Abstract

The fundamental unit of chromatin is the nucleosome, which consists of a core of eight proteins wrapped by DNA. This core is composed of four pairs of histone proteins: H2A, H2B, H3, and H4. The DNA wraps around the protein core ~1.7 times, facilitating compaction of DNA length in the cell. Further, the location of nucleosomes makes genomic elements encoded in the DNA, such as promoters or enhancers, accessible or inaccessible to RNA polymerase and transcription factors. Thus, where nucleosomes are located (or positioned), can play a major role in transcription or other cellular processes. Additionally, histone proteins are frequently post-translationally modified, and these modifications further play a role in cellular processes, and in some cases are even required for specific protein function. What positions nucleosomes, and the downstream results of positioning or post-translational modifications (PTMs) is a topic of prolific study. Nucleosome formation is not random. In vivo it is believed that chromatin remodelers are the primary determinant of where nucleosomes form, while in vitro the DNA itself is the primary determinant. Formation of nucleosomes in vitro is a potent tool to elucidate fundamentals of chromatin. Considering that in vitro nucleosome formation is dependent on free energy, morphology and base composition of the DNA influence the free energy of formation. We found that the ends of linear DNA fragments were much more likely to have in vitro nucleosomes form on them. While this has the potential to bias results, based on our observations we could not find any significant alteration of the overall underlying DNA sequence composition due to the end preference observed. Histone proteins frequently receive the PTMs of methylation or acetylation. Histone methylation is typically indicative of repressed genes, while histone acetylation is typically indicative of active genes. In vivo the addition and removal of methylation and acetylation is highly dynamic. We hypothesized that the histone PTMs of methylation and acetylation also played a role in where nucleosomes formed. Comparing both in vivo and in vitro datasets, we observed strikingly similar patterns of nucleosomes for several histone methylations and acetylations, suggesting that these PTMs do indeed direct nucleosome formation. Upon further investigation, the underlying DNA sequence preferences change when compared to unmodified nucleosomes. This suggests that the genome is encoded to position these marks in locations where they are likely to be needed.

Degree

PhD

College and Department

Life Sciences; Microbiology and Molecular Biology

Rights

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