In 2019, the severe acute respiratory syndrome coronavirus (SARS CoV-2) emerged across the world with devastating effects. In severe cases of infection, SARS CoV-2 elicits an overactive immune response leading to extensive damage to lung tissue potentially through overproduction of proinflammatory cytokines. To further our understanding of this novel coronavirus, our project studies the effects of SARS CoV-2 on the innate immune response in lung epithelial cells. Innate immunity mediates antiviral defense in response to virus infection and other pathogens, wherein innate immune activation is triggered by virus infection and induces type I and III interferons that play an important role in antiviral defense though expression and actions of interferon stimulated genes (ISGs). Our project investigates which SARS CoV-2 protein is responsible for antagonizing virus-induced innate immune activation of the infected host cell. We screened the individual SARS-CoV-2 proteins in a lung epithelial cell transfection/innate immune activation model using Sendai virus as an innate immune activator to examine interferon induction in cells expressing each SARS-CoV-2 protein. We hypothesize that one or more viral proteins will antagonize innate immune activation. Our preliminary results show that increasing amounts of the viral membrane protein (M) decreases the innate immune activation as marked by reduced ISG induction. Our findings have implications for future treatments of SARS CoV-2 by providing insight on new potential viral targets to mitigate virus-mediate innate immune blockades.

Cystic fibrosis (CF) is a genetic disorder characterized by chronic lung infections involving various organisms, including the gram-positive pathogen Staphylococcus aureus. Antibiotics, such as trimethoprim-sulfamethoxazole (SMX), play key roles in treating CF infections; these drugs inhibit bacterial growth by disrupting important bacterial metabolic processes. SMX specifically inhibits folate metabolism, causing DNA damage that results in bacterial cell death. However, S. aureus is able to persist in CF pulmonary infections despite treatment with antibiotics, and evidence suggests that S. aureus does so through adaptive mutations. Our goal is to identify the adaptive mutations of S. aureus during SMX exposure in vitro, to understand how this pathogen persists and to prevent the emergence of resistance. We grew S. aureus in the presence of super-inhibitory SMX concentrations for 24 hours in Luria Bertani broth. We sampled the culture at specific timepoints and measured viable bacterial counts on chocolate agar; we evaluated all resulting colonies for SMX susceptibility and associated genetic changes. Surprisingly, we identified mutants that survived SMX treatment carrying diverse adaptive changes not associated with folate metabolism or DNA repair, suggesting previously-unknown lethal effects of SMX against S. aureus. These mutants carried mutations predicted to decrease production of reactive oxygen species (ROS) - toxic compounds produced by all cells during aerobic respiration and in response to stress. Our results indicate ROS may play a role in SMX-mediated S. aureus cell death, suggesting that treatments that augment the effects of ROS could improve antibiotic efficacy. We are now exploring the involvement of ROS in S. aureus killing by SMX using engineered S. aureus strains with knockout and overproducing mutations in ROS detoxification genes. This study will help us better understand SMX’s mechanism of action and S. aureus’ response to this drug, in order to improve the treatment of diverse infections caused by this pathogen.

