Protein dynamics during cellular responses to extracellular stimuli has been widely studied to understand how cells respond to change. Here, we employ two distinct techniques to look at two separate aspects of cellular response in Saccharomyces cerevisiae. First, we use a transcriptomics and proteomics approach to examine the pheromone response of yeast, using RNA-sequencing and mass spectrometry, respectively. We find that in general, mRNA abundance is not sufficient as a predictor of protein levels during dynamic responses such as pheromone response, but by using a mass-action kinetics model, we can reconcile these discrepancies. Performing this experiment in both a lab strain and the clinical isolate, YJM145, allows us to further identify possible genes where post-transcriptional regulation is affected by polymorphisms, allowing possible mechanisms of post-transcriptional regulation to be further explored. Second, we used an extreme bulk segregant analysis approach to map alleles in wild yeast strains, L-1374 and YPS606, which cause a difference in molecular response during the yeast’s response to hyper-osmotic shock by tracking the protein, STL1p. We find that many alleles have transient effects on STL1p levels during the osmotic stress response, but only a few have an impact on the cellular state after adaptation to the new environment. In addition, we found that alleles in the wild strain affect STL1p levels in both directions, suggesting possible compensatory mutations. Most genes were not found to be in the known osmotic stress response pathway suggesting regulation mechanisms of STL1p that were previously unknown.

It is difficult to research the evolution of cell differentiation in nature because of the unavailability of a suitable model organism and the advanced time since the divergence of single and multicellular organisms. Instead of looking at a natural model we decided to explore this issue with a computer model. We based our model on the one proposed by Andreas Wagner in 1994 to look at the evolution of gene networks. We modified the model to account for selection goals that promoted cell differentiation. Because of the prevalence of modular components in more advanced biological systems we expected modularity to form in the evolved gene

networks. Therefore, we tested the gene networks for modularity using the K-Means clustering test. The model was completed after many editions that swapped between concepts introduced in previous scientists’ adaptations of the Wagner model. Increased modularity was observed in the evolved populations of the model over the unevolved populations, 0.149 over 0.136 p < 2.2e-16. However, this change was so minor there may be no biological significance. Additionally, whether the model evolved towards one fitness goal then the other or both at the same time was tested and found to be both. Finally, whether connectivity increased when selecting for differentiation was tested and the results are inconclusive at best.

A long-term goal of biology has been to understand how DNA sequences specify complex multicellular living organisms. Understanding this mapping of sequence to form requires identification of cis-regulatory elements and parsing their roles in gene regulatory networks (GRNs). We have tackled this problem experimentally through the study of the C. elegans gut GRN, and by developing machine learning tools for predicting and visualizing cis-regulatory sites.

For the C. elegans GRN, we examined the function of \textit{cis}-regulatory GATA motifs upstream of the gut master regulator elt-2. We found that despite similar conservation and binding capability to upstream activators, different GATA cis-regulatory motifs within the promoter of the C. elegans endoderm regulator elt-2 play distinctive roles in activating and modulating gene expression throughout development. By using single copy insertion promoter-reporter constructs and high resolution single-molecule RNA FISH, we determined that a single primary dominant GATA motif located -527 bp upstream of the elt-2 start codon was necessary for both embryonic activation and later maintenance of transcription, while nearby secondary GATA motifs played largely subtle roles in modulating postembryonic levels of elt-2. Mutation of the primary activating site increased low-level spatiotemporally ectopic stochastic transcription, indicating that this site acts repressively in non-endoderm cells. Our results reveal that CREs with similar GATA factor binding affinities in close proximity can play very divergent context-dependent roles in regulating the expression of a developmentally critical gene in vivo.

In order to better understand CREs in a context dependent manner, we developed a convolutional neural network (CNN) based tool, DeepNuc, to explore how deep learning techniques can be used to examine cis-regulation. We have used DeepNuc to demonstrate that much of the information needed to define a worm promoter sequence is present in the primary DNA sequence, and we have also applied our tool for exploring miRNAs.

Biologists have long been interested in the dynamic relationship between organism development and environmental temperature. Coined in nature as Bergmann size clines, and in the lab the Temperature Size Rule, the vast majority of animals exhibit larger body sizes at cooler temperatures (~lower latitudes) and smaller body sizes are warmer temperatures (~higher latitudes). While this can be considered a rule for endotherms which have evolved thermogenesis, ectotherms, who are at the mercy of the temperature of their immediate environment, have unique and disparate responses. Using the humble roundworm genus Caenorhabditis, we show that indeed there is no robust pattern of developmental response to temperature. And moreover, the temperature size rule can be better thought of as simply a reflection of rearing temperatures compared, which can be modeled as a bell distribution unique to an individual organism. We also look towards changes in reproductive strategy as the main driving factor effecting the responses shown. I propose a possible signaling pathway connecting changes in environmental temperature to changes in reproduction and longevity. Continuing prior research, we provide evidence in support of a Single Nucleotide Polymorphism (SNP) in a gene encoding a calcium dependent protease known as tra-3 attenuating C. elegans ability to maintain larger body sizes as temperatures decrease. I propose a reasonable mechanism by which the aforementioned SNP may be exerting its effects.

