With advances in sequencing technology it has become feasible to sequence a patient’s genome in a clinical setting, but our ability to interpret the effect of genetic variants on health and disease remains a large challenge. Genome-wide association studies (GWAS) have helped to interpret DNA variants by correlating thousands of variants in the genome with disease. However, many of these variants fall in noncoding DNA (making it hard to predict their effects) and are often in tight linkage with neighboring single nucleotide polymorphisms (SNPs) (meaning these neighboring SNPs may also be responsible for driving the correlation with disease). Consequently, most disease-associated variants still need functional validation. There is mounting evidence that a large number of these variants may be effecting gene regulation (when, where, and to what degree a gene is expressed), and several groups have tried testing whether these SNPs effect regulatory activity with traditional reporter assays. In this study, we aimed to validate GWAS candidates linked to osteoarthritis, a joint disorder with several SNPs previously associated. We compiled a list of these variants as well as all SNPs in linkage disequilibrium, for a total of 1,605 variants. To screen this list for functional candidates, we leveraged “self-transcribing active regulatory region sequencing” (STARR-seq), a reporter assay that can test thousands of sequences for regulatory activity in a single experiment. Our screen identified two variants with statistically significant differences between the major and minor alleles. To validate these findings in the genome, we introduced the minor allele of one variant into cell lines via CRISPR/Cas9. We found a similar difference in transcription between the major and minor allele in the genome as our initial episomal screen. We are currently working with our collaborators to further validate these findings in patient samples.

Gene amplification can be associated with genetic disorders such as cancer and varying types of autism. One such form of amplification is a neochromosome where a chromosomal segment is amplified and these two segments are joined at an inverted repeat. There are two models to explain how this event could occur: double stranded break and repair and origin dependent inverted repeat amplification (ODIRA). ODIRA hypothesizes that a replication fork error occurring near short inverted repeats could cause the leading strands to erroneously become ligated to the lagging strands and produce an extrachromosomal palindromic DNA intermediate. Because both models produce identical neochromosomes, I have worked to find which pathway produces them. To do so I utilized CRISPR/cas9 to induce breaks at commonly observed junctions, and analyzed the resulting intermediates using gel analysis of whole yeast chromosomes. If the neochromosome were to occur via the double stranded break and repair model, I expected that providing the break should increase the frequency with which these neochromosomes arise. Alternatively, if the neochromosomes were occurring due to the ODIRA model, I expected the frequency to remain unchanged. I followed this initial analysis with DNA sequencing to give further support to either outcome. In particular I verified that the CRISPR system was working by sequencing across the junctions where cas9 was cutting. If cas9 was indeed cutting, then when comparing the sequence from the cas9 survivors to that of the yeast genome, the survivors had an altered cut site. My work provides insight to the etiology to this fascinating class of chromosome rearrangements.

Our project is on urothelial urinary bladder cancer, one of the more common types of cancers. Our lab focuses on protein synthesis control of ARID1A, a known tumor suppressor in the context of urothelial urinary bladder cancer. Our initial workup led us to believe that ARID1A loss is not important for tumor initiation but may be important for tumor progression. To prove this, we used CRE recombinase editing tool to knock out ARID1A after treatment with BBN, a known carcinogen that causes bladder cancer. The results confirmed our hypothesis. In our investigation of this mouse model, we discovered an unexpected relationship between ARID1A loss and lowered protein synthesis. There is a growing body of literature showing that high levels of protein synthesis are required for transformation. As such, we conducted an experiment with rpL24+/- mouse model to test this hypothesis. We found that rpL24+/- mice showed a significant delay in cancer initiation compared to wild type mice when treated with BBN, thus establishing the role of protein synthesis in cell transformation. Our focus now is to figure out the relationship between ARID1A loss and protein synthesis. Based on preliminary results that we have conducted, I hypothesize that ARID1A loss and decreased protein synthesis leads to a weakened cell state which makes them vulnerable to carcinogen induced DNA damage and cell death. This was shown by measuring DNA damage, apoptosis, and cell proliferation as cellular markers of cancer and then comparing them with protein synthesis levels in ARID1A knock out (KO) cells and WT cells upon short treatments with BBN. We used both a mouse model and in vitro model to address this hypothesis.

