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

dc.contributor.authorLi, Andra Jia Jia
dc.contributor.supervisorAusio, Juan
dc.date.accessioned2010-04-30T15:17:35Z
dc.date.available2010-04-30T15:17:35Z
dc.date.copyright2009en
dc.date.issued2010-04-30T15:17:35Z
dc.degree.departmentDept. of Biochemistry and Microbiologyen
dc.degree.levelDoctor of Philosophy Ph.D.en
dc.description.abstractThe 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 post-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 C-terminal domain has been shown to significantly weaken its affinity for the chromatin 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.en
dc.identifier.bibliographicCitationLi, A., Eirin-Lopez, J. M., and Ausio, J. (2005). H2AX: tailoring histone H2A for chromatin-dependent genomic integrity. Biochem Cell Biol 83, 505-515.en
dc.identifier.bibliographicCitationLi, A., Maffey, A. H., Abbott, W. D., Conde e Silva, N., Prunell, A., Siino, J., Churikov, D., Zalensky, A. O., and Ausio, J. (2005). Characterization of nucleosomes consisting of the human testis/sperm-specific histone H2B variant (hTSH2B). Biochemistry 44, 2529-2535.en
dc.identifier.bibliographicCitationRose, K. L., Li, A., Zalenskaya, I., Zhang, Y., Unni, E., Hodgson, K. C., Yu, Y., Shabanowitz, J., Meistrich, M. L., Hunt, D. F., and Ausio, J. (2008). C-Terminal Phosphorylation of Murine Testis-Specific Histone H1t in Elongating Spermatids. J Proteome Res. 7(9),4070-4078.en
dc.identifier.bibliographicCitationThambirajah, A. A., Li, A., Ishibashi, T., and Ausio, J. (2009). New developments in post-translational modifications and functions of histone H2A variants. Biochem Cell Biol 87, 7-17.en
dc.identifier.bibliographicCitationIshibashi, T., Li, A., Eirin-Lopez, J. M., Zhao, M., Missiaen, K., Abbott, D. W., Meistrich, M., Hendzel, M. J., and Ausio, J. (2009). H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis. Nucleic Acids Res. 38(6), 1780-1789.en
dc.identifier.bibliographicCitationLi, A., Yu, Y., Lee, S.C., Ishibashi, T., Lees-Miller, S.P., Ausio, J. (2010). Phosphorylation of histone H2A.X by DNA-dependent Protein Kinase (DNA-PK) is not affected by core histone acetylation but it alters nucleosome stability and histone H1 binding. J Biol Chem, [Epub ahead of print].en
dc.identifier.urihttp://hdl.handle.net/1828/2681
dc.languageEnglisheng
dc.language.isoenen
dc.rightsAvailable to the World Wide Weben
dc.subjectBiochemistryen
dc.subjectMolecular Biologyen
dc.subject.lcshUVic Subject Index::Sciences and Engineering::Chemistry::Biochemistryen
dc.subject.lcshUVic Subject Index::Sciences and Engineering::Biology::Microbiologyen
dc.titleCharacterization of spermatogenic histone variants with special emphasis on histone H2A.X and double stranded break repairen
dc.typeThesisen

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