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During spermatogenesis of many species the histone-based chromatin structure of the paternal genome becomes disassembled. It is replaced by a heavily condensed structure based on protamines. Correct completion of this histone to protamine switch (H-P switch) is essential to male fertility. The H-P switch is also found in developing spermatids of Drosophila. The time frame of the H-P switch during postmeiotic sperm development is still unknown. To this end, a culture system for isolated, pupal testes and isolated spermatid bundles (cysts) was established in this work. Using histone H2AvD-RFP and protamineB-eGFP expressing flies the development of isolated cysts through meiosis and the H-P switch was observed in in vivo-time-lapse fluorescence microscopy. In these cultures the H-P switch was completed after five hours.
In vertebrates as well as in Drosophila an increase in histone H4 acetylation precedes the H-P switch, while the switch is accompanied by introduction of DNA breaks. In the mammalian model systems the H4 acetylation is proposed as the start-signal for the H-P switch and the DNA breaks may be related to the activity of topoisomerases involved in the relaxation of histone deprived DNA. Here, this model was now tested in the Drosophila background. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity was manipulated via inhibitor treatment of cultured pupal testes and isolated cysts. Histone acetylation Histone acetylation turned out to be essential to the correct development of postmeiotic cysts. However, HDAC-inhibitor induced, premature histone acetylation did not lead to premature signs of the H-P switch. Thus, histone H4 acetylation alone is not sufficient to be the start-signal of the H-P switch in Drosophila. Inhibition of type I and II topoisomerases had no effect on the development of postmeiotic spermatids and the completion of the H-P switch. A connection between DNA breaks and topoisomerase activity appears not to exist in Drosophila spermatids.
The HAT responsible for histone H4 acetylation in the H-P switch of Drosophila is not known so far. Here, the postmeiotic expression pattern of a few HATs was determined by stage-specific RT-PCR on RNA purified from manually isolated single cysts. One candidate HAT was identified.
The results of this work were used in the assembly of a working model for the molecular mechanism of the H-P switch in Drosophila.