SUMMARYIn both mammalian and Drosophila spermatids, the completely histone-based chromatin structure is reorganized to a largely protamine-based structure. During this histone-to-protamine switch, transition proteins are expressed, for example TNP1 and TNP2 in mammals and Tpl94D in Drosophila. Recently, we demonstrated that in Drosophila spermatids, H3K79 methylation accompanies histone H4 hyperacetylation during chromatin reorganization. Preceding the histone-to-protamine transition, the H3K79 methyltransferase Grappa is expressed, and the predominant isoform bears a C-terminal extension. Here, we show that isoforms of the Grappa-equivalent protein in humans, rats and mice, that is DOT1L, have a C-terminal extension. In mice, the transcript of this isoform was enriched in the post-meiotic stages of spermatogenesis. In human and mice spermatids, di-and tri-methylated H3K79 temporally overlapped with hyperacetylated H4 and thus accompanied chromatin reorganization. In rat spermatids, trimethylated H3K79 directly preceded transition protein loading on chromatin. We analysed the impact of bacterial infections on spermatid chromatin using a uropathogenic Escherichia coli-elicited epididymo-orchitis rat model and showed that these infections caused aberrant spermatid chromatin. Bacterial infections led to premature emergence of trimethylated H3K79 and hyperacetylated H4. Trimethylated H3K79 and hyperacetylated H4 simultaneously occurred with transition protein TNP1, which was never observed in spermatids of mock-infected rats. Upon bacterial infection, only histone-based spermatid chromatin showed abnormalities, whereas protaminecompacted chromatin seemed to be unaffected. Our results indicated that H3K79 methylation is a histone modification conserved in Drosophila, mouse, rat and human spermatids and may be a prerequisite for proper chromatin reorganization.

Much of spermatid differentiation takes place in the absence of active transcription, but in the early phase, large amounts of mRNA are synthesized, translationally repressed, and stored. Most nucleosomal histones are then degraded, and chromatin is repackaged by protamines. For both transcription and the histone-to-protamine transition in differentiating spermatids, chromatin must be opened. This raises the question of whether two different processes exist. It is conceivable that for initiation of the histone-to-protamine transition, the already accessible, actively transcribed chromatin regions are utilized or vice versa. We analyzed the enrichment of different canonical TATA-boxbinding, protein-associated factors and their variants in murine spermatids, diverse bromodomain-containing proteins, and components of the Polycomb repressive complexes PRC1 and PRC2 using quantitative PCR. We compared the enrichment of corresponding proteins in human and murine spermatids and analyzed the time frame of postmeiotic transcription and expression of histones, transition proteins, and protamines in human and murine spermatids using immunohistology. We correlated the expression of different transcription factors and bromodomain-containing proteins and the pattern of acetylated histones to active transcription and to the histone-to-protamine transition in both human and murine spermatids. Our findings suggest that differentiating spermatids use both common and specific features to open chromatin first for transcription and subsequently for histone-to-protamine transition.