This thesis examines the effects of nerve growth factor (NGF) and lithium on the regulation of neurotensin/neuromedin N (NT/N) gene expression in PC12 pheochromocytoma cells. In PC12 cells, the expression of the rat NT/N gene is strictly dependent on simultaneous exposure to combinations of NGF, glucocorticoids, activators of adenylate cyclase, and lithium. Transient transfection experiments indicated that a consensus AP-1 site located within the NT/N promoter is the principal target of NGF and lithium action. NGF rapidly, but transiently, induces the expression of several AP-1 genes in PC12 cells, suggesting that the effect of NGF on NT/N gene expression results from increased AP-1 activity. These results led to the prediction that the induction of NT/N gene expression should be rapid, transient and dependent on de novoprotein synthesis. These experiments also suggested that the NT/N gene is principally regulated through the initiation of transcription. However, post-transcriptional mechanisms may also be involved. Experiments in this thesis were designed to examine the regulatory mechanisms responsible for increased NT production in PC12 cells when treated with different inducer combinations and whether AP-1 factors could act as mediators in responses to NGF and lithium. Results described in this thesis indicate that the principal mechanism by which NGF and lithium regulate NT biosynthesis is by activating NT/N gene transcription. Comparison of NT/N mRNA, pro NT/N synthetic rates, proNT/N proteins and mature NT levels in induced PC12 cells, demonstrated that NGF and lithium had no effect on the translation of NT/N mRNA and had only a modest effect on post-translational processing. Nuclear run-on assays showed that NT/N transcription is transicntly activated in maximally induced cells. A rapid RNase protection assay was developed to examine both the kinetics of NT/N gene activation and whether activation requires newly synthesized proteins. Quantitation of nuclear NT/N precursor RNA. using a probe spanning the junction between exon onc and intron one, provides a sensitive measure of NT/N gene activity and by several criteria provides an accurate measure of NT/N transcription. When either NGF or lithium was combined with dexamethasone and forskolin, nuclear NT/N precursor RNA transiently accumulated, although each inducer displayed different kinetics, rapid and delayed, respectively. De novo protein synthesis was not required for activating NT/N transcription when NGF was used as the permissive agent, although newly synthesized proteins secm to be needed for subsequent down-regulation. The response to lithium displayed a marked requirement for new protein synthesis, consistent with the involvement of newly synthesized AP-1 factors. RNA blot analysis showed that lithium either alone or in combination with dexamethasone and forskolin induced c-jun and fra-1 gene expression with delayed kinetics, consistent with c-Jun/Fra-1 complexes mediating the effects of lithium on NT/N gene transcription. The pathway identified by lithium does not activate or require protein kinase C. This pathway is also active in neuronally-differentiated PC12 cells suggesting that it could be involved in the regulation of NT/N gcne exprcssion in the intact nervous system. These results and order of addition experiments demonstrate that NGF and lithium activate distinct pathways required for NT/N gene induction.

ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA. Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament. The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes. In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.

The JNK signal transduction pathway mediates a broad, complex biological process in response to inflammatory cytokines and environmental stress. These responses include cell survival and apoptosis, proliferation, tumorigenesis and the immune response. The divergent cellular responses caused by the JNK signal transduction pathway are often regulated by spatial and cell type contexts, as well as the interaction with other cellular processes. The discovery of additional components of the JNK signal transduction pathway are critical to elucidate the stress response mechanisms in cells. This thesis first discusses the cloning and characterization of two novel members of the JNK signal transduction pathway. JIP1 and JMP1 were initially identified from a murine embryo library through a yeast Two-Hybrid screen to identify novel JNK interacting proteins. Full length cDNAs of both genes were cloned and analyzed. JIP1 represents the first member of the JIP group of JNK scaffold proteins which were characterized. The JNK binding domain (JBD) of JIP1 matches the D-domain consensus of other JNK binding proteins, and it demonstrates JNK binding both in vitro and in vivo. This JNK binding was demonstrated to inhibit JNK signal transduction and over-expression of JIP1 inhibits the JNK mediated pre-B cell transformation by bcr-abl. Over-expressed JIP1 also sequesters JNK in the cytoplasm, which may be a mechanism of the inhibition of JNK signaling. A new, high-resolution digital imaging microscopy technique using deconvolution demonstrated the absence of JNK1 in the nucleus of co-transfected JIP1 and JNK1 cells. The other protein discussed in this thesis is JMP1, a novel JNK binding, microtubule co-localized protein. There is a JBD in the JMP1 carboxyl end and a consensus D-domain within this region. The JMP1 JBD demonstrates an increased association with phospho-JNK from UV irradiated cells compared to un-irradiated cells in vivo. JMP1 also has 12 WD-repeat motifs in its amino terminal end which are required for microtubule co-localization. JMP1 demonstrates a cell cycle specific localization at the mitotic spindle poles. This co-localization is dependent on intact microtubules and the amino-terminal WD-repeats are required for this localization. JMP1 mRNA is highly expressed in testis tissues. Immunocytochemistry on murine testis sections using an affinity purified anti-JMP1 antibody demonstrates JMP1 protein in the lumenal compartment of the seminiferous tubules. JMP1 protein is expressed in primary and secondary spermatocytes, cells which are actively undergoing meiosis. The results obtained from the localization of JMP1 in meiotic spermatocytes led to an investigation of the roles of JNK signal transduction in the testis. The testis is an active region of cellular proliferation, apoptosis and differentiation, which make it an appealing model for studying JNK signal transduction. However, the roles JNK signaling have in the testis are poorly understood. I investigated the reproduction capability of Jnk3-/- male mice and discovered older Jnk3-/- males had a reduced capacity to impregnate females compared to younger animals and age-matched wild type controls. The testis morphology and sperm motility of these animals were similar to wild-type animals, and there was no alteration of apoptosis in the testis. The final section of this thesis involves the study of this breeding defect and investigating for cellular defects that might account for this age-related Jnk3-/- phenotype.

