Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. This folding, and the subsequent organization of the genome, reflects both the transcription profile of the cell and of the specific cell type. A dispersed, mesh-like chromatin configuration, for example, is characteristic of a pluripotent stem cell. Here we show that the acquisition of the pluripotent state during somatic cell reprogramming is coincident with the disruption of compact heterochromatin domains. Using Electron Spectroscopic Imaging (ESI), I made the surprising observation that the heterochromatin domains of the induced pluripotent and of the parental somatic cell contained 10 nm chromatin fibres. Since ESI generates projection images, the precise three-dimensional organization of all chromatin fibres within these domains could not be elucidated. To circumvent this limitation, I developed an electron microscopy technique that combines ESI with tomography. Using this approach, I found that both heterochromatin domains and the surrounding euchromatin of murine pluripotent cells, fibroblasts, and somatic tissues are in fact organized entirely as 10 nm chromatin fibres. This challenges the current paradigm that most, if not all, of the genome exists as 30 nm and higher-order chromatin fibre assemblies. Rather than transitions between 10 nm and 30 nm fibres, I propose that the organization and thus the regulation of the genome is achieved by the bending and folding of 10 nm chromatin fibres into discrete domains in a cell type-specific manner.

Marked by a defect in the production of the Beta (β)-globin chain that make-up hemoglobin, Beta (β)-thalassemia is the most prevalent form of inherited single-gene disorders in the world. To understand the molecular mechanisms that govern the expression of the β-globin polypeptide encoded by the β-globin locus, we examined closely the enzymes involved in the epigenetic regulation of gene expression through histone 3 lysine 9 mono and di-methylation (H3K9 me1/2). G9a-like protein (GLP), a mammalian methyltransferase involved in the establishment and maintenance of H3K9 me1/2 mark at euchromatin, regions was found to be critical for the full activation of the adult β-globin genes in vivo during Murine erythroleukemia cell line (MEL) differentiation. Though it was initially hypothesized that GLP binding to H3K9 me1/2 mark through its Ankyrin domain was key to its activating function, we found that Flag- GLP ankyrin mutants E817R and W791A unable to bind to the methyl mark, are able to activate β-globin genes as well as their wild-type counterpart. Additionally, this study found that the embryonic gene εγ, known to be re-activated after G9a KD at the mRNA level, was effectively transcribed at the protein level using Triton Urea Acetic acid (TAU) western blots, thereby identifying potential therapeutic applications for treatment for β-thalassemia patients.

Recent studies have focused their attention on the structure and function of histone chaperones involved in different aspects of somatic chromatin assembly and disassembly. However, one of the most dramatic chromatin remodeling processes takes place immediately after fertilization and is mediated by proteins in metazoan eggs that function as histone chaperones and histone storage proteins. These include members of the nucleoplasmin (NPM) family and the nuclear autoantigenic sperm protein (NASP) families. While it had been know for some time that these proteins function as “histone sinks”, new studies are shedding light on their role as histone chaperones. NASP was first identified in Xenopus laevis eggs where it accumulated in the nucleoplasm and was found to bind histones H3 and H4 at which time it believed to act simply as a histone storage protein. Interestingly, in addition to binding and providing storage to H3-H4 in the egg and in somatic cells, our studies have shown NASP to be the first characterized chaperone for histone H1. The members of the histone H1 family (linker histones) are essential to maintaining the structure of chromatin with respect to the folding of the chromatin fiber, nucleosome spacing, chromatin remodeling, gene transcription and progression through the cell cycle. Until recently there has been no histone H1 chaperone characterized and no evidence of a storage protein to which linker histones are bound, when not associated with DNA of NCPs. By using recombinant NASP, to incorporate linker histones onto the nucleosome arrays in a chromatin fiber, we studied the dynamics and conformation of chromatin in a more biologically relevant and precise method than presented in any previous chromatin research. Like NASP, nucleoplasmin was identified as a factor in X. laevis eggs that binds histones and loads them onto DNA. Subsequently, the nucleoplasmin protein family has been to be universally represented throughout metazoans where it plays a similar role in chromatin metabolism. Members of this family include nucleophosmin (NPM1), nucleoplasmin (NPM2, NPM or Np), the newly characterized NPM3 and nucleoplasmin-like proteins (NPM-like or NLP). We have been able to demonstrate that NPM2 that is highly phosphorylated in X. laevis eggs can unfold sperm and somatic chromatin by facilitating the removal of linker histones and other chromosomal proteins from linker DNA regions between nucleosomes in the absence of any stable interaction with the nucleosome core particle (NCP) itself. In addition, our studies reveal that NPM2 is a pentameric chaperone, as opposed to a decameric chaperone, that regulates the condensation state of sperm and somatic chromatin by removing linker histones and specific nuclear basic proteins and depositing histone H2A-H2B dimers on the distal face of the NPM2. / Graduate

