Opioids such as morphine are frequently used in the clinic to treat pain. However, the perennial bane of chronic opioid use is the rapid development of tolerance to the analgesic effects but delayed development of tolerance to the respiratory depressant and constipating effects. As constipation is one of the most common opioid-related adverse effects in humans, it is important to delineate mechanisms that drive opioid tolerance in the ileum and lack of it in the colon. The overarching goal of this thesis was to investigate mechanisms of morphine tolerance in the ileum by comparing the mechanism of morphine tolerance in ileum myenteric plexus neurons and dorsal root ganglia (DRG) neurons. Myenteric plexus neurons are integral to the motor function of the ileum, whereas DRG neurons are important components of peripheral nociceptive sensation. We also examined the mechanism of morphine tolerance development to small intestinal transit and to antinociception at the systemic level in male and female mice. Studies presented in this dissertation demonstrate that the mechanism of morphine tolerance in the mouse ileum is not contingent on b-arrestin 2. In fact, tolerance in the small intestine might be mediated by a b-arrestin 2-independent mechanism following protein kinase C-induced phosphorylation of the m-opioid receptor. We also demonstrate that morphine tolerance to antinociception is not solely dependent on b-arrestin 2, and is mediated by b-arrestin 2-dependent and-independent mechanisms. Lastly, we have shown how sex of the animal can influence mechanisms underlying the development of morphine tolerance. Collectively, the findings presented here increase our understanding of the mechanisms by which morphine tolerance develops in the ileum and to antinociception.

Tissue-resident stem cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells self-renew or differentiate are highly context dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem cells (McSCs) are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified adult McSCs in the zebrafish. To elucidate mechanisms governing McSC self-renewal and differentiation fates we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for McSCs, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of McSC direct differentiation. Analysis of the scRNAseq dataset also revealed a population of mitfa/aox5 co-expressing cells that divides following melanocyte destruction, likely corresponding to cells that undergo self-renewal. Our findings show how different subpopulations of mitfa-positive cells underlie regeneration and differentiation of at least one subpopulation requires reactivation of developmental KIT signaling to properly reconstitute the melanocyte stripe.

Integrins have the ability to impact major aspects of epithelial biology including adhesion, migration, invasion, signaling and differentiation, as well as the formation and progression of cancer (Hynes 2002; Srichai and Zent 2010; Anderson et al. 2014). This thesis focuses on how integrins are regulated and function in the context of mammary epithelial biology and breast cancer with a specific focus on the α6 integrin heterodimers (α6β1 and α6β4). These integrins function primarily as receptors for the laminin family of extracellular matrix (ECM) proteins and they have been implicated in mammary gland biology and breast cancer (Friedrichs et al. 1995; Wewer et al. 1997; Mercurio et al. 2001; Margadant and Sonnenberg 2010; Muschler and Streuli 2010; Nistico et al. 2014). The first project investigates how alternative splicing of the α6 subunit impacts the genesis and function of breast cancer stem cells (CSCs). This work revealed that the α6Bβ1 splice variant, but not α6Aβ1, is necessary for the function of breast CSCs because it activates the Hippo transducer TAZ (Zhao et al. 2008a), which is known to be essential for breast CSCs (Cordenonsi et al. 2011). My work also led to the discovery that laminin (LM) 511 is the specific ligand for α6Bβ1 and that autocrine LM511, which is mediated by TAZ, is needed to sustain breast CSCs by functioning as a ‘ECM niche’. An important aspect of this study is the finding that surface-bound LM511 characterizes a small population of cells in human breast tumors with CSC properties. The second project of my thesis concentrated on identifying transcription factors that regulate expression of the β4 subunit. The expression of the α6β4 integrin is repressed during the epithelial-mesenchymal transition (EMT) (Yang et al. 2009) but the contribution of specific transcription factors to this repression is poorly understood. This study revealed that Snai1 is a transcriptional repressor of β4, which is responsible for establishing the PRC2 (Polycomb complex 2)- associated repressive histone mark H3K27Me3. However, I also found that the ability of Snai1 to repress transcription is abrogated by its interaction with Id2. Specifically, I identified the biochemical mechanism for how Id2 regulates Snai1. Id2 binds the SNAG domain of Snai1 that is the docking site for several corepressors (Peinado et al. 2004; Lin et al. 2010b; Dong et al. 2012a). One important consequence of Id2 interacting with Snai1 on the β4 promoter is that it prevents repressive epigenetic modifications. This finding may explain why some epithelial cells express Snai1 and β4 because they also express Id2 (Vincent et al. 2009; Bastea et al. 2012). The repression of the α6β4 integrin during the EMT is consistent with data indicating that this integrin is not expressed in CSCs (Mani et al. 2008; Goel et al. 2012; Goel et al. 2013; Goel et al. 2014). An important question going forward is to understand how the α6β4 integrin contributes to tumor formation. In summary, my thesis provides novel insights into the biology of the α6 integrins that has important implications for the function of these integrins in mammary gland biology and breast cancer, especially our understanding of breast CSCs.

