Human Topoisomerase 1 (TOP1) is a dynamic enzyme that functions to overcome topological constraints on the DNA that are caused by strand separation. In this study, we demonstrate that TOP1 is an RNA binding protein (RBP) as revealed by ultraviolet-crosslinked RNA immunoprecipitation (UV-RIP) analyses at intergenic and promoter regions in colon cancer cells treated with acute doses of tumor necrosis alpha (TNF-. Through biochemical methods, we found that TOP1 directly interacts with different RNAs. Furthermore, proteomic analyses reveal that RNAs add an additional layer of regulation by contributing to the maintenance of TOP1 ribonucleoprotein complexes.

While the dynamic crosstalk between cancer and immune cells within the tumor microenvironment have been extensively investigated, the mechanisms by which immune signaling drives alterations in the cancer cell transcriptome remain to be fully examined. Notably, cytokines such as TNF-α that are released by immune cells can serve as tumor promoting signals to trigger activation of pro-oncogenic networks by the master inflammatory regulator, NFκB. Importantly, tumor promoting properties of inflammation are further regulated by various genetic aberrations in cancer cells. Of special interest are gain-of-function (GOF) mutations in the tumor suppressor gene, p53 which occur in approximately 50% of all human cancers and have been established to promote inflammation-induced oncogenesis.

in this dissertation, I provide mechanistic insights into the various modes by which mutant p53 fuels proinflammatory gene expression programs in colon cancer cells. First, I established that mutant p53 and NFκB redirect each other’s binding to distinct subsets of distal regulatory elements, commonly referred to as enhancers in response to chronic TNF signaling. Notably, this co-occupancy by mutant p53 and NFκB results in activation of such pro-tumorigenic enhancers. I also found that mutant p53 regulates the enhancer occupancy of the RNA polymerase II machinery and supports the biogenesis of nascent transcripts known as enhancer RNAs (eRNAs). Importantly, this pro-inflammatory eRNA signature was dependent on mutant p53 and not detected in cancer cell lines or human nonneoplastic tissues that expressed wild type p53.

Our group has since revealed the consequences of mutant p53-dependent eRNAs in gene regulation by establishing that these transcripts form complexes with the bromodomain and extra-terminal motif (BET) protein, BRD4 which often occupies active enhancers and regulates inflammatory and oncogenic programs. In this dissertation, I further explore the significance of transcriptional cofactors also functioning as noncanonical RNA binding proteins by establishing DNA topoisomerase 1 (TOP1) as an RNA interacting factor and demonstrating TOP1’s dependency on RNA associations to maintain its native protein interactome. Collectively, these findings define previously unrecognized mechanisms by which mutant p53 promotes inflammation-induced tumorigenesis through modulation of enhancer activity and underscore critical consequences of RNA-protein complexes.

As e-cigarette use continues to rise in popularity, many have questioned how e-cigarette vapor inhalation actually affects mammalian systems. Because >99% of commercially available e-cigarettes contain nicotine, and nicotine has been found to have effects on cardiac physiology, we hypothesized that daily inhalation of e-cigarette vapor (EV) would lead to alterations in both heart rate (HR) and blood pressure (BP). This hypothesis is supported by recent human data showing increases in HR and BP after inhaling EV once (acute exposure). HR variability has been highlighted as a sign of cardiac dysfunction, with increased risk of adverse cardiac outcomes. These experiments were designed to evaluate HR, HR variability and BP of mice exposed to nicotine containing e-cigarette vapor daily for 3 months, compared to controls.

10 week-old C57BL/6 mice were obtained from Jackson Labs. Mice were placed into the full body SciReq inExpose system where they were exposed for 60min daily to freshly made EV for 3 months. The e-liquid used contained 70% propylene glycol, 30% vegetable glycerin and 6 mg/mL nicotine. At least once weekly during each exposure time point, mice were measured for their heart rate (HR), heart rate variability (HRV), and blood pressure (BP). The mouse’s temperature was taken to ensure temperatures were between 35-37C during observation. Two cuffs were placed on the tail to measure both BP (Kent Scientific) over 5-10min. The EMKA tech ECGtunnel was used to measure HR and HRV in 15 minute intervals both pre-exposure and post-exposure.

The results from our data suggest that chronic inhalation of nicotine containing e-cigarette vapor (EV) affects cardiac function. Although it is know that in humans heart rate increases acutely during nicotine inhalation (demonstrated by both cigarette smoke and EV inhalation studies), our results from a mouse model of chronic inhalation imply that over repeated exposures, HR tend to decrease. E-cigarette exposed mice also showed signed of higher HR variability, SBP, DBP, and MAP. These results imply that e-cigarette users may be at risk for adverse cardiac physiological effects.

Traditional use of the yeast two-hybrid (Y2H) system is primarily for identifying unknown protein interactors to proteins of interest. Alternatively, Y2H can be adapted to make quantitative measurements of interaction strength, but there are some obstacles. For example, unequal expression of the proteins of interest will lead to false comparisons and using slightly toxic proteins can decrease cell viability. To alleviate this issue, we sought to control expression by implementing an inducible CUP1 promoter design using the major nuclear export factor, Nxf1, as bait. We observed that induction increased expression levels above that of the traditionally used constitutive ADH1 promoter in the pGBK vector. Furthermore, we showed that CUP1 driven expression levels are titratable and dependent on copper concentrations. Lastly, our findings suggest that we could quantify the changes in growth and therefore interaction strength using a liquid growth assay. Thus, this modified assay has the potential to allow for comparisons of protein interaction strength using the Y2H system.