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Understanding the chromatin structure as the dynamic, active nuclear environment, and its influence on transcription

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The last decade has witnessed a rapid transformation in our understanding of the structure of chromatin, the nuclear complex of DNA and its structural proteins. While, barring mutations, the DNA sequence in each cell of the human body is the same, it is the structure of the chromatin complex that is different and determines cell type and function. Moreover, as chromatin occupies a significant fraction of the nucleus and is the most abundant constituent, it is not only the primary functional component, but also comprises the physical nanoenvironment in which nuclear processes occur. Due to the confined nature of the cell, the nucleus is highly crowded by chromatin and other macromolecules, and these crowders not only participate directly in target reactions, but also exclude volume from, and thereby influence, adjacent reactions. This thesis presents an examination of the chromatin structure, including how it is altered when undergoing experimental observation, and how, as part of the nuclear environment, it influences transcription. For the study of chromatin biology, it is critical to use a combination of both theory and experiments to understand the extent to which molecular interactions, which cannot be resolved experimentally, propagate to affect the global chromatin structure and function, which occur on a length-scale several orders of magnitude larger. Herein, with our Self-Returning Random Walk model, I first examine how population-averaged study, a common practice of experimental methods, could alter our understanding of chromatin at the single-cell level. Next, I examine the ways in which the chromatin structure directly integrates with transcription kinetics. Toward this pursuit, I developed two models of transcription. The first directly compares transcriptional efficiency between the hypothesized transcription factory (proteins form a hub that recruits genes) and the classical, protein-lead search. The second model investigates how dynamics within chromatin, as the primary component of the nuclear environment, affect transcription kinetics. Finally, to understand how experimental observation alters the observed chromatin structure, I quantified how the chromatin structure is altered when undergoing gold standard experimental techniques for chromatin imaging, specifically fluorescence in situ hybridization (FISH) and CRISPR/dCas9-based labelling. Altogether, this work highlights the importance of considering that chromatin as a dynamic, active part of the nuclear environment affects gene expression and must be considered from multiple angles to fully understand its impact on cell function.

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