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Browsing by Author "Stargell, Laurie A., advisor"

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    In vivo regulation of chromatin dynamics by Saccharomyces cerevisiae histone chaperone Nap1
    (Colorado State University. Libraries, 2011) Barker, Kristi Leigh, author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Wilusz, Carol J., committee member
    Eukaryotic cells must organize massive amounts of DNA into the nucleus. In order to accomplish this, the DNA must be compacted into a highly ordered structure known as chromatin. The basic, repeating unit of chromatin is the nucleosome, which consists of two copies of each histone (H2A, H2B, H3 and H4) and organizes 147 base pairs of DNA. Due to its highly compact nature, nucleosomes must be removed during gene expression in order for the transcription machinery to access the DNA. Shuttling of nucleosomes on and off DNA is mediated by a group of proteins known as histone chaperones. Importantly, histone chaperones interact with another family of chromatin remodeling complexes known as histone acetyltransferases (HATs). Acetylation of histones is correlated with the active transcription of genes. The work presented here explores the dynamics and kinetics of histone H3 occupancy and acetylation of histone H3-K9 and H3-K14 in a wild-type strain and strains deleted for three known histone chaperones (Nap1, Vps75 and Asf1) of the yeast Saccharomyces cerevisiae at the well characterized galactose inducible genes. This data offers insight into the epigenetic regulation of chromatin as well as possible mechanisms for the histone chaperones surveyed.
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    Spn1, a highly conserved and essential node of RNA polymerase II dependent functions
    (Colorado State University. Libraries, 2011) Almeida, Adam Raymond, author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Woody, Robert, committee member; Suchman, Erica, committee member
    A multitude of proteins are responsible for regulating the activity of RNA Polymerase II (Pol II) in the nucleus of a eukaryotic cell. Two types of themes are used by these proteins to control transcription: recruitment-regulation and postrecruitment-regulation. The main difference between the two is the rate-limiting step for producing transcript. This rate-limiting step for the first mechanism is the recruitment of Pol II to the promoter. For the second mechanism, Pol II constitutively occupies the promoter, is "poised", and an unknown rate-limiting postrecruitment step prevents transcription from commencing. The highly conserved and essential transcription factor Spn1 was identified as a protein that functions postrecruitment of Pol II and has been characterized for having a direct role at regulating the poised CYC1 gene in Saccharyomyces cerevisiae. This activity has been determined from mutations made within the most conserved portion of Spn1 made up of a highly folded central domain. Little is known about the functions of the N-and C-terminal regions flanking this central domain, which is the focus of the work done here. Genetic characterization indicates that these regions have physiologically relevant and important functions within the cell outside of optimum growth conditions, but do not involve significant regulation of the CYC1 gene. A broader approach of experimentation is likely required to understand all of the Spn1 protein's functions regarding transcription. This led to the observation that Spn1 is able to bind to nucleosomes in vitro and that this interaction is dependent on the N-and C-terminal regions of the protein. The possibility that Spn1 could affect nucleosome dynamics in the cell is consistent with the physical and genetic interactions observed between Spn1 and the Spt6 and Swi/Snf histone chaperone and chromatin remodeling complexes. This result will provide several new avenues for future Spn1 research. A genomic ChIP-chip experiment performed by two independent groups revealed that Spn1 is recruited to a majority of the genes in the yeast genome. Evidence indicates that there are multiple, evolutionarily conserved pathways within the cell that are responsible for determining the rate at which an organism will age that include: ribosome biogenesis, protein translation, mitochondrial activity and function, heterochromatic stability, maintenance of the genome, and apoptosis. The possibility that Spn1 regulates the genes involved in these pathways is highly suggestive that this protein could be an aging factor within the cell. Chronological aging assays revealed that the removal of the N-and C-terminal regions of the Spn1 protein dramatically increase the lifespan of the BY4741 strain of yeast. These results further verify the physiological importance of this protein and the need for further Spn1 research.
