Tag: Eric Selker

Regulation of the Kynurenine Pathway in Neurospora crassa

Presenter: Haley Speed – Biology

Faculty Mentor(s): Eric Selker

Session: (In-Person) Poster Presentation

The kynurenine pathway is a metabolic pathway that degrades tryptophan into NAD+ and several other essential biomolecules. In humans, the kynurenine pathway is closely tied to healthy and disease states, and current research indicates that altering the activity of the pathway could have therapeutic benefits. This ancient pathway is also conserved across all eukaryotes. Previous work from the Selker lab using the filamentous fungus Neurospora crassa as a model organism suggested that the regulation of this pathway is intertwined with general chromatin control processes, including H3K36me and chromatin remodelers. My research aimed to identify novel regulatory factors affecting this pathway. I used a phenotypic screen of the Neurospora Functional Genomics Project strains. In addition, RT-qPCR analyses of RNA for kynurenine pathway enzymes helped reveal which strains were abnormally inducing the pathway. In total, five genes of interest have been identified as potential regulators of the kynurenine pathway, including one factor also tied to MAPK pathway induction. Further research is needed to characterize these genes to understand how they may function to regulate the kynurenine pathway, or if their regulatory function is tied to chromatin control.

Identification and Study of Genes Involved in DNA Methylation in Fungal Species Neurospora crassa

Presenter: Ariel Morrison, Biology

Panel: Genes, Cells & Jaws

Mentor: Eric Selker, Institute of Molecular Biology

AM Session Panels

Time: 11:00am – 12:00pm

Location: Century A

DNA methylation is an essential feature for proper development in many mammalian and plant species. However it is absent or nonessential in some organisms, including the fungal species Neurospora crassa. I am studying the effects of DNA methylation on Neurospora, with hopes to isolate all known genes involved in the process. Neurospora is sensitive to the drugs Basta and Hygromycin, due to two inactive genes, bar and hph. However, a loss of methylation activates these genes, creating a resistancy to these drugs. By mutagenising samples of Neurospora, I can selectively isolate dim (defective in methylation), mutants by their response to the drugs. Their genome can then be studied and the information gained can be applied to the eukaryote genome.

Saturating the Neurospora Genome for Mutants Defective in Methylation

Presenter: Calvin Summers

Mentor: Eric Selker

Poster: 31

Major: Biology

Cytosine methylation, a fundamental form of epigenetic regulation, is found in many eukaryotes and plays a significant role in cancer and other diseases. Using the genetically tractable model organism Neurospora crassa, the Selker laboratory has identified genes that when mutated, cause the strains to be defective in methylation (dim). The process of DNA methylation in Neurospora has been shown to be dependent on DCDC, a five member complex that directs the histone methyltransferase DIM-5 to trimethylate Lysine 9 on histone H3 (H3K9me3). This mark is recognized by HP1, which directs DIM-2 to methylate DNA. Another silencing complex, HCHC, employs HDA-1, CDP-2, HP1, and CHAP to deacetylate lysines on the histone. While we know a good deal about DNA methylation, it is still unclear whether we have identified all genes involved in the process. Thus this research focuses on our search for dim mutants, and asks whether our current model for DNA methylation is saturated. Using a selection for reactivation of resistance genes silenced by DNA methylation we aimed to answer this question. Interestingly, we predominantly identified known dim genes, including dim-5, dim-7, dim-8, dim-9, chap, cdp-2, and hda-1, suggesting our model may be saturated. We localized mutations in these known dim genes by DNA sequencing. The dim mutant collection generated should be a useful resource for further investigation into the roles of these genes and their protein products in DNA methylation. Understanding epigenetic pathways in Neurospora is an auspicious first step in elucidating mechanisms of complex genetic regulation in our own cells.

