Type 1 diabetes (T1D) is an autoimmune disease in which the pancreas is unable to produce enough insulin to effectively regulate sugar into the body’s cells. Recent literature suggests that T1D has a prevalence and incidence of 9.5% and 15 per 100,000 people respectively. T1D risk is multifactorial but is heavily dependent on genetics and the environment. Twin studies have shown that although disease risk for the general population is 0.4%, children of diabetic patients are 2 to 9% at risk and identical twins are up to 70% at risk of developing T1D. Genome-wide association studies have shown that many genetic variants associated with T1D risk are found in gene regulatory regions such as enhancer elements. As T1D is an autoimmune disease with a strong genetic component particularly in non-coding, regulatory regions, it follows that thorough genomic profiling of immune cells such as T cells can help identify functional non-coding genetic variants that alter gene regulation for target genes associated with disease risk. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) allows for unbiased identification of cis-regulatory elements (CREs). By utilizing ATAC-seq on various T cell types of both healthy control donors and T1D patients, chromatin accessibility can be collected, and the underlying sequence data can be used to determine allelic differences in transcription factor binding due to T1D-associated genetic variants. I am currently applying ATAC-seq to three T cell subtypes isolated from both donor cohorts. Through these data functional links will be made between non-coding genetic variants and associated target genes to better understand how they impact disease risk in T1D.

Target of rapamycin (TOR) signaling is a nutrient-sensitive pathway that plays a role in cell growth and aging. Caffeine is a factor that inhibits TOR signaling and growth, compromising overall cellular fitness. The budding yeast S. cerevisiae is an ideal organism to study TOR signaling in relation to caffeine tolerance as there are many resources available to study yeast genetics, and yeast shares many basic biological properties with other eukaryotic cells. Additionally, yeast allows us to study TOR signaling using experimental evolution, where under a defined selective pressure - here, caffeine - we observe what rare beneficial mutations arise and increase in frequency. While many inputs to the TOR signaling pathway are known, others are yet to be identified. Furthermore, how other pathways compensate for TOR signaling inhibition is not completely understood. Our goal is to identify factors involved in TOR signaling by growing yeast in inhibitory concentrations of caffeine to select for better growing mutants with increased resistance. We will then sequence the resistant strains’ genomes and study the resultant mutations to determine how they connect to TOR signaling and caffeine tolerance. We evolved yeast for 5-10 weeks in increased doses of caffeine, and sequenced clones from the evolved populations. Our yeast evolved increased caffeine tolerance, and mutations arose in drug-response pathways including in the Pdr1 transcription factor. We also observed mutations in processes regulated by TOR, such as nutrient sensing. With further genome sequencing of more evolved populations we aim to identify novel mutations and factors involved in caffeine tolerance and TOR signaling. Ultimately, these findings increase our understanding of how caffeine impacts TOR signaling and how other cellular processes are regulated by TOR signaling. More broadly, this research can aid in the continued development of how cell signaling pathways are related to nutrient response, aging, and growth.

The advent of next generation sequencing (NGS) has revolutionized how we study both the genome and epigenome. However, current approaches are limited by the short read lengths associated with NGS, which limits our ability to discern genetic and epigenetic features across nearly 10% of the human genome containing repetitive genomic elements, such as telomeres and centromeres. Recent developments in long-read sequencing methodologies have enabled longer, high-quality reads with significantly more information at the single-molecule level than ever before. Our group has developed a methodology called Fiber-seq that uses an exogenous, non-specific adenine methyltransferase to ‘spray paint’ the genome with m6A methylation marks in an effort to stencil the chromatin architecture of the epigenome onto underlying DNA fibers in a manner that is interpretable by long-read sequencing. Each molecule is then sequenced using PacBio Single Molecule Real-Time (SMRT) sequencing. Methylated adenines are subsequently identified and used to directly resolve the chromatin architecture overlying that molecule of DNA, such as nucleosome footprints and transcription factor binding events. However, calling these methylated adenines on single molecules is computationally intensive, revealing the need for more efficient computational frameworks for base modification detection using PacBio sequencing. To address this, we have developed a statistical framework for identifying m6A-modified bases using raw PacBio sequencing data, leveraging the fact that each molecule is sequenced multiple times during the process. Specifically, we utilized raw data from an unmodified control sample to generate a background methylation model. We then scored data from Fiber-seq test samples against our background distributions to determine the methylation status of individual adenines across single molecules. Our initial model shows high specificity but lacks sensitivity, driving the need for further model development.We aim to generate a Deep Learning framework co-opting the data processing framework built for the statistical model.

Paraoxonases (PONs) are a family of three closely related genes found on the long arm of chromosome 7. The genes encode PON1, PON2, and PON3, which are primarily involved in metabolizing oxidized lipids and modulating oxidative stress. However, each of the PON enzymes are involved in important secondary reactions. PON2 is an intracellular enzyme localized in the mitochondria that plays a vital role in modulating oxidative stress and inactivating microbial quorum sensing factors. Individuals with PON2 deficiencies are sensitive to oxidative stress. It may be possible to restore PON2 function by creating an injectable protein for individuals with a PON2 deficiency. Our goal is to actively express recombinant PON2 in an E. coli expression system and inject PON2 into PON2 knockout mice to determine if PON2 function can be restored. To express PON2 in E. coli we designed a synthetic DNA sequence by removing the transmembrane sequence of PON2 and replacing it with the signaling sequence from PON1, which facilitates the purification of the chimeric protein. We transformed the synthetic PON1/PON2 plasmid in E. coli and are currently performing gel electrophoresis and activity assays to analyze the expression of PON2. If we see protein expression as expected, then we will purify the recombinant PON2 for injection using a histidine tag that we added to the end of the protein coding sequence in the construct. The histidine tag allows for single-step purification via affinity chromatography, which we will then inject into PON2 knockout mice to observe their response to oxidative stress. Findings from this experiment will allow for further understanding of PON2 function and its restoration via an injectable protein, as well establishing that E. coli expression systems can be used as a more cost-effective method for pursuing further PON2 related research.

 Mutations are the main source of diversity due to the random changes in genetic sequence. Mutation rate can be influenced by genetic modifiers, DNA damage, and the environment. Mutator alleles, which cause high mutation rates, are less known about how often they arise in the natural populations and how they interact with other genomic changes, such as recombination and introgression. Using the natural isolates of the 1,011 strains of the budding yeast Saccharomyces cerevisiae, we examined the relative frequencies of different types of mutation calculated from natural polymorphisms, i.e. mutation spectrum, to determine potential historical impacts of S. cerevisiae mutation rate modifiers that affect certain mutation rates disproportionately. From the mutation spectra analysis, we observed African Beer strains are outliers and some French Dairy strains show a mutation spectra more similar to African beer strains than the rest of the strains. Our initial analysis of the two groups of strains suggests potential mutator allele introgression from the African Beer to some of the French dairy strains. We set out to examine de novo mutation spectra from African beer, French dairy strains with and without African beer Introgression. The natural strains have a variety of ploidies so we repurposed our existing mutation accumulation pipeline with haploid strains. Using CAN1 as a reporter gene, we deleted the endogenous locus using CRISPR-Cas9 and inserted a sensitive copy to measure the mutation spectra in these strains to test the hypothesis. We expect to find the introgressed French dairy strains’ mutation spectra resemble that from African beer strains if our prediction is correct. Ultimately, our results will shed light on how mutators behave in natural populations of budding yeast and how they contribute to evolution.

The genetic makeup of beer-brewing yeast plays an essential role in determining the flavor profile during production of beer. With functional copies of genes on the MAL locus, beer yeasts, namely Saccharomyces cerevisiae, can utilize maltose and maltotriose as their carbon source. The three genes in this locus are responsible for the regulation (MALx3), transport across membrane (MALx1), and breakdown of sugars (MALx2) in brewers’ wort. If a strain lacks a functional copy of any of these three genes, it cannot digest these alternative sugars. Alternatively, some strains have more than one MAL locus, but which loci are functional, remains unknown. Apart from previous studies that have investigated a handful of MAL alleles, the function of the genes in these duplicated loci (or paralogs) cannot be determined based on sequences alone. To address this problem, I experimentally tested the function of alleles from 1,011 natural isolates. I focused on MALx3 because the reference strain lacks a functional MALx3 allele, preventing its growth in maltose. Therefore, introduction of any functional MALx3 alleles should permit growth in maltose. To test this, I cloned ~250 MALx3 alleles from three different loci (MAL1, MAL3, MAL7) and transformed the reference strain to generate three yeast libraries. The library with the MALx3 gene of the MAL3 locus, or MAL33, successfully grew to saturation after ~2.5 days of incubation in 2% maltose, showing this approach can be used to determine the function of MAL33 alleles. Looking forward, I will use DNA barcodes to track the growth of alleles in maltose to pinpoint which are functional. With the MAL loci serving as a great candidate for understanding paralog differences, by identifying the functional paralogs, we can better understand the evolutionary history of MAL genes and what role these loci play in the brewery and across all natural isolates.

Biological tubes are the foundation of most animal organs, and thus, tube formation is an important developmental process. We study tube formation in Drosophila melanogaster egg chambers by analyzing the formation and elongation of dorsal appendages (DAs), eggshell structures that facilitate gas exchange in the fully developed egg. To form these tubes, a subset of follicle cells (the DA-patch) must rearrange, change shape, and migrate towards the anterior of the egg chamber. Polarity proteins, which establish cell directionality, are important for mediating these behaviors, but the mechanisms through which they do so are not fully understood. My research aims to enhance our understanding of these mechanisms by establishing how the polarity protein Crumbs (Crb) is involved in the formation of the DAs. To address this question, I used the mosaic analysis with a repressible cell marker method to generate mosaic clones in egg chambers that are null for crb and marked with green fluorescent protein. To determine how the loss of Crb affects DA formation, I used immunostaining to assess cell shape and position by visualizing the membrane protein E-cadherin, which participates in a regulatory network with Crb. I found that Crb is important for proper DA-patch cell shape changes during early tube formation, and that losing Crb causes delayed DA-patch cell migration, leading to shortened DAs. I also discovered that E-cadherin is mislocalized when Crb is lost, indicating that the interaction pathway involving E-cadherin and Crb may be responsible for the defects observed during tube formation. To test this hypothesis, I will stain for other proteins in this interaction pathway. By understanding this pathway, we can gain insight into how Crb functions in DA formation. Since tube-forming mechanisms are highly conserved, this work will suggest how polarity proteins regulate tube formation in all animals, including humans.