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First published online April 23, 2007
doi: 10.1242/10.1242/jcs.004663

Journal of Cell Science 120, 1689-1700 (2007)
Published by The Company of Biologists 2007
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Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila

Christina Rathke1, Willy M. Baarends2, Sunil Jayaramaiah-Raja1,*, Marek Bartkuhn3, Rainer Renkawitz3 and Renate Renkawitz-Pohl1,{ddagger}

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


Figure 1
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  		Fig. 1 (A-E) Tpl94D is transiently expressed during the switch between histones and protamines. (A,D) Anti-core histone antibody staining of transgenic flies (A) expressing protamine-eGFP and (D) bearing tpl94D-eGFP to compare histone and protamine expression in the same fly tissue. Core histones are detectable in young elongating nuclei and in early canoe stage spermatids but not in later stages. (B) Protamine-eGFP expression starts at the late canoe stage when histones have already gone and stays in the individualising spermatids. (C) Anti-Flag antibody staining of flies expressing histone H3.3-Flag. Histone H3.3 is detectable in young elongating nuclei and in early canoe stage spermatids and shows the same pattern as the core histones (A,D). (E) Expression of Tpl94D-eGFP starts in the early canoe stage and becomes highest in late canoe stage spermatids. In individualising sperm, it is not detectable (see D for histone staining of the identical slide). Bars, 5 µm. (F) Tpl94D is a HMG protein. The deduced 164 amino acid protein sequence of the tpl94D transcript is shown. The N-terminal HMG domain is underlined in red and lasts from amino acid 4 to 84. The HMG box was determined using the Ensembl genome browser (www.ensembl.org). (G) tpl94D expression is testes specific. RT-PCR of tpl94D from wild-type testes (t), carcass males (m), embryos (e), adult females (f) and larvae (l). The tpl94D-specific primers amplified a 361 bp cDNA fragment from the open reading frame of tpl94D in testes and in larvae. In carcass males and in adult females a 429 bp DNA fragment was amplified, whereas in embryos no fragment was detectable. A 397 bp cDNA fragment of the beta3-tubulin gene was amplified as control. The cDNA beta3-tubulin fragment was visible in all samples, whereas DNA contamination shown by the 492 bp fragment was visible only in adult females and larvae.

 

Figure 2
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  		Fig. 2. Ubiquitin and proteasomes accumulate in the nuclei during spermiogenesis. (A) Anti-ubiquitin antibody staining on testes squashes of wild-type flies. Ubiquitin is detectable in young elongating nuclei (arrow) as well as in canoe stage nuclei (arrowhead and double arrow) with the highest expression in early canoe stage nuclei. (B) Expression of the proteasome subunit Pros{alpha}3-eGFP is very strong in young elongating nuclei (arrow), decreases during the canoe stage (arrowhead and double arrow) and is no longer detectable after the canoe stage. (C) Expression of the proteasome subunit Pros{alpha}3T-eGFP (isoform of Pros{alpha}3-eGFP) starts in young elongating nuclei (arrow) and stays in the nucleus during all later stages (arrowhead and double arrow). Bars, 5 µm.

 

Figure 3
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  		Fig. 3. H3K4 methylation is removed during the early canoe stage. (A) Hoechst staining to visualise chromatin. (B) Anti-methylated H3K4 antibody staining on squashed testes of protamine-eGFP expressing flies. A high level of H3K4 trimethylation is detectable in spermatids with young elongating nuclei (double arrow). During the early canoe stage, the signal decreases (arrowhead) and vanishes completely from the late canoe stage (not shown) onwards (arrow). (C) There is no protamine-eGFP expression in young elongating nuclei and early canoe stage spermatids, whereas high expression is observed in individualising spermatids (arrow). (D) Merged images. Bars, 20 µm.

 

Figure 4
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  		Fig. 4. Histone H4 hyper-acetylation and de novo H2A ubiquitylation characterise the stages before histone removal. (A-C) Double immunostaining of core histones and acetylated histone H4 on squashed testes of protamine-eGFP flies. (D-F) Double immunostaining of core histones and mono-ubiquitylated histone H2A on squashed testes of protamine-eGFP flies. (A,D) Histones are abundant in round spermatids (arrows), whereas the staining decreases in early canoe stage spermatids (arrowhead in A). In later stages when protamines are present (double arrow in C), histones are no longer detectable. (B) Nuclei of round spermatids show a faint staining of acetylated histone H4 (arrow). In early canoe stage spermatids, acetylation of histone H4 increases (arrowhead) and has vanished completely from the late canoe stage onwards. (C,F) Protamine-eGFP expression starts in late canoe stage spermatids (double arrow in C). (E) Mono-ubiquitylated histone H2A is detectable in spermatids with round nuclei (arrow). Mono-ubiquitylated histone H2A is no longer detectable during the canoe stage and in later stages when protamines are present (double arrow in C). Bars, 5 µm.

 

Figure 5
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  		Fig. 5. DNA breaks, UbcD6 and SUMO are abundant at the late canoe stage. (A-C) Anti-core histone antibody staining together with TUNEL staining on testes squashes of protamine-eGFP flies to visualise DNA breaks in comparison to histone deposition and protamine incorporation during the canoe stage in the same animal. (A) Anti-histone staining is detectable in early canoe stage spermatids (arrow), but not in any later stages. (B) Protamine-eGFP expression is detectable in late canoe stage (arrowhead) and individualising (double arrow) spermatids. (C) DNA breaks marked by TUNEL staining are detectable only at a low level in early canoe stage spermatids (arrow) reaching their highest level in late canoe stage spermatids (arrowhead). During individualisation, DNA breaks are no longer detectable. (D) Anti-HR6A/B antibody staining is detectable in early (arrow) and late (arrowhead) canoe stage spermatid nuclei, but not in later stages. (E) Anti-SUMO staining is detectable in early (arrow) and late (arrowhead) canoe stage spermatid nuclei, but not in later stages. Bars, 5 µm.

 

Figure 6
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  		Fig. 6. CTCF, a zinc finger DNA binding protein, and RNA polymerase II are expressed when protamines start to accumulate at the late canoe stage. (A-C) Anti-CTCF (C) and anti-histone antibody (A) staining on testes squashes of flies expressing protamine-eGFP (B). (A) Core histones are detectable in young elongating nuclei (arrow) and in early canoe stage spermatids (arrowhead), but not in later stages. (B) Protamine-eGFP expression starts in late canoe stage spermatids (double arrow). (C) Anti-CTCF is detectable in young elongating nuclei (arrow) and early canoe stage spermatids (arrowhead). During the late canoe stage, expression of CTCF overlaps with that of protamine-eGFP (double arrow), whereas CTCF is no longer visible in individualising spermatids. (D) Active RNA polymerase II is detectable by antibody staining in late canoe stage nuclei (double arrow), but not in any other post-meiotic stage. (E) Hoechst staining of the same cells as in D. Bars, 5 µm.

 

Figure 7
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  		Fig. 7. Key chromatin remodelling events in Drosophila spermiogenesis. Hoechst stainings visualise the morphogenesis of the spermatid and/or sperm nuclei in the order of events after meiosis. Below is a scheme of histone degradation and Tpl94D, Mst77F and protamine deposition in comparison to general and specific histone modifications, ubiquitylation, SUMOylation, DNA breaks, expression of UbcD6 and CTCF, active RNA polymerase II and the presence of proteasomes in the nucleus.

 

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© The Company of Biologists Ltd 2007