The genome is hierarchically organized into DNA-looped domains ranging from kilobase to megabase in size, largely shaped by two architectural protein factors: cohesin and CCCTC-binding factor (CTCF). DNA loops impact gene expression either directly via looping between genes and regulatory elements (i.e., enhancers) or indirectly by limiting available regulatory element interactions. In mouse liver, there are ~1,000 sex- biased genes whose activity and expression are modulated by ~5,000 mostly distal sex- biased enhancers. The impact of distal enhancer-promoter communication on sex-biased gene expression in mouse liver is explored in this thesis. First, I present a computational method to predict tissue-conserved DNA loops mediated by CTCF and cohesin using ChIP-seq and sequence information alone. Functionally, these loops indirectly guide enhancer-promoter interactions by insulating chromatin interactions, as is shown for the v Albumin gene. Next, I directly compared male and female CTCF and cohesin binding in mouse liver to identify ~1,000 sex-biased binding sites for each factor, the majority of which are distal to sex-biased genes. Chromatin interaction analysis revealed sex-biased enhancer-promoter and promoter-promoter interactions neighboring highly sex-biased genes. CTCF and cohesin contribute directly (as for A1bg and Sult3a2) and indirectly (as for C9, Nudt7, and Nox4) to chromatin interactions, and cohesin may be necessary for proper expression of distally-regulated male-biased genes. Finally, I identified a core subset of sex-biased genes and enhancers common between two genetically distant mouse strains, C57Bl/6J and CAST/EiJ, as well as genes and enhancers unique to each strain, reflecting their divergent evolution. The shared sex-biased genes are highly liver- specific in their function and expression and likely are the consequence of sex-specific selective evolutionary pressure. Strain-specific genetic variants were used to identify enhancers important for the expression of 481 sex-biased genes associated with genetic variants at 491 significantly contributing loci. A substantial fraction (36%) of all strain- specific, sex-biased enhancers overlap genetic variants within these 491 loci, evidencing the role of genetic disruption at distal sex-biased enhancers. Together, this research characterizes distal enhancer function in relation to gene expression using sex differences in mouse liver as a framework, with both DNA looping and genetic variation as useful tools to better understand genomic regulation. / 2022-02-14T00:00:00Z

Factor Quinolinone Inhibitor 1 (FQI1) is a novel anti-cancer compound, having shown efficacy against hepatocellular carcinoma (HCC) in multiple murine tumor models with no detectable toxicity at effective doses. FQI1 exerts its anti-proliferative activity against HCC cells by disrupting mitotic spindles and inhibiting the attachment between mitotic microtubules and kinetochores, thereby causing a mitotic arrest culminating in cell death or senescence. As patients suffering from HCC are challenged with a high mortality rate and HCC treatment options are limited, there is a dire need for developing new chemotherapeutics against HCC. In light of this, FQI1s are particularly promising for further development towards HCC chemotherapeutics. Previous studies focused on the impact of FQI1 solely in mitosis. In this dissertation, I report on the cytoskeletal and morphological effects of FQI1 in interphase immortalized fetal hepatocytes and retinal pigment epithelial cells. I established that FQI1 drastically and rapidly reduces cell spreading and increases circularity in non-mitotic cells. These morphological phenotypes are preceded by a sudden breakdown of microtubules. FQI1 also decreases the range of locomotion of interphase cells and reduces their ability to close wound-gaps, supporting an impact of FQI1-induced microtubule breakdown on cell migration. The FQI1-induced microtubule destabilization activates the RhoA/ROCK contractility pathway, which plays a role in FQI1-mediated reduction of cell spreading. Taken together, I demonstrated that FQI1 interferes with microtubule-associated functions outside of mitosis that specifically regulate cell morphology, cell motility and contractility. As cancer cells rely on non-mitotic processes, most notably cell migration, to exert their malignancy, the findings presented here expand the potential of FQIs as effective and clinically successful anti-cancer compounds. Finally, I document the potential utility of nanoparticles for intravenous delivery of lipophilic compounds to the liver, using FQI1 as an example. When FQI1 was encapsulated into expansile nanoparticles (eNPs), the eNPs were effectively internalized by hepatocytes in vitro and released FQI1 to exert growth inhibition. In addition, intravenously injected eNPs predominantly targeted the liver with minimal, if any, acute toxicity. As a proof of concept, I therefore show that lipophilic compounds including FQIs could be formulated with eNPs for treatment against liver diseases, including HCC. / 2022-11-01T00:00:00Z

