Chromatin is organized dynamically to accommodate different biological processes. One of the factors required for proper chromatin organization is a group of complexes called condensins. Most eukaryotes have two conserved condensins (I and II) required for chromosome segregation. C. elegans has a third condensin (IDC) that specializes in dosage compensation, a process that down-regulates X gene dosage in XX hermaphrodites to match the dosage in XO males. How the three condensins are regulated is not well understood. Here, I present the discovery and characterization of a novel condensin regulator, SMCL-1. We identified SMCL-1 through purification of a MAP-tagged condensin subunit. Condensins are comprised of SMC ATPases and regulatory CAP proteins; SMCL-1 interacts most abundantly with condensin SMC subunits and resembles the ATPase domain of SMC proteins. Interestingly, the SMCL-1 protein has residues that differ from SMC consensus and potentially render SMCL-1 incapable of hydrolyzing ATP. Worms harboring smcl-1 deletion are viable and show no detectable phenotype. However, deleting smcl-1 in a condensin hypomorph mildly suppresses condensin I and IDC mutant phenotypes, suggesting that SMCL-1 functions as a negative regulator of condensin I and IDC. Consistent with this, overexpression of SMCL-1 leads to condensin loss-of-function phenotypes such as lethality, segregation defects and disruption of IDC localization on the X chromosomes. Homology searches based on the unique ATPase domain of SMCL-1 reveal that SMCL-1-like proteins are present only in organisms also predicted to have condensin IDC. Taken together, we conclude that SMCL-1 is a negative modulator of condensin functions and we propose a role for SMCL-1 in helping organisms adapt to having a third condensin by maintaining the balance among three condensin complexes.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

Pleckstrin homology (PH) domains, which play an essential role in membrane trafficking and signal transduction, recognize phosphoinositides with a diverse range of affinities and specificities. The PH domains of the Grp1 family of Arf GTPase exchange factors recognize a select group of phosphoinositides with dramatic differences in specificity, despite 90% sequence identity. The work described in this thesis has focused on the structural basis for these differences. The structure of the Grp1 PH domain revealed structural determinants for phosphoinositide recognition. Through a wide range of crystallographic and biochemical means, the structural basis that accounts for the differential binding affinities amongst the Grp1 family PH domains has also been determined. Furthermore, examination of the structural details of these PH domains bound to different inositol phosphate groups have aided in understanding the structural mechanisms by which all PH domains recognize phosphoinositides.

Alcoholism is responsible for more than 6% of deaths internationally per annum. The development of acute tolerance to ethanol (EtOH) is a critical component of alcoholism. Previous studies identified large conductance calcium-activated potassium (BK) channels as potential EtOH targets in a variety of species and cells. In order to elucidate mechanisms underlying tolerance development, I used inside-out patch clamp techniques to measure EtOH induced changes in channel activity (measured as open probability) of hSlo, hSlo+β1, and hSlo+β4 channels exogenously expressed in HEK 293 cells. I show that the human BK channels have subunit dependent responses to acute application of EtOH, and the magnitude of potentiation was dependent on the concentration of ethanol used and the type of β-subunit expressed. In addition the subunit dependent effects on the channels were a function of cytosolic calcium concentration. Furthermore, to determine if BK channels in ripped-off patches can become tolerant to EtOH, I monitored changes in channel activity in response to a second application of the drug, 10-minutes after washing-out the first exposure. I found that channels were less responsive to the second exposure, indicative of tolerance. I examined long-term consequences of EtOH exposure by repeating these experiments on cells cultured in 25 mM EtOH in the culture medium for 24-hours. Under these conditions, all three channel types show chronic tolerance has developed as revealed by the response to acute EtOH applications. Subunit-dependent differences to the development of acute tolerance were apparent, however. In response to a second application to EtOH, hSlo+β4 channels were now inhibited. Overall, these results indicate that BK channels respond to and develop tolerance to EtOH in the absence of cellular context, suggesting the possibility that alcohol tolerance within organisms may be in part mediated by changes imparted by EtOH on BK channels directly.

Stress-induced JNK activity has been implicated in apoptosis. Gene disruption studies have established that JNK signaling is required for some forms of apoptosis. However, it was not clear whether and how JNK was able to deliver an apoptotic signal, because JNK and its regulated-downstream transcriptional factors control a variety of gene activities and multiple biological functions. I have studied this question by using constitutively activated JNK that is independent of upstream signaling. The results indicate that activated JNK is sufficient to deliver an apoptotic signal that causes cytochrome c release from mitochondria. Significantly, this apoptotic signal requires pro-apoptotic Bc12 proteins of Bax and Bak to mediate the downstream apoptotic program. This part of work established the apoptotic activity of JNK signal transduction and the key downstream components of JNK-stimulated apoptotic signal. Two pathways are known to mediate apoptosis in response to apoptotic stimulations: death receptor pathway and mitochondrial pathway. It has been established that JNK is required for the apoptosis mediated by mitochondria in response to ultraviolet irradiation and some genetic stress. However, the mechanisms are not fully understood. It is well known that Bax and Bak are indispensable downstream components leading to apoptotic mitochondrial changes and that other Bc12 family members can regulate the relative apoptotic activity of Bax and Bak. In conjunction with the first part of the research, I have investigated the hypothesis that JNK-mediated regulation of BH3-only Bc12 members contributes to its apoptotic activity. These results indicate that JNK-mediated phosphorylation of Bim and Bmf promotes the release of these proapoptotic BH3-only proteins from their sequestration and these factors become free to initiate apoptosis. This part of work established one mechanism of activated JNK-stimulated apoptosis. This mechanism may contribute to the phenomenon that Jnk1-/-Jnk2-/- fibroblasts are resistant to ultraviolet irradiation-induced apoptosis.

TNFα-induced programmed necrosis is a caspase-independent cell death program that is contingent upon the formation of a multiprotein complex termed the necrosome. The association of two of the components of the necrosome, receptor interacting protein 1 (RIP1) and RIP3, is a critical and signature molecular event during necrosis. Within this complex, both RIP1 and RIP3 are phosphorylated which are consequential for transmission of the pro-necrotic signal. Namely, it has been demonstrated that RIP3 phosphorylation is required for binding to downstream substrates. Nevertheless, the regulatory mechanisms governing necrosome activation remain unclear. Since necrosis is implicated in a variety of different diseases, understanding the biochemical signaling pathway can potentially yield future drug targets. I was interested in identifying other regulators of necrosis in hope of gaining a better understanding of the necrosis signaling pathway and regulators of the necrosome. To address this, I screened a cancer gene siRNA library in a cell line sensitive to necrosis. From this, I independently identified CYLD as a positive regulator of necrosis. Previous studies suggest that deubiquitination of RIP1 in the TNF receptor (TNFR)-1 signaling complex is a prerequisite for transition of RIP1 into the cytosol and assembly of the RIP1-RIP3 necrosome. The deubiquitinase cylindromatosis (CYLD) is presumed to promote programmed necrosis by facilitating RIP1 deubiquitination in this membrane receptor complex. Surprisingly, I found that TNFα could induce RIP1-dependent necrosis in CYLD-/- cells. I show that CYLD does not regulate RIP1 ubiquitination at the receptor complex. Strikingly, assembly of the RIP1-RIP3 necrosome was delayed, but not abolished in the absence of CYLD. In addition to the TNFR-1 complex, I found that RIP1 within the necrosome was also ubiquitinated. In the absence of CYLD, RIP1 ubiquitination in the NP-40 insoluble necrosome was greatly increased. Increased RIP1 ubiquitination correlated with impaired RIP1 and RIP3 phosphorylation, a signature of kinase activation. My results show that CYLD regulates RIP1 ubiquitination in the NP-40 insoluble necrosome, but not in the TNFR-1 signaling complex. Contrary to the current model, CYLD is not essential for necrosome assembly. Rather, it facilitates RIP1 and RIP3 activation within the necrosome and the corollary is enhancement of necrosome functionality and subsequent necrosis. My results therefore indicate that CYLD exerts its pro-necrotic function in the NP-40 insoluble necrosome, and illuminates the mechanism of necrosome activation.

Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders. Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease. Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.

Hox genes encode a conserved family of homeodomain containing transcription factors essential for metazoan development. The establishment of overlapping Hox expression domains specifies tissue identities along the anterior-posterior axis during early embryogenesis and is regulated by chromatin architecture and retinoic acid (RA). Here we present the role nucleosome positioning plays in hox activation during embryogenesis. Using four stages of early embryo development, we map nucleosome positions at 37 zebrafish hox promoters. We find nucleosome arrangement to be progressive, taking place over several stages independent of RA. This progressive change in nucleosome arrangement on invariant sequence suggests that trans-factors play an important role in organizing nucleosomes. To further test the role of trans-factors, we created hoxb1b and hoxb1a mutants to determine if the loss of either protein effected nucleosome positions at the promoter of a known target, hoxb1a. Characterization of these mutations identified hindbrain segmentation defects similar to targeted deletions of mouse orthologs Hoxa1 and Hoxb1 and zebrafish hoxb1b and hoxb1a morpholino (MO) loss-of-function experiments. However, we also identified differences in hindbrain segmentation as well as phenotypes in facial motor neuron migration and reticulospinal neuron formation not previously observed in the MO experiments. Finally, we find that nucleosomes at the hoxb1a promoter are positioned differently in hoxb1b-/- embryos compared to wild-type. Together, our data provides new insight into the roles of hoxb1b and hoxb1a in zebrafish hindbrain segmentation and reticulospinal neuron formation and indicates that nucleosome positioning at hox promoters is dynamic, depending on sequence specific factors such as Hox proteins.

Asparagine (N)-linked glycosylation occurs on 90% of membrane and secretory proteins and drives folding and trafficking along the secretory pathway. The N-glycan can be attached to an N-X-T/S-Y (X,Y ≠ P) consensus site by one of two oligosaccharyltransferase (OST) STT3 enzymatic isoforms either during protein translation (co-translational) or after protein translation has completed (post-translational). While co-translational N-glycosylation is both rapid and efficient, post-translational N-glycosylation occurs on a much slower time scale and, due to competition with protein degradation and forward trafficking, could be detrimental to the success of a peptide heavily reliant on post-translational N-glycosylation. In evidence, mutations in K+ channel subunits that shift N-glycosylation kinetics have been directly linked to cardiac arrhythmias. My thesis work focuses on identifying primary sequence factors that affect the rate of N-glycosylation. To identify the molecular determinants that dictate whether a consensus site acquires its initial N-glycan during or after protein synthesis, I used short (~ 100-170 aa) type I transmembrane peptides from the KCNE family (E1-E5) of K+ channel regulatory subunits. The lifetime of these small membrane proteins in the ER translocon is short, which places a significant time constraint on the co-translational N-glycosylation machinery and increases the resolution between co- and post-translational events. Using rapid metabolic pulse-chase experiments described in Chapter II, I identified several molecular determinants among native consensus sites in the KCNE family that favor co-translational N-glycosylation: threonine containing-consensus sites (NXT), multiple N-terminal consensus sites, and long C-termini. The kinetics could also be shifted towards post-translational N-glycosylation by converting to a serine containing-consensus site (NXS), reducing the number of consensus sites in the peptide, and shortening the C-termini. In Chapter III, I utilized an E2 scaffold peptide to examine the N-glycosylation kinetics of the middle X residue in an NXS consensus site. I found that large hydrophobic and negatively charged residues hinder co-translational N-glycosylation, while polar, small hydrophobic, and positively charged residues had the highest N-glycosylation efficiencies. Poorly N-glycosylated NXS consensus sites with large hydrophobic and negatively charged X residues had a significantly improved co-translational N-glycosylation efficiency upon conversion to NXT sites. Also in Chapter III, I adapted a siRNA knockdown strategy to definitively identify the OST STT3 isoforms that perform co- and post-translational N-glycosylation for type I transmembrane substrates. I found that the STT3A isoform predominantly performs co-translational N-glycosylation while the STT3B isoform predominantly performs post-translational N-glycosylation, in agreement with the roles of these enzymatic subunits on topologically different substrates. Taken together, these findings further the ability to predict the success of a consensus site by primary sequence alone and will be helpful for the identification and characterization of N-glycosylation deficiency diseases.

The innate immune system is an evolutionarily conserved primary defense system against microbial infections. One of the central components of innate immunity are the pattern recognition receptors which sense infection by detecting various conserved molecular patterns of pathogens and trigger a variety of signaling pathways. In this dissertation, the signaling pathways of several classes of these receptors were dissected. In chapters II and III, the role of two NOD-like receptors, NLRP3 and NLRC4 were investigated in the context of infection with the fungal pathogen, C. albicans. C. albicans is an opportunistic pathogen that causes diseases mainly in immunocompromised humans and innate immunity is critical to control the infection. In chapters II and III, we demonstrate that a multiprotein-inflammasome complex formed by the NLR protein, NLRP3 and its associated partners, ASC and caspase-1 are critical for triggering the production of mature cytokine IL-1β in response to C. albicans. NLRC4, another inflammasome forming NLR that is activated by intracellular bacterial pathogens, was not required for this process in macrophages. Thus, our data indicates that NLRP3 inflammasome responds to fungal infections in addition to its known stimuli such as bacterial and viral infections, toxic, crystalline and metabolic signals. Interestingly, this NLRP3 dependent inflammasome response was maintained even when the pathogen is not viable, and is either formalin fixed or heat-killed (HK). Hence, in chapter III, we examined β-glucans, a structural cell wall component, as the potential immunostimulatory component of C. albicans and dissected the inflammasome responses to β -glucans. We observed that NLRP3-ASC-caspase-1 inflammasome was critical for commercially obtained particulate β-glucans similar to the case of C. albicans. β-glucan sensing C-lectin receptor dectin-1 and the complement receptor CR3 mediated inflammasome activation, IL-1β production in response to the glucan particles. Interestingly, CR3 which recognizes glucans as well as complement opsonized pathogens was strongly required for HK C. albicans induced IL-1β, and partially required for that of live C. albicans, while dectin-1 was not required. Consistent with the receptor studies, blocking of β -glucan receptors by pre-incubating cells with nonstimulatory, soluble glucans led to decreased IL-1β production in response to HK C. albicanswith no effect on IL-1β in response to the live fungus. Dectin-1, CR3 and β-glucan sensing also triggered a moderate dendritic cell death response to β-glucans and HK C. albicans. Live C. albicans induced cell death requires phagocytosis but not the inflammasome, β-glucan sensing, dectin-1 or CR3. The Drosophila caspase-8 like molecule DREDD plays an essential, nonapoptotic role in the Drosophila NF-κB pathway called the ‘IMD’ pathway. Owing to the remarkable evolutionary conservation between Drosophila and mammalian innate immune NF-κB pathways, we explored the potential role of caspase-8 in inflammasomes and in TLR signaling. Using casp8-/- Rip3-/- macrophages and dendritic cells, we observed that caspase-8, specifically augments β-glucan and HK C. albicans induced IL-1β as well as cell death in a caspase-1 independent manner, but not that of live C. albicans, in chapter III. We also found that caspase-8 differentially regulates TLR4 and TLR3 induced cytokine production (chapter IV). Caspase-8 specifically promotes TLR4 induced production of cytokines such as TNF, IL-1β in response to LPS and E. coli. On the other hand, caspase-8 negatively regulates TRIF induced IFNβ production in TLR4 and TLR3 signaling in response to LPS and dsRNA. Caspase-8 executed a similar mode of regulation of the cytokine RANTES in MEFs, in part, by collaborating with RIP3. Strikingly, caspase-8 deficiency alone triggers higher macrophage death and IL-1β production in response to TLR ligands, due to the presence of RIP3. Thus, in addition to its conventional roles in apoptosis, caspase-8 modulates TLR4 and TLR3 induced cytokine production and prevents RIP3 mediated hyper inflammation in response to TLR signals. Together, our findings provide valuable information on fungal pattern recognition and inflammasome pathways and define the contribution of β-glucan sensing to C. albicans induced inflammasome responses. In addition, we demonstrate how caspase-8 adds a layer of specificity to inflammasome as well as TLR signaling. Overall, these results also shed light on the cross talk between death signaling components and innate immune pathways to mount a specific and potentially effective innate immune response against microbial pathogens.