Browsing by Subject "Transcription Factors"
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Item Biochemical and Functional Analysis of Members of the Myocardin Family During Cardiovascular Development(2006-12-20) Oh, Jiyeon; Olson, Eric N.The various stages of muscle development are characterized by distinct patterns of gene expression precisely controlled by combinatorial interaction between a large number of muscle-specific and ubiquitous transcription factors. Myocardin is a cardiac and smooth muscle-specific transcriptional coactivator of serum response factor (SRF) that forms a ternary complex with SRF on DNA and provides its strong transcriptional activation domain (TAD) to SRF. SRF has been shown to stimulate expression of smooth and cardiac muscle genes in association with GATA transcription factors, which play important roles in cardiac and smooth muscle development. I show that GATA transcription factors can either stimulate or suppress the transcriptional activity of myocardin, depending on the target gene. Modulation of myocardin activity by GATA4 is mediated by the physical interaction of myocardin with the DNA binding domain of GATA4 but does not require binding of GATA4 to DNA. The ability of GATA transcription factors to modulate myocardin activity provides a potential mechanism for fine tuning the expression of serum response factor target genes in a gene-specific manner. Two Myocardin Related Transcription Factors, referred to as MRTF-A and B, are expressed in numerous embryonic and adult tissues, implying their potential to modulate SRF target genes in a wide range of tissues. To determine the functions of MRTF-B in vivo, I generated MRTF-B mutant mice by targeted inactivation of the MRTF-B gene. I show that mice homozygous for an MRTF-B loss-of-function mutation die during mid-gestation from a spectrum of cardiovascular defects. These abnormalities are accompanied by a failure in differentiation of smooth muscle cells within the branchial arch arteries, which are derived from the neural crest. The phenotype of MRTF-B mutant mice is distinct from that of mice lacking myocardin and MRTF-A, revealing unique roles for these SRF coactivators in the development of different subsets of smooth muscle cells in vivo.Item Biochemical Characterization Of Delta FosB(2006-12-19) Carle, Tiffany Lynn; Phillips, MegDeltaFosB, the truncated splice variant of FosB, is an important mediator of the long-term plasticity induced in brain by chronic exposure to many types of stimuli, such as repeated administration of drugs of abuse, stress, or compulsive running. Once induced, DeltaFosB persists in the brain for weeks or months following cessation of the chronic stimulus. In addition, DeltaFosB both activates and represses transcription. The biochemical basis of DeltaFosB's persistent expression and dual transcriptional regulation has remained unknown. Both the enhanced protein stability and transcriptional properties are unique to DeltaFosB, compared to FosB, and are critical for its role in neural plasticity. DeltaFosB lacks the C-terminal 101 amino acids of FosB as a result of alternative splicing. The purpose of this work is to biochemically characterize DeltaFosB relative to FosB, to determine how truncation of the FosB C-terminus directs its function. Here, I show that the FosB C-terminus contains two destabilizing elements that promote the degradation of FosB by both proteasome dependent and independent mechanisms. Pulse chase experiments of FosB C-terminal truncation mutants indicate that removal of these C-terminal degrons increases the FosB half-life ~5 fold and prevents its proteasome-mediated degradation and ubiquitylation, properties similar to FosB. These data indicate that alterative splicing specifically removes two destabilizing elements from FosB in order to generate a longer-lived transcription factor, DeltaFosB, in response to chronic perturbations to the brain. Truncation of the C-terminus from FosB also results in differing interaction partners for FosB and DeltaFosB that may contribute to the varying functions of each protein. Specifically, using co-immunoprecipitation assays both in vitro and in vivo, I determined that HDAC1 (histone deacetylase 1) is the preferential binding partner of DeltaFosB compared to FosB. These data suggest an intriguing hypothesis that DeltaFosB interactions with specific HATs and HDACs may be one mechanism by which DeltaFosB mediates both activating and repressive transcriptional activities. DeltaFosB is a unique transcription factor compared to its Fos family members. Truncation of the FosB C-terminal domain liberates DeltaFosB, enabling long-term protein stability and promoting specific interactions with protein partners that are critical for gene regulation important for neural plasticity.Item Controlling Gene Expression With Synthetic Molecules(2006-08-11) Alluri, Prasanna G.; Kodadek, ThomasAberrant gene expression patterns have been implicated in several pathological states. Synthetic