Characterization of spermatogenic histone variants with special emphasis on histone H2A.X and double stranded break repair

Supervisory Committee Dr. Juan Ausió, (Department of Biochemistry and Microbiology) Supervisor Dr. Robert D. Burke, (Department of Biochemistry and Microbiology) Departmental Member Dr. Claire G. Cupples, (Department of Biochemistry and Microbiology) Departmental Member Dr. John S. Taylor, (Department of Biology) Outside Member The fundamental subunit of chromatin, known as a nucleosome, is comprised of DNA wrapped around two H2A-H2B dimers and one H3-H4 tetramer. This structure perpetuates itself and together with linker histones (histone H1) give rise to the chromatin fibre. This causes compaction of DNA within the nucleus of a eukaryotic cell, which have inhibitory effects in terms of both its accessibility and metabolism. By modifying chromatin structure, cells can regulate and fine-tune different cellular processes, such as DNA repair, replication, transcription and spermatogenesis. Chromatin structure can be modulated by three main mechanisms: covalent post-translational modification of histone tails, incorporation of histone variants and recruitment of chromatin remodelling complexes. In this thesis, the contribution of histone variants and their post-translational modifications to chromatin structure will be discussed. In Chapter 1, I review the role of H2A.X in DNA double stranded break (DSB) repair and other less studied cellular processes, such as transcription and cell cycle. In addition, this chapter also introduces a putative model for the role of H2A.X phosphorylation in DNA DSB repair. In Chapter 2, our results demonstrate that S139 and T136 of H2A.X are both phosphorylated during DNA DSB repair. These two postiv translational modifications are functionally different in that S139E and T136A/S139E mutants partition to different chromatin fractions. Furthermore, we show that nucleosomes containing H2A.X are less stable compared to nucleosomes with canonical H2A. The destabilizing effect is more prominent in the nucleosomes containing H2A.X phosphorylated by DNA-dependent protein kinase suggesting that the post-translational modifications of histone variants and histone variant itself have a direct structural role in chromatin integrity. Recombinantly expressed H2A.Bbd has also been shown to modify chromatin structure by destabilizing nucleosomes in a way that resembles that of H2A.X. However, the native form of this H2A.Bbd has never been identified in vivo. Chapter 3 provides evidence for the presence of native H2A.Bbd in mammalian testis and human sperm. Histone variant hTSH2B, which was found in only a fraction of mature human sperm, has been characterized most recently. In Chapter 4, we report the structural characterization of this variant in the context of other core histones (histone octamer) and in a nucleosome. Although an hTSH2B-containing nucleosome did not show structural alterations compared to its canonical counterpart, hTSH2B octamers were shown to be less stable. Finally, we addressed the disagreement in the literature as to whether or not H1t, a linker histone variant specific to mammalian testis, is phosphorylated during spermatogenesis. Chapter 5 shows that native H1t is phosphorylated. The sites of phosphorylation of H1t were determined. The phosphorylation of histone H1 at the Cterminal domain has been shown to significantly weaken its affinity for the chromatin v fibre thus inferences chromatin structure. It is not surprising that the newly identified phosphorylation sites of H1t within this region also serve similar function. The four histone variants analyzed in this thesis: H2A.X, H2A.Bbd, hTSH2B and H1t, are all expressed in mammalian germ cells and hence play an important role in spermiogenesis. Their structural contribution may help explain some of the complex chromatin transitions involved in this multifaceted cell differentiation process.