Browsing by Author "Selker, Eric"

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    A New Mechanism for Genomic Nucleosome Positioning by Native ISWI and Engineered CHD Chromatin Remodeling Proteins is Driven by Sequence-Specific DNA Binding.
    (University of Oregon, 2021-09-13) Donovan, Drake; Selker, Eric
    Encoded within DNA are all the instructions that a cell needs to survive, and these instructions must be regulated such that cells take the appropriate actions in response to environmental and developmental cues. One way that the DNA is regulated is that a portion of it is wrapped around proteins to form structures known as nucleosomes, which makes the wrapped DNA inaccessible to the machinery that reads the genetic code. Thus, the location of these nucleosome structures on the DNA plays a large role in which genes are active at what times. The location of nucleosomes on the DNA is partially regulated by a family of enzymes called chromatin remodeling proteins. While the function of chromatin remodeling proteins to position nucleosomes is well understood, the mechanisms by which they do so have not been thoroughly tested and are thought to be non-specific. It is unknown, for example, how chromatin remodeling proteins know when and where to position a nucleosome on a given DNA sequence. Reconciling how these supposedly non-specific molecular machines result in highly specific chromatin structures is the focus of this dissertation. The first part of this work challenges the idea that a chromatin remodeling protein in yeast, Isw2, acts as a non-specific nucleosome spacer. Instead, the use of biochemical and genomic techniques shows us that Isw2 is directly targeted to particular nucleosomes in a sequence-specific manner through interactions with other proteins. Further, it suggests that this mechanism may potentially exist in humans. In the second part of this work, we use the idea of sequence-specific targeting seen in Isw2 to engineer another chromatin remodeling protein, Chd1. This allows us to directly target particular nucleosomes in a synthetic manner both in test tubes and in living cells. Excitingly, we show that the use of these engineered proteins allows us to control DNA access and downstream biological outputs in yeast. Overall, this dissertation contributes a completely new understanding of how chromatin remodeling proteins can be directed to act on specific target nucleosomes both natively in eukaryotes and synthetically by researchers. This dissertation includes previously published co-authored material.
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    Characterization of LSD Complex Function, Histone Exchange, and Regulation of a Tryptophan Catabolism Gene Pair in Neurospora crassa
    (University of Oregon, 2020-09-24) Storck, William; Selker, Eric
    DISSERTATION ABSTRACT William K. Storck Doctor of Philosophy Department of Chemistry and Biochemistry March 2020 Title: Characterization of LSD Complex Function, Histone Exchange, and Regulation of a Tryptophan Catabolism Gene Pair in Neurospora crassa Gene expression is regulated by a plethora of factors associated with chromatin, such as histone proteins. DNA can be methylated and histones can be marked with various chemical tags, which can influence expression of underlying DNA. Chromatin is organized into distinct domains: transcriptionally-active euchromatin and transcriptionally-silent heterochromatin. My dissertation involved investigating different aspects of chromatin regulation in the filamentous fungus Neurospora crassa. I examined heterochromatin spreading in Neurospora mutants defective in lysine-specific demethylase 1 (LSD1). Loss of either LSD1 or its associated complex members, PHF1 or BDP-1, results in variable spreading of trimethylation of H3K9 (H3K9me3) and DNA methylation, and this spreading is dependent on DNA methylation, which is typically not involved in H3K9me3 establishment, and on the catalytic activity of the histone deacetylase complex, HCHC. Though there are gene expression changes present in ∆lsd1 strains, these changes do not appear to be driven by spreading H3K9me3 and DNA methylation. Though their relative positioning within chromatin appears to be regulated, histones are not static structures embedded within DNA, but are subject to constant exchange. I characterized a light-inducible histone turnover reporter strain and used it to build a simple protocol for profiling DNA replication-independent histone turnover in Neurospora. I investigated how histone turnover correlates with gene expression, and observed similarities to other models in turnover profiles over genes. I also examined turnover at heterochromatin domains in heterochromatin mutants, which revealed that loss of H3K9me3 or its binder, HP1, or histone deacetylation by HCHC results in turnover increases. Lastly, I investigated the regulation of a pair of genes involved in tryptophan catabolism, kyn-1 and iad-1. I demonstrate that these genes are induced through the exposure of extracellular tryptophan. Though this locus is enriched for the conserved repressive mark, H3K27me3, loss of which does not appear to significantly affect the activity of this locus. Rather, I show that this locus is repressed by the H3K36 methyltransferase, ASH1, and chromatin remodelers, CRF4-1 and CRF6-1. Another H3K36 methyltransferase, SET-2, is required to overcome this repression. This dissertation contains previously unpublished coauthored material.
