Single-Molecule Investigation of Chromatin-Associated Factors in Genome Organization and Epigenetic Maintenance

The central dogma of biology has laid the foundation for understanding gene expression through the mechanisms of transcription and translation. However, another layer of eukaryotic gene regulation lies in the complex structure of chromatin. This scaffold of structural proteins and enzymatic regulators determines what genes are expressed at what times, leading to cell differentiation, cell fate, and often disease. Currently, the field of chromatin biology has relied on basic biochemistry and cellular assays to identify key epigenetic regulators and their role in genomic maintenance. For this thesis work, I have developed a biophysical platform to study chromatin-associated factors at the single-molecule level (Chapter 2). This methodology allows us to extract key mechanistic details often obscured by standard bulk methodologies. Using this platform, we posed the question of how epigenetic factor, Polycomb repressive complex 2 (PRC2) engages with chromatin (Chapter 3). PRC2 is a major epigenetic machinery that maintains transcriptionally silent heterochromatin in the nucleus and plays critical roles in embryonic development and oncogenesis. It is generally thought that PRC2 propagates repressive histone marks by modifying neighboring nucleosomes in a strictly linear progression. However, the behavior of PRC2 on native-like chromatin substrates remains incompletely characterized, making the precise mechanism of PRC2-mediated heterochromatin maintenance elusive. Our understanding of this process was limited by the resolution of structural techniques that fail to identify PRC2-binding modes on long chromatin substrates. In short, we found direct evidence that PRC2 can simultaneously engage nonadjacent nucleosome pairs. The demonstration of PRC2\u27s ability to bridge noncontiguous chromosomal segments furthers our understanding of how Polycomb complexes spread epigenetic modifications and compact chromatin. In addition to this single-molecule chromatin binding technology, I also created a singlemolecule platform harnessing correlative force and fluorescence microscopy to assay the material properties of phase separated condensates (Chapter 2). This assay combined methodology to visualize condensate formation at the single-molecule level, in addition to optical trapping of individual droplets to investigate their material properties. Utilizing this technology, we interrogated the role of linker histone H1 (Chapter 4). The linker histones are the most abundant group of chromatin-binding proteins that bind and organize eukaryotic chromatin. However, roles for the diverse and largely unstructured H1 proteins beyond chromatin compaction remain unclear. We used correlative single-molecule force and fluorescence microscopy to directly visualize the behavior of H1 on DNA under different tensions. Unexpectedly, our results show that H1 preferentially coalesces around nascent, relaxed singlestranded DNA. In vitro bulk assays confirmed that H1 has a higher propensity to form phaseseparated condensates with single-stranded DNA than with double-stranded DNA. Furthermore, we dissected the material properties of different H1:DNA condensates by controlled droplet fusion with optical tweezers, and found that increased DNA length and GC content result in more viscous, gel-like H1 condensates. Overall, our findings suggest a potential role for linker histones to sense and coacervate single-stranded nucleic acids in the nucleus, forming reaction hubs for genome maintenance. This work also provides a new perspective to understand how various H1 subtypes and disease-associated mutations affect chromatin structure and function. In summary, we have gained a greater understanding of the biophysical basis for chromatin regulation by both PRC2 and histone H1. Both of the biophysical platforms created for these studies can be applied to various new targets in chromatin biology. They will enable the investigation of a multiplicity of binding interactions, regulatory mechanisms, and material properties of protein-nucleic acid complexes (Chapters 5 & 6). I believe single-molecule techniques will become a major toolset to study chromatin biology, identifying the intricacies and interactions between epigenetic factors and our genome

The structure and development of mammalian enamel.

PhDEnamel development and structure have been studied in a number of placental and marsupial mammals, by light microscopy; electron-microscopy; and scanning electron microscopy. The relationship between the formative cells of the enamel and its structural organisation into "prisms" and interprismatic regions has been studied in particular. The crystallites in developing enamel tend to be oriented perpendicular to its mineralising front; but their orientation may be modified by either the translatory movement which may occur between certain surfaces of the TOMES' processes of the ameloblasts and the mineralising front, or the self directed growth of groups of groups of crystallites. The presence of a repetitive (prism) pattern of crystallite orientation in formed enamel is determined by changes of orientation of and within the mineralising front: these changes are 1) the result of the peculiar mode of secretion of the enamel precursor substances from and about projections from the ameloblasts; and 2) absent during the formation of the first and last layers of enamel (formed at the enamel-dentine junction and the true enamel surface respectively) by a given group of ameloblasts: hence there are no prisms in these regions. Abrupt changes in orientation of the mineralising front determine abrupt changes in crystallite orientation in the enamel (equivalent to the "prism-sheaths" of adult enamel). The secretory territories of individual ameloblasts are only equivalent to prisms in one particular pattern: one ameloblast may be related to more than one prism. Decussation of prisms is associated with the depressions in the mineralising front filling in from alternate sides in "zones". Zone formation begins as a spiral over cusp centres. Light scattering from enamel depends on 1) the size; and 2) the orientation of its ultrastructural elements and 3) the wavelength of the incident radiation; blue light being scattered preferentially; hence the visibility of: - 1) the incremental striae; and 2) the decussating zones of prisms; and 3) the brown colour of the incremental striae when viewed by transmitted light. The calcium content in developing enamel measured by the x-ray emission microanalytical method was found to increase steadily, from the surface of the developing enamel inwards

IDENTIFICATION AND CHARACTERIZATION OF THE GENETIC AETIOLOGY OF A RARE DISEASE KNOWN AS ORAL-FACIAL-DIGITAL SYNDROME TYPE II OR MOHR SYNDROME

Ph.DDOCTOR OF PHILOSOPH