Stage-specific testes proteomics of Drosophila melanogaster identifies essential proteins for male fertility.

Spermiogenesis in Drosophila melanogaster is a highly conserved process and essential for male fertility. In this haploid phase of spermatogenesis, motile sperm are assembled from round cells, and flagella and needle-shaped nuclei with highly compacted genomes are formed. As transcription takes place mainly in spermatocytes and transcripts relevant for post-meiotic sperm development are translationally repressed for days, we comparatively analysed the proteome of larval testes (only germ cell stages before meiotic divisions), testes of 1-2-day-old pupae (germ cell stages before meiotic divisions, meiotic and early spermatid stages) and adult flies (germ cell stages before meiotic divisions, meiotic and early spermatid stages, late spermatids and sperm). We identified 6,171 proteins; 61 proteins were detected solely in one stage and are thus enriched, namely 34 in larval testes, 77 in pupal testes and 214 in adult testes. To substantiate our mass spectrometric data, we analysed the stage-specific synthesis and importance for male fertility of a number of uncharacterized proteins. For example, Mst84B (gene CG1988), a very basic cysteine- and lysine-rich nuclear protein and was present in the transition phase from a histone-based to a protamine-based chromatin structure. CG6332 encodes d-Theg, which is related to the mouse tHEG and human THEG proteins. Mutants of d-Theg were sterile due to the lack of sperm in the seminal vesicles. Our catalogue of proteins of the different stages of testis development in D. melanogaster will pave the road for future analyses of spermatogenesis.

Nejire/dCBP-mediated histone H3 acetylation during spermatogenesis is essential for male fertility in Drosophila melanogaster.

Spermatogenesis in many species including Drosophila melanogaster is accompanied by major reorganisation of chromatin in post-meiotic stages, involving a nearly genome-wide displacement of histones by protamines, Mst77F and Protamine-like 99C. A proposed prerequisite for the histone-to-protamine transition is massive histone H4 hyper-acetylation prior to the switch. Here, we investigated the pattern of histone H3 lysine acetylation and general lysine crotonylation in D. melanogaster spermiogenesis to elucidate a possible role of these marks in chromatin reorganisation. Lysine crotonylation was strongest prior to remodelling and the deposition of this mark depended on the acetylation status of the spermatid chromatin. In contrast to H4 acetylation, individual H3 acetylation marks displayed surprisingly distinct patterns during the histone-to-protamine transition. We observed that Nejire, a histone acetyl transferase, is expressed during the time of histone-to-protamine transition. Nejire knock down led to strongly reduced fertility, which correlated with misshaped spermatid nuclei and a lack of mature sperm. protA and prtl99C transcript levels were reduced after knocking down Nejire. ProtB-eGFP, Mst77F-eGFP and Prtl99C-eGFP were synthesized at the late canoe stage, while histones were often not detectable. However, in some cysts histones persist in parallel to protamines. Therefore, we hypothesize that complete histone removal requires multiple histone modifications besides H3K18ac and H3K27ac. In summary, H3K18 and H3K27 acetylation during Drosophila spermatogenesis is dependent on Nejire or a yet uncharacterized acetyl transferase. We show that Nejire is required for male fertility since Nejire contributes to efficient transcription of protA and prtl99C, but not Mst77F, in spermatocytes, and to maturation of sperm.

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

Male germ cell differentiation proceeds to a large extent in the absence of active gene transcription. In Drosophila, hundreds of genes whose proteins are required during post-meiotic spermatid differentiation (spermiogenesis) are transcribed in primary spermatocytes. Transcription of these genes depends on the sequential action of the testis meiotic arrest complex (tMAC), Mediator complex, and testis-specific TFIID (tTFIID) complex. How the action of these protein complexes is coordinated and which other factors are involved in the regulation of transcription in spermatocytes is not well understood. Here, we show that the bromodomain proteins tBRD-1 and tBRD-2 regulate gene expression in primary spermatocytes and share a subset of target genes. The function of tBRD-1 was essential for the sub-cellular localization of endogenous tBRD-2 but dispensable for its protein stability. Our comparison of different microarray data sets showed that in primary spermatocytes, the expression of a defined number of genes depends on the function of the bromodomain proteins tBRD-1 and tBRD-2, the tMAC component Aly, the Mediator component Med22, and the tTAF Sa.

The HMG-box-containing proteins tHMG-1 and tHMG-2 interact during the histone-to-protamine transition in Drosophila spermatogenesis.

Spermatogenesis is accompanied by a remarkable reorganization of the chromatin in post-meiotic stages, characterized by a near genome-wide displacement of histones by protamines and a transient expression of transition proteins. In Drosophila, the transition-protein-like protein Tpl94D contains an HMG-box domain and is expressed during chromatin reorganization. Here, we searched for additional HMG-box-containing proteins with a similar expression pattern. We identified two proteins specifically expressed in the testis, tHMG-1 and tHMG-2, whose expression levels were highest during the histone-to-protamine transition. Protein-protein interaction studies revealed that tHMG-1 and tHMG-2 form heterodimers in vivo. We demonstrated that Tpl94D, tHMG-1 and tHMG-2 localize to chromatin of the male germ line, with the most abundant levels observed during post-meiotic chromatin reorganization. Analysis of a tpl94D mutant showed that the C-terminal region of Tpl94D is dispensable for fertility. These data strongly suggested either that the truncated protein, which still contains the N-terminal HMG-box domain, is functional or that other proteins act in functional redundancy with Tpl94D during spermiogenesis. A thmg-1/thmg-2 null mutant also had no detectable specific phenotype, but hmgz, which encodes the major somatic HMG-box-containing protein HMGZ, was transcriptionally up-regulated. Our results showed that Drosophila spermatogenesis is characterized by continuous and overlapping expression of different HMG-box-containing proteins. We hypothesize that the mechanism of chromatin reorganization is a process highly secured by redundancies.

Ex vivo culture of Drosophila pupal testis and single male germ-line cysts: dissection, imaging, and pharmacological treatment.

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin-the histone-to-protamine switch. Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages. The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

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

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.

H3K79 methylation: a new conserved mark that accompanies H4 hyperacetylation prior to histone-to-protamine transition in Drosophila and rat.

During spermiogenesis, haploid spermatids undergo extensive chromatin remodeling events in which histones are successively replaced by more basic protamines to generate highly compacted chromatin. Here we show for the first time that H3K79 methylation is a conserved feature preceding the histone-to-protamine transition in Drosophila melanogaster and rat. During Drosophila spermatogenesis, the Dot1-like methyltransferase Grappa (Gpp) is primarily expressed in canoe stage nuclei. The corresponding H3K79 methylation is a histone modification that precedes the histone-to-protamine transition and correlates with histone H4 hyperacetylation. When acetylation was inhibited in cultured Drosophila testes, nuclei were smaller and chromatin was compact, Gpp was little synthesized, H3K79 methylation was strongly reduced, and protamines were not synthesized. The Gpp isoform Gpp-D has a unique C-terminus, and Gpp is essential for full fertility. In rat, H3K79 methylation also correlates with H4 hyperacetylation but not with active RNA polymerase II, which might point towards a conserved function in chromatin remodeling during the histone-to-protamine transition in both Drosophila and rat.