molecules capable of functionally mimicking native transcription factors and regulating gene expression in a specific and predictable manner may represent a new paradigm in drug development. Native transcription factors are minimally composed of two domains, a DNA-binding domain (DBD) and an activation domain (AD). Several synthetic DBDs capable of recognizing DNA in a sequence specific manner have been reported in the literature. Furthermore, studies have demonstrated that coupling of these synthetic DBDs to peptides that are capable of acting as activation domains results in chimeric molecules that are capable of activating target gene expression. Since peptides and other biomolecules generally have poor cell-membrane permeability and are prone to rapid enzymatic inactivation inside cells, it is highly desirable to develop artificial molecules that are capable of mimicking native ADs. Towards this goal, a comprehensive methodology for the synthesis, screening and characterization of large peptoid libraries has been developed. Peptoids are a new class of peptidomimetic compounds that are resistant to proteolytic cleavage and are relatively simple and cheap to synthesize. One of the combinatorial libraries was screened against CBP (CREB-binding protein), an important transcriptional coactivator, and three novel, low micromolar affinity ligands were isolated. A cellbased reporter gene assay was employed to assess the cell permeability and transcription activation potential of the synthetic ligands in live mammalian cells. The assay consists of transfecting into HeLa cells a luciferase reporter gene harboring Gal4 binding sites and a construct in which the ligand binding domain of the Glucocorticoid receptor has been fused to Gal4 DBD. The cells are treated with the CBP-binding peptoids that have been chemically coupled to a dexamethasone derivative. Among the three peptoids tested, one of the molecules as a steroid conjugate, has been found to activate the transcription of a reporter gene nearly 1000-fold suggesting that it may be acting as an activation domain surrogate. The mechanistic aspects of the observed transcriptional activity of the peptoid-steroid conjugate remain to be elucidated.Item Defining the Constellation of RNA Elements That Associate with Bacillus Subtilis HFQ(2013-01-17) Dambach, Michael David; Winkler, Wade C., Ph.D.Bacteria utilize a wide variety of genetic regulatory strategies in order to sense and respond to various environmental fluctuations in nutrient availability, temperature, salinity, and oxygen among others. As such, bacterial species have evolved highly coordinated and tightly regulated systems as a means of efficiently responding to potentially deleterious changes in environmental conditions. Traditionally DNA binding transcriptions factors were thought to be the primary means by which the cell executes a selective genetic response. However, the advent of microarray and next generation sequencing platforms, coupled with the wealth of sequenced genomes and powerful bioinformatics have revealed that RNA mediated post transcriptional gene regulation is wide spread in bacterial species and may in fact rival protein based regulatory systems in scope and breadth. RNA mediated post transcriptional gene regulation is broadly divided into two categories-those in which the RNA element is transcribed with the mRNA it regulates (cis-acting regulatory RNAs) or those which are transcribed independently from the gene that they regulate (trans-acting regulatory RNAs). In general cis-acting RNA elements are embedded within a 5’ UTR of a gene that they regulate and may or may not require a protein cofactor to execute genetic regulation. Whereas, trans-acting regulatory RNAs, also known as sRNAs, function via base pairing with their target mRNA and this usually requires the protein chaperone Hfq. Hfq mediated gene regulation is poorly understood in Gram-positive organism, thus I undertook studies of this protein in the model Gram-positive organism Bacillus subtilis. I used co-immunoprecipitation and deep-sequencing to define the suite of RNA elements that associate with this regulatory protein. In addition I performed global transcriptomic studies on an Hfq deletion mutant in order to identify genes that are regulated via Hfq. These studies identified sRNAs that may be involved with sporulation. This led me to analyze the transcriptomic profile of Bacillus subtilis spores in an attempt to identify new sRNA regulators.Item Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the HTERT Locus(2011-02-01T19:36:40Z) Wilson, Kimberly Anne; Porteus, MatthewThe ability to direct hTERT expression through genetic control or tunable regulatory factors would advance our understanding of the transcriptional regulation of hTERT, and also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and zinc finger nucleases (ZFNs) to target sequences at the hTERT promoter. We first explored expansions to the repertoire of sites that can be targeted by ZFNs and modifications of ZFN architecture to accommodate such sites. A ZFN is made of a zinc-finger DNA binding domain (ZFP) linked to the FokI nuclease domain by a short amino acid “inter-domain linker”. The general sequence motif of a ZFN target is 5’-(ZFN site1)-(6 bp spacer)-(ZFN site2)-3’ and each half-site is 5’-GNNGNNGNN-3’. Variations of this motif come in the forms of variable spacer lengths, extra basepairs in-between triplets, and the inclusion of non-GNN triplets. To explore these types of target sites, we created ZFN variants that contained different inter-domain linkers, lengthened inter-finger linkers, and DNA binding domains created through hybridizing the modular assembly and OPEN methodologies. We show that through altering ZFN architecture, target sites with 5-7-bp spacers and those with ANN, CNN, and TNN triplets can be efficiently recognized and cut by ZFNs. We then generated new ZFPs to five ZFN target sites with 5- or 6-bp spacers in the hTERT locus based on those findings and made ZFTFs by linking the ZFPs to the VP16 transcriptional activation domain. We were able to identify several active ZFTFs that demonstrate a dose-dependent response. The same ZFPs were also converted into ZFNs and screened in combinatorial pairs in cell-based single-strand annealing assays and gene targeting assays. These screening strategies have pinpointed several ZFN pairs that may be useful in genomic editing of the hTERT locus. Our findings provide guidelines for modifying ZFP architecture to a wider array of potential target sites for use in developing ZFTFs and ZFNs at the hTERT promoter, which may be applicable towards inheritable, telomerase-based diseases and answering basic science questions about hTERT transcriptional regulation.Item FHL2 Inhibits Calcineurin and Represses Pathological Cardiac Hypertrophy(2010-11-02T18:11:08Z) Hojayev, Berdymammet; Hill, Joseph A.Stress-induced cardiac hypertrophy is a hallmark feature of pathological remodeling which, left unchecked, predisposes hearts to arrhythmia and failure. FHL2 is a member of the four-and-a-half LIM domain (FHL) family of proteins expressed predominantly in the heart. Targeted disruption of FHL2 leads to an exaggerated response to beta-agonist (isoproterenol)-induced cardiac hypertrophy. Isoproterenol-induced hypertrophy relies on activation of the calcineurin-NFAT pathway, and inhibition of calcineurin is sufficient to block growth in response to isoproterenol. I also observed that FHL2 is up-regulated in mouse hearts after isoproterenol treatment. Based on this, we hypothesized that FHL2 negatively regulates the calcineurin-NFAT pathway and consequently, the hypertrophic growth response. To determine whether calcineurin signaling is enhanced in the absence of FHL2, wild type (WT) and FHL2 knockout (FHL2-/-) mice were treated with isoproterenol (32 mg/kg/day). We observed a significant increase in isoproterenol-induced expression of the NFAT target genes RCAN1.4 and BNP in FHL2-/- hearts as compared to WT. To determine whether the effect of FHL2 on the abundance of NFAT target gene transcripts was mediated by calcineurin- NFAT-dependent transcription, HEK 293 cells were transfected with luciferase reporter constructs containing the NFAT-driven promoters of either RCAN1 or IL-2. Consistent with the in vivo data, knockdown of FHL2 message using siRNA led to increases in both RCAN1 and IL-2 promoter activities elicited by constitutively active calcineurin or the calcium ionophore, ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporin A, confirming the calcineurin dependence of the response. Over-expression of FHL2 in HEK 293 cells inhibited the activation of both NFAT reporters triggered by either constitutively active calcineurin or ionomycin. Furthermore, neonatal rat ventricular myocytes over-expressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin (measured by cell crosssectional area and fetal gene expression). Finally, immunostaining of adult cardiomyocytes revealed co-localization of FHL2 and calcineurin predominantly at the sarcomere, and activation of calcineurin by endothelin-1 treatment resulted in interaction between FHL2 and calcineurin as demonstrated by coimmunoprecipitation. These observations demonstrate that FHL2 represses calcineurin-NFAT signaling and thereby suppresses hypertrophic cardiac growth at least in part by interacting with calcineurin and inhibiting its activation.Item Functional Domains in the Multigene Regulator of the Group A Streptococcus(2006-05-16) Vahling, Cheryl M.; McIver, Kevin S.The group A streptococcus (GAS) is a human pathogen capable of causing a broad range of symptoms from mild erythema to severe tissue destruction. The multigene regulator of the GAS, or Mga, has been shown to bind DNA and activate transcription of virulence genes implicated in colonization and immune evasion in response to environmental conditions. Several different avenues of investigation were undertaken in the present study with an overall goal of identifying and characterizing functional domains within the protein. The first section of this study uses a naturally occurring mutant, previously identified to be deficient in transcriptional activation, as a guide to map functional residues within Mga. Electrophoretic mobility shift assays using purified proteins determined that the defect was not a result of the inability to bind DNA, so gain-of-function mutants were used to restore transcriptional activity to the mutant and pinpoint the residues necessary for full activation. In the second section, a genetic screen was undertaken to identify novel domains within the protein. From this screen, a domain was found within the extreme N-terminus. Sequence homology revealed several proteins in other pathogenic treptococci that shared this motif. As a result, it was named CMD-1 for conserved Mga domain 1, while the group of proteins was subsequently designated as the Mga family of putative virulence gene regulators. Alanine scanning mutagenesis demonstrated the importance of CMD-1 for transcriptional activation in several members of this family. This established that the results yielded from investigations of Mga could be broadened beyond the GAS to provide global insight into possible mechanisms of virulence regulation in other pathogenic streptococci. Finally, an in silico analysis of Mga was performed, revealing several areas with differing degrees of structural similarity to known domains of other bacteria including a receiver domain and a PTS-regulatory domain. Each region was explored to determine if it was functionally active in Mga. Since both domains have been implicated in forming higher order structures, the oligomeric state of Mga was also determined. Overall, characterization of Mga has helped unravel the mechanisms of virulence regulation in pathogenic streptococci.Item Insig-Mediated Regulation of Hepatic Lipid Synthesis(2007-05-22) Engelking, Luke James; Brown, Michael S.Cholesterol synthesis in mammals is tightly regulated by end-product feedback inhibition. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a rate determining reaction that is highly regulated by transcriptional and post-transcriptional mechanisms. As cellular cholesterol accumulates, the transcription of HMGR mRNA is suppressed and the proteosomal degradation of HMGR protein is accelerated. The sterol-regulated transcription of HMGR and other lipogenic genes is controlled by sterol regulatory element binding proteins (SREBPs). These membrane-bound transcription factors are escorted by SREBP cleavage activating protein (SCAP) from the endoplasmic reticulum (ER) to the Golgi apparatus where SREBPs are proteolytically processed to their active forms. In cultured cells, feedback inhibition of SREBP processing is mediated by Insigs. When sterols accumulate, Insigs block SREBP activation by retaining SCAP in the ER. Insigs also mediate rapid, sterol-dependent turnover of HMGR protein. When sterols accumulate, Insigs bind to HMGR and stimulate its ubiquitination and degradation. Although Insigs are key regulators of cholesterol homeostasis in cultured cells, their role in the intact mammal was undefined. To explore this question, gain-of-function and loss-of-function analyses were performed by studying the livers of genetically engineered mice. First, transgenic mice that overexpress Insig-1 in liver (TgInsig-1) were generated. In the livers of TgInsig-1 mice, nuclear SREBPs (nSREBPs) were reduced and SREBP processing was supersensitive to inhibition by feeding high-cholesterol diets. The block in SREBP processing reduced the mRNA levels of SREBP target genes. Levels of HMGR protein were reduced and declined further with cholesterol feeding. Next, knockout mice that lack Insig-1, Insig-2, or both Insigs were generated. In the livers of Insig double knockout mice, cholesterol and triglycerides accumulated to high levels, and despite their accumulation, nSREBPs and mRNAs of SREBP target genes were not suppressed. SREBP processing was insensitive to inhibition by feeding high-cholesterol diets. HMGR protein levels were increased and failed to decline with cholesterol feeding. As a consequence of Insig overexpression or deficiency and the respective effect on SREBPs and HMGR, hepatic cholesterol and fatty acid synthesis in living animals was decreased in TgInsig-1 mice and increased in Insig double knockout mice. These studies indicate that Insigs are essential regulators of hepatic lipid synthesis.Item Mechanistic Analysis of SRF and the Myocardin Family of Coactivators During Muscle Development(2005-12-20) Li, Shijie; Olson, Eric N.The precise mechanism of how specification and differentiation of different muscle types are controlled by a large number of transcription factors has been a long-standing question in developmental biology. Using animal models with tissue-specific deletions of various transcription factors, coupled with biochemical studies, the molecular mechanisms regulating muscle development and growth are being elucidated. Serum response factor (SRF), a muscle-enriched transcription factor, activates the expression of numerous muscle genes by recruiting a variety of partner proteins. The function of SRF in each muscle type in vivo is clouded by the fact that SRF mutant mice die before gastrulation without the formation of mesoderm. Generating a tissue-specific deletion of the SRF gene, I found that SRF is required for skeletal muscle growth and maturation. Myocardin was identified as a cardiac and smooth muscle-specific transcriptional coactivator of SRF. Mice lacking myocardin die during early embryogenesis due to cardiovascular defects, which are caused by the failure of vascular smooth muscle to differentiate. Together with the data that overexpression of myocardin in non-muscle cells can activate the smooth muscle gene program, we demonstrate that myocardin is both required and sufficient for smooth muscle differentiation. Two Myocardin Related Transcription Factors, referred to as MRTF-A and B, which also interact with SRF and stimulate its transcriptional activity, are expressed in numerous embryonic and adult tissues, implying their potential to modulate SRF target genes in a wide range of tissues. Consistent with the role of SRF during skeletal muscle development, a dominant-negative form of MRTF-A interferes with skeletal muscle development in transgenic mice. To further elucidate MRTF-A's function, I generated MTTF-A mutant mice by gene homologous recombination. Female MRTF-A mutant mice fail to nurture their offspring due to mammary defects. While milk is produced at a normal level, mammary myoepithelial cells, which are similar to smooth muscle cells and required for milk ejection, fail to differentiate and undergo programmed cell death during lactation. Taken together, these data indicated that SRF regulates specification or maturation of different muscle types by interacting with various members of the myocardin family of coactivators.Item Mechanistic Dissection of Insig-1, a Master Regulator of Cholesterol Homeostasis(2006-05-15) Gong, Yi; Brown, Michael S.Insigs are polytopic membrane proteins of the endoplasmic reticulum (ER) that regulate lipid synthesis by controlling the sterol-mediated vesicular transportation of sterol regulatory element binding proteins (SREBPs). SREBPs are ER bound transcription factors that form complexes with Scap. In sterol-depleted cells, Scap escorts SREBPs from the ER to the Golgi apparatus, where SREBPs are proteolytically cleaved to liberate the nuclear fragments that activate genes for cholesterol synthesis and uptake. When sterols overaccumulate in cells, the Scap/SREBP complex is retained in the ER by the anchor proteins called Insigs. In this thesis I describe the formation of a complex between Insig-1 and Scap in a sterol regulated fashion which facilitates the ER retention of Scap. To understand the molecular basis of the interactions between Insig-1 and Scap, I use a site-directed mutagenesis approach to select residues in Insig-1 that are essential for Insig-1/Scap complex formation. This study reveals a functional role for the amino acid Asp-205, which is located at the beginning of the fourth loop of Insig-1. Mutation of this aspartic acid to alanine produces an inactive Insig-1 that no longer binds to Scap, and leads to sterol-resistant processing of SREBPs. Mammalian cells express two Insig proteins differ in their mode of control. Insig-1, but not Insig-2, is an SREBP target gene. Also, Insig-1 protein is degraded more rapidly than Insig-2. Thus, Insig-1 is the focus of the study. I further demonstrate that degradation of Insig-1 is regulated by sterols. When ER cholesterol content is low, Insig-1 is ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Sterol-induced binding of Insig-1 to Scap prevents Insig-1 ubiquitination and degradation. The dynamic change in Insig-1 protein stability, together with its transcriptional control by nuclear SREBPs, creates a new model for the convergent inhibition of SREBP processing and cholesterol supply in animal cells. Taken together, these studies established Insig-1 as the master regulator in the cholesterol homeostasis.Item MEF2 and HDAC Proteins Regulate Striated Muscle Development and Remodeling(2007-12-18) Potthoff, Matthew Joseph; Olson, EricThe establishment of different tissues during embryogenesis requires coupling of upstream signal transduction pathways with networks of transcription factors that govern cell differentiation and morphogenesis. The myocyte enhancer factor 2 (MEF2) transcription factor acts as a lynchpin in the transcriptional circuits that control differentiation of diverse cell types including skeletal, cardiac and smooth muscle cells, neurons, chondrocytes, lymphocytes, endothelial cells and neural crest cells. Class II histone deacetylase (HDAC) proteins bind to MEF2 and regulate MEF2 activity in response to various signaling cascades. To understand the role of MEF2 and class II HDAC proteins in skeletal muscle development and remodeling, we analyzed individual MEF2 knockout mice, HDAC knockout mice, and compound mutant mice. We discovered that skeletal muscle-specific deletion of Mef2c in mice results in disorganized myofibers and perinatal lethality. In contrast, neither Mef2a nor Mef2d are required for normal skeletal muscle development in vivo. Skeletal muscle deficient in Mef2c differentiates and forms normal myofibers during embryogenesis, but myofibers rapidly deteriorate after birth due to disorganized sarcomeres and a loss of integrity of the M-line. We discovered that MEF2C directly regulates important structural genes required for the maintenance of sarcomere integrity and postnatal maturation of skeletal muscle. To address the function of class II HDACs and MEF2 proteins in adult skeletal muscle remodeling, we discovered that class II HDAC proteins, which function as transcriptional repressors of the MEF2 transcription factor, fail to accumulate in the soleus, a slow-twitch muscle, compared to fast-twitch muscles (eg.., white vastus lateralis). Using gain- and loss-offunction approaches in mice, we discovered that class II HDAC proteins suppress slow, oxidative myofiber identity through the repression of MEF2 activity. Conversely, expression of a hyperactive form of MEF2 in skeletal muscle of transgenic mice promotes the slow fiber phenotype and enhances running endurance, enabling mice to run almost twice the distance of wild type littermates. Thus, the selective degradation of class II HDACs in slow skeletal muscle provides a mechanism for enhancing physical performance and resistance to fatigue by augmenting the transcriptional activity of MEF2. To understand the functions of class I HDACs in cardiac development and remodeling, we generated cardiac-specific HDAC1 and HDAC3 transgenic mice. Overexpression of HDAC1 resulted in a dilated cardiomyopathy, while overexpression of HDAC3 produced a stress-induced cardiac phenotype. We establish an important role for these proteins in cardiac remodeling and provide potential mechanisms regulating these enzymes in vivo. Taken together, these studies demonstrate an important role for MEF2 and HDAC proteins in muscle development and function. Moreover, these results provide important mechanistic insights into the regulation of MEF2 and HDAC proteins in vivo.Item Modulation of Transcription Factor Activity by Mono-ubiquitin(2008-09-12) Archer, Chase Tanner; Kodadek, ThomasThe Ubiquitin-Proteasome Pathway plays both proteolytic and non-proteolytic roles in the regulation of transcription. We recently reported that the ATPases of the 26S proteasome can destabilize activator-DNA complexes in a non-proteolytic manner that requires direct interactions between the Rpt4 and 6 subunits with the activation domain of the activator. Remarkably, mono-ubiquitylation of the activator blocks this repressive activity. In this study, we probe the mechanism of this protective effect. Using novel label transfer and chemical cross-linking techniques, we show that ubiquitin contacts the ATPase complex directly, apparently via Rpn1 and/or Rpt1, and that this interaction results in the dissociation of the activation domain-ATPase complex via an allosteric process. We also provide in vivo evidence demonstrating the importance of monoubiquitylation in inhibition of activator-DNA destabilization. A model is proposed in which activator mono-ubiquitylation serves to limit the lifetime of the activator-ATPase complex interaction and thus the ability of the ATPases to unfold the activator and dissociate the protein-DNA complex.Item Multiple Functions of BRD4 in E2 Mediated HPV Transciptional Regulation(2009-09-04) Lee, A-Young; Chiang, Cheng-MingHuman papillomaviruses (HPVs) are DNA viruses that cause benign and malignant tumors of epithelial origins. Expression of HPV-encoded E6 and E7 oncoproteins is controlled by the viral E2 protein, which plays a dual role in gene activation and repression. Recently, we identified bromodomain-containing protein 4 (Brd4) as a cellular corepressor for E2-mediated inhibition of HPV transcription. Brd4 contains two bromodomains which function as acetyl-lysine-binding modules that facilitate chromatin targeting via their interactions with acetylated histones. Although Brd4 has been known to be involved in E2-mediated transcriptional regulation, it was unclear how Brd4 regulates E2 function and whether the involvement of Brd4 in transactivation and transrepression is common to different types of E2 proteins. Here, we show that Brd4 enhances E2 binding to its cognate sequences through Brd4's bromodomains and the E2-interacting region in chromatin. We further demonstrate that the corepressor function of Brd4 is common to E2 proteins encoded by cancer-inducing high-risk HPV, wart-causing low-risk HPV, and bovine papillomavirus type 1. The general cofactor function of Brd4 on E2-mediated transcription is in part controlled by enhancing the protein stability of E2, which is normally degraded via the ubiquitin-dependent