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    Characterization of the Cohesin Complex in Neurospora crassa
    (University of Oregon, 2020) Hart, Chaney; Bicocca, Vincent; Selker, Eric; Selker, Eric
    The cohesin complex is a conserved protein complex that plays an important role in multiple aspects of genomic function. Of particular interest is cohesin’s demonstrated role in influencing 3D genomic structure. While previous work has identified basic elements of 3D genomic structure in the model organism Neurospora crassa, the undermining factors that contribute to these structures are unclear. We hypothesize that the cohesin complex may interact with heterochromatin to shape genomic architecture in N. crassa. Features of the cohesin complex such as where it is recruited, its contributions to gene regulation and its presence at topologically associated domains are widely divergent amongst model organisms in which it has been studied, making it important to establish basic features of this complex in N. crassa. In this study I took the first steps towards characterizing the cohesin complex in N. crassa by showing that cohesin shares features with well characterized yeast species such as enrichment over 3’ untranslated regions and intergenic regions of convergent genes across the genome. I also developed a strain of N. crassa that has a mutation in cohesin component RAD21 which leads to temperature-sensitive lethality. My findings and the strains I generated will be useful for further characterization of the cohesin complex in N. crassa and for exploration of the role this complex plays in genomic structure and function.
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    Chromatin Regulation: How Nucleosomes Find their Positions and the Role of Chromatin Binding Proteins in Cellular Quiescence
    (University of Oregon, 2023-03-24) Bailey, Thomas; Selker, Eric
    Chromatin is made up of an organism’s DNA and DNA binding proteins inside of the nucleus. Nucleosomes are the fundamental unit of chromatin, consisting of a 147 base pair DNA sequence and a protein octamer comprised of 8 histone proteins, two copies of histone H2A, H2B, H3, and H4. Nucleosomes are used to regulate gene expression in both the positioning of the nucleosome along DNA, or the post-translational histone modifications. In the first chapter, we will demonstrate that the chromatin binding protein, Tup1 is essential for nucleosome positioning and H3K23 deacetylation, which is required for entry into cellular quiescence. In the next chapter, we will address how the chromatin remodeler complex, Isw2, precisely positions nucleosomes through direct interactions with transcription factors. In the next chapter, we present an optimized protocol for measuring nucleosome positioning. Finally, we will end on some preliminary data and discussion on the next steps in researching Tup1 and Isw2.This dissertation contains previously published co-authored material.
