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La stabilité de l’information génétique est d’une importance cruciale pour la fonction normale et la reproduction de tous les êtres vivants. Or, la capacité de fertilisation chez l’homme est habituellement mesurée en considérant la concentration, la motilité et la morphologie des spermatozoïdes. Cependant, ces paramètres ne prennent pas en considération l’intégrité du matériel génétique. Pourtant, de fortes évidences démontrent que la spermiogenèse, qui est la phase haploïde de la spermatogenèse durant laquelle se produit un important remodelage de la chromatine, serait une importante source d’instabilité génétique. En effet, des bris transitoires de l’ADN surviennent durant la spermiogenèse au même moment que l’hyperacétylation des histones H4 et la stimulation de l’hyperacétylation de H4 par traitement à la trichostatine A stimule la formation de cassures dans l’ADN. Ainsi, des histones acétyltransférases (HATs) pourraient affecter la compaction et l’intégrité de l’ADN et par conséquent le potentiel fertilisant du gamète mâle. Il est donc important d’identifier l’histone acétyltransférase impliquée dans l’hyper acétylation des histones H4 durant la spermiogenèse, puisqu’il s’agit d’un processus possiblement important pour la fertilité de l’homme. À la suite d'analyses par spectrométrie de masse d’échantillons protéiques de testicules de souris possédant la propriété d'acétyler l’histone H4 aucune HAT n’a été identifée. Par contre, la protéine mitochondriale ACAT1, qui catalyse la transformation réversible de deux acétyl-CoA en CoA et acétoacétyl-CoA, a été détectée. Ces observations permettent d’émettre l’hypothèse que cette protéine pourrait jouer un rôle dans la spermiogenèse en augmentant le niveau d’acétyl-CoA chez les spermatides en élongation. En effet, puisque selon mes résultats les histones H4 sont en mesure de s’auto-hyperacétyler, on peut supposer qu’une augmentation du niveau d’acétyl-CoA causerait une acétylation de ces histones à l’échelle du génome, permettant ainsi la poursuite de la spermiogenèse et éventuellement la formation de spermatozoïdes matures et fonctionnels.

The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.

[Truncated abstract] Protein phosphorylation, one of the most important forms of post-translational modification, has been demonstrated to play crucial roles in regulation of cell function. Phosphorylation of protein serine, threonine and tyrosine residues has been the most thoroughly investigated, taking advantage of the acid-stable character of these phosphohydroxyamino acids. Whereas, the cellular occurrence of acid-labile phosphoamino acids, such as phosphohistidine, phosphoarginine and phospholysine was often underestimated due to the acid treatments employed by most of the traditional phosphoamino acid analysis methods. The biological roles of histidine kinases (HKs) in prokaryotes are well understood in contrast to those of HKs in eukaryotes, especially in mammalian cells. However, the evidence has shown that phosphohistidine comprised 6% of phosphoamino acids of the basic nuclear proteins in eukaryotes (Matthews, 1995) and there was more phosphohistidine than phosphoserine in rat liver mitochondria (Bieber and Boyer, 1966). More significantly, phosphohistidine was revealed to be the major phosphoamino acid in phosphorylated histone H4 in regenerating liver in vivo (Chen et al., 1974) and the Walker-256 carcinosarcoma cells in vitro (Smith et al., 1974). Recently, the histone H4 histidine kinase (HHK) activity of human hepatocellular carcinoma (HCC) tumour tissue was measured to be 400 times higher than the normal liver tissue surrounding the tumour. HepG2 cells (HCC cell line) and PIL-2 cells (a p53 knockout mouse tumorigenic liver progenitor cell line) also displayed high HHK activity (Tan et al., 2004). The above observations suggested that HKs and HHKs are playing important roles in both prokaryotes and eukaryotes, including mammals. One major obstacle in the study of HHK study has been the lack of knowledge of the amino acid sequence of an HHK. Attempts at purifying and identifying the HHK from yeast led to the partial purification of a yeast HHK protein(s) at 32kDa (Huang et al., 1991). However, the amino acid sequence of the HHK has not yet been established. ... The success of the separation was demonstrated by the MALDI-TOF-MS and/or ESI-MS spectra of the RP-HPLC fractions. These achievements suggested that it is possible to detect phosphohistidyl histone H4 in vivo using MS under experimental conditions where phosphohistidine is relatively stable. The study in this thesis represents the progression of HHK research in various aspects, including the yeast HHK purification and identification, mammalian HHK partial purification and the methodological developments in detecting histone H4 histidine phosphorylation using MS. Furthermore, new information regarding the physical characteristics of yeast HHKs and its potential role in cellular biology have been documented. It is anticipated that knowledge generated in these studies will contribute to the insight and the understanding of the biological significance of HHK in yeast and mammalian cells.

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