The fundamental repeating unit of all eukaryotic chromatin is the 147bp DNA:histone complex known as the nucleosome. Genome-wide studies have demonstrated that nucleosomes are organized with the 5’ promoter being nucleosome depleted and the transcribed region is occupied by a periodic array of positioned nucleosomes. While this organization is well described, the determinants, particularly trans-acting factors that contribute to this architecture are only partly described with gene expression, however, while the connection between chromatin and the various facets of gene expression regulation, especially in evolution, is apparent the detailed mechanisms remain to be described. In this thesis, I describe 1) The role of nucleosomes in gene expression evolution in closely related yeast species 2) The role of trans-acting factors (particularly transcription factors and co-factors) in determining the nucleosome depleted region of promoters and 3) The role of trans-acting factors in nucleosome spacing within genes.

The fundamental repeating unit of all eukaryotic chromatin is the 147bp DNA:histone complex known as the nucleosome. Genome-wide studies have demonstrated that nucleosomes are organized with the 5’ promoter being nucleosome depleted and the transcribed region is occupied by a periodic array of positioned nucleosomes. While this organization is well described, the determinants, particularly trans-acting factors that contribute to this architecture are only partly described with gene expression, however, while the connection between chromatin and the various facets of gene expression regulation, especially in evolution, is apparent the detailed mechanisms remain to be described. In this thesis, I describe 1) The role of nucleosomes in gene expression evolution in closely related yeast species 2) The role of trans-acting factors (particularly transcription factors and co-factors) in determining the nucleosome depleted region of promoters and 3) The role of trans-acting factors in nucleosome spacing within genes.

Time-resolved fluorescence spectroscopy was used to monitor the effects of varying ionic strength on nucleosome core particle structure. Two main methods were used in these studies. First, the fluorescence anisotropy decay of bound ethidium was measured and was shown to reflect the rotational tumbling of the core particle through solution, the longest recovered decay time being a measure of the rotational correlation time of the particle. A rotational correlation time of 165 ns was recovered for the native core particle at 10 mM ionic strength, in excellent agreement with that predicted by hydrodynamic calculations based on the particle's known size and shape. This technique was then used to measure the rotational correlation time of the core particle as a function of ionic strength. Below 1 mM salt the recovered rotational correlation times suggested little change in shape throughout the region of the reversible low salt transition. At very low ionic strengths (below 0.2 mM), where the low salt transition becomes irreversible, the rotational correlation time increased sharply to ~330 ns, suggesting a major change in the core particle structure. Computer modeling was performed to show that this increase was most likely due to a substantial elongation in the core particle structure, to at least a 5:1 axial ratio. At elevated ionic strengths, the rotational correlation time was seen to increase from the initial value of ~165 ns to ~240 ns as the salt concentration was raised from 10 mM to 0.35 M, with further increases being observed only above 0.65 M; we term this initial increase the moderate salt transition. Trypsinization of the core particles to remove the Nterminal histone domains completely abolished the increase, demonstrating that the moderate salt transition as measured by this technique involves the release of these protein domains from the body of the core particle. The second method used involved the measurement of the fluorescence decay of the intrinsic tyrosine residues of the core particle. This decay proved to be very complex, and was best represented by a distribution of lifetimes, suggesting different environments for the tyrosines. This distribution changed as the ionic strength of the solution changed, suggesting the movement of tyrosine residues to differing environments as the particle undergoes the low and moderate salt transitions, as well as the high salt dissociation. / Graduation date: 1993

163 leaves : ill. ; 28 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1973

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Physics and Astronomy." Includes bibliographical references (p. 77-79).

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Univ., 2004. / Härtill 3 uppsatser.