Skin cancer is the most prevalent cancer in the United States, and its annual incidence exceeds all other cancers combined. There is thus a pressing need to develop novel approaches to inhibit skin cancer. Inactivation of tumor suppressor genes is a frequent event in carcinogenesis. In skin cancer, the CDKN2A gene, which encodes the p16^INK4A tumor suppressor protein, is often silenced by epigenetic abnormalities such as promoter DNA methylation and histone deacetylation at this genomic locus. The p16^INK4A tumor suppressor functions as a cell cycle regulator and plays an important role in tumor growth and metastasis. Unlike gene loss, gene silencing by aberrant epigenetic modifications can be reversed by small-molecule compounds such as DNA methyltransferase inhibitors and histone deacetylase inhibitors. However, these small-molecule inhibitors are nonspecific, affecting epigenetic modifications globally. Here we use CRISPR-Cas9-based epigenome editing tools for targeted histone acetylation at specific genomic loci. To induce histone acetylation at the CDKN2A promoter, nuclease-deactivated Cas9 (dCas9) is fused to histone acetyltransferase p300, and the dCas9-p300 fusion protein can be recruited to the target site by using guide RNA (gRNA) that binds to the CDKN2A promoter. After dCas9-p300 with gRNA was introduced into the A431 skin cancer cell line to target the CDKN2A promoter, RT-qPCR revealed that p16^INK4A mRNA expression levels increased by approximately 30-fold. Cell proliferation assays further revealed that proliferation of A431 cells decreased by approximately 20%. To fully inhibit proliferation of skin cancer cells, further investigations are needed to evaluate the combined effects of targeted histone acetylation and DNA demethylation at the CDKN2A promoter. This study provides insight into how epigenetic abnormalities can be targeted by novel epigenome editing tools in order to reactivate dormant tumor suppressors and inhibit cancer phenotypes.

Over the past fifty years, survival rates for most cancers have risen as innovative treatments have been developed. However, Diffuse Intrinsic Pontine Glioma (DIPG), a rare pediatric brainstem tumor, has seen no such improvement and remains one of the deadliest cancers. DIPGs are genetically distinguished from adult gliomas by a lysine-to-methionine mutation in a variant of histone H3 called H3.3 (H3.3K27M), found in 80% of DIPG tumors. This mutation is absent in canonical H3.1 and H3.2, yet it triggers a global reduction in the levels of polycomb repressive complex 2 (PRC2) mediated H3K27me3 (tri-methylation) marks, which is traditionally considered a driving event in tumorigenesis. However, genome-wide studies of H3K27me3 marks in DIPG cells have revealed that this global loss of H3.3K27me3 is accompanied by a sharp increase in H3K27me3 repressive marks at certain genes. These trimethylation spikes represent regions with high transcriptional repression and may be key in understanding and treating DIPG. Our lab has previously described the inhibition of PRC2 using a computationally designed protein EBdCas9. Using a complementary guide-RNA tiled to the promoter region of a gene, I can target EBdCas9 to that specific region on the genome, remove existing H3K27me3 marks, and increase gene transcription. Identification of candidate genes which are repressed in DIPG cells under these H3K27me3 spikes remains an ongoing project that I am working on. Results of one target, tumor-suppressor gene p16, demonstrate that I can transfect primary DIPG cells with EBdCas9 plasmid and a p16-specific gRNA to trigger a 20-fold increase in p16 expression. I hypothesize that restoration of critical cell-cycle genes like p16 will reduce DIPG viability and offer potential therapeutic targets. I’m currently working to improve transfection efficiency in order to increase p16 expression, and future steps include in-vivo testing of this system, either via mouse model or a brainstem organoid.

Atherosclerosis is the buildup of fats, calcium, and other substances within the walls of arteries. Localized atherosclerotic deposits, known as plaques, can obstruct arterial blood flow. Obstructed arteries are often treated by surgical implantation of vein grafts, which bypass the obstructed arteries and restore blood flow. In our rabbit vein graft gene therapy model, we consistently observed diminished blood flow over time. We hypothesized that this decrease was caused by narrowing at the ends (anastomoses) of the vein grafts, a phenomenon that is known as anastomotic stenosis and can be caused by vascular injury or poor surgical technique. Because decreased blood flow can interfere with vein graft function, we designed an experiment to test whether anastomotic stenoses were present and—if so—whether the stenoses were the cause of decreased blood flow. To test this hypothesis, in a new set of grafted veins, we harvested the anastomoses, embedded, sectioned, and stained them, and measured the percentage stenosis using computer-assisted image analysis. We measured the stenoses in both anastomoses of 44 vein grafts, harvested from 22 rabbits (88 total anastomoses). Blood flow (mL/minute) in each vein graft was measured with a flow meter 1 month after grafting and again 12 weeks later (at harvest). The mean change in blood flow was -16 mL/minute (range: -55 to 14) and the mean stenosis was 39% (range 2.3% to 89%). After confirming the presence of the stenoses, we used a nonparametric correlation test to examine our hypothesis that percentage stenosis and blood flow change were inversely correlated. We found that percentage stenosis did not correlate with blood flow change (r=0.059; P=0.8). Therefore, decreased blood flow in our vein graft model is not caused by anastomotic stenoses. The cause of the decreased blood flow remains uncertain, and other explanations may be considered.

