Graduation Date

Fall 12-16-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Yuri L. Lyubchenko

Abstract

Nucleosomes are the basic repeating unit defining the assembly and function of chromatin. Understanding the fundamental mechanisms of nucleosome structure and dynamics is critical to elucidating the chromatin assembly process. This dissertation describes my work in elucidating the role of different factors that drive the nucleosome dynamics.

In my first study, we characterized, for the first time, the effect of sequence on nucleosome assembly. We then characterized the role of internucleosomal interactions, discovering a critical role internucleosomal interactions in the assembly of higher order structures.

Based on the previous study and literature regarding histone tails, we hypothesized the histone H4 tail is critical in internucleosomal interactions. We characterized nucleosomes lacking the H4 tail on different sequences and discovered the H4 tail, thought to facilitate internucleosomal interaction, is responsible for nucleosome stability in a sequence dependent manner. We also discovered that internucleosomal interactions rescue nucleosome instability due to H4 truncation.

To test the hypothesis that DNA sequence and internucleosomal interactions are the factors that determine nucleosomal compaction, we assembled tetranucleosomes on DNA substrates with different sequences and simulated random nucleosome placement. In a first of its kind study, our data, supported by a theoretical model, revealed that nucleosomes adopt a condensed conformation at a predictable rate significantly higher than seen in simulations, confirming nucleosomes communicate and interact to form heterogeneous higher order structures, as opposed to the theorized homogenous structure.

In forward-looking studies, we addressed unanswered questions in complex nucleosome systems. In the first, we studied the nucleosome variant containing centromere protein A (CENP-A) on centromeric DNA, hypothesizing this unique region contains structures distinct from bulk chromatin due to these two features. Our results indicate that CENP-A nucleosomes assemble similarly to canonical (H3) nucleosomes on centromeric and other DNA, indicating assembly is dictated by interplay with other factors. In the second, we studied the interaction of transcription factor (TF) nuclear factor-κB (NF-κB) with nucleosomes, hypothesizing that it can alter nucleosome structure. For the first time, we discovered this TF induces nucleosome unraveling and identified it as a pioneer factor. We proposed two models explaining the interaction of NF-κB with nucleosomes.

This dissertation also describes advances we have made in methodology for studying nucleosomal systems. We successfully designed a DNA nanoring to induce mechanical strain upon the DNA, which can be used to mimic the strain imposed by DNA wrapping around nucleosomes. Another structure we designed, a three-way junction as a DNA end-label, was applied to nucleosome assemblies, demonstrating its usefulness in the study of nucleosomal systems.

Comments

Copyright 2022, the author

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