proteasome pathway. These findings indicate that a chromatin adaptor can enhance the binding of a sequence-specific transcription factor to chromatin and further promote the stability of a labile transcription factor via direct protein-protein interaction. Also, we identify two additional E2-interacting regions of Brd4: the E2-interacting domain (E2ID) and phosphorylation-dependent interacting domain (PDID). While E2ID binds to all different types of E2 proteins, PDID interacts only with high risk E2 (HRE2) in a casein kinase 2 (CK2) phosphorylation-dependent manner. In addition to HRE2-specific interaction, the phosphorylation of PDID also induces intramolecular interaction between PDID and E2ID, which blocks the E2-interaction of E2ID. Finally, we show that the PDID-HRE2 interaction is important for the HRE2-mediated transcriptional activation. Collectively, our data show that the posttranslational modification of the cellular protein Brd4 confers selective recognition of HRE2, thereby providing a unique regulation mechanism for the protein encoded by cancer-inducing HPV.Item New Roles of the Transcription Factor NKX6.1 in Beta Cell Biology(2006-05-15) Schisler, Jonathan Cummings; Johnston, Stephen A.Pancreatic islet beta cells play a critical role in the maintenance of metabolic fuel homeostasis. Type 1 diabetes results from autoimmune destruction of the beta cells, whereas type 2 diabetes involves loss of beta cell function, particularly glucose-stimulated insulin secretion (GSIS), and a gradual diminution of beta cell mass. To identify genes that are involved in GSIS and contribute to the mature beta cell phenotype, a panel of beta cell lines with varying capacities for GSIS was employed for a candidate gene screen. Expression of the homeodomain transcription factor Nkx6.1 was found to be positively correlated with the capacity for GSIS in these lines. Although previously identified as an essential factor for beta cell differentiation, little was known about the function of Nkx6.1 in mature beta cells. The purpose of this dissertation research was to investigate the role of Nkx6.1 in the biology of mature beta cells via manipulation of its expression in beta cell lines and primary pancreatic islets and measurement of a variety of functional outcomes. These studies led to identification of three novel roles of Nkx6.1 in the mature beta cell. First, Nkx6.1 was found to suppress expression of the glucagon gene via a direct interaction with its promoter, although the studies also demonstrate a role for Pdx1 and Mafbeta in mediating this suppression. Second, the silencing of Nkx6.1 expression in pancreatic beta cells results in a severe impairment in GSIS, suggesting that Nkx6.1 target genes are critical for robust GSIS. Third, Nkx6.1 is shown to regulate beta cell proliferation, in part via direct interaction with the cyclin B1 gene and stimulation of its expression. Thus, whereas the majority of primary beta cells are quiescent, overexpression of Nkx6.1 is sufficient to initiate DNA synthesis and cell division. Importantly, and different from the common experience, this manipulation also enhances GSIS. These studies suggest that Nkx6.1 should be further investigated as a gene involved in development of diabetes and as a potential new therapeutic target.Item The p13-kinase/Tsc pathway: a role in neural and renal development and pathology(2008-09-19) Zhou, Jing; Parada, Luis F.PTEN is a tumor suppressor gene and its protein product negatively regulates the PI3K/AKT pathway through counteracting the kinase function of PI3-Kinase. Loss of function of PTEN results in overactivation of AKT and in turn activates multiple AKT downstream pathways. One AKT substrate is the TSC1/2 protein complex, which controls protein synthesis and cell growth through regulating mTOR activity. AKT inhibits TSC1/2 complex by directly phosphorylating TSC2, and in turn releases the inhibition of TSC1/2 complex on mTOR. Thus, loss of either PTEN or TSC1 or TSC2 can result in increased mTOR activity. However, regulation of TSC1/2 complex by AKT could be context dependent and the TSC/mTOR pathway is regulated by upstream regulators other than AKT in different cell types. In this study, I characterized the functions of PTEN and TSC1 in both post-mitotic neurons and renal tubule cells, and evaluated the relationship of these two tumor suppressor genes in two distinct contexts. Previously, a conditional Pten knockout mouse line was generated with Pten loss in limited post-mitotic neurons in the cortex and hippocampus. These mice develop macrocephaly accompanied by neuronal hypertrophy and loss of neuronal polarity. The mutant mice also exhibit behavioral abnormalities reminiscent of certain features of human autism. Biochemical analysis indicates that multiple AKT downstream pathways including the TSC/mTOR pathway are activated in neurons that lose Pten. In the current study, I demonstrate that rapamycin, a specific inhibitor of mTOR, can prevent or reverse neuronal