Most organisms, from cyanobacteria to humans are equipped with circadian clocks. These endogenous and self-sustained pacemakers allow organisms to adapt their physiology and behavior to daily environmental variations, and to anticipate them. The circadian clock is synchronized by environmental cues (i.e. light and temperature fluctuations). The fruit fly, Drosophila melanogaster, is well established as a model for the study of circadian rhythms. Molecular mechanisms of the Drosophilacircadian clock are conserved in mammals. Using genetic screens, several essential clock proteins (PER, TIM, CLK, CYC, DBT, SGG and CK-II) were identified in flies. Homologs of most of these proteins are also involved in generating mammalian circadian rhythms. In addition, there are only six neuronal groups in the adult fly brain (comprising about 75 pairs of cells) that express high levels of clock genes. The simplicity of this system is ideal for the study of the neural circuitry underlying behavior. The first half of this dissertation focuses on a genetic screen designed to identify novel genes involved in the circadian light input pathway. The screen was based on previous observations that a mutation in the circadian photoreceptor CRYPTOCHROME (CRY) allows flies to remain rhythmic in constant light (LL), while wild type flies are usually arrhythmic under this condition. 2000 genes were overexpressed and those that showed a rhythmic behavior in LL (like crymutants) were isolated. The candidate genes isolated in the screen present a wide variety of biological functions. These include genes involved in protein degradation, signaling pathways, regulation of transcription, and even a pacemaker gene. In this dissertation, I describe work done in order to validate and characterize such candidates. The second part of this dissertation focuses on identifying the pacemaker neurons that drive circadian rhythms in constant light (LL) when the pacemaker gene period is overexpressed. We found that a subset of pacemaker neurons, the DN1s, is responsible for driving rhythms in constant light. This attractive finding reveals a novel role for the DN1s in driving behavioral rhythms under constant conditions and suggests a mechanism for seasonal adaptation in Drosophila.

The Innate immune system is the first line of defense against invading microbial pathogens. It is a fast-acting and non-antigen-specific defense system, which employs germline encoded surveillance systems capable of responding to a broad-spectrum of pathogens. The innate immune system involves a variety of immune cells, which express different profiles of surveillance or detection receptors. Upon sensing pathogens, these receptors trigger cell signalling to turn on transcription of inflammatory cytokines, chemokines, anti-microbial peptides and type I Interferons. These effectors have direct effects on the control of pathogen load and also activate the adaptive immune system, which is ultimately required to clear infections. The type I interferons (IFNs) are the principal cytokines strongly induced during infection with viruses and are required for direct control of viral replication and modulation of cells of the adaptive immune response. The signalling pathways induced in order to activate type I IFNs are dependent on the interferon regulatory factors (IRFs). Striving for survival, microbes have evolved various strategies to subvert/impair these critical defense molecules. In this thesis work, I have used Human Metapneumoviruses (HMPVs), a relatively newly described family of paramyxoviruses as model viruses to explore the role of pattern recognition receptors (PRRs) and the IRF family of transcription factors in the innate immune response. These studies revealed that the recognition of HMPV viral pathogen-associated molecular patterns (PAMPs) by immune cells is different in different cell types. Retinoic acid-inducible gene-I (RIG-I), a cytosolic RNA helicases senses HMPV-A1 virus for triggering type I IFN activation by detecting its 5’- triphosphate viral RNA in most human cells, including cell lines and primary monocytes. An exception to these findings was plasmacytoid dendritic cells (PDCs), where Toll-like receptor (TLR)-7 is the primary sensor involved in detecting HMPV viruses. By comparing the innate immune response to two HMPV strains, we found that these two closely related strains had very different immune stimulatory capabilities. HMPV-1A strain triggered type I IFNs in monocytes, PDCs and cells of epithelial origin. In contrast, a related strain, HMPV-B1 failed to trigger IFN responses in most cell types. Our studies suggested that the phosphoprotein (P) of HMPV-B1 could prevent the viral RNA from being detected by RIG-I, thus inhibiting the induction of type I IFN production in most cell type examined. This finding adds to our understanding of the mechanisms by which viruses are sensed by surveillance receptors and also unveils new means of viral