Tyrosine kinases are critical drug targets in oncology due to their role in tumorigenesis. Accordingly, the treatment of specific cancers has been revolutionized by the development of tyrosine kinase inhibitors (TKIs). However, the long-term effectiveness of TKIs is undermined by the emergence of drug resistance, often caused by mutations that prevent drug binding. Understanding whether a given mutation confers drug resistance can enable physicians to tailor treatment based on the patient’s tumor genotype. Current methods to identify resistance mutations are laborious and can only interrogate a small subset of possible mutations. To overcome this limitation, I have designed an assay to identify all possible single drug resistance mutations in the oncogenic kinase ALK in a single experiment. A key feature of this functional assay is that it can accurately discriminate between drug resistant and drug sensitive mutations. Moreover, this assay leverages the Ba/F3 cell line, a mammalian cell line that only proliferates when an active oncogenic tyrosine kinase variant is expressed. Thus, when treated with a TKI, cells expressing drug resistant kinases continue to proliferate while cells expressing sensitive kinases die. As a first step, I have genetically engineered the Ba/F3 cell line to enable single-copy integration of tens of thousands of ALK variants. Additionally, I have cloned ALK into a mammalian expression vector and have optimized transfection conditions for the Ba/F3 cell line. Next, I will transfect the cloned vectors into Ba/F3 cells and measure the growth rates of each transfected cell line. I anticipate that only cells harboring active ALK will grow, while those with inactive variants will not. This result would lay the foundation for further development of a system for the exhaustive identification of drug resistance mutations in oncogenic kinases.

We are using yeast to investigate how the DNA helicase mutation mcm4^Chaos3 interferes with the early events leading to DNA replication and how these replication defects may lead to its role in cancer development. Mcm4^Chaos3 encodes a mutation in a subunit of the MCM helicase, an essential complex required for unwinding double stranded DNA during replication. Mcm4^Chaos3 mice exhibit genomic instability and more than 80% of female mice homozygous for Mcm4^Chaos3 developed mammary tumors (Shima et. al 2007). Further work in yeast identified an origin-specific minichromosome loss phenotype, suggesting the mcm4^Chaos3 mutation may have particular sequence requirements at origins, where DNA replication initiates. To determine the basis of origin sequence specificity in mcm4^Chaos3 function, we had previously performed a plasmid maintenance competition assay using a mutARS317-seq library, containing 500+ plasmids with random single mutations within the ARS317 origin sequence. This assay identified 5 origin sequence variants that performed better in mcm4^Chaos3 yeast than wild type ARS317. To investigate mcm4^Chaos3 interaction with ARS317 variants, I first measured the loss rate for wild type ARS317 plasmids in mcm4^chaos3 vs wild type yeast. As predicted, wild type cells maintained the plasmids better than the mcm4^Chaos3 mutants (16.8% loss rate/generation compared to 5% in wild type). I am currently characterizing the 5 sequence variants, and based on the competition assay data, am predicting to see differences in plasmid loss rates across the ARS317 variants. Understanding the cause for this origin specificity could help us develop a greater understanding of the mechanics involved in DNA replication, genome stability, and cancer-causing mutations.

Hybridization is an event that occurs when different species or populations reproduce to create offspring, and represents a possible way to fast track adaptation by introducing an abundance of genetic variation all at once. Investigating the adaptive potential of hybrids is especially relevant under our current conditions where stresses from climate change and global warming are increasing the frequency of hybridization, and putting increased pressure on species to rapidly adapt to new conditions. We have used yeast as a model organism to look at how hybrids adapt to new environments. We used Saccharomyces cerevisiae, a strain that prefers warm temperatures, and Saccharomyces uvarum, a strain that prefers cold temperatures, and evolved their hybrid offspring in both warm and cold temperatures. Using whole genome sequencing, we discovered mutations which may be important in temperature and nutrient adaptation, including a mutation involving the gene PHO84 that has differential allele selection depending on temperature. I then tested the fitness effects of these mutations by measuring a strain’s competitive growth over time compared to a common ancestor in both cold and warm temperatures. I expect to find other specific genes that are involved in temperature sensitivity, and possibly reveal ways these and other species have adapted to withstand divergent temperatures.

The development of tubes is a conserved process observed in a wide variety of species; tubes contribute to structures such as neural tubes, digestive systems, and vascular systems. For a tube to form correctly, individual cells within a sheet of cells (epithelium) must move and change shape in very specific ways. My goal is to understand what controls this movement. Polarity proteins help to establish the directional identity of cells and tissues. This identity is important to ensure that cells coordinate their behaviors. I hypothesize that the proteins that help establish polarity also play key roles in the development of tubes, including in proper closing of the tubes and correct directional elongation. To study the involvement of polarity proteins during tube formation, I am using the dorsal appendages (DAs) on Drosophila melanogaster egg shells. Because the appendages form as a direct readout of morphology, I can see how tissues developed earlier on. These appendages, which provide the developing laid egg with oxygen, are formed from an epithelium that wraps and elongates into a tube, then fills with eggshell protein; the initial epithelium sloughs off, leaving the appendages as a visualization of the earlier tube formation. To test my hypothesis, I am using RNA interference (RNAi) to knock down the expression impact of various proteins and evaluate the impact on DA formation. The RNAi system induces transcript loss, eventually revealing the impact of protein loss. I am using RNAi in epithelial follicle cells to test 20 candidate polarity genes. This analysis will help me identify a few genes to then characterize further. These results will inform future studies and add to the knowledge of the role of polarity proteins in the patterning and subsequent growth of epithelial tubes across many species.

The PTEN (phosphatase and tensin homolog) protein negatively regulates growth-promoting PI3K-Akt signaling in cells. Due to its function as a tumor suppressor, PTEN is often mutated in diverse cancers. Unfortunately, most PTEN variants have not been individually studied, making it difficult to ascertain their functionality within cells. Our lab recently demonstrated that thousands of PTEN missense variants exhibit decreased steady-state abundance when expressed in human cell lines and likely have reduced function. However, the mechanism behind their lower abundance is currently unknown. We hypothesized that many low-abundance PTEN variants are thermodynamically unstable and possess a reduced melting temperature. To test this, we fused EGFP, Enhanced Green Fluorescent Protein, to a panel of PTEN single amino acid variants, and these fusion proteins were expressed within human cell lines. Then, we cultured these cells at their standard growth temperature (37°C) and two lower temperatures (33°C and 30°C). We found that a subset of variants of intermediate abundance at 37°C exhibit WT-like abundance at decreased temperatures, while variants of extremely low-abundance remain unchanged. These results suggest that we can identify missense variants with reduced thermodynamic stability using this method. Next, we will repeat this experiment at high throughput to identify hundreds of temperature-dependent PTEN variants. We will determine biochemical properties shared by partially stable variants and compare them to computational predictors of protein folding. We will also identify variants of intermediate abundance that are not temperature-dependent, which may reveal other mechanisms by which variants lower a protein’s abundance. Our results demonstrate that we can characterize the thermodynamic stability of PTEN variants by measuring their abundances in cells grown at different temperatures. These results might be more physiologically relevant because the variants were studied in a cellular environment. Furthermore, our methods may be applied to other proteins that cannot be studied as purified protein.

In the context of the cell, molecular crowding can be described as the summation of molecular forces acting on polypeptides at the high protein concentrations that exist naturally in membrane-bound structures. This phenomenon of molecular crowding can affect protein conformations and interactions, particularly for proteins that require high flexibility to recognize and bind a large number of interactors like the chaperone Hsp90. To observe changes in protein conformations and interactions, we cross-link proteins with lysine-reactive linker molecules, then identify and quantify cross-linked sites using mass spectrometry. The linkage sites provide physical distance constraints useful for structural predictions, and the observed changes in cross-link abundance levels is informative of conformation and interaction changes. In this project, we apply chemical cross-linking to proteins under conditions that exert different levels of molecular crowding. These conditions include in vivo cross-linking of in-tact cells where high protein density causes high levels of crowding, and cross-linking of cellular lysates where protein density and molecular crowding effects are reduced. This presentation will highlight cross-linked peptides in Hsp90 and how these are being used to improve our understanding of the effects of molecular crowding on the function of this important chaperone.

