Doctor of Philosophy, The Ohio State University, 2022, Molecular, Cellular and Developmental Biology

Maintenance of genome stability and faithful transmission of genetic and epigenetic information comprises the foundation of cellular, and thus organismal fitness, within their environment. The greatest challenge to genome integrity is the process of genome duplication in preparation for cell division. This process is highly destructive to the genome's epigenetic and 3D organization information, yet accurately rebuilt after DNA sequence copying. The dynamic nature of genome replication requires a vast number of proteins and complexes, one of which is Histone acetyltransferase 1 (HAT1). HAT1 is responsible for acetylating Histone H4 Lysines 5 and 12 in the cytoplasm before H3:H4 dimers are translocated to and imported into the nucleus and deposited on nascent chromatin during DNA replication. HAT1 loss in mice is neonatal lethal with pups exhibiting developmental lung defects and craniofacial defects. Mouse embryonic fibroblasts (MEFs) isolated from HAT1 -/- mice experience slowed growth, heightened sensitivity to DNA damage, and genome instability indicators such as chromosome breaks and fusions and changes in chromosome number. Recently, multiple lines of evidence suggest that histone acetyltransferase 1's (HAT1) purpose extends far beyond its activity in new histone editing. In this study, we focus on HAT1's role at replication forks and explore its possible role in promoting the nascent chromatin-nuclear periphery relationship. First, we developed a proximity-based Chromatin Assembly Assay (CAA) to study replication fork dynamics with standardized data collection and analysis procedures. We then used this protocol and HAT1 +/+ and HAT1 -/- immortalized Mouse Embryonic Fibroblasts (iMEFs) to investigate HAT1's role in the maintenance of genomic integrity. We show that HAT1 transiently associates with nascent DNA and that loss of HAT1 slows replication fork progression, due at least in part to an increase in fork stalling. In addition, fork stalling stabilizes HAT1' (open full item for complete abstract)

Committee: Mark Parthun (Advisor); Dmitri Kudryashov (Committee Member); Kirk Mykytyn (Committee Member); Jeff Parvin (Committee Member) Subjects: Biology; Cellular Biology; Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2021, Molecular, Cellular and Developmental Biology

Faithful transmission of epigenetic information through cell divisions is critical for control of cell proliferation as well as maintenance of cell identity and genome integrity. Misregulation of epigenetic states is characteristic of normal ageing, neurodegenerative diseases, and cancer. Epigenetic information can be transmitted by a variety of mechanisms, which include patterns of histone posttranslational modifications. When cells divide, maintenance of these patterns partially relies on recycling of parental histones, but they represent only half the histones necessary to maintain proper nucleosome density on the two daughter DNA strands. To supplement parental histones, newly synthesized histones must also be incorporated into nascent chromatin. However, newly synthesized histones do not get deposited onto nascent DNA in an unmodified state, rather, they acquire a set of specific post-translational modifications during the process of replicationcoupled chromatin assembly. These modifications have to be removed before the pattern of the parental pattern of epigenetic marks can be propagated to the newly synthesized histones. Histone acetyltransferase 1 (HAT1) has been shown to acetylate lysines 5 and 12 in the tail of the newly synthesized histone H4 during replication-coupled chromatin assembly. While this pattern of post-translational modifications has been conserved extremely well across the eukaryotic evolution, its exact biological function remains an open question. This dissertation attempts to describe the role iii of HAT1 and HAT1-dependent acetylation of newly synthesized histones in epigenetic inheritance of specific chromatin states. Chapter 1 of this document introduces the subject of epigenetic inheritance and provides a more detailed look into the HAT1's role in the replication-coupled chromatin assembly. Chapter 2 reports that HAT1 and acetylation of newly synthesized histones regulate chromatin acessibility of specific he (open full item for complete abstract)

Committee: Mark Parthun PhD (Advisor); Michael Freitas PhD (Committee Member); Paul Herman PhD (Committee Member); Jeffrey Parvin MD, PhD (Committee Member) Subjects: Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2006, Molecular, Cellular, and Developmental Biology

Acetylation of the N-terminal tails of newly synthesized histones H3 and H4 by type-B histone acetyltransferases (HATs) is thought to play a role in chromatin assembly. While originally known as a cytoplasmic factor, Hat1p, the catalytic subunit of the first identified type-B HAT, has been shown to exist in both the cytoplasm and the nucleus. Hat1p and specific lysine residues in the histone H3 N-terminal tail have been shown to be redundantly required for telomeric silencing, and multiple protein factors have been found to be involved in both telomeric silencing and DNA damage repair. This raised the possibility that Hat1p might also be involved in DNA damage repair. Substitution of specific lysine residues in the histone H3 N-terminal tail, as well as combination of specific lysine residue replacement in H3 and HAT1 deletion resulted in enhanced sensitivity to methyl methanesulfonate. This sensitivity was specific for DNA double Strand beraks (DSBs), as these mutants were sensitive to endonuclease digestion, but not to UV irradiation. While histone H3 mutations showed minor effects on nonhomologous end joining, the primary defect in the histone H3 and HAT1 mutants was in pathway of homologous recombination. Subsequent epistasis analysis indicates that the histone H3 and Hat1p may contribute to DSB repair through an Asf1p-dependent chromatin assembly pathway. Hat1p was then found to become associated with damaged DNA. Further kinetic analysis showed that Hat1p localization to DSB occurs after the phosphorylation of histone H2A S129 and concomitantly with the recruitment of the recombinational repair factor Rad52p. In addition, the nuclear Hat1p-associated histone chaperonee Hif1p was also recruited to DSB and followed similar kenetics as that of Hat1p. Moreover, the acetylation of all four histone H4 N-terminal domain lysine residues including 5, 8, 12 and 16 was increased following DSB generation on the MAT locus, but only acetylation of lysine 12, the primary tar (open full item for complete abstract)

Committee: Mark Parthun (Advisor) Subjects: Biology, Molecular