evasion of host immune responses. Although IRFs are extensively studied for their role in regulating type I IFN activation, especially in TLR and RIG-I like receptor (RLR) signalling pathways upon viral infection, a clear understanding of how this family of transcription factors contributes to anti-viral immunity was lacking. Studies conducted as part of this thesis revealed that in addition to IRF3 and IRF7, which play a central role in anti-viral immunity downstream of most PRRs (e.g. TLRs, RLRs, DNA sensors), the related factor IRF5 was also an important component of innate anti-viral defenses. Using IRF5-deficient mice we studied in detail the role of IRF5 in coordinating antiviral defenses by examining its involvement in signalling downstream of TLRs. These studies led us to examine the role of IRF5 in the regulation of type I IFNs as well as inflammatory cytokines in different cell types. While most TLRs that induced IFNβ showed normal responses in IRF5-deficient mice, CpG-B-induced IFNβ production in CD11c+CDCs isolated from mouse spleen but not those generated in vitro from bone marrow required IRF5. This was in contrast to responses with lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid (polyIC), ligands for TLR4 and 3, respectively. Moreover, we found that in contrast to IRF3 and/or IRF7, IRF5 was important in coordinating the expression of inflammatory cytokines such as TNFα downstream of some TLRs. In addition to our studies to examine the requirement for IRF5 in TLR signaling, we also showed that muramyl peptide (MDP) from Mycobacterium tuberculosis (Mtb) could activate type I IFNs via IRF5. This was the first evidence linking IRF5 to a non-TLR-driven pathway. IRF5 activation in this case was downstream of a novel nucleotide-binding oligomerization domain containing (NOD)-2/receptor-interacting serine-threonine kinase (RIP)-2 signaling pathway. Collectively, the studies outlined in this thesis have assisted in providing a framework to understand the role of TLRs, RLRs and IRFs in the immune response to paramyxoviruses and have unveiled new mechanisms of activation of the IRFs as well as new mechanisms by which pathogens subvert or evade these important innate defense mechanisms.

The KCNE1 and KCNE3 type I transmembrane-spanning β-subunits assemble with the KCNQ1 voltage-gated K+ channel to afford membrane-embedded complexes with dramatically different properties. Assembly with KCNE1 produces the very slowly activating and deactivating IKs current that shapes the repolarization phase of cardiac action potentials. Genetic mutations in KCNQ1 or KCNE1 that reduce IKs current cause long QT syndrome and predispose affected individuals to potentially fatal cardiac arrhythmias. In contrast, complexes formed between KCNQ1 and KCNE3 produce rapidly activating and mostly voltage-independent currents, properties that are essential for function in K+ recycling and Cl−secretion in gastrointestinal epithelia. This thesis addresses how these two homologous accessory peptides impart their distinctive effects on KCNQ1 channel gating by examining two important protein regions: 1) a conserved C-terminal motif in the β-subunits themselves, and 2) the voltage sensing domain of KCNQ1 channels. Sequences in both the transmembrane domain and C-terminus of KCNE1 and KCNE3 have been identified as contributing to the divergent modulatory effects that these β-subunits exert. The homology of transmembrane-abutting C-terminal residues within the KCNE family and the presence of long QT-causing mutations in this region highlight its importance. A bipartite model of modulation was proposed that suggests the transmembrane domain of KCNE1 is passive, allowing the C-terminal domain to control modulation. Chapter II builds on this model by investigating the effect of mutating specific amino acids in the KCNE1 C-terminal domain. Point mutants that produce ‘high impact’ perturbations in gating were shown to cluster in a periodic fashion, suggesting an alpha-helical secondary structure that is kinked by a conserved proline residue and interacts with the Q1 channel complex. In Chapter III, the voltage sensing domain of Q1 channels is examined in the presence of either KCNE1 or KCNE3. To determine the influence of these two peptides on voltage sensing, the position of the S4 voltage sensor was monitored using cysteine accessibility experiments. In the slowly opening KCNQ1/KCNE1 complexes, voltage sensor activation appears to occur much faster than the onset of current, suggesting that slow channel activation is not due to slowly moving voltage sensors. KCNE3, on the other hand, shifts the voltage sensor equilibrium to favor the active state, producing open channels even at negative voltages. Taken together, these findings provide mechanistic detail to illustrate how two homologous peptides radically alter the gating properties of the same K+ channel and present a structural scaffold to map protein-protein interactions.

Haemophilus influenzae encounters niches within the human host that are predicted to differ in availability of oxygen and reactive nitrogen species (RNS: nitrite and nitric oxide), which influence the environmental redox state. Previously reported data has indicated that an altered redox condition could serve as a signal recognized by H. influenzae to optimize its survival within host microenvironments. To elucidate the role of redox signaling in virulence, we examined regulation by the FNR homolog of H. influenzae, whose counterpart in E. coli has been reported to be a direct oxygen sensor and a regulator of genes responsible for RNS metabolism and resistance. Many members of the FNR regulon are subject to coordinated transcriptional control by NarP, a regulator in E. coli that is activated by cognate sensor NarQ in response to environmental nitrite. To study the regulatory activities of FNR and NarQ-NarP in H. influenzae, I targeted a gene predicted to be FNR-regulated, nrfA, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration. The fnr, narP and nrfA mutants were assayed for nitrite reduction, which implicated the roles of FNR, NarP and NrfA in RNS metabolism. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous nrf promoter (Pnrf-HA), I demonstrate that FNR and NarP, but not NarQ, are required for full activation of the nrf promoter. Additionally, Pnrf-HA expression increases as oxygen becomes depleted and decreases when exposed to high concentrations of nitrite, implying that the nrfpromoter is modulated by environmental redox signals. FNR of E. coli has been implicated in regulation of resistance mechanisms to a reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection as a host defense mechanism. A mutant lacking FNR is more sensitive to NO exposure and killing by activated macrophages than wild type H. influenzae after anaerobic pre-growth. Mutants of nrfA and narP have been tested and initial experiments have shown both mutants have a lesser NO sensitivity phenotype as compared to the fnr mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in H. influenzae. Upon examination of potential factors that might be involved to this phenotype, we discovered FNR-regulated gene, ytfE, which contributes to defense against nitrosative stress. The fnr and ytfE mutants are more susceptible to killing by activated macrophages indicating that FNR regulation of ytfE might be important for in vivo infection.

The main focus of my work has been the role of the MRN in the S-phase DNA damage checkpoint. The MRN plays many roles in cellular metabolism; some are checkpoint dependent and some are checkpoint independent. The multiple roles in cellular metabolism complicate study of the role of the MRN in the checkpoint. MRN mutations in budding yeast and mammals may display separation of function. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5’ resection to create single stranded DNA that is required for both signaling and homologous recombination. However, it is unclear if resection is essential for all of the cellular functions of MRN. Therefore I have made mutations to mimic those in budding yeast and mammals. I found that several alleles of rad32, as well as ctp1Δ, are defective in double-strand break repair and most other functions of the complex but maintain an intact S-phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads me to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function. One of the potential roles of Rad32 and the rest of the MRN complex is in sister chromatid exchange. The genetic requirements of sister chromatid exchange have been examined using unequal sister chromatid assays which only are able to assay exchanges that are illegitimate and produce changes in the genome. Most sister chromatid exchange must be equal to maintain genomic integrity and thus far there is no good assay for equal sister chromatid exchange. Yeast cells expressing the human equilibrative nucleoside transporter 1 (hENT1) and the herpes simplex virus thymidine kinase (tk) are able to incorporate exogenous thymidine into their DNA. This strain makes it possible for the fission yeast DNA to be labeled with halogenated thymidine analogs. This strain is being used to design an assay that will label one sister with BrdU and then DNA combing will be used to see equal sister chromatid exchange.

Transcriptional regulation of gene expression is critical for all unicellular and multicellular organisms. The ability to selectively induce or repress expression of only a few genes from the entire genome gives cells the ability to respond to changing environmental conditions, grow and proliferate. Multicellular organisms begin life as a single totipotent cell, which undergoes many cell divisions during embryonic and later postnatal development. During this process, the dividing cells of the embryo progressively lose their pluripotency and adopt restricted cell fates. Cell fate restriction leads different cell types to gain unique transcriptional profiles. This transcriptional profile or gene expression pattern not only defines the cell types and restricts the ways in which they can respond to signals, it also has to be faithfully re-established in the progeny of these fate-restricted cells when they divide. Different mechanisms have evolved in multicellular organisms to propagate transcriptional memory of cell identity. Most of mechanisms involve modifications of chromatin such as epigenetic modification of DNA or alterations of associated histones. In contrast to multicellular organisms which have considerable cellular diversity and a long lifespan for which cell fates and transcriptional memory needs to be maintained, single celled budding yeast, Sachharomyces cerevisiae have a life cycle of about 90 minutes in normal nutrient rich conditions. However, even budding yeast have tremendous potential to respond to changing environmental conditions like nutrient availability by inducing expression of various genes. We observed that members of the GAL gene cluster, which encodes genes induced in response to and for metabolizing the sugar galactose, showed heritable transcriptional memory of previous activation. This dissertation thesis describes the studies I have done for my graduate research to define this phenomenon of transcriptional memory at the yeast GALgenes and to determine the mechanism by which it can be formed and inherited. Chapter I gives an introduction to different mechanisms of establishing transcriptional memory in unicellular and multicellular organisms. Chromatin based mechanisms have been well studied in multicellular organisms but not observed in budding yeast. We compare chromatin based or nuclear inheritance with cytoplasmic inheritance that can be observed in yeast. Chapter II describes work done to define the phenomenon of transcriptional memory at GAL1 gene. We define this as a faster rate of induction of the GAL1 gene, compared to a naïve gene, after a brief period of repression. We show that this cellular memory persists through mitosis and can be passed on to the next generation. We also show that chromatin remodeling enzymes appear to be required for rapid reinduction, raising the question if yeast may also possess chromatin associated, nuclear mechanisms for cellular memory. Chapter III describes experiments that show that cellular memory observed at GAL1 is cytoplasmic in nature and also compares our work with similar examples observed recently by other groups. Finally, Chapter IV offers a perspective of the significance of such cellular memory mechanisms in budding yeast and outlines some potential further experiments to better understand the control of GAL1 induction kinetics.

Metazoan genomes contain thousands of protein-coding and non-coding RNA genes, most of which are differentially expressed, i.e., at different locations, at different times during development, or in response to environmental signals. Differential gene expression is achieved through complex regulatory networks that are controlled in part by two types of trans-regulators: transcription factors (TFs) and microRNAs (miRNAs). TFs bind to cis-regulatory DNA elements that are often located in or near their target genes, while microRNAs hybridize to cis-regulatory RNA elements mostly located in the 3’ untranslated region (3’UTR) of their target mRNAs. My work in the Walhout lab has centered on understanding how these trans-regulators interact with each other in the context of gene regulatory networks to coordinate gene expression at the genome-scale level. Our model organism is the free-living nematode Caenorahbditis elegans, which possess approximately 950 predicted TFs and more than 100 miRNAs Whereas much attention has focused on finding the protein-coding target genes of both miRNAs and TFs, the transcriptional networks that regulate miRNA expression remain largely unexplored. To this end, we have embarked in the task of mapping the first genome-scale miRNA regulatory network. This network contains experimentally mapped transcriptional TF=>miRNA interactions, as well as computationally predicted post-transcriptional miRNA=>TF interactions. The work presented here, along with data reported by other groups, have revealed the existence of reciprocal regulation between these two types of regulators, as well as extensive coordination in the regulation of shared target genes. Our studies have also identified common mechanisms by which miRNAs and TFs function to control gene expression and have suggested an inherent difference in the network properties of both types of regulators. Reverse genetic approaches have been extensively used to delineate the biological function of protein-coding genes. For instance, genome-wide RNAi screens have revealed critical roles for TFs in C. elegans development and physiology. However, reverse genetic approaches have not been very insightful in the case of non-coding genes: A single null mutation does not result in an easily detectable phenotype for most C. elegans miRNA genes. To help delineate the biological function of miRNAs we sought to determine when and where they are expressed. Specifically, we generated a collection of transgenic C. elegans strains, each containing a miRNA promoter::gfp (Pmir::gfp) fusion construct. The particular pattern of expression of each miRNA gene should help to identify potential genetic interactors that exhibit similar expression patterns, and to design experiments to test the phenotypes of miRNA mutants.

Chromatin has a compact organization in which most DNA sequences are structurally inaccessible and functionally inactive. Reconfiguration of thechromatir required to activate transcription. This reconfiguration is achieved by the action of enzymes that covalently modify nucleosomal core histones, and by enzymes that disrupt histone-DNA interactions via ATP hydrolysis. TheSWI/SNF family of ATP-dependent chromatin remodeling enzymes has been implicated not only in gene activation but also in numerous cellular processes including differentiation, gene repression, cell cycle control, recombination and DNA repair. PPARγ, C/EBPα and C/EBPβ are transcription factors with well established roles in adipogenesis. Ectopical expression of each of these factors in non-adipogenic cells is sufficient to convert them to adipocyte-like cells. To determine the requirements of SWI/SNF enzymes in adipocyte differentiation, we introduced PPARγ, C/EBPα or C/EBPβ into fibroblasts that inducibly express dominant-negative versions of the Brahma-Related Gene 1 (BRG1) or human Brahma (BRM), which are the ATPase subunits of the SWI/SNF enzymes. We found that adipogenesis and expression of adipocyte genes were inhibited in the presence of mutant SWI/SNF enzymes. Additionally, in cells expressing C/EBPα or C/EBPβ, PPARγ expression was SWI/SNF dependent. These data indicate the importance of these remodeling enzymes in both early and late gene activation events. Subsequently, we examined by chromatin immunoprecipitation (ChIP) assay the functional role of SWI/SNF enzymes in the activation of PPARγ2, the master regulator of adipogenesis. Temporal analysis of factors binding to the PPARγ2 promoter showed that SWI/SNF enzymes are required to promote preinitiation complex assembly and function. Additionally, our studies concentrated on the role of C/EBP family members in the activation of early and late genes during adipocyte differentiation. During adipogenesis, C/EBPβ and δ are rapidly and transiently expressed and are involved in the expression of PPARγ and C/EBPα, which together activate the majority of the adipocyte genes. Our studies determined the temporal recruitment of the C/EBP family at the promoters of early and late genes by ChIP assay during adipocyte differentiation. We found that all of the C/EBP members evaluated are present at the promoters of early and late genes, and the binding correlated with the kinetics of the C/EBPs expression. Binding of C/EBPβ and δ is transient, subsequently being replaced by C/EBPα. These studies demonstrated that C/EBPβ and δ are not only involved in the regulation of PPARγ and C/EBPα, but also in the activation of late expressed adipocyte genes.

