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Date of Award
Campus Access Dissertation
Doctor of Philosophy in Chemistry (PhD)
Administrative Home Department
Department of Chemistry
Committee Member 1
Committee Member 2
Committee Member 3
Four different histone proteins comprise the octameric histone core, a key component of DNA compaction into chromatin. The N-terminal tails of each histone protein contain a variety of post-translational modifications that can help modulate gene expression. Mutations are rare in these key proteins, but when found they are often linked to very serious and lethal diseases. In 2012, mutations in histone genes HIST1H2B and H3F3A were found to be implicated in brain cancer. The protein products of these genes produced four point mutants in two proteins: H3.1K27M, H3.3K27M, and H3.3G34R/V. The positions of these mutations are located at or adjacent to known sites of post-translational modification. Trimethylation of H3K27 is a known mark of gene repression and tumors harboring the K27M mutation have been found to have globally reduced levels of this mark. G34R/V mutations have been shown to produce locally reduced levels of H3K36 trimethylation as well. H3K36 trimethylation has been tied to transcription regulation and DNA repair. While these mutations are clearly disrupting the histone post-translational landscape they may also be perturbing the nucleosome structure itself. Histone proteins interact with DNA through basic residues. As these mutations are directly changing the basic residue content of the proteins, DNA-histone interactions may be altered. Research examining these mutations thus far have focused on secondary interactions between protein complexes and the nucleosome. No studies have examined how these mutations and changes in post-translational modifications could be effecting overall nucleosome structure.
The main purpose of this research was to develop a system to examine the effects that these mutations have on nucleosome structure. To address this, Aim 1 of the project involved cloning eleven genes to produce the four canonical histone proteins (H2A, H2B, H3, and H4), an H3 variant (H3.3), three H3.3 point mutants (H3.3 K27M, H3.3 G34R, H3.3 G34V), one tailless H3.1 mutant (H3.1 Δ5), and two tailless H3.3 mutants (H3.3 Δ32 and H3.3 Δ45). A new, 2-step purification method was developed for the simple and inexpensive purification of histone proteins. Purified histones were then reconstituted into nucleosomes using a salt-gradient method. Four nucleosome constructs were reconstituted, differing only in the H3 protein included (H3.1, H3.3, H3.3 K27M, or H3.3 Δ32). Atomic Force Microscopy images of the four nucleosome constructs were acquired and analyzed.
The location of these histone mutations is at or adjacent to known sites of post-translational modification. Due to this, it was necessary to determine how modifications at these sites effected the nucleosome structure as well to get a full scope of the impact of the mutations. Studying individual post-translational modifications is difficult as extracting histone proteins from tissue samples produces a heterogeneous population of modifications. To generate a homogenous population of modified proteins native chemical ligation methods were explored with the goal of producing histone proteins containing site-specific modifications. Preliminary ligation studies successfully created peptide dimers and trimers through a salicylaldehyde ester ligation technique.
Nikula, Chelsea, "DEVELOPMENT OF A SYSTEM TO STUDY THE EFFECTS OF HISTONE MUTATIONS AND POST-TRANSLATIONAL MODIFICATIONS ON NUCLEOSOME STRUCTURE VIA ATOMIC FORCE MICROSCOPY", Campus Access Dissertation, Michigan Technological University, 2017.