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

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.

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

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.

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

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.

Replacement by Drosophila melanogaster protamines and Mst77F of histones during chromatin condensation in late spermatids and role of sesame in the removal of these proteins from the male pronucleus.

Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little is known for Drosophila melanogaster. Here we characterize three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame.