Abstract: Regulation of RNA splicing is critical for proper gene expression levels and generation of transcript diversity, and its misregulation is the basis for numerous diseases. It is now well established that a majority of RNA splicing takes place as the RNA is still being transcribed by RNA polymerase II. Consequentially, regulation of transcription and splicing are intertwined which presents a novel framework for the study of disease-associated mutations in either process. However, connecting splicing rate and with transcription regulation is difficult due to numerous technical challenges. In addition, splicing efficiency is difficult to measure since mis-spliced transcripts are often undetectable due to their rapid degradation. Here, we propose to address these issues by combining precision run-on sequencing (PRO-seq) with full length RNA sequencing on oxford nanopore’s technology. The adaptations allow us to map the position of RNA polymerase II when co-transcriptional splicing occurs. Our approach achieves near base pair resolution and initial results from Drosophila S2 cells suggest that splicing occurs within seconds of the downstream splice sites becoming accessible to the spliceosome.
ISG Core Used: CGI

"Does urbanization increase host resistance to invasive parasites in Darwin’s finches of the Galapagos Islands?"
Abstract: Human population size is increasing exponentially and, in turn, the urban environment is one of the few ecosystems that is rapidly expanding. A recent example of urbanization is in the Galapagos Islands, which is home to the endemic Darwin’s finches. The Galapagos currently hosts 225,000 tourists each year and is home to over 21,300 permanent residents. Consequently, humans have altered the natural habitat and introduced parasites to the Galapagos, such as the parasitic fly Philornis downsi, which causes up to 100% mortality in nestling Darwin’s finches. Adult flies are non-parasitic but the larval stage feeds on the blood of nestlings and brooding females. However, our recent work shows that small ground finches (Geospiza fortis, a species of Darwin’s finch) in urban areas are healthier and less affected by P. downsi compared to finches in non-urban areas, which suggests that birds are better defended against the parasite in urban areas. Specifically, my team found that small ground finches are more resistant to P. downsi in urban areas compared to non-urban areas on the island of San Cristobal, Galapagos. The aim of the proposed research is to compare overall gene expression in the blood of parasitized and nonparasitized finches in urban and non-urban areas and determine whether any immune genes (i.e. potential resistance mechanisms) differ in finches from each treatment and location. In 2019, we conducted the field work in which we experimentally manipulated P. downsi abundance in the nests of finches in an urban and non-urban area. We then collected and preserved 95 blood samples from nestling and female finches across treatments and locations. We have also successfully extracted RNA from the blood samples. Therefore, the proposed funding would cover the RNA library preparation and sequencing at CGI with Dr. Bo Reese and the RNA-Seq model and non-model systems bioinformatics workshop withDr. Jill Wegrzyn. Overall, the proposed work will increase our understanding of the benefits of urban living for endemic island species, especially in response to novel parasites, and provides preliminary data for an NSF proposal.
ISG Cores Used: CBC and CGI

Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) have proven to be the most effective clinical agents for suppressing inflammation and cancer, primarily by reducing the production of prostaglandin E2 (PGE2)1. Unfortunately, long-term treatment with NSAIDs is often associated with varying toxicities, including most commonly gastroduodenal ulcerations2. The likelihood of developing clinically significant NSAID-induced intestinal injury, however, is non-uniform within the general population, suggesting that there may be underlying genetic and/or environmental factors that influence susceptibility to NSAID-induced GI toxicity3.
We have recently found that genetic deletion of mPGES-1, the terminal enzyme that generates inducible PGE2, in strain A mice (A/J:KO) causes spontaneous ulcerations throughout their colons, while the same gene deletion in C57BL/6 mice (B6:KO) failed to induce a comparable phenotype. This differential response to PGE2 abrogation suggested that these mice may serve as a useful model to study the varying susceptibility to NSAIDs in human population. In initial studies to characterize this phenotype, we found that A/J mice harbor a significantly lower fecal abundance of Akkermansia muciniphila, a mucin-degrading bacterium, when compared to B6 mice, regardless of mPGES-1 genotype. A. muciniphila produces mucin-derived short-chain fatty acids (SCFAs), such as propionate and acetate, which can modulate host gene expression4. Furthermore, a membrane protein of A. muciniphila, Amuc_1100, has been shown to activate Toll-Like receptor 2 (TLR2), which enhances the gut barrier function in mice5. Given that PGE2 is also critical for maintaining mucosal homeostasis, we hypothesize that strain-specific abundance of A. muciniphila, in collaboration with mPGES-1 deficiency, may impart significant effects on host gene expression and that these epigenetic changes may ultimately affect mucosal integrity in the colon. For a future grant application, we plan to propose the following aims; 1) examine the gut microbiota in the feces and colon tissues of A/J:KO and B6:KO, 2) determine whether the a subset of bacteria affect the development of colonic ulceration in A/J:KO mice, and 3) examine the potential mechanisms by which A. muciniphila or Amuc_1100 affects epithelial barrier function in intestinal organoids generated from A/J and B6 mice. For this seed grant, we propose to perform a genome-wide expression profiling of colons from these mice to identify specific pathways associated with the sensitivity to colonic ulceration. We anticipate that the data generated by this pilot study will strengthen the future proposal by providing mechanistic links for understanding the interplay between the levels of PGE2 and the abundance of A. muciniphila in mucosal homeostasis.
ISG Cores Used: CBC and CGI

Abstract: The use of electronic cigarettes (e-cigarette) is increasing and has already surpassed conventional cigarette use among youth. E-cigarettes have been considered safer than conventional cigarettes under the premises that they contain lower concentrations of chemicals than cigarettes. However, recent news reports have provided evidence of the adverse effects that using e-cigarette, in particular to pulmonary health. In-vitro studies and animal studies have provided evidence of the potential harms of “vaping”, yet the mechanisms by which e-cigarettes leads to pulmonary disease remain unclear. A recent study showed that exposure to e-cigarette vapor induces gene expression changes in pathways associated with inflammation, cell metabolism, apoptosis and DNA repair in ex-vivo human bronchial epithelial cells. A different study, evaluated the gene expression from participants exposed to e-cigarettes using nasal epithelial cells, showed that e-cigarettes suppress immune and inflammatory-response genes more than conventional cigarettes and to our knowledge, the only study reported in induced sputum of e-cigarette users showed proteomic changes related to altered innate immunity. Although highly valuable, most of these reports were performed in-vitro or in animal models, which may not reflect the changes occurring in the airways and respiratory system of e-cigarette users, and no study has been reported on the transcriptome of e-cigarette users. It is also likely that e-cigarettes have effects at the systemic level on not limited to the respiratory system since a dynamic cross-talk between the lungs and circulatory system has been well described. Therefore, I hypothesize that e-cigarette use alters the airway and systemic transcriptomes involved in response to the noxious stimuli they deliver.
ISG Cores Used: CBC and CGI

Abstract: One of the major unanswered questions in sex chromosome evolution is why some sex chromosome pairs diverge while others do not. Study of amphibian genomes provides a transformative system for the study of heteromorphic sex chromosome evolution in vertebrates because over 95% have sex chromosomes that are hardly divergent (homomorphic), whereas 5% have highly divergent, heteromorphic sex chromosomes that have evolved 20-30 independent times. The main objectives of this proposal are to use the Center for Genome Innovation’s (CGI’s) Nanopore PromethION and to work with the CBC to generate long read data that will assemble, annotate, and distinguish the complete Z and W sex chromosomes of the African bullfrog (Pyxicephalus adspersus), an amphibian with heteromorphic sex chromosomes. The results generated from this seed grant will serve as the foundation for a proposal to NHGRI’s Comparative Genomics Research Program (PAR-17-482) to identify transcriptional changes for genes that change from autosomes to sex chromosomes and the links to sex chromosome dosage compensation that occur in some vertebrate lineages. The ultimate goal is understand how sex chromosome evolution molds gene network changes and how these changes impact genome function across vertebrate evolution.
ISG Cores Used: CBC and CGI

