Distinct CoREST complexes act in a cell-type-specific manner.

CoREST has been identified as a subunit of several protein complexes that generate transcriptionally repressive chromatin structures during development. However, a comprehensive analysis of the CoREST interactome has not been carried out. We use proteomic approaches to define the interactomes of two dCoREST isoforms, dCoREST-L and dCoREST-M, in Drosophila. We identify three distinct histone deacetylase complexes built around a common dCoREST/dRPD3 core: A dLSD1/dCoREST complex, the LINT complex and a dG9a/dCoREST complex. The latter two complexes can incorporate both dCoREST isoforms. By contrast, the dLSD1/dCoREST complex exclusively assembles with the dCoREST-L isoform. Genome-wide studies show that the three dCoREST complexes associate with chromatin predominantly at promoters. Transcriptome analyses in S2 cells and testes reveal that different cell lineages utilize distinct dCoREST complexes to maintain cell-type-specific gene expression programmes: In macrophage-like S2 cells, LINT represses germ line-related genes whereas other dCoREST complexes are largely dispensable. By contrast, in testes, the dLSD1/dCoREST complex prevents transcription of germ line-inappropriate genes and is essential for spermatogenesis and fertility, whereas depletion of other dCoREST complexes has no effect. Our study uncovers three distinct dCoREST complexes that function in a lineage-restricted fashion to repress specific sets of genes thereby maintaining cell-type-specific gene expression programmes.

Tumour-associated missense mutations in the dMi-2 ATPase alters nucleosome remodelling properties in a mutation-specific manner.

ATP-dependent chromatin remodellers are mutated in more than 20% of human cancers. The consequences of these mutations on enzyme function are poorly understood. Here, we characterise the effects of CHD4 mutations identified in endometrial carcinoma on the remodelling properties of dMi-2, the highly conserved Drosophila homologue of CHD4. Mutations from different patients have surprisingly diverse defects on nucleosome binding, ATPase activity and nucleosome remodelling. Unexpectedly, we identify both mutations that decrease and increase the enzyme activity. Our results define the chromodomains and a novel regulatory region as essential for nucleosome remodelling. Genetic experiments in Drosophila demonstrate that expression of cancer-derived dMi-2 mutants misregulates differentiation of epithelial wing structures and produces phenotypes that correlate with their nucleosome remodelling properties. Our results help to define the defects of CHD4 in cancer at the mechanistic level and provide the basis for the development of molecular approaches aimed at restoring their activity.

Blocking promiscuous activation at cryptic promoters directs cell type-specific gene expression.

To selectively express cell type-specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. Here, we show in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. Our results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation.

RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.

Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.

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.

Chromatin dynamics during spermiogenesis.

The function of sperm is to safely transport the haploid paternal genome to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made during the post-meiotic stages of spermatogenesis. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodeling complexes, as well as transient DNA strand breaks. The consequences of mutations have revealed that a protamine-based chromatin is essential for fertility in mice but not in Drosophila. Nevertheless, loss of protamines in Drosophila increases the sensitivity to X-rays and thus supports the hypothesis that protamines are necessary to protect the paternal genome. Pharmaceutical approaches have provided the first mechanistic insights and have shown that hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. We conclude with a model that illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.

The bromodomain-containing protein tBRD-1 is specifically expressed in spermatocytes and is essential for male fertility.

By a conserved cellular differentiation process, spermatogenesis leads to formation of haploid sperm for successful reproduction. In Drosophila and in mammals, post-meiotic spermatid differentiation depends on several translationally repressed and stored mRNAs that are often expressed exclusively in the testis through a cell type specific transcriptional program. In Drosophila, the mRNAs of proteins required for post-meiotic chromatin reorganisation, like ProtB and Mst77F, are transcribed in meiotic spermatocytes and subjected to translational repression for days. Transcription of many of these translationally repressed mRNAs depends on testis-specific homologs of TATA box binding protein-associated factors (tTAFs). Here, we identified the testis-specific bromodomain protein, tBRD-1, that is only expressed in primary spermatocytes. Bromodomain proteins are able to recognise and bind acetylated histones and non-histone proteins. We generated tbrd-1 mutant flies and observed that function of tBRD-1 is required for male fertility. tBRD-1 partially colocalised with tTAFs, TAF1 and Polycomb to a Fibrillarin-deficient region within the spermatocyte nucleolus. The nucleolar localisation of tBRD-1 depended on tTAF function but not the other way round. Further, we could show that ectopically expressed tBRD-1-eGFP is able to bind to the interbands of polytene chromosomes. By inhibitor treatment of cultured testis we observed that sub-cellular localisation of tBRD-1 may depend on the acetylation status of primary spermatocytes.

Histone H4 acetylation is essential to proceed from a histone- to a protamine-based chromatin structure in spermatid nuclei of Drosophila melanogaster.

In humans, other mammals, and also in Drosophila, the paternal genome in the sperm is highly condensed and organized mainly in a protamine-based chromatin structure. However, the timing and mechanism of the switch from a histone- to the protamine-based chromatin configuration is still poorly understood. We therefore established Drosophila in vitro cultures of cysts with 64 synchronously developing spermatids genetically marked with histone H2AvD-RFP and ProtamineB-eGFP. Live cell imaging showed that the switch from H2AvD-RFP to Protamine-eGFP chromatin takes approximately five hours, with a short but clear overlap of the presence of both histones and protamines. Moreover, cultured pupal testes showed H4 hyperacetylation at the canoe stage shortly before histone removal; a feature previously observed in the intact animal. We then used TSA to inhibit histone deacetylation and found that premature hyperacetylation was already induced at the round nuclei stage of spermatids. However, this premature hyperacetylation did not lead to a premature switch to the protamine-based chromatin structure, showing that histone hyperacetylation is not the sole inducer of the histone to protamine switch. Importantly, we observed that inactivation of histone acetyltransferases by anacardic acid blocks further differentiation and thus prevents the degradation of histones and the switch to a protamine-based chromatin. Thus, we conclude that H4 hyperacetylation is an essential feature but not the sole inducer of the histone to protamine switch during spermiogenesis.