ex-vivo-culture-drosophila-pupal-testis-single-male-germ-line-cysts

<em>Ex vivo</em> Culture of <em>Drosophila</em> 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 ...

Philipps-Universität Marburg

The genes don juan (dj) and don juan like (djl) encode basic proteins expressed in the male germline. Both proteins show a similar expression pattern being localized in the sperm heads during chromatin condensation and along the flagella. Prematurely expressed Don Juan-eGFP and Myc-Don Juan Like localize to the cytoplasm of spermatocytes and in mitochondrial derivatives from the nebenkern stage onward suggesting that both proteins associate with the mitochondria along the flagella in elongated spermatids. Premature expression of Myc-Don Juan Like does not impair spermatogenesis where-as Don Juan-eGFP when prematurely expressed causes male sterility as spermatids fail to individualize. In spite of the sequence identity of 72% on the nucleotide level and 42% on the protein level, the presumptive promoter regions and the untranslated regions of the mRNA are diverged. Our in vivo analysis revealed that don juan and don juan like are transcriptionally and translationally controlled by distinct short cis regulatory regions. Transcription of don juan and don juan like depends on the male germ line specific TAF(II)80, Cannonball (Can). Translational repression elements for both mRNAs are localized in the 5' UTR and are capable to form distinct secondary structures in close proximity to the translational initiation codon.

In Drosophila, don juan and don juan like encode proteins of the spermatid nucleus and the flagellum and both are regulated at the transcriptional level by the TAF II80 cannonball while translational repression is achieved by distinct elements.

In higher organisms, the chromatin of sperm is organised in a highly condensed protamine-based structure. In pre-meiotic stages and shortly after meiosis, histones carry multiple modifications. Here, we focus on post-meiotic stages and show that also after meiosis, histone H3 shows a high overall methylation of K9 and K27 and we hypothesise that these modifications ensure maintenance of transcriptional silencing in the haploid genome. Furthermore, we show that histones are lost during the early canoe stage and that just before this stage, hyper-acetylation of histone H4 and mono-ubiquitylation of histone H2A occurs. We believe that these histone modifications within the histone-based chromatin architecture may lead to better access of enzymes and chromatin remodellers. This notion is supported by the 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.

Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila.

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 genome-wide 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.

Active promoters and insulators are marked by the centrosomal protein 190.

Chromatin reorganisation is a major event towards the end of mammalian and Drosophila spermatogenesis. In Drosophila, we previously identified protamine A, protamine B and Mst77F as major chromatin components of the mature sperm. Here, an antibody against Mst77F reveals a dual expression pattern of Mst77F as a chromatin component and in association with microtubules during nuclear shaping. Spermatids of ms(3)nc3 (Mst77F(1)) mutants show disturbed nuclear shaping, instability of perinuclear microtubules but no obvious chromatin condensation defects. Furthermore, we generated a deletion including both protamine genes (prot Delta) and observed that in Drosophila, protamine genes are not haploinsufficient in contrast to those of mice and humans. Moreover, we show that in prot Delta mutants, histone degradation, distribution of DNA breaks and Tpl(94D)-eGFP and Mst77F expression proceed as in wild-type males. Surprisingly, in homozygous prot Delta mutants, males are fertile and sperm are motile, while about 20% of sperm show abnormally shaped nuclei. The latter phenotype can be rescued by supplying protamine-eGFP but not by supplying Mst77F-eGFP. Finally, we demonstrate a 21% increase in X-ray-induced mutation rate of prot Delta sperm. These data support the long-standing hypothesis that the switch from a histone- to protamine-based chromatin protects the paternal genome from mutagens.

Distinct functions of Mst77F and protamines in nuclear shaping and chromatin condensation during Drosophila spermiogenesis.

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.

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

Differentiation from a haploid round spermatid to a highly streamlined, motile sperm requires temporal and spatial regulation of the expression of numerous proteins. One form of regulation is the storage of translationally repressed mRNAs. In Drosophila spermatocytes, the transcription of many of these translationally delayed mRNAs during spermiogenesis is in turn directly or indirectly regulated by testis-specific homologs of TATA-box-binding-protein-associated factors (tTAFs). Here we present evidence that expression of Mst77F, which is a specialized linker histone-like component of sperm chromatin, and of protamine B (ProtB), which contributes to formation of condensed sperm chromatin, is regulated at three levels. Transcription of Mst77F is guided by a short, promoter-proximal region, while expression of the Mst77F protein is regulated at two levels, early by translational repression via sequences mainly in the 5' part of the ORF and later by either protein stabilization or translational activation, dependent on sequences in the ORF. The protB gene is a direct target of tTAFs, with very short upstream regulatory regions of protB (-105 to +94 bp) sufficient for both cell-type-specific transcription and repression of translation in spermatocytes. In addition, efficient accumulation of the ProtB protein in late elongating spermatids depends on sequences in the ORF. We present evidence that spermatocytes provide the transacting mechanisms for translational repression of these mRNAs, while spermatids contain the machinery to activate or stabilize protamine accumulation for sperm chromatin components. Thus, the proper spatiotemporal expression pattern of major sperm chromatin components depends on cell-type-specific mechanisms of transcriptional and translational control.

Three levels of regulation lead to protamine and Mst77F expression in Drosophila.

One of the most dramatic forms of chromatin reorganization occurs during spermatogenesis, when the paternal genome is repackaged from a nucleosomal to a protamine-based structure. We assessed the role of the canonical histone chaperone CAF1 in Drosophila spermatogenesis. In this process, CAF1 does not behave as a complex, but its subunits display distinct chromatin dynamics. During histone-to-protamine replacement, CAF1-p180 dissociates from the DNA while CAF1-p75 binds and stays on as a component of sperm chromatin. Association of CAF1-p75 with the paternal genome depends on CAF1-p180 and protamines. Conversely, CAF1-p75 binds protamines and is required for their incorporation into sperm chromatin. Histone removal, however, occurs independently of CAF1 or protamines. Thus, CAF1-p180 and CAF1-p75 function in a temporal hierarchy during sperm chromatin assembly, with CAF1-p75 acting as a protamine-loading factor. These results show that CAF1 subunits mediate the assembly of two fundamentally different forms of chromatin.

Subunits of the histone chaperone CAF1 also mediate assembly of protamine-based chromatin.

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.

Chromatin dynamics during spermiogenesis.

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.

Christina Rathke

Fachbereich Biologie,
Entwicklungsbiologie,
Fachbereich Biologie, Entwicklungsbiologie

Philipps-Universität Marburg

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Christina Rathke

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Philipps-Universität Marburg

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Fachbereich Biologie,
Entwicklungsbiologie,
Fachbereich Biologie, Entwicklungsbiologie