Transcription factors regulate gene expression. The coordination of gene expression is fundamental for specifying, determining, and maintaining cell fates. Here I investigate the evolution of a family of developmental transcription factors in the genus of nematodes Caenorhabditis. In the model organism Caenorhabditis elegans, GATA factors primarily function in either endoderm or hypoderm (epidermal) development. The genomes of Caenorhabditis species have diverged extensively however, anatomically these species are barely distinguishable. Which raises the question, have GATA factors evolved while the phenotype of these worms has remained stable? To address this question, Scott Rifkin and I searched for GATA factor orthologs in the proteomes of 58 Caenorhabditis species and two outgroup nematode species using the GATA-type zinc finger domain profile from the PROSITE database. Using maximum likelihood approaches, we estimated the evolutionary history of these GATA-domain-containing sequences. We found that GATA factors have radiated extensively within the Elegans supergroup. For example, the core of the endodermal cell specification network – med-1, med-2, end-3, end-1, and elt-7 – are unique to the Elegans supergroup, suggesting that this expansion rewired Caenorhabditis development. Our phylogenetic analyses, gene structure comparisons, and gene expression studies all support an evolutionary scenario in which a duplication of elt-3 produced the ancestor of elt-7, end-1, and end-3 in the Elegans supergroup ancestor. If this hypothesis is true, it would be an example of subfunctionalization after gene duplication that resulted in at least three Elegans supergroup paralogs that are expressed endoderm-specifically with another, elt-3, only expressed in the hypoderm. These gene duplications coincided with an increase in conserved GATA binding sites in the promoter of another endoderm-specifically expressed GATA factor gene, elt-2, which acts downstream of and is a transcriptional target of elt-7, end-1, and end-3 during endoderm development in C. elegans. One of these sites was found to be necessary for elt-2 expression. Since ELT-2 also regulates itself, there may be a brief time when all four of these GATA factors are expressed and competing for binding to sites in the elt-2 promoter. Using bio-layer interferometry I started quantifying the in vitro binding kinetics of these GATA factors to a functional GATA site and permutations of this site. My preliminary results show subtle differences in binding kinetics and specificity. These findings give insights into how three closely related transcription factors likely partitioned the ancestral role of a single transcription factor in a conserved developmental process.

While transcription factors are a crucial part of gene regulatory networks, their role in evolutionary processes is still not fully understood by researchers. Recent advancements in studying protein-DNA interactions have revealed that transcription factors have a binding affinity profile where there are both primary and secondary binding sites. The existence of these binding profiles hints to the possibility that transcription factors may have more potential for change than previously thought. How binding profiles may be affected by changing environmental conditions or mutations in the binding domains and binding sites remains to be seen. Existing in vitro and in vivo methodologies do not have the ability to produce the high throughput, informative data required to answer these questions. In this paper, an inducible platform, integrated in the yeast genome, is described that allows for the study of multiple transcription factors in vivo. By measuring an observable phenotype (fluorescence), how introduced mutations and altered environmental conditions affect the binding abilities of a chosen transcription factor can be investigated. The platform plasmid has already been assembled and the inducible system has been transformed into the yeast cells. The next steps are to transform the platform plasmid into the yeast and to insert a target transcription factor system for a pilot study. Once completed, this system will allow for more comprehensive studies of the binding space of various transcription factors.

Middle school and high school students are often confronted with the problem of textbook memorization as the singular form of biological learning, rather than acquiring knowledge by performing lab work. Middle and high school teachers may lack experience teaching biology by guiding a class through a protocol in the lab, as opposed to lecturing. This protocol uses CRISPR technology, an advanced genome editing application. This wet-lab approach not only teaches students the reality of molecular biology research, but also works through the scientific method and realistic problem based thinking researchers must apply. Most notably, this protocol’s purpose is to ignite a scientific passion in these students beginning at a young age. This exciting protocol is simple to use and can be completed in minimum time and is inexpensive. If used correctly, students may develop a life-long passion for science and one day research disease ending medicines, such as CRISPR/Cas9. Experienced researchers can also use this protocol as an introduction to CRISPR/Cas9. This protocol uses Saccharomyces cerevisiae, a medium that displays broad phenotypic change after mutation. The visual nature of this mutation allows students to see that the tiny biomolecules organisms are made of really are the cause of phenotypes.