Doctor of Philosophy, Case Western Reserve University, 2006, Pathology

The possible existence of a lamina which determines nuclear shape in yeast is controversial since clear lamin sequence homologs do not exist. Nevertheless S. cerevisiae Esc1p resembles the lamins in that it contains coiled-coil domains and localizes to the nuclear periphery where it anchors heterochromatin. Increased expression of Esc1p causes dramatic elaborations of the nuclear envelope (NE), which extend into the cytoplasm and are reminiscent of modifications of nuclear shape seen in human laminopathies. These double membrane extensions, here referred to as "ESCapades", include nuclear pores, but little or no nucleoplasm. They generally contact the nucleolus, originate at the nucleus-vacuole junction and wrap around the vacuole. Examination of cells in anaphase shows that escapades are not inherited by daughter nuclei. Contrary to expectation, exclusion of these structures from daughters is independent of septin function and the presence of vacuoles. Moreover, they are excluded even when nuclear division is restricted to the maternal cytoplasm. Thus, this behavior may reflect the existence of an intrinsically coherent “maternal domain” of the nuclear periphery. The exclusion of atypical structures provides a striking example of “phenotypic normalization” which may make it possible to sustain continued cell growth when parental nuclei have acquired potentially deleterious characteristics. Consistent with this, retention of escapades slows the pace of bud formation in mothers while the overall speed of growth is only moderately affected.

Committee: Alan Tartakoff (Advisor) Subjects: Biology, Cell

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