1689 Research Article Introduction The switch from a nucleosome- to a protamine-based chromatin structure is a characteristic feature of sperm maturation in many vertebrates including humans. First, testis- specific linker histones appear (Catena et al., 2006; Martianov et al., 2005; Tanaka et al., 2005; Yan et al., 2003), and then histones are replaced by transition proteins (major types: TP1 and TP2), which in turn are replaced by protamines, leading to condensed chromatin with a doughnut structure (reviewed by Braun, 2001; Kimmins and Sassone-Corsi, 2005; Sassone- Corsi, 2002). Recently, we have shown that Drosophila sperm also contain protamines as well as Mst77F, representing at least one further chromatin component, whereas transition protein- like chromatin components have not been found in Drosophila so far (Jayaramaiah-Raja and Renkawitz-Pohl, 2005). In Drosophila, GFP-tagged versions of the histone variant H2AvD are degraded during the canoe stage of spermatid development when protamines and Mst77F are starting to accumulate (Jayaramaiah-Raja and Renkawitz-Pohl, 2005). This raises the question of whether the histones and histone variants are depleted from the chromosomes simultaneously. However, to date, little is known about the mechanism of histone displacement and degradation, and it is also unclear whether protamine synthesis is a signal for histone degradation. Multiple histone modifications have been analysed that mark transcriptionally active and inactive chromatin either on the level of euchromatin versus heterochromatin or on the level of individual genes (Lachner et al., 2003; Peterson and Laniel, 2004; Shilatifard, 2006). In Drosophila spermatogenesis, transcription nearly ceases with the entry into meiotic division (for review see Renkawitz-Pohl et al., 2005), which is long before the protamine-based chromatin status is established. Histone modifications in the haploid phase might play a fundamental role in keeping the genome largely transcriptionally inactive. In addition, we also expected that histones to be displaced from the chromatin would be marked by characteristic modifications, as is known to be the case for histone H4 that is acetylated in mammals. Furthermore, phosphorylation and ubiquitylation were also suggested to be important during this stage (reviewed by Braun, 2001). Here, we show for Drosophila that histones carry multiple modifications, which also characterise the transcriptionally highly active spermatocyte stage. Interestingly, we found de novo H2A mono-ubiquitylation and a tremendous increase in H4 acetylation shortly before histone degradation in the spermatid nucleus. This finding might indicate an opening of the chromatin which is accompanied by the expression of a transition protein such as chromatin 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. Key words: Drosophila, Histone acetylation, Histone methylation, Histone ubiquitylation, Transition protein, Protamine, CTCF, DNA breaks, Rad6, UbcD6, RNA polymerase II Summary Transition from a nucleosome-based to a protamine- based chromatin configuration during spermiogenesis in Drosophila Christina Rathke 1 , Willy M. Baarends 2 , Sunil Jayaramaiah-Raja 1, *, Marek Bartkuhn 3 , Rainer Renkawitz 3 and Renate Renkawitz-Pohl 1,‡ 1 Philipps-Universität Marburg, Fachbereich Biologie, Entwicklungsbiologie, 35043 Marburg, Germany 2 University Medical Center Rotterdam, Department of Reproduction and Development, Erasmus MC, 3000 DR Rotterdam, Netherlands 3 Justus Liebig-Universität, Institut für Genetik, 35390 Giessen, Germany *Present address: EMBL, Heidelberg, Germany ‡ Author for correspondence (e-mail: renkawit@staff.uni-marburg.de) Accepted 14 March 2007 Journal of Cell Science 120, 1689-1700 Published by The Company of Biologists 2007 doi:10.1242/jcs.004663 Journal of Cell Science