Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis inDrosophila

Abstract

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...

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

“…Next, we used HIS2AV-RFP and Protamine B-GFP to mark early spermatids and mature sperm, respectively (Rathke et al 2007(Rathke et al , 2010. The round uncondensed sperm of CR44420 −/− , tadpoleshape sperm of lncRNA:TS1 −/− , and bent sperm of CR43416 −/− were positive for Protamine B-GFP but not HIS2AV-RFP, confirming that they corresponded to the late elongate and/or mature stage (Fig.…”

The Drosophila DAXX-Like Protein (DLP) Cooperates with ASF1 for H3.3 Deposition and Heterochromatin Formation

“…Interestingly, high dlp expression levels are observed in primary spermatocytes, in meiotic spermatocytes, and also in the germinal vesicle, suggesting that it may have important functions during the development of germ cells. In Drosophila testis, H3.3 disappears with the bulk of histones, prior to the accumulation of protamine and other sperm-specific nuclear basic proteins, leading to sperm DNA compaction at late stages of spermiogenesis (48). At fertilization, the assembly of nucleosomes on paternal DNA immediately follows the rapid loss of protamines from the decondensing male nucleus and is dependent on maternally provided factors like Hira and YEM (14,16,17).…”

The Impact of Bacterial Infections on Human Spermatozoa

“…Hyperacetylation of histone H4 occurs during the histone-to-protamine transition, perhaps causing a more open chromatin structure to facilitate histone replacement (93)(94)(95)(96) and serving as a signal for the bromodomain protein BRDT to initiate the histone-to-protamine transition (97). In mammals, reduced levels of histone H4 hyperacetylation correlates with impaired fertility (95,98).…”