::eGFP is localized in nucleolus and chromatin. In tbrd-12 homozygous (ii), tBRD-2::eGFP signal is barely detected. (c) RT-PCR analysis. tbrd-1 and tbrd-2 mRNA levels are not affected in tbrd-2 and tbrd-1 mutant testes, respectively. rp49 transcripts were analysed as a control. RT, reverse transcriptase. (d) A scheme of the expression stage of tBRD-1 and tBRD-2 proteins in Drosophila spermatogenesis. Their expressions were limited in the primary spermatocyte. MI, meiosis I; MII, meiosis II. (e) A cooperation model for tBRD-1 and tBRD-2 proteins. Although tBRD-1 and tBRD-2 are unstable if they are alone, their interaction is required for their stability and the heterodimer functions as a single BRDT-like protein. BD, bromodomain; ET, ET domain; SEED, seed motif.
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