Flowering plants exhibit striking diversity in their floral symmetry due to independent genetic changes throughout their evolutionary history. Diversity is also observed within the flowers of genus Rhododendron, which displays a variety of radially and bilaterally symmetric flowers across its approximately 1000 species. Floral symmetry is governed by an interaction between one TCP transcription factor, CYCLOIDEA (CYC), and two MYB-class genes, RADIALIS (RAD) and DIVARICATA (DIV). This interaction initiates when CYC is expressed in the floral meristem, leading to the activation of RAD in the dorsal domain. RAD then antagonizes DIV activity in the dorsal regions, leading to radial symmetry. In other flowering plants, duplications in CYC have been associated with changes in floral symmetry, but the role of RAD and DIV remain unstudied. A number of transitions to and reversals from radial symmetry have occurred throughout Rhododendron from a bilaterally symmetric ancestor. Focusing on RAD and DIV, the goal of this study is to better understand the evolutionary history of these floral symmetry genes in rhododendrons, and their correlation to floral symmetry changes. I sampled eight different species throughout Rhododendron in addition to various outgroups and reconstructed phylogenies of these genes. I obtained sequences from genomic and transcriptomic databases, and used these sequences to design primers for amplifying and sequencing these genes from laboratory samples from wild and cultivated specimens. I found two distinct copies resulting from one duplication, in both RAD and DIV. In future studies, we will expand our sampling of species to investigate the phylogenetic placement of the origin of these duplications in tandem with changes in flower symmetry, to determine whether duplications in RAD or DIV are associated with symmetry changes as has been shown for CYC.

Wound healing is an essential physiological process that repairs damaged tissues through a collaboration of molecular and cellular events.  For a wound to heal properly, hemostasis (blood clotting), inflammatory, proliferative, and maturation phases must occur sequentially at specific times and at optimal levels.  I’ve been using the fruit fly Drosophila melanogaster as a model organism to study wound healing and the potential role of a family of six growth factors called Imaginal Disc Growth Factors (IDGFs). IDGFs are related to human chitinase-like proteins, which are upregulated in cancer and other diseases associated with inflammation; their function, however, is not yet understood.  We hope to gain some insight by studying their role in wound healing in the Drosophila wing imaginal disc, a larval tissue that will become the adult wing.  Using a method called in situ hybridization, which reveals the patterns of RNA localization in fixed tissue, I found that while IDGFs are normally expressed in wing imaginal discs, their expression is immediately turned off upon wounding.  However, using transgenic fly strains that have IDGF2 and IDGF6 proteins tagged with a green fluorescent protein and an extended culture assay that allowed me to track protein expression over time, I found that IDGFs are upregulated at the wound sites after several hours.  Combining these observations, I hypothesize that IDGFs are first turned off in the hemostasis phase and later are upregulated in the inflammatory or proliferative phase, when new tissue begins to form.  To determine the cell types, cellular dynamics, and precise timing of IDGF upregulation, I will use confocal live imaging and label cell types that are known to be involved in wound healing, such as hemocytes.  These studies will contribute to our understanding of the genes that regulate tissue healing after mechanical injury.

As an airborne virus, influenza A is a widespread threat to global economies and a consistent danger to public health. Through high reassortment and evolutionary rates, influenza is even able to infect those who have been previously vaccinated against the virus. The innate immune system serves as a key first line of defense against this pathogen, with the signaling components, called interferons, driving the production of a potent cellular antiviral response. Studies have indicated that viral populations replete in defective virus particles, virions with a deletion in a portion of their genome, are less efficient at blocking the antiviral response, as shown by increased interferon in the host. Our project seeks to explore this phenomenon of RNA deletions leading to increased interferon expression in host cells by testing the hypothesis that deletions in the three polymerase genes of influenza alone are sufficient to cause an increase in the interferon response. In addition, we are currently testing if mutational deactivation of one of the other genome segments, or absence of such segments, is capable of producing a more robust immune response when combined with polymerase gene deletions. In order to support this analysis, I began by creating pure populations of PA defective influenza particles grown on PA expressing host cells. Similar to results observed by my mentor Dr. Alistair Russell with PB1 and PB2 defective populations, it was found that these PA defective influenza particles were sufficient to induce the interferon response. Recently, I have assisted in the creation of multiple influenza protein expressing cell lines and influenza populations with simultaneous modifications to the HA, NS, and polymerase genes. It is hoped that immune stimulation data derived from these custom viruses, in combination with previous findings, will improve current antiviral therapies and models of the human immune response to influenza.