Maintaining cellular homeostasis is a complex task, which involves monitoring energy states and essential nutrients, regulating metabolic fluxes to accommodate energy and biomass needs, and preventing buildup of potentially toxic metabolic intermediates and byproducts. Measures aimed at maintaining a healthy cellular economy inherently depend on the composition of nutrients available to the organism through its diet. We sought to delineate links between dietary composition, metabolic gene regulation, and physiological responses in the model organism C. elegans. As a soil-dwelling bacterivore, C. elegans encounters diverse bacterial diets. Compared to a diet of E. coli OP50, a diet of Comamonas aquatica accelerates C. elegans developmental rate, alters egg-laying dynamics and shortens lifespan. These physiological responses are accompanied by gene expression changes. Taking advantage of this natural, genetically tractable predator-prey system, we performed genetic screens i) in C. elegans to identify regulators of diet-responsive genes, and ii) in E. coli and Comamonas to determine dietary factors driving transcriptional responses in C. elegans. We identified a C. elegans transcriptional program that regulates metabolic genes in response to vitamin B12 content in the bacterial diet. Interestingly, several B12- repressed metabolic genes of unknown function are highly activated when B12- dependent propionyl-CoA breakdown is impaired, and inactivation of these genes renders animals sensitive to propionate-induced toxicity. We provide genetic and metabolomic evidence in support of the hypothesis that these genes form a parallel, B12-independent, β-oxidation-like propionate breakdown shunt in C. elegans, similar to the pathway utilized by organisms like yeast and plants that do not use vitamin B12.

Sterile particles underlie the pathogenesis of numerous inflammatory diseases. These diseases can often become chronic and debilitating. Moreover, they are common, and include silicosis (silica), asbestosis (asbestos), gout (monosodium urate), atherosclerosis (cholesterol crystals), and Alzeihmer’s disease (amyloid Aβ). Central to the pathology of these diseases is a repeating cycle of particle-induced cell death and inflammation. Macrophages are the key cellular mediators thought to drive this process, as they are especially sensitive to particle-induced cell death and they are also the dominant producers of the cytokine responsible for much of this inflammation, IL-1β. In response to cytokines or microbial cues, IL-1β is synthesized in an inactive form (pro-IL-1β) and requires an additional signal to be secreted as an active cytokine. Although a multimolecular complex, called the NLRP3 inflammasome, controls the activation/secretion of IL-1β (and has been thought to also control cell death) in response to particles in vitro, the in vivo inflammatory response to particles occurs independently of inflammasomes. Therefore, I sought to better understand the mechanisms governing IL-1β production and cell death in response to particles, focusing specifically on the role of lysosomal cathepsin proteases. Inhibitor studies have suggested that one of these proteases, cathepsin B, plays a role in promoting inflammasome activation subsequent to particle-induced lysosomal damage, however genetic models of cathepsin B deficiency have argued otherwise. Through the use of inhibitors, state-of-the-art biochemical tools, and multi-cathepsin-deficient genetic models, I found that multiple redundant cathepsins promote pro-IL-1β synthesis as well as particle-induced NLRP3 activation and cell death. Importantly, I also found that particle-induced cell death does not depend on inflammasomes, suggesting that this may be why inflammasomes do not contribute to particle-induced inflammation in vivo. Therefore, my observations suggest that cathepsins may be multifaceted therapeutic targets involved in the two key pathological aspects of particle-induced inflammatory disease, IL-1β production and cell death.

Regulation of gene expression comprises a wide range of mechanisms that control the abundance of gene products in response to environmental and developmental changes. These biological processes can be modulated by posttranslational modifications including arginine methylation. Among the enzymes that catalyze the methylation, protein arginine methyltransferase 7 (PRMT7) is known to modify histones to repress gene expression. Jumonji domain-containing protein 6 (JMJD6) is a putative arginine demethylase that potentially antagonize PRMT7. However, the biological significance of these enzymes is not well understood. This thesis summarizes the investigation of both PRMT7 and JMJD6 in cell culture models for adipocyte differentiation. The results suggest that PRMT7 is not required for the differentiation, whereas JMJD6 is necessary for the differentiation by promoting the expression of the lineage determining transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancerbinding proteins (C/EBPs). The underlying mechanisms by which JMJD6 regulate differentiation involve transcriptional and post-transcriptional control of gene expression. Unexpectedly, the adipogenic function of JMJD6 is independent of its enzymatic activity. Collectively, the present research reveals a novel role of JMJD6 in gene regulation during the differentiation of adipocytes.

Chronic exposure of humans or rodents to high calorie diets leads to hypertriglyceridemia and ectopic lipid deposition throughout the body, resulting in metabolic disease. Cellular lipids are stored in organelles termed lipid droplets (LDs) that are regulated by tissue-specific LD proteins. These proteins are critical for lipid homeostasis, as humans with LD protein mutations manifest metabolic dysfunction. Identification of novel components of the LD machinery could shed light on human disease mechanisms and suggest potential therapeutics for Type 2 Diabetes. Microarray analyses pinpointed the largely unstudied Hypoxia-Inducible Gene 2 (Hig2) as a gene that was highly expressed in obese human adipocytes. Imaging studies demonstrated that Hig2 localized to LDs in mouse hepatocytes and the human SGBS adipocyte cell line. Thus, this work examined the role of Hig2 as a LD protein in liver and adipose tissue. Hig2 deficiency reduced triglyceride deposition in hepatocytes; conversely, ectopic Hig2 expression promoted lipid deposition. Furthermore, liver-specific Hig2-deficient mice displayed improved glucose tolerance and reduced liver triglyceride content. Hig2 deficiency increased lipolysis and -oxidation, accounting for the reduced triglyceride accumulation. Similarly, adipocyte-specific Hig2-deficient mice displayed improved glucose tolerance, reduced adipose tissue weight and brown adipose tissue that was largely cleared of lipids. These improvements were abrogated when the animals were placed in thermoneutral housing and brown adipocyte-specific Hig2-deficient mice also displayed improved glucose tolerance, suggesting that active brown fat largely mediates the metabolic phenotype of Hig2 deletion. Thus, this work demonstrates that Hig2 localizes to LDs in liver and adipose tissue and promotes glucose intolerance.

Adipose tissue is one of the most dynamic tissues in the body and is vital for metabolic homeostasis. In the case of excess nutrient uptake, adipose tissue expands to store excess energy in the form of lipids, and in the case of reduced nutrient intake, adipose tissue can shrink and release this energy. Adipocytes are most functional when the balance between these two processes is intact. To understand the molecular mechanisms that drive insulin resistance or conversely preserve the metabolically healthy state in obese individuals, our laboratory performed a screen for differentially regulated adipocyte genes in insulin resistant versus insulin sensitive subjects who had been matched for BMI. From this screen, we identified the type II transmembrane protein tenomodulin (TNMD), which had been previously implicated in glucose tolerance in gene association studies. TNMD was upregulated in omental fat samples isolated from the insulin resistant patient group compared to insulin sensitive individuals. TNMD was predominantly expressed in primary adipocytes compared to the stromal vascular fraction from this adipose tissue. Furthermore, TNMD expression was greatly increased in human preadipocytes by differentiation, and silencing TNMD blocked adipogenic gene induction and adipogenesis, suggesting its role in adipose tissue expansion. Upon high fat diet feeding, transgenic mice overexpressing Tnmd specifically in adipose tissue developed increased epididymal adipose tissue (eWAT) mass without a difference in mean cell size, consistent with elevated in vitro adipogenesis. Moreover, preadipocytes isolated from transgenic epididymal adipose tissue demonstrated higher BrdU incorporation than control littermates, suggesting elevated preadipocyte proliferation. In TNMD overexpressing mice, lipogenic genes PPARG, FASN, SREBP1c and ACLY were upregulated in eWAT as was UCP-1 in brown fat, while liver triglyceride content was reduced. Transgenic animals displayed improved systemic insulin sensitivity, as demonstrated by decreased inflammation and collagen accumulation and increased Akt phosphorylation in eWAT. Thus, the data we present here suggest that TNMD plays a protective role during visceral adipose tissue expansion by promoting adipogenesis and inhibiting inflammation and tissue fibrosis.

Cellular senescence is thought to play an essential role in many biological functions including tumor suppression and organismal aging. Senescent cells, which are permanently removed from the cell cycle, can be found both in vivo in many different tissue types and in vitro within cultures of non-immortalized cells. Despite their inability to proliferate, these cells persist and remain metabolically active for indefinite periods of time. This physiologic process occurs in response to a variety of cellular insults including oxidative stress, shortened telomeres, constitutive oncogene expression, and DNA damage, and can be initiated by upregulation of one of the two known senescent pathways, involving p16/Rb or p53/p21. The senescent cell phenotype is also characterized by changes to cell and nuclear morphology and to the secretory profile of the cell. Related to changes in nuclear morphology, epigenetic modifications to the packaging of DNA are thought to be key to the initiation and maintenance of the senescence program. While a large number of earlier studies focused on the findings that senescent cells gain regions of condensed heterochromatin, often in the form of Senescent Associated Heterochromatin Foci (SAHF), this thesis work shows that there is a marked loss of heterochromatin in the peri/centromeric regions of the genome. In fact, both α-satellite and satellite II sequences across the genome distend in a striking and unanticipated fashion; this can be readily visualized by fluorescence in situ hybridization (FISH) as their structure changes from a condensed spot to highly elongated and fine thread-like signals. We have termed this exceptional decondensation of constitutive heterochromatin Senescence Associated Distension of Satellites (SADS). Importantly, a series of experiments shows that SADS is both a consistent and an early event in the cell senescence process, which occurs as a result of every senescence induction method examined. We also observed that this distension was characteristic of both human and murine cells and in vivo in human benign Prostatic Intraepithelial Neoplasia (PIN) tissue. Furthermore, unlike SAHF formation, SADS can occur due to the activation of either of the two senescence pathways, p16/Rb or p53/p21. Additionally, the cytological dimensions of the thread-like satellite signals indicates that SADS represents “unraveling” of DNA on an unprecedented scale. Thus, it was surprising that this event was not facilitated by changes to several canonical histone modifications associated with condensed heterochromatin, namely H3K9Me3, H3K27Me3, or H3K4Me3, nor is it caused by loss of DNA methylation. Consequently, we believe that this marked distension of satellite DNA is due to changes in higher-order folding of the chromatin fiber. This is important for understanding fundamental events in the cell senescence process, but also provides a unique system for study of chromatin packaging that may provide new insights into the organization of DNA well beyond nucleosome packaging and the ten nanometer fiber. In fact, initial super resolution images of SADS suggest that the satellite sequences may be organized into domains or “globules”. Hence, we suggest that the changes to satellite sequence packaging may be facilitated by changes to higher-order nuclear structural proteins, such as LaminB1, which is reduced in senescent cells. Finally, this work provides analysis of the literature and preliminary experiments to consider the possibility that there are increased levels of cell senescence in Down syndrome (trisomy 21) cells. As individuals with Down syndrome (DS) experience many manifestations of premature aging (including early-onset Alzheimer’s Disease), have a resistance to solid tumor formation, are more susceptible to oxidative stress, and are trisomic for several genes implicated in causing senescence, our analysis provides plausibility for the hypothesis that accelerated rates of senescence may play a significant role in DS physiology. We also provide results of preliminary studies and outline the next steps for experimentation, using DS fibroblasts and a unique genetically engineered DS iPS cell system. As a final note, the quantification of cell senescence in trisomic versus disomic cells for these experiments relies substantially on the new single-cell marker of senescence discovered and established by this theses work, the Senescence-Associated Distension of Satellites.

The following work demonstrates that chromatin regulators play far more pronounced roles in dynamic gene expression than they do in steady-state. Histone modifications have been associated with transcription activity. However, previous analyses of gene expression in mutants affecting histone modifications show limited alteration. I systematically dissected the effects of 83 histone mutants and 119 gene deletion mutants on gene induction/repression in response to diamide stress in yeast. Importantly, I observed far more changes in gene induction/repression than changes in steady-state gene expression. The extensive dynamic gene expression profile of histone mutants and gene deletion mutants also allowed me to identify specific interactions between histone modifications and chromatin modifiers. Furthermore, by combining these functional results with genome-wide mapping of several histone modifications in the same time course, I was able to investigate the correspondence between histone modification occurrence and function. One such observation was the role of Set1-dependent H3K4 methylation in the repression of ribosomal protein genes (RPGs) during multiple stresses. I found that proper repression of RPGs in stress required the presence, but not the specific sequence, of an intron, an element which is almost unique to this gene class in Saccharomyces cerevisiae. This repression may be related to Set1’s role in antisense RNA-mediated gene silencing. Finally, I found a potential role for Set1 in producing or maintaining uncapped mRNAs in cells through a mechanism that does not involved nuclear exoribonucleases. Thus, deletion of Set1 in xrn1Δ suppresses the accumulation of uncapped transcripts observed in xrn1Δ. These findings reveal that Set1, along with other chromatin regulators, plays important roles in dynamic gene expression through diverse mechanisms and thus provides a coherent means of responding to environmental cues.