It is shown here that the ability of B lymphocytes to act as supportive host cells for virus infections requires they be activated from the resting Gostage of the cell cycle. I have used a series of activation regimens, which allow B cells to progress to different stages in their activation/differentiation pathway toward antibody secretion, in order to evaluate the extent of activation required to support vesicular stomatitis or Newcastle disease virus infections. At least three distinct phases during B cell activation which affected VSV infection were defined. Freshly isolated resting murine splenic B cells in the Go phase of the cell cycle do not support VSV, assessed by protein synthesis, infectious center formation, and PFU production. Small B cells cultured for 48 hours without stimulation still do not support VSV. B cells stimulated with the lymphokines found in Con A activated supernatants from splenic T cells or cloned T cell lines transited into the G1 phase of the cell cycle but remain refractory to VSV. These VSV non-supportive B cell populations do take up virus particles and transcribe viral mRNAs which can be translated in vitro, suggesting a translational block to VSV. B cells stimulated into the S phase of the cell cycle with anti-immunoglobulin synthesize VSV proteins and increased numbers of infectious centers, but only low level PFU synthesis (center) is observed. Co-stimulation with anti-Ig and lymphokines, which supports differentiation to antibody secretion, enhanced PFU synthesis without further increasing the number of infected B cells. LPS, which activates B cells directly to antibody secretion by a pathway different from anti-Ig, induced infectious centers, and PFUs at levels comparable to those seen when stably transformed permissive cell lines are infected. Co-stimulation of LPS activated B cells with the same lymphokine populations that enhance PFU production when anti-Ig is used as a stimulator suppresses PFU production completely, suggesting that anti-Ig and LPS activated B cells are differentially responsive to lymphokines. NDV infection of murine B cells differed markedly from VSV infection, as all B cell populations examined gave a similar response pattern. NDV viral proteins were synthesized by B cells in each of the activation states previously described, even freshly isolated B cells. Infectious center formation increased up to 5-fold over the levels observed with unstimulated B cells after anti-Ig or LPS activation. However, PFU synthesis was low (center) for all B cell populations. These results suggest that these two similar viruses may be dependent on different host cell factors and that these factors are induced for VSV but not NDV by the B cell activators employed here or that the process of infection of B cell by these two viruses induces different cellular responses.

The inherently repressive nature of chromatin presents a sizeable barrier for all nuclear processes in which access to DNA is required. Therefore, eukaryotic organisms ranging from yeast to humans rely on a battery of enzymes that disrupt the chromatin structure as a means of regulating DNA transactions. These enzymes can be divided into two broad classes: those that covalently modify histone proteins, and those that actively disrupt nucleosomal structure using the free energy derived from ATP hydrolysis. The latter group, huge, multisubunit ATP-dependent chromatin remodeling factors, are emerging as a common theme in all nuclear processes in which access to DNA is essential. Although transcription is the process for which a requirement for chromatin remodeling is best documented, it is now becoming clear that other processes like replication, recombination and DNA repair rely on it as well. A growing number of ATP-dependent remodeling machines has been uncovered in the last 10 years. Although they differ in their subunit composition, organism or tissue restriction, substrate specificity, and regulating/recruiting partners, it has become increasingly evident that all ATP-dependent chromatin remodeling factors share a similar underlying mechanism. This mechanism is the subject of the studies presented in this thesis. Chromatin-remodeling factors seem to bind both the histone and DNA components of nucleosomes. From a fixed position on nucleosomes, the remodeling factors appear to translocate on the DNA, generating torsional stress on the double helix. This activity has several consequences, including the distortion of the DNA structure on the surface of the histone octamer, the disruption of histone-DNA interactions, and the mobilization of the nucleosome core with respect to the DNA. The work presented in this thesis, along with data reported by other groups, supports the hypothesis that yeast SWI/SNF chromatin remodeling complex and the recombinational repair factor, Rad54p, both employ similar mechanisms to regulate gene transcription, and facilitate homologous DNA pairing and recombination, respectively.