Gene amplifications are an often-overlooked source of genetic variation frequently associated with genetic disorders, including cancer and developmental delays. However, the mechanism by which they arise is still unclear. There are currently two competing models for how gene amplifications arise. The first model requires a double-stranded break in DNA that results in chromosome recombination. In the second model, an error in DNA replication results in the formation of an extra-chromosomal DNA intermediate that can reinsert into the genome. To distinguish between these two models, I am using CRISPR/Cas9 to induce double stranded breaks in a strain of yeast with two fragments of the URA3 gene on separate chromosomes. I then select for strains with restored URA3 function, indicating that the two fragments have recombined to form the full URA3 gene. If the genome rearrangements are occurring through the double-stranded break mechanism, inducing these breaks should increase the frequency of recombination events that result in functional URA3 genes. However, the preliminary results suggest that these events are occurring through the replication-error mechanism rather than through double-stranded breaks. Clarifying the mechanism of gene amplifications is a significant step toward understanding the how these genetic disorders arise and suggests further research in preventing disease-causing gene amplifications.

Antibiotic resistance is a growing threat across the planet. This resistance, coupled with a dearth of new antibiotics, makes development of new antibiotics of critical importance. Antibiotics derive from microbial pathways that synthesize complex natural products, and engineering these pathways to produce unique products is an exciting prospect. We will use such an engineering strategy to produce variants of the antibiotic Microcin J25 (MccJ25) in E. coli. MccJ25 belongs to a unique set of compounds known as lasso peptides. It is produced by three biosynthetic enzymes that modify the precursor peptide, McjA, and then export the mature lasso. MccJ25 functions by blocking the nucleoside triphosphate (NTP) uptake channel of RNA polymerase, interfering with transcription. Moreover, a single amino acid change in RpoC, one component of RNA polymerase, confers resistance to MccJ25. Extensive studies on the MccJ25 biosynthetic pathway suggests that the biosynthetic enzymes are tolerant of mutations in McjA. We plan to identify variants of MccJ25 that overcome resistance due to rpoC mutation. We will construct a library of protein variants of McjA and transform this library into a strain of E. coli carrying the rpoC T931I mutation. After induction of the MccJ25 biosynthesis pathway, cells with biologically active variants of MccJ25 should drop out of the population. By deep sequencing the population before and after selection, we can infer which variants successfully inhibited growth. This strategy of altering a precursor peptide to create novel antibiotic structures may provide a framework for future antibiotic engineering efforts.

The budding yeast is a popular model organism for aging research. Amino acids are a fundamental building block of biology with crucial roles in metabolism and intracellular signaling, but effects of amino acid levels and ratios in the growth media on yeast aging has never before been tested. We measured the effects of supplementation of different amino acids on yeast replicative lifespan by imaging hundreds of trapped mother cells in a microfluidic device during the course of replicative aging. We find that increased absolute levels of threonine can increase yeast lifespan through reduction of toxic biosynthetic intermediates. Conversely higher relative levels of cysteine depress lifespan. These effects are regulated by the status of the vacuole, which becomes less acidic with age. Thus, we find that longevity promoting optimal amino acid composition can optimized in an age-specific manner.

The pathologies of yeast replicative aging are extensively studied as a simple model organism for human aging. The golden standard in measuring yeast lifespan has been microdissection, a time- and labor- intensive process. We have developed a novel microfluidic microscopy system to facilitate more efficient and inexpensive collection of lifespan data. By counting replication through time-lapse imaging of trapped mother cells using consumer-grade microscopes coupled with an in-house auto-focusing system, we can research yeast pathways with significant time- and cost-savings over traditional microdissection and microscopy methods. I applied this system to study the effects of probiotic metabolites on replicative lifespan and have discovered lifespan-extending metabolite treatments.