p38 mitogen-activated protein (MAP) kinases represent a subgroup of MAP kinases that respond to environmental stress and inflammatory cytokines. p38 MAPK is activated by two upstream kinases, MKK3 and MKK6, by dual phosphorylation on threonine and tyrosine in conserved kinase subdomain VII. Until recently the relative roles of MKK3 and MKK6 have remained unclear. I have undertaken two strategies in an effort to understand the importance of MKK3 as a p38 MAPK activator. First, I cloned and characterized the murine mkk3 gene and determined the structure of the 5'-terminus. Comparison of the murine and human mkk3 genes revealed that the mouse gene encodes a single MKK3 isoform, MKK3b, and the human gene encodes two isoforms, MKK3a and MKK3b. Comparison of the mouse and human mkk3 genes suggests that expression of MKK3a and MKK3b is regulated from different promotors. Analysis of the mkk3 promoter demonstrates that muscle specific expression of murine MKK3b is controlled, in part, by the transcription factors MEF2 and MyoD. Second, I have utilized a gene targeting strategy to disrupt the murine mkk3 gene and to examine the effect on p38 MAPK signaling. I found that there is a p38-specific signaling defect in MKK3 deficient primary mouse embryo fibroblasts (MEF) which correlates with deficits in interleukin (IL)-1 and IL-6 production in response to tumor necrosis factor-α (TNFα) stimulation. In addition there is a defect in TNFα mediated expression of TNFα and macrophage inflammatory proteins (MIP) 1α, MIP1β and MIP2. p38 MAPK-specific signaling defects were also observed in lipopolysaccharide (LPS) stimulated mkk3 (-/-) macrophages. Additionally, mkk3 (-/-) macrophages exhibit defects in LPS and CD40-ligand (CD40L) stimulated IL-12 biosynthesis. Similar data were obtained from CD40L-stimulated mkk3 (-/-) dendritic cells. I also observe that interferon (Ifn)-γ production is diminished during T-helper-1 (TH1) differentiation of CD4+ T-cells derived from mkk3 (-/-) mice. Taken together these data demonstrate a crucial role for p38 MAPK activation by MKK3 in response to the inflammatory cytokine, TNFα and during a TH1 inflammatory response.