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    Control and Function of Histone H3 Lysine 27 Methylation in Neurospora crassa
    (University of Oregon, 2020-02-27) McNaught, Kevin; Selker, Eric
    In higher eukaryotes, proper development and maintenance of cellular identity requires the activity of Polycomb repressive complexes (PRCs). PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), which serves as a repressive chromatin mark. PRC2 is conserved in the filamentous fungus, Neurospora crassa, and H3K27 methylation covers around 7% of the genome, repressing scores of genes. My research focused on two basic questions: 1. What controls the distribution of H3K27 methylation? and 2. How does H3K27 methylation function as a repressive mark? As one approach to identify factors that influence the deposition of H3K27 methylation, I screened gene knock-outs of nst (neurospora sir two) homologues for defects in H3K27 methylation. I discovered that loss of NST-3, a putative H3K56 deacetylase, results in accumulation of H3K27 methylation at centromeres, and that this is suppressed by strains lacking rtt109 or bearing histones with the H3K56R substitution. In collaboration with Kirsty Jamieson, we investigated what role chromosome position had on the deposition of H3K27 methylation by examining chromosomal rearrangement strains. We identified both position-dependent and position-independent H3K27 methylation. We further found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive H3K27 methylation, and that this is effect is mediated by telomere repeats, (TTAGGG)n. As a part of a forward-genetics selection for factors necessary for Polycomb silencing, spearheaded by Elizabeth Wiles, I identified two alleles of a gene, NCU04278, that is required for the telomere-dependent H3K27 methylation uncovered in our previous work. Immunoprecipitation followed by mass spectrometry analyses of NCU04278 and known PRC2 members established that NCU04278 is a PRC2 accessory subunit and was therefore designated PAS. The aforementioned forward genetic selection identified another gene, epr-1 (effector of polycomb repression 1; NCU07505). EPR-1 associates with H3K27 methylation in vivo and in vitro, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR-1 was a component of a primitive Polycomb repression pathway. This dissertation includes both previously published and unpublished co-authored material.
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    The contributions of Polycomb Repressive Complex 2 and H3K27me3 on gene repression in Neurospora crassa
    (University of Oregon, 2020) Kulawiec, Anna; Storck, Will; Selker, Eric; Selker, Eric
    Though DNA contains our genes, the expression of genes varies during development and across different cellular conditions. Gene expression can be regulated by the post-translational protein modification of chromatin, such as the trimethylation of lysine 27 of histone 3 (H3K27me3). This mark, catalytically deposited by the protein complex Polycomb Repressive Complex 2 (PRC2), represses associated genes. Such repression is crucial for establishing gene expression patterns for proper development, and aberrant activity of PRC2 can cause disease, such as cancer. Here I present Neurospora crassa as a model organism for studying the repressive effects of PRC2, independent of its catalytic mark, H3K27me3. I generated a catalytically inactive SET-7, the catalytic component of PRC2 in N. crassa, demonstrating that elimination of H3K27me3 is sufficient to depress genes it normally marks despite the physical presence of PRC2. I further show that, in contrast to SET-7 knockout, catalytic inactivation of SET-7 does not alter the stability of PRC2. Moreover, catalytic inactivation of SET-7 enriches a higher molecular weight form of the core PRC2 member SUZ12. Overall, these results indicate that the physical form of PRC2 in itself does not act repressively and suggests that studies focusing on its repressive effects should consider that methods of H3K27me3 elimination, either knockout or catalytic inactivation, differentially affect PRC2 complex stability. This work provides valuable insights into the appropriate methodologies for studying developmental processes and disease related to PRC2 and H3K27me3.
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    Using Fluorescence Assays to Explore the Regulation of the Kynurenine Pathway in Neurospora crassa
    (University of Oregon, 2021) Speed, Haley; Selker, Eric
    In Neurospora crassa (a filamentous fungus), there are several enzymes that cause the breakdown of tryptophan into fluorescent anthranilic acid, several of whose genes have the chromatin markers that our lab studies, specifically methylation of lysine 27 of histone H3. If we give a N. crassa culture tryptophan and it fluoresces, this indicates that the genes are “turned on” normally; if it doesn't, they could be abnormally “turned off.” Since July of 2020, I have been using this convenient system to gain insights into the mechanism controlling tryptophan degradation, which may illuminate general chromatin control processes. Utilizing the FGSC knockout library, I have screened 13,000 mutants using fluorescence assays, leading me to identify eleven that fluoresce atypically. I used 150 μL liquid cultures for my primary screening and 1 mL liquid cultures for my secondary screening of the collection. I have also confirmed the knockouts via PCR and am currently complementing the genes of interest to determine if the knockouts are causing the phenotype. The next steps of this research is to find out why my mutants are behaving abnormally, and to determine if this is because of abnormalities in their chromatin markers. Studying chromatin markers is essential to understanding the eukaryotic genome at large because of their ubiquity throughout most eukaryotic organisms.