Although RNA is most commonly regarded as a passive carrier of genetic information, throughout biology RNA molecules exhibit an exceptionally broad scope of other, non-coding functions. Exciting recent work has demonstrated that RNA plays diverse and fundamental roles in helping to pattern subcellular architecture, including that of numerous subnuclear structures that are essential to cellular function. Many such architectural RNAs are also causally dysregulated in human diseases, suggesting that they may represent a novel resource for untapped therapeutic targets. Yet, the mechanisms by which these architectural RNAs function have remained elusive. An essential first step towards deciphering RNA’s cellular function is to elucidate the complex networks of proteins, RNAs, and genomic loci with which that RNA interacts. Yet, this kind of analysis is impossible using conventional approaches. To address this critical need, the Shechner lab has been developing Oligonucleotide-directed biotinylation (ODB), a straightforward and universal method for mapping RNA interaction networks without taking them out of their native cellular context. Our recent results demonstrate that ODB enables precise targeting of individual RNA interactions in situ, enabling high-resolution proteomic, transcriptomic, and genome-interaction analysis in situ. The two goals of my project are to: (1) develop generalized and extensible ODB strategies that can be applicable to a wide range of RNA targets of interest, and (2) expand ODB's range of in situ labeling chemistries, enabling higher-precision analysis. To achieve these goals, I am performing a series of ODB experiments on 9 selected long non-coding RNAs of interest and establishing a high-precision localization pattern across all the RNA targets within the cellular context. The establishment of this powerful and general method can enable unprecedented dissection into RNAs that have been notoriously difficult to analyze by traditional approaches, and may potentially reveal new avenues for novel therapeutic target-discovery.

Tuft cells are a lineage of chemosensing epithelial cells that contribute to intestinal “type 2” immune responses during parasitic worm (helminth) infection. Tuft cells are thought to monitor the intestinal lumen, looking for type 2 agonists. While rare at steady state, during a helminth infection the number of tuft cells in the gut increases greatly (a phenomenon called hyperplasia), directly proportionate to the underlying immune response. Nippostrongylus brasiliensis is a common model of hookworm infection. This parasite is injected under the skin, migrates to the intestine, and can be cleared within 10 days, with a tuft cell response mediated by the cytokine interleukin (IL) 25 and largely independent of the IL33 receptor, ST2. In contrast, the roundworm Heligmosomoides polygyrus is ingested orally and has a lifecycle in which it matures inside the intestinal tissue rather than in the lumen, then re-enters the lumen and establishes chronic infection. Given their distinct lifecycles, we hypothesized that N. brasiliensis and H. polygyrus are sensed differently by tuft cells. Since the kinetics of tuft cell hyperplasia during N. brasiliensis are well defined, we created a comprehensive timeline of tuft cell hyperplasia in wild type mice infected with H. polygyrus for comparison. Intestinal tissue was collected from three mice on every other day of infection, until 20 days post infection. We used immunofluorescent staining to quantify the tuft cells on each harvest day and discovered distinct waves of tuft cell hyperplasia that align with the two separate times that H. polygyrus worms enter the lumen. This timecourse highlighted two peak timepoints of hyperplasia, which we studied further to test the hypothesis that IL33 is critical in the H. polygyrus immune response. Through this work, we strive to better understand how and why our bodies respond to the various type 2 agonists, and how this response can be most effective.