Exposure to external stimuli promotes a variety of cellular responses including changes in morphology, gene expression and cell division status. These responses are promoted by signaling pathways composed of modules that are conserved from lower to higher eukaryotes. In Saccharomyces cerevisiae response to the external stimuli provided by mating pheromone is governed by the pheromone response pathway. This pathway is composed of a G protein coupled receptor/heterotrimeric G protein (Gαβγ) module and a MAP kinase cascade. Activation of this pathway allows the heterotrimeric G protein βγ dimer (Gβγ) to recruit polarity proteins to promote changes in cell morphology and to activate signaling through the MAP kinase cascade. Here we investigate the regulation of these pheromone-induced responses. We first examine how an asymmetric polarization response is generated. Normally, a gradient of pheromone serves as a spatial cue for formation of a polarized mating projection, but cells can still polarize when pheromone is present uniformly. Here we show that an intact receptor/Gαβγ module is required for polarization in response to both a gradient and uniform concentration of pheromone. Further investigation into regulation of Gβγ by Gα revealed that the two interaction interfaces between Gα and Gβ have qualitatively different roles. Our results suggest that one interface controls signaling whereas the other governs coupling to the receptor. Overall our results indicate that communication between the receptor and Gαβγ is required for proper polarization. We then examine how G1 CDKs regulate MAP kinase signaling. Response to pheromone is restricted to the G1 stage of the cell cycle. Once cells commit to a round of division they become refractory to mating pheromone until that round of division is complete. One contributor to this specificity involves inhibition of signaling through the MAP kinase cascade by G1 CDKs, but it was not known how this occurs. Here, we show that the MAP kinase cascade scaffold Ste5 is the target of this inhibition. Cln/CDKs inhibit signaling by phosphorylating sites surrounding a small membrane-binding domain in Ste5, thereby disrupting the membrane localization of Ste5. Furthermore, we found that disrupting this regulation allows cells to arrest at an aberrant non-G1 position. Our findings define a mechanism and a physiological benefit for restricting pheromone-induced signaling to G1. This thesis describes findings related to generation of an asymmetric polarization response, heterotrimeric G protein function, and coordination of differentiation signaling with cell division status. Lessons learned here might be applicable to the regulation of polarization and differentiation responses in other systems as the signaling modules are conserved.

Circadian clocks are biological timekeepers that help maintain an organism’s behavior and physiological state optimally timed to the Earth’s day/night cycle. To do this, these internal pacemakers must accurately keep track of time. Equally importantly, they must be able to adjust their oscillations in response to external time cues to remain properly synchronized with the environment, and correctly anticipate environmental changes. When the internal clock is offset from its surrounding day/night cycle, clinically relevant disruptions develop, ranging from inconveniences such as jet-lag to more severe problems such as sleep disorders or mood disorders. In this work, I have used the fruit fly, Drosophila melanogaster, as a model organism to investigate how light and temperature can synchronize circadian systems. My initial studies centered on an intracellular photoreceptor, CRYPTOCHROME (CRY). CRY is a blue light photoreceptor previously identified as a major component of the primary light-input pathway into the Drosophila circadian clock. We used molecular techniques to show that after light-activation, CRY binds to the key circadian molecule TIMELESS (TIM). This interaction irreversibly targets TIM, but not CRY, for degradation. Further studies characterizing a newly isolated cry mutant, crym, showed that the carboxyl-terminus of CRY is not necessary for CRY’s ability to impart photic information to the molecular clock. Instead, the C-terminus appears to be necessary for normal CRY stability and protein-protein interactions. Thus, we conclude that in contrast to previous reports on CRYs of other species, where the C-terminal domain was required for transduction of photic information, the C-terminus of DrosophilaCRY has a purely modulatory function. During the second part of my dissertation work, I focused my studies on circadian thermoreception. While the effects of light in synchronization of the Drosophilaclock to environmental cycles have been extensively characterized, significantly less is known about temperature input pathways into the circadian pacemaker. I have used two approaches to look at how temperature affects the circadian system. First, I conducted a series of behavioral analyses looking at how locomotor rhythms can be phase-shifted in response to temperature cycles. By examining the behavior of genetically ablated flies, we determined that the well-characterized neurons controlling morning and evening surges of activity during light/dark cycles are also implicated in morning and evening behaviors under temperature cycles. However, we also find evidence of cells that contribute to modulating afternoon and evening behavior specifically under temperature cycles. These data contribute to a growing number of studies in the field suggesting that pacemaker cells may play different roles under various environmental conditions. Additionally, we provide data showing that intercellular communication plays an important role in regulating circadian response to temperature cycles. When the morning oscillator is absent or attenuated, the evening cells respond abnormally quickly to temperature cycles. My work thus provides information on the roles of different cell groups during temperature cycles, and suggests that beyond simply synchronizing individual oscillating cells, intercellular network activity may also have a role in modulating proper response to environmental time cues. Finally, I present some preliminary work looking at effects of temperature on known circadian molecules. Using a combination of in vivo and cell culture techniques, I have found that TIM protein levels decrease at higher temperatures. My cell culture data suggest that this is a proteasome-independent degradation event. As TIM is also a key molecule in the light-input pathway, the stability of TIM proteins may be a key point of integration for light and temperature input pathways. While additional research needs to be conducted to confirm these effects in vivoin wild-type flies, these preliminary results identify a possible avenue for further study. Taken together, my work has contributed new data on both molecular and neuronal substrates involved in processing light and temperature inputs into the Drosophila circadian clock.

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 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.

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.