The Role of Post-Translational Modifications in Regulating Distinct Heterochromatin Protein One Functions

Presenter: Collin Hickmann

Faculty Mentor: Tereza Ormsby, Eric Selker

Presentation Type: Oral

Primary Research Area: Science

Major: Biochemistry

Funding Source: Presidential Undergraduate Research Scholars Program (PURS), Undergraduate Research Opportunity Program (UROP), $5,000; Oregon Undergraduate Researchers in SPUR (OURS), National Institute of Child Health and Human Development (NICHD), $5,000

Heterochromatin is a minimally transcribed, densely bundled complex of DNA and associated factors comprising large regions of the eukaryotic genome. It is essential for chromosome stability, genome integrity, gene regulation, and the silencing of transposons. The filamentous fungus Neurospora crassa is often employed as a model organism to study the epigenetic regulation of heterochromatin. In Neurospora, the conserved scaffolding protein heterochromatin protein 1 (HP1) binds H3 histones marked by lysine nine trimethylation (H3K9me3) and recruits other proteins to form at least three distinct complexes. HP1 recruits the DIM-2 DNA methyltransferase, which catalyzes DNA methylation. HP1 is also an essential component of both the HCHC histone deacetylation complex, which facilitates centromeric silencing, and the DMM complex, which limits aberrant heterochromatin spreading. However, it’s unclear how these disparate functions are coordinated. We hypothesized that they are modulated by post-translational modifications (PTMs) of HP1. Previously, we used mass spectrometry to identify HP1 sites harboring methylation, acetylation, formylation, and phosphorylation. I used amino acid substitutions at a subset of these sites to prevent individual PTMs in vivo. Substitutions at multiple sites were found to cause a substantial decrease in centromeric silencing independent of DNA methylation. These results suggest that the recruitment of HCHC to incipient heterochromatin may be selectively mediated by specific PTMs.

The BAH Domains of the DIM-2 DNA methyltransferase Are Required for DIM-2 Localization and Normal DNA methylation

Presenter: Sabrina Abdulla

Faculty Mentor: Eric Selker, Vincent Bicocca

Presentation Type: Poster 42

Primary Research Area: Science

Major: Biology

Funding Source: OURS (Oregon Undergraduate Researchers in SPUR), NICDH, $4500

The regulation of epigenetic marks, including histone modifications and DNA methylation, is critical for normal development. Defects in the processes that regulate epigenetic marks can lead to the development of diseases, including cancer. To better understand these processes, we utilize the model organism Neurospora crassa, a filamentous fungus that possesses many epigllmerenetic marks that are common to higher order eukaryotes, including humans. Using this model, we have investigated the functional requirements of the DNA methyltransferase DIM-2, which is responsible for all DNA methylation in Neurospora. Using site-directed mutagenesis to systematically disrupt regions of DIM-2, and Southern blotting to assay DNA methylation, we discovered that the bromo-adjacent homology (BAH) domains of DIM-2 are required for normal DNA methylation activity. Based upon these observations, we hypothesized that the BAH domains are essential for making specific interactions with histone residues, and that these interactions are necessary for DIM-2 methyltransferase activity. To test this hypothesis, we utilized a DNA adenine methyltransferase (DAM) construct that allowed us to test the ability of the protein to localize to regions that are normally methylated based on the presence of adenine methylation. These experiments revealed that disruption of the BAH domains is sufficient to eliminate DIM-2 chromatin localization. Thus, the BAH domains prove to be essential for DNA methylation due to their role in DIM-2 localization.

The Contributions of Polycomb Repressive Complex 2 and H3K27me3 in Gene Repression

Presenter(s): Anna Kulawiec—Biology

Faculty Mentor(s): Eric Selker, William Storck

Session: Prerecorded Poster Presentation

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 .

Characterization of the Cohesin Complex in the Model Organism Neurospora crassa

Presenter(s): Chaney Hart—Biology

Faculty Mentor(s): Eric Selker, Ken-ichi Noma

Session: Prerecorded Poster Presentation

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 .