"Identifying Novel miRNAs to Investigate as Diagnostic Biomarkers in Patients with Statin-Associated Muscle Symptoms"
Abstract: Statin drugs are lifesaving drugs that reduce the incidence of cardiac events in men and women by approximately ~40%. They are among the most frequently prescribed drugs in the U.S and world. Statins have few side effects but can produce statin-associated muscle symptoms (SAMS) consisting of pain, cramps and weakness that reduce medication adherence and quality of life. SAMS are difficult to diagnose since there are no validated questionnaires or biomarkers, and at least 30-50% of patients on statins with self-reported SAMS actually have non-specific muscle complaints unrelated to statin use. Despite this, the majority (~60%) of adults who discontinue statins report SAMS as the primary reason for statin non-adherence and discontinuation. The absence of predictors and markers for SAMS makes its diagnosis and management difficult, forcing clinicians to rely entirely on clinical complaints and drug cessation. The absence of validated methods to diagnose SAMS also has major public health importance because patients with statin intolerance have an increased risk of cardiovascular disease events and incur substantially higher healthcare costs as a result of these events. Resultantly, the need for an effective biomarker of SAMS that can be used clinically to diagnose and confirm SAMS is critical for improving statin adherence and managing SAMS. We recently received American Heart Association (AHA) funding (#17GRNT33661247) to investigate the effectiveness of eight muscle-specific miRNAs, nonprotein-coding RNA molecules that regulate cellular function at the posttranscriptional level, as biomarkers for the detection of muscle damage and possibly SAMS. We seek to utilize the current funding mechanism to first perform Illumina small RNA sequencing in order to expand our investigation beyond muscle-specific miRNAs to all circulating miRNAs and ultimately refine our proposed candidate panel of miRNAs implicated in SAMS. The final candidate panel of eight miRNAs will then be examined as planned in the AHA proposal via real-time reverse transcription polymerase chain reaction (RT-qPCR) analysis of archived samples from an ongoing randomized, cross-over clinical trial of 40 patients with a previous history of SAMS treated with 20 mg simvastatin and placebo for two, 8 week treatment periods separated by a 4 week washout. Specific Aim 1 will use Illumina small RNA sequencing to explore and identify novel circulating miRNAs related to SAMS in a small subset of patients (n=6) with confirmed SAMS. Specific Aim 2 (as funded in the AHA proposal) will determine whether a targeted panel of eight candidate miRNAs, chosen from the quantitative analyses in Specific Aim 1, can then differentiate between patients with confirmed SAMS and those with non-specific muscle complaints. The addition of Specific Aim 1 greatly improves our scientific approach over the current funded AHA proposal, which, due to funding constraints, limits us to a generalized panel of a priori muscle-specific miRNAs. We hypothesize that patients with confirmed SAMS will be distinguished from all other patients by significant alterations in one or more miRNAs during simvastatin treatment relative to placebo, demonstrating efficacy of selected miRNAs as biomarkers that may eventually be utilized by clinicians and researchers to diagnose, treat and study SAMS.
ISG Cores Used: CBC and CGI

"RNAseq to ascertain hepatic differentiation pathways mediated by lncRNAs and hepatic exosome responses to drug-induced toxicity."
Abstract: We propose to use RNAseq to generate additional preliminary data to support two related but distinct lines of research having to do with hepatocyte biology. The overall goal is to enhance my lab’s position to obtain significant extramural funds and publication. The projects are as follows: Project 1: Analysis of transcriptional regulatory pathways governed by a novel long noncoding RNA (lncRNA) required for differentiation to hepatocytes. Project 2: Identification of lncRNA and miRNA biomarkers of hepatotoxicity in exosomes and extracellular vesicles (EX/EVs) secreted by hepatocytes. For Project 1, we have completed all research needed for a significant publication now in preparation. Developmentally-primitive hepatoblasts are bi-potent and differentiate into both mature hepatocytes and biliary epithelial cells. My lab discovered a novel lncRNA (lnc76) that is required for the development of mature hepatocytes using an inducible shRNA approach. We propose here to use RNAseq to determine which mRNAs are perturbed in the human transcriptome in lnc76-deficient versus control cells. In project 2, we aim to discover biomarkers consisting of lncRNAs and microRNAs present in EX/EVs secreted from human hepatocytes exposed to acetaminophen and ethanol. There is a significant unmet medical need for human “liquid biopsies” to detect hepatocellular toxicity in response hepatotoxic levels of both acetaminophen and ethanol.
ISG Core Used: CGI

Abstract: Aging is the greatest risk factor for most common chronic diseases including cancer, heart disease, osteoporosis, dementia and diabetes, conditions that are collectively responsible for most morbidity and health care costs in older adults. Growing evidence indicates that targeting fundamental aging mechanisms such as cellular senescence has the potential to slow the progression of these chronic conditions as a group. My research goal is to design novel interventions to target cellular senescence in order to enhance life quality and lifespan in older adults by slowing the onset or progression of multiple chronic diseases. To achieve this, we must have a better understanding of cellular senescence. In this proposal, we will leverage single cell transcriptomic (SCT) technology to comprehensively compare young versus aged mouse tissues at single cell level in an unbiased manner. We will specifically examine naturally occurring senescent (SEN) cells in these tissues, with the goal of finding a common transcriptomic signature for these cells in different cell types, thus allowing the identification of pathways to target precisely.
ISG Core Used: SCGC