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    Spn1, Spt4, Spt5, and Spt6 preserve chromatin structure over promoters and open reading frames
    (Colorado State University. Libraries, 2024) Tonsager, Andrew Jordan, author; Stargell, Laurie A., advisor; Hansen, Jeffrey C., committee member; Santangelo, Thomas, committee member; Argueso, Juan Lucas, committee member
    The eukaryotic chromatin landscape presents formidable nucleosomal barriers for processes that require access to DNA, such as transcription. These barriers are overcome through the action of many factors, including histone chaperones Spn1, Spt5, Spt6, and FACT and transcription elongation factor Spt4. However, it is poorly understood how each contributes to this process. To ascertain the role that these factors play on preserving chromatin structure over the genome, this thesis has utilized micrococcal nuclease digestion followed by sequencing (MNase-seq) to analyze chromatin protections in the yeast genome in cells expressing numerous mutant alleles of these factors. Extensive characterization of MNase-protected fragments in a wide range of sizes established that the essential histone chaperone Spn1 preserves both nucleosomal and subnucleosomal structures over both promoters and open reading frames across the genome. Additional analyses from existing MNase-seq datasets demonstrated the extent to which Spn1 and other RNAPII-associated factors maintain nucleosome features over genes of varied characteristics. The study of factors described in this thesis is performed in living cells, which have been genetically modified to express mutant alleles of chromatin factors. This thesis also describes a course-based undergraduate research experience (CURE) developed to introduce upper-level biochemistry students to techniques in yeast genome engineering in an authentic research setting.
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    TATA binding protein dynamics within the cellular chromatin landscape
    (Colorado State University. Libraries, 2013) Yearling, Marie N., author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Nyborg, Jennifer K., committee member; Yao, Tingting, committee member; Slayden, Richard A., committee member
    RNA polymerase II (RNAPII) is a twelve subunit enzyme that catalyzes messenger (mRNA) in eukaryotic organisms. A number of essential transcription factors associate with RNAPII to form the pre-initiation complex (PIC) at gene promoter regions. TATA binding protein (TBP) is one member of the transcription machinery indispensable for transcription. At some genes, the formation of the PIC correlates strongly with the transcription output (Ptashne, 2005). These genes have a low occupancy of TBP and other PIC components prior to activation. Upon activation, these factors assemble onto the promoter and transcriptional output increases. Genes that become active upon PIC formation are termed recruitment regulated because their transcription is regulated at the level of recruitment of the PIC to the promoter. While recruitment of the PIC is required for transcription, in many cases promoter-occupancy is not correlated with transcription output. Post-recruitment gene regulation has been conserved across evolution from prokaryotes to humans (Choy et al., 1997; Guenther et al., 2007). At these genes, TBP and RNAPII and other transcription-related factors occupy the promoter region regardless of whether transcription is occurring. Upon gene activation, the occupancy increases only slightly when compared to the increase in transcript level. These genes are described as being poised. At poised genes, these transcription proteins constitutively occupy the promoter region, but it is unknown if the promoter interaction is stable or dynamic. One principal objective of my work was to investigate TBP-promoter dynamics at the poised CYC1 gene in yeast. Due to the genetic and biochemical amenability of the yeast system, studies of the transition of poised CYC1 gene to the active form have provided key insights into the sophisticated molecular requirements involved in this post-recruitment process. To describe the dynamics of the transcription complex bound at the CYC1 promoter I developed a TBP exchange assay. The results suggest that the TBP within the RNAPII transcription complex exists in a relatively stable configuration at the poised gene prior to activation. Upon induction, TBP-promoter dynamics increased at the CYC1 gene promoter. Rapid exchange during activated transcription was also observed at other genes, including at recruitment regulated gene promoters. Overall, we found rapid TBP-promoter exchange to be associated with active transcription. From my findings I propose a model where frequently clearing the promoter offers a functional advantage to support activated transcription.