Characterizing the Fundamental Chromatin Structure and Function in a Realistic Nuclear Environment

Virk, Ranya Kaur Aphrodite

doi:https://doi.org/10.21985/n2-gkt5-en09

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Characterizing the Fundamental Chromatin Structure and Function in a Realistic Nuclear Environment

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Chromatin is the biological material that packages our genetic information. In humans, 2 meters of linear DNA is compacted into an approximately 6 μm nucleus. Our DNA is transcribed into RNA, which is then translated into proteins. Cellular phenotype, the composite of all cellular functions, is defined by the entire transcriptome and proteome of the cell. Thus, the organization of chromatin, which controls accessibility of DNA to transcriptional machinery, helps to dictate cellular function. Recent developments in technology, ranging from high-throughput sequencing, to super-resolution optical imaging and electron microscopies, to increases in computing power, have greatly expanded our understanding of chromatin structure and function. It is not just the genome, the sequence of A’s, T’s, C’s, and G’s, that influences cellular phenotype. DNA wraps around histone octamers to form nucleosomes, which are strung together by linker DNA to form the ‘beads-on-a-string’ chromatin fiber. The epigenome - including chemical modifications such as DNA methylation and histone tail acetylation/methylation - modulates cell function by controlling local chromatin structure and accessibility. Epigenetic marks can recruit chromatin readers, which are capable of nucleosome remodeling and activating transcription or compacting nucleosomes and repressing transcription. In contrast to the previously accepted textbook view of a highly ordered 30 nm fiber, chromatin also exhibits a highly disordered structure at the level of the primary fiber. Higher-order chromatin domains have also been observed at the level of 100’s of kilobase pairs to megabase pairs. The existence of these dynamic structureshas been shown to modulate transcriptional efficiency by influencing four-dimensional enhancer-promoter contacts and the spreading of epigenetic marks. Despite these recent advances in our understanding, there remain several key open questions in the chromatin field that this thesis aims to address. Chapter 2 identifies and investigates the fundamental units of chromatin folding. Chapter 2 begins by employing a unique combination of high-resolution electron microscopy imaging and polymer physics-based analysis techniques to characterize the morphological and functional properties of higher-order chromatin packing domains. Next, in Chapter 2 we uncover fundamental organizational principles of the genome using nanoimaging and chromosome conformation capture experimental approaches to validate and better understand a statistical model of chromatin structure. Altogether, we demonstrate that the topology of chromatin can be represented by branching, tree-like network structures and that statistical rearrangements in connectivity and mass density distribution occur upon heat stress. Chapter 3 then transitions into the functional implications of the fundamental statistical chromatin organization identified in Chapter 2. The Chromatin Packing Macromolecular Crowding (CPMC) model, which combines a kinetic model of transcription with the statistical packing descriptors of chromatin packing domains, is able to faithfully predict the phenotypic plasticity of cancer cells. The initial model is then extended to predict cancer cell death in response to cytotoxic chemotherapy treatment. Altogether, the initial chromatin packing state of cells is shown to directly influence cellular adaptability to external stressors. Chapter 4 then focuses on developing and employing a molecular theoretical approach to characterize the effects of the physicochemical intranuclear environment, including bulk ions, pH, and density, on the structure and charge of DNA-like and chromatin-like systems. We identify bridging of multivalent cations as an important mechanism for both neutralizing the strongly negative charges of DNA-phosphates and increasing compaction of DNA-phosphate loops. We extend our approach to investigate the effects of the physicochemical environment on individual nucleosomes and 8-mer nucleosome arrays, and determine the importance of chromatin density on the effects of the monovalent electrolyte environment on chromatin structure. Finally, Chapter 5 provides a summary of this thesis work and an outlook for future directions. Overall, this thesis combines physics-based modeling, nanoimaging, and sequencing-based molecular approaches to better understand fundamental mechanisms underlying chromatin structure and function in a realistic nuclear environment.

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