Human pulmonary alveolar type II cells (AT2s) are facultative progenitors of the distal lung epithelium and secrete surfactant, a protein-lipid mixture that lowers alveolar surface tension and prevents alveolar collapse during expiration. AT2s exist at a physiological interface between inhaled air and pulmonary vasculature, and are among the pulmonary cell types that are directly exposed to inhaled environmental stimuli such as respiratory viruses and cigarette smoke. Primary human AT2s can be cultured in vitro in three-dimensional organoids known as alveolospheres, but are difficult to culture in the physiologically relevant air-liquid interface (ALI) format due to their tendency to lose their AT2 phenotype and senesce in culture. Human induced pluripotent stem cells (iPSCs) can be directed to differentiate to iPSC-derived AT2s (iAT2s) in three-dimensional spheres, where they transcriptomically resemble primary human fetal lung. Here we report the successful adaptation of iAT2s to ALI culture, which promotes their maturation and permits exposure to inhaled environmental stimuli. We transcriptomically profile iAT2s cultured at ALI and find that they upregulate key markers of AT2 maturation as they downregulate cell cycle-associated transcripts. We then evaluate the extent of iAT2 maturation at ALI within the developmental context by transcriptomic comparison to cultured and freshly isolated primary AT2s. We find that iAT2s cultured at ALI are more similar to primary AT2s than iAT2s cultured as spheres, and that the main differences between iAT2s at ALI and primary AT2s are due to primary AT2s’ response to immune stimuli. We then test the capacity of iAT2s to respond to immune stimuli and serve as useful in vitro model system for human respiratory viral infections by infecting iAT2s at ALI with SARS-CoV-2, the virus that causes Coronavirus Disease 2019 (COVID-19). We find that iAT2s are permissive to SARS-CoV-2 infection, mount an epithelial-intrinsic interferon and inflammatory response to infection, and can serve as an in vitro platform for testing antiviral therapeutics. Finally, we demonstrate that iAT2s at ALI also have utility as a system for modeling the response to cigarette smoke and electronic cigarette vapor, enabling the direct comparison of these two common inhaled noxious stimuli. Overall, we describe a novel disease modeling platform that enables future exploration of gene-environment interactions unique to inhaled exposures of the alveolar epithelium.

Human and animal waste are prominent anthropogenic contaminants in aquatic ecosystems which come from a variety of sources including septic tanks, stormwater, and agricultural runoff. The Virginia Eastern Shore (VAES) is a rural coastal landscape with numerous potential sources of fecal contamination, including an expanding poultry industry and a historical reliance on septic systems. The small watersheds of the VAES combined with agricultural and residential land use in close proximity to tidal creeks make this a model landscape for examining the impacts of upland land use and watershed characteristics on human and animal waste contamination in adjacent waters. However, traditional methods such as the use of fecal indicator bacteria (FIB) to monitor contamination in waterbodies cannot identify the source of fecal contamination. This study developed an environmental DNA (eDNA) source-tracking method using quantitative PCR (qPCR) to detect the 16S rRNA gene from the chicken mitochondrial genome and the NADH dehydrogenase 5 (ND5) gene from the human mitochondrial genome. This new method demonstrated 100% sensitivity and specificity in chicken and human feces and was subsequently applied to six tidal creeks on the VAES with varying levels of upstream poultry operations and residential development. Overall, chicken and human fecal contamination was widespread across the VAES, with levels of human contamination exceeding those of chicken contamination. Metabarcoding analysis of mammalian mitochondrial 16S rRNA genes identified cattle and domestic dogs as additional sources of fecal contamination. Chicken fecal contamination was found in creeks with poultry operations and manure application upstream, while human fecal contamination was highest in a creek with high population density and failing septic systems. Temporal patterns in chicken fecal contamination followed the general trends of broiler chicken production and manure application in Virginia, with a decrease in summer. Human fecal contamination was highest in summer, which coincided with the influx of tourists to the VAES; contamination and tourism were lower in Summer 2020 compared to Summer 2021, most likely due to the COVID-19 pandemic. Data were used in an Information-Theoretic approach to develop multiple linear regression models to predict human and chicken fecal contamination as a function of watershed characteristics; the human model was driven by temperature and population density, while the chicken model was driven by agricultural land cover and fecal coliforms. Models were used to scale fecal contamination across the entire VAES and estimate the impact of changes in anthropogenic land use. While both models predicted increases in contamination with increasing anthropogenic activity, human fecal contamination was less sensitive to these changes. Although these models are in the beginning stages of development, this proof-of concept approach provides a framework for scaling fecal contamination measured at specific sites to a larger region, and to estimate fecal contamination as a function of changes in anthropogenic land use. The integrative use of eDNA analysis and statistical modeling, together with traditional water quality monitoring data and GIS analysis, is a powerful approach for examining fecal contamination and analyzing the effects of anthropogenic land use changes on the water quality of downstream coastal ecosystems.

Thyroid hormone receptor (TR) is responsible for mediating the expression of genes involved in growth, development, and homeostatic regulation, in response to thyroid hormone. There are two main subtypes of TR, TRα1 and TRβ1, that mediate these physiological processes. Dysregulation of TR mediated processes is a contributing factor in disease pathology. Our prior studies show that TR is shuttled rapidly between the nucleus and cytosol, while localizing primarily to the nucleus. TRα1 contains two nuclear localization signals (NLSs) that act together to confer strong nuclear localization, while TRβ1 only contains one NLS (NLS-1). Recently, we found that the intracellular localization of TR is regulated, in part, by post-translational modification of lysines within NLS-1 by acetylation. We constructed GFP or mCherry-tagged TRα1 and TRβ1 acetylation-mimic and nonacetylation-mimic (unable to be endogenously acetylated) expression plasmids, transfected them into HeLa cells, and used fluorescence microscopy to determine the nuclear/cytosolic (N/C) ratio of the mutant receptors by measuring fluorescence intensity. Data show that the TR acetylation mimic has a significantly lower N/C ratio compared to wild-type TR, indicating a striking decrease in nuclear localization, whereas the TR nonacetylation mimic's N/C ratio was the same as wild-type TR. Previously, we showed that TR nuclear import is mediated by importins 7, β1, and α1. We hypothesized that acetylation state alters either TR's ability to bind importins or its intranuclear mobility, and that inhibition of the acetyltransferase CBP/p300 would lead to enhanced TR nuclear localization, while inhibition of the deacetylase SIRT1 would lead to TR cytoplasmic localization. Using GFP-Trap co-immunoprecipitation, we found that importins 7, β1, and α1 interact with both the TRα1 acetylation and nonacetylation mimics, suggesting that the TRα1 acetylation mimic's decreased nuclear localization is due to factors other than reduced importin binding. Intriguingly, fluorescence recovery after photobleaching (FRAP) revealed that the GFP-TRα1 nonacetylation mimic has a significantly slower rate of recovery than wild-type TRα1, suggesting that this reduced mobility correlates with greater nuclear retention. in contrast, the GFP-TRα1 acetylation mimic had the same rate of recovery as wild-type TRα1. Lastly, pharmacological inhibition of CBP/p300 by C646 increased GFP-TRβ1's nuclear localization in a dose-dependent manner (GFP-TRβ1 has a slight cytosolic population due in part to having one NLS); however, inhibition of SIRT1 by EX-527 did not alter GFP-TRα1's localization. Taken together, these data provide further evidence that acetylation plays a key regulatory role in TR shuttling and retention and extends our understanding of how acetylation interplays with TR-regulated processes of growth and development.

No description available.

This dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and the leading cause of infectious disease related deaths worldwide. The control of TB is limited by the lack of sensitive and specific diagnostic tools available at the point of care (POC). The studies presented here illustrate the advances in our technology for the detection and differentiation of M.tb and NTM. The use of BiDz sensors enables the selective recognition of DNA/RNA analytes containing single nucleotide polymorphisms associated with species-specific identification, drug susceptibility testing (DST) and strain typing. First, we developed a platform for the detection of M.tb and drug susceptibility using multiplex PCR and BiDz sensors. However, this method relies on the use of expensive instrumentation which is often not available in high TB burden countries. Therefore, additional studies focused on the development of tools for the detection of isothermal amplification products and the direct detection of rRNA. Based on these findings, we also developed an NTM species typing tool using BiDz sensors for species identification in ~1 hour. Despite the advantages of BiDz sensor technology, their use is limited to the detection of a few selected mutations. To address this limitation, we developed a 15-loci multiplex PCR followed by analysis with a custom microarray for high-throughput identification of SNPs. The work presented in this dissertation has the potential to enable the rapid, specific and sensitive identification of mycobacterial species necessary to reduce the diagnostic delay, ensure initiation of effective therapy, and prevent further transmission.

Cellular immortalization is a prerequisite of cancers and depends upon evasion of telomeric erosion that would otherwise lead to replicative senescence. A subset of cancers achieves telomere maintenance via a pathway known as Alternative Lengthening of Telomeres (ALT), which relies on homologous recombination that is driven by chronic DNA replication stress and allows for telomeric elongation events. Here, we sought to further elucidate the role of the annealing helicase SMARCAL1, which reverses and stabilizes stalled replication forks, in cancers that utilize ALT. SMARCAL1 is crucial to resolve the replication stress at ALT telomeres, but paradoxically is lost in a subset of ALT-positive tumors. Other common ALT-related mutations occur in the ATRX/DAXX complex, which deposits the histone variant H3.3 at telomeres. In contrast to loss of ATRX, we found that SMARCAL1 depletion does not affect H3.3 deposition, but does lead to changes in trimethylation of histone 3 lysine 9 that may create an ALT-permissive state. We also found that ALT-positive cell lines are more sensitive to combined depletion of SMARCAL1 and ATRX. Furthermore, we discovered that SMARCAL1 interacts with heterochromatin protein 1 (HP1), and that loss of SMARCAL1 deregulates the presence of HP1 at telomeres, providing a link to changes in histone methylation. We also identified a novel complex between SMARCAL1, HP1, and the histone methyltransferase SETDB1. Overall, our results indicate that SMARCAL1 is an important factor in telomeric chromatin formation, indicating a previously undescribed role for SMARCAL1 in genome maintenance.

This study focuses on the gene (glnA) encoding glutamine synthetase from the cyanobacterium Synechococcus sp. strain PCC 7942. A molecular analysis of the gene was initiated to test the hypothesis that expression of glnA is regulated at the level of transcription and that this regulation is reflective of global control of nitrogen metabolism in Synechococcus 7942. The method used for testing the hypothesis involved first isolating and sequencing the glnA gene and its flanking regions, and second, analyzing the RNA produced by the glnA gene by transcript mapping. Sequence analysis of the glnA gene revealed high homology within the open reading frame at the nucleotide level when compared with glnA from the cyanobacterium Anabaena 7120, and lower homology with glnA from other bacteria. Comparisons of the deduced amino acid sequences showed a similar pattern of highest homology between the cyanobacterial glutamine synthetases, with lower homologies in comparison with other bacteria. Northern analysis using the Synechococcus glnA gene as a hybridizing probe revealed a transcript of 1.6 kb, verifying expression of glnA. It was upon these three criteria--heterologous hybridization with the glnA gene of Anabaena 7120, sequence comparisons with several other bacterial glnA genes, and identification of a 1.6 kb transcript--that the open reading frame was identified as the structural gene (glnA) of glutamine synthetase. The results of transcript mapping indicate that transcription begins at a start site 142 nucleotides upstream of the translational start when cells are grown under either nitrogen sufficient or nitrogen deficient (N for 10 hours) conditions. When cells are grown in a medium lacking nitrogen for only 4.5 hours, the transcriptional start site maps to a position 139 nucleotides upstream of the translational start. The identification of two transcriptional start sites, 3 base pairs apart, might be an artifact inherent in the experimental strategy, or it may imply transcriptional control. A sequence consisting of TAGGAT is present 14 or 17 bases upstream of the two transcriptional starts, respectively, and is similar to other Synechococcus promoters (TAGAAT in psbA1 and TATTAT in psbA2) as well as the $-$10 promoter, (TATAAT), found in enteric bacterial $\sigma\sp{70}$ promoters. Two overlapping sequences, GTTACA and CAAAAG, are positioned in the $-$35 promoter region. The first of these resembles the $-$35 region for three light responsive genes (TTTACA for psbA1 and psbA2, and TTCACA for psbA3) from Synechococcus. It also resembles, TTGACA, found at $-$35 in unregulated promoters of enteric bacteria. The other sequence, CAAAAG, resembles two Anabaena 7120 $-$35 promoter regions, CAAAAC in glnA, and CATAAC in nifH, which are nitrogen regulated. Taken together, the presence of two potential $-$35 promoter regions and two transcriptional start sites, indicates transcriptional regulation of glnA in Synechococcus 7942.

MRP13 was defined by biochemical criteria as a 35-kilodalton small subunit protein of the yeast mitochondrial ribosome. The MRP13 gene was identified by immunological screening of a yeast genomic library in $\lambda$gt11 and a functional copy of the gene has been cloned on a 2.2-kilobase BglII fragment. Sequencing of this fragment showed that the MRP13 coding region specifies as 324-amino acid basic protein with a calculated M$\sb{\rm r}$ of 37,366. Computer searches failed to reveal any significant sequence similarity to previously identified ribosomal proteins or to the sequences in the current National Biomedical Research Foundation data base. Cells carrying disrupted copies of MRP13 lacked the MRP13 protein but were not impaired for growth on nonfermentable carbon sources. However, in comparison to the wild type, mrp13-$\Delta$2::TRP1 mutant cells had a lower rate of whole cell respiration, an unusual profile on in vivo labeled mitochondrial translation products and an abnormal profile of ribosomal subunits in sucrose gradient centrifugation. Mutants lacking MRP13 were also impaired in their ability to undergo the transition from growth on high concentrations of glucose to growth on nonrepressing carbon sources. This mutant phenotype suggests an important role for the MRP13 protein under conditions where cells are actively increasing their capacity for the synthesis of mitochondrial encoded proteins. Analysis of the sequence in the MRP13 5$\sp\prime$-flanking region revealed the closely linked gene for the cytoplasmic ribosomal protein RP39A. The RP39A coding region begins at nucleotide $-$846 and ends at $-$325 with respect to the MRP13 translational start. The steady-state levels of the MRP13 mRNA were determined in response to carbon catabolite repression, variation in the mitochondrial genetic background, and increased gene dosage of MRP13. In $\rho\sp+$ cells, transcript levels were repressed severalfold by growth in glucose as compared with growth in either galactose or nonfermentable carbon sources. In respiratory-deficient strains ($\rho\sp{\rm o,}$ mit$\sp-$), however, transcription appeared to be largely derepressed even in the presence of high concentrations of glucose. Thus, the MRP13 mRNA is a member of a class of yeast nucleus-encoded RNAs whose transcription responds to changes in the mitochondrial genetic background. Despite high levels of the MRP13 transcripts in $\rho\sp{\rm o}$ cells, the MRP13 protein did not accumulate, suggesting that the protein is relatively unstable in the absence of ribosome assembly. Cells carrying the MRP13 gene on a multiple-copy plasmid overproduced the mRNA in rough proportion to the gene dosage and the protein accumulated to a significant but lesser extent. The results indicate that MRP13 expression is regulated predominantly at the transcriptional level in response to catabolite repression and the cellular capacity for respiration. In addition, the levels of the MRP13 protein appear to be modulated posttranscriptionally by degradation of excess, unassembled polypeptides.