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The yeast Saccharomyces cerevisiae is a prevalent system for studying gene regulation because of the ease of experimental methods and the simplicity of its gene structure. Here, I describe my work that aims to identify the sequences and factors responsible for demarcating genes within the genome sequence. With comparative genomics and RNA-Seq, we are quite adept at identifying gene structure. However, the cell does not have access to this kind of information. Instead, it uses the specificities of DNA- and RNA-binding proteins to read and interpret the sequence of the genome; it is this process that I have studied in my thesis. In the first chapter, I describe my work collecting yeast transcription factor specificities. I evaluated these specificities using available confirmatory data to determine which one best represents the transcription factor; this gave me a high-confidence description of what DNA sequences yeast transcription factors recognize. Next, I look for over- and under-represented DNA words within and surrounding gene structures and attempt to explain these in terms of the specificities of known factors or other known biological phenomena. I found that the sequences in the 5' and 3' gene ends are very similar and can often be explained by similar phenomena. I also provide evidence that several factors may be involved in regulating transcription in non-canonical ways. In the final chapter, I describe my efforts to build a model that uses my collection of transcription factor specificities as well as DNA structural features to identify gene structure as we think the cell would. This model is comprised of two classifiers that identify mRNA initiation and termination sites, and these are used to provide evidence to a hidden Markov model that predicts gene structure. I test that the predicted determinants of promoter structure are sufficient to initiate transcription, and that the transcription arising from randomly-generated DNA is correctly predicted. Overall, my work demonstrates that the sequence elements demarcating yeast genes are relatively simple in nature, which has implications for how transcription is regulated and how genes evolve.

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The ascomycete Fusarium verticillioides (Sacc.) Nirenberg (teleomorph: Gibberella moniliformis Wineland) causes stalk and ear rots on maize worldwide. In addition to the economic losses due to reduced yield, the fungus produces fumonisins on infected corn. One of the unanswered questions in mycotoxin research is how fungi perceive and respond to various extracellular stimuli and produce mycotoxins. To date, extensive research has been performed on important signaling pathways that regulate mycotoxin biosynthesis, but little is known about the downstream target genes, notably transcription factors (TFs). While the roles of TFs have shown to be critical in eukaryotic transcription regulation, only a few have been characterized in F. verticillioides. TFs with zinc fingers have been reported in all living organisms, and in fungal species, members of the Cys2-Hys2 (C2H2) zinc finger TF family are predicted to be involved in cell differentiation, carbon utilization, and development. Using the available genomic resources, I constructed a library of C2H2 TF deletion mutants, and identified SDA1, FvFLBC and CHT1 genes with a potential role in carbon utilization, development and fumonisin B1 (FB1) biosynthesis. The Δsda1 strain showed complete growth inhibition when using sorbitol as the sole carbon source and produced higher levels of FB1 when grown on corn kernels. In addition, the Δsda1 strain produced less number of conidia compared to the wild-type progenitor. Through gene complementation, I also demonstrated that F. verticillioides SDA1 and Trichoderma reesei ACE1 are functionally conserved. FvFLBC acts as a regulator of asexual development but not FB1 biosynthesis. I also discovered that the FvFlbC N-terminus is critical for conidia production. CHT1 is associated with asexual development, fumonisin biosynthesis and pigmentation. Characterization of key signal transduction pathways, and more importantly the function of SDA1, FvFLBC and CHT1, should facilitate the elucidation of the mechanisms and regulations of growth, development, and secondary metabolism in F. verticillioides. The outcome of this study may help us determine how to minimize F. verticillioides contamination of crops and the resulting mycotoxins, providing safer and higher value corn in the US and worldwide.