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Browsing by Author "Hansen, Jeffrey C., advisor"

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    Dynamics of H3 and H2B octamer variants
    (Colorado State University. Libraries, 2020) McVay, Abigail Lea, author; Hansen, Jeffrey C., advisor; Quackenbush, Sandra, committee member; DeLuca, Jennifer, committee member
    Introductions of alterations within a nucleosome can lead to drastic changes to the accessibility and stability of genetic material during various phases of the cell cycle. To understand these changes, three octamer mutants were recombined, reconstituted with a high affinity tandem repeat sequence, and tested using a combination of in vitro experimental methods. All Tailless, H2BTL and an H2BTL & H3TL nucleosomal array mutants were sedimented, digested, re-associated, and visualized in order to determine the role of histone tails and their influence on chromatin condensate structure. The All Tailless octamer mutant expressed an inability to form complex molecular structures, suggesting that histone tails are necessary to further the process of chromatin condensate association and subsequent folding. The H2BTL mutant expressed high levels of concentration and an increased level of association compared to the other mutants. This lead to the assumption that the exclusion of only one histone tail lead to a greater ability to associate compared to mutants lacking two or more tails. The H2BTL & H3TL mutant had a possibility of two distinct populations within solution, suggesting that the exclusion of at least two tails led to a loosely compacted and easily accessible chromatin condensate structure. In summary, this data suggests that as histone tails are excluded from octamer mutants, the chromatin condensates expresses a decreased ability to form higher degrees of compaction. Exclusion of histone tails from octamer mutants also resulted in a more accessible 601-12 tandem repeat sequence susceptible to various changes.
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    Exploring induced secondary structure and unmethylated DNA binding domains of methyl CpG binding protein 2 (MeCP2)
    (Colorado State University. Libraries, 2011) Hite, Kristopher Charles, author; Hansen, Jeffrey C., advisor; Woody, Robert W., committee member; Ross, Eric D., committee member; Mykles, Donald L., committee member
    Our understanding of Methyl CpG binding protein 2 (MeCP2) structure and function has changed and expanded considerably over the last two decades. Mutations along the entirety of the human MeCP2 gene product lead to a disease state - Rett syndrome. The clinical connection of this protein has continued to drive intense research into the nature of MeCP2 structure and function. There is now considerable and corroborated evidence that proves MeCP2 is an archetypical intrinsically disordered protein acting as a global ATP independent chromatin architectural protein. The ubiquity of MeCP2 in vertebrate neuronal nuclei has only recently been realized and has focused my investigations. Results from my work demonstrate a clear relationship between predicted α-molecular recognition features and inducible α- helical structure. From these data I suggest that inducible α-helices and maintained intrinsic disorder participate in binding the pool the twenty reported MeCP2 binding partners. In addition to structural studies I have identified two non-specific unmethylated DNA binding domains unreported in the literature at the onset of my work. I have also shown that MeCP2 acquires some secondary structural stability when bound to DNA and relatively little additional stability when bound to methylated DNA. The results presented here improve the fine resolution functional understanding of MeCP2 by observing isolated fragments of MeCP2 using both structural and functional methods. This approach is significant in and of itself as, like the large disordered subset of all eukaryotic proteins, the full-length MeCP2 molecule has proven impossible to crystallize thus far. Therefore narrowing the amino acid residues responsible for DNA binding activity or any other measurable functionality in a solution state is valuable.
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    Nucleosomal array condensation: new insights into an old "tail"
    (Colorado State University. Libraries, 2011) Sorensen, Troy C., author; Hansen, Jeffrey C., advisor; Stargell, Laurie, committee member; Luger, Karolin, committee member; Bailey, Susan, committee member
    The DNA present within the nucleus of each human somatic cell, when extended end to end, would span a distance of about one meter. The first level of compaction critical to fitting the entire genome into the nucleus is the nucleosome, consisting of 147 base pairs of DNA wrapped 1.7 times around an octameric structure composed of the four core histones H2A, H2B, H3 and H4. Nucleosomes separated by up to 80 base pairs of linker DNA called nucleosomal arrays compact the DNA further through short range intra-array and long range inter-array contacts that generate different levels of higher order condensed structures. This dissertation investigates the involvement of the core histone "tail" domains as well as the influence of the H3 centromeric variant CENP-A in nucleosomal array condensation events. In vitro, 12-mer nucleosomal arrays condense intra- and inter-molecularly through nucleosome-nucleosome interactions driven primarily by the core histone tail. This dissertation details the contributions and the molecular determinants of the histone tail domains to the condensation processes. Importantly, we found that the H3 and H4 tail domains were the largest contributors to array condensation. The mode of action used by the H4 tail domain in intra- and intermolecular condensation centered on the following determinants: 1) position of the H4 tail, 2) amino acid composition, 3) positive charge density and 4) tail domain length. Importantly, the primary sequence of the H4 tail was found to not be an important molecular determinant. To date no study has been performed to determine short-range compaction between "bulk" H3 containing and H3 centromeric specific variant, CENP-A chromatin. 12-mer nucleosomal arrays containing either H3 or CENP-A histones were reconstituted and tested for their ability to fold intra-molecularly. Major finding include that CENP-A containing nucleosomal arrays assemble in the same stepwise manner as conical arrays and were always more compact than H3 containing arrays at every salt concentration tested. The increased compaction was found to be in part due to a lysine to arginine mutation at position 49 of CENP-A.
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    The influence of histone orthologues, histone variants and post-translational modifications on the structure and function of chromatin
    (Colorado State University. Libraries, 2008) Resch, Michael George, author; Hansen, Jeffrey C., advisor; Luger, Karolin, advisor
    Two meters of DNA is packaged into the nucleus of each eukaryotic cell in the form of chromatin. DNA wraps around a protein histone octamer to form a nucleosome, the fundamental repeating unit of chromatin. The highly basic histone octamer contains two copies each of H2A, H2B, H3 and H4 to form the protein core of the nucleosome. There is a dynamic interplay of accessibility which compacts DNA yet allows access for fundamental cellular processes like transcription and DNA replication. This thesis investigates how histone variants and post-translational modifications contribute to the level of chromatin compaction. I demonstrated that defined nucleosomal arrays made with histones from multiple species oligomerize at different concentrations of MgCl2. A comparison of endogenous and recombinant Drosophila melanogaster histone octamers showed that this is unlikely due to posttranslational histone modifications, but likely a result of subtle changes in the sequences constituting the histone tails and structured surface of the histone octamer. I investigated the effect of incorporation of the centromere specific H3 histone variant centromere protein - A (CENP-A) into nucleosomes and nucleosomal arrays. Despite the fact that CENP-A shares only 60% sequence homology within the structured domain of major-type H3 (15% in the N-terminal domain), CENP-A (together with the other three core histones) forms nucleosomes and condensed nucleosomal arrays comparable to major-type H3. Post-translational modifications (PTM) contribute to the regulation of chromatin structure. I have analyzed the effect of H3 lysine 56 acetylation on nucleosome structure and chromatin condensation. This modification was previously thought to disrupt nucleosome structure. I developed methods to enzymatically acetylate large amounts of H3 specifically at Lys 56, and demonstrated that histone octamers containing H3-K56Ac form canonical nucleosomes. However, nucleosomal array condensation is compromised by this particular PTM. Together, these studies suggest that even subtle variations in histone sequence or post-translational modifications result in differences in chromatin higher order structure.