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.

presence of the architectural protein CTCF, numerous DNA breaks, SUMO, UbcD6 and high content of ubiquitin, as well as testes-specific nuclear proteasomes at this time. Moreover, we report the first transition protein-like chromosomal protein, Tpl 94D, to be found in Drosophila. We propose that Tpl 94D -an HMG box protein -and the numerous DNA breaks facilitate chromatin unwinding as a prelude to protamine and Mst77F deposition. Finally, we show that histone modifications and removal are independent of protamine synthesis. ), the presence of CTCF and DNA breaks. We furthermore show that histone removal is independent of the presence of protamines. Both this histone removal and protamine accumulation are essential for transmission of the male genome to the oocyte, and therefore of fundamental importance for the persistence of species. ResultsCore histones and their variants are removed simultaneously from the DNA of spermatid nuclei prior to protamine accumulation In Drosophila melanogaster, sperm morphogenesis, i.e. from meiosis until sperm individualisation, lasts 3.5 days. After meiosis, the nucleus initially is round and then gradually changes its shape accompanied by reorganisation of the chromatin during the canoe stage (Jayaramaiah-Raja and Renkawitz-Pohl, 2005), resulting in sperm containing a slim nucleus in which the nuclear volume is decreased by a factor of 200 (for review see Fuller, 1993;Renkawitz-Pohl et al., 2005).In the work reported here we concentrated on post-meiotic sperm morphogenesis with particular focus on chromatin reorganisation from the nucleosomal-to the protamine-based structure, which is a dramatic switch. Previously, we have reported that the histone variant H2AvD-GFP vanishes at the canoe stage while protamines begin to accumulate simultaneously (Jayaramaiah-Raja and Renkawitz-Pohl, 2005). To analyse the timing of histone removal and protamine accumulation we brought protamine-eGFP and H2AvD-RFP (Clarkson and Saint, 1999) into one genetic background to enable a study in the same individual. We found that H2AvD-RFP disappeared before protamine-eGFP accumulation took place (data not shown). We then went on to immunostain testes of protamine-eGFP flies with an antibody recognising all histones. This antibody was raised against total histones of humans and detects all core histones and the linker histone H1 in mammals. As -in contrast to core histones -H1 is not well conserved between mammals and Drosophila, we presumably detect solely core histones with this antibody. Our findings show that core histones (Fig. 1A) are detectable up to the canoe stage whereas protamines start to be synthesised at the canoe stage (Fig. 1B) but with no apparent positional overlap. As the canoe stage is quite long, we defined the early canoe stage by the start of histone removal, and the late canoe stage by the start of protamine accumulation (see Fig. 1A-E, second and third columns). With histone H3.3, a further replacement variant is expressed in the testis and disappears in post-meiotic stages togethe...

For the compact Drosophila genome, several factors mediating insulator function, such as su(Hw) and dCTCF, have been identified. Recent analyses showed that both these insulator-binding factors are functionally dependent on the same cofactor, CP190. Here we analysed genomewide binding of CP190 and dCTCF. CP190 binding was detected at CTCF, su(Hw) and GAF sites and unexpectedly at the transcriptional start sites of actively transcribed genes. Both insulator and transcription start site CP190-binding elements are strictly marked by a depletion of histone H3 and, therefore, a loss of nucleosome occupancy. In addition, CP190/dCTCF double occupancy was seen at the borders of many H3K27me3 'islands'. As before, these sites were also depleted of H3. Loss of either dCTCF or CP190 causes an increase of H3 and H3K27 trimethylation at these sites. Thus, for both types of cis-regulatory elements, domain borders and promoters, the chromatin structure is dependent on CP190.

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SUMMARYIn both mammalian and Drosophila spermatids, the completely histone-based chromatin structure is reorganized to a largely protamine-based structure. During this histone-to-protamine switch, transition proteins are expressed, for example TNP1 and TNP2 in mammals and Tpl94D in Drosophila. Recently, we demonstrated that in Drosophila spermatids, H3K79 methylation accompanies histone H4 hyperacetylation during chromatin reorganization. Preceding the histone-to-protamine transition, the H3K79 methyltransferase Grappa is expressed, and the predominant isoform bears a C-terminal extension. Here, we show that isoforms of the Grappa-equivalent protein in humans, rats and mice, that is DOT1L, have a C-terminal extension. In mice, the transcript of this isoform was enriched in the post-meiotic stages of spermatogenesis. In human and mice spermatids, di-and tri-methylated H3K79 temporally overlapped with hyperacetylated H4 and thus accompanied chromatin reorganization. In rat spermatids, trimethylated H3K79 directly preceded transition protein loading on chromatin. We analysed the impact of bacterial infections on spermatid chromatin using a uropathogenic Escherichia coli-elicited epididymo-orchitis rat model and showed that these infections caused aberrant spermatid chromatin. Bacterial infections led to premature emergence of trimethylated H3K79 and hyperacetylated H4. Trimethylated H3K79 and hyperacetylated H4 simultaneously occurred with transition protein TNP1, which was never observed in spermatids of mock-infected rats. Upon bacterial infection, only histone-based spermatid chromatin showed abnormalities, whereas protaminecompacted chromatin seemed to be unaffected. Our results indicated that H3K79 methylation is a histone modification conserved in Drosophila, mouse, rat and human spermatids and may be a prerequisite for proper chromatin reorganization.

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.

SummaryBy 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.

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.