hypertrophy resulting in the amelioration of PTEN-associated abnormal behaviors. In addition, loss of Tsc1 in a same context results in similar neuronal hypertrophy. Thus, the study provides evidence that the mTOR pathway is critical for the neuronal phenotype observed in Pten mutant mice. In the second part of the study, I demonstrate that severe polycystic kidneys disease develops in Tsc1 mutant mice, but not in Pten mutant mice. Apparently, overactivation of mTOR signaling only occurs in the kidneys of Tsc1 mutant mice, suggesting distinct activities for PTEN and TSC1 in mTOR activation in renal tubule cells compared to that found in neurons.Item Post-Transcriptional Regulation by microRNAS in Pregnancy and Parturition(2012-08-15) Renthal, Nora Edwards; Mendelson, Carole R.Throughout most of pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) transcriptional activity, while spontaneous labor is initiated/facilitated by a concerted series of biochemical events that activate inflammatory pathways and negatively impact PR function. In this study, we uncovered a new regulatory pathway whereby miRNAs serve as hormonally-modulated and conserved mediators of contractile gene regulation in the pregnant uterus from mouse to human. Using miRNA and gene expression microarray analyses of uterine tissues, we identified a conserved family of miRNAs, the miR-200 family, that is highly induced at term in both mice and humans, as well as two coordinately downregulated targets, zinc finger E-box binding homeobox proteins, ZEB1 and ZEB2, which act as transcriptional repressors. We also observed upregulation of the miR-200 family and downregulation of ZEB1 and ZEB2 in two different mouse models of preterm labor. We further demonstrated that ZEB1 is directly upregulated by the action of P4/PR at the ZEB1 promoter. Excitingly, we observed that ZEB1 and ZEB2 inhibited expression of the contraction-associated genes, oxytocin receptor and connexin-43 and blocked oxytocin-induced contractility in human myometrial cells. Together, these findings implicate the miR-200 family and their targets ZEB1 and ZEB2 as novel P4/PR-mediated regulators of uterine quiescence and contractility during pregnancy and labor, and shed new light on the molecular mechanisms involved in preterm birth. [Keywords: microRNA; pregnancy; parturition; uterus; myometrium; progesterone; hormonal regulation; ZEB1; ZEB2; miR-200]Item Recognition Mechanisms of Nuclear Localization and Export Signals(2009-06-19) Suel, Katherine Elizabeth; Chook, Yuh MinThe transport of proteins between the nucleus and cytoplasm of cells is mediated by the Karyopherin beta family of proteins. Karyopherin betas recognize their substrates through either a nuclear localization or export signal depending on the direction of transport. Even though there are ten yeast import Karyopherin betas, for the past thirteen years there was only one well characterized nuclear localization signal, the classical nuclear localization signal. However, a second signal, the proline-tyrosine nuclear localization signal recognized by Karyopherin beta2, was recently identified through X-ray crystallography and biochemical studies of Karyopherin beta2 bound to one of its substrates. These studies identified rules for the recognition of the proline-tyrosine nuclear localization signal by Karyopherin beta2. The signal must have overall basic charge, structural disorder and a weak consensus sequence of an amino-terminal basic or hydrophobic-enriched region followed by a carboxyl-terminal arginine residue separated from a proline and tyrosine residue by two to five residues. The proline-tyrosine nuclear localization signal is also recognized by the Saccharomyces cerevisiae homolog of Karyopherin beta2, Karyopherin 104, demonstrating the generality of this import mechanism across eukaryotes. Thermodynamic analyses of the two known substrates of Karyopherin 104, Hrp1p and Nab2p, revealed physical properties governing its binding. The proline-tyrosine nuclear localization signal is an extended signal with significant sequence diversity. The signal is comprised of three binding epitopes, each of which can have varying energetic strengths in different substrates. The multivalent nature of the signal increases the diversity of the signal as well as the difficulty of identifying new substrates. A bioinformatics search identified putative proline-tyrosine nuclear localization signals which were validated through biochemical studies. Additionally, one of the proteins identified, Tfg2p, was verified as a bona fide Karyopherin 104 substrate. Analysis of Tfg2p's cellular localization revealed that its nuclear localization was not solely determined by the presence of a nuclear localization signal, but was also dependent on its retention in the nucleus. Furthermore, crystallographic studies of substrate Snurportin1 bound to the export karyopherin CRM1 revealed that its nuclear export signal has two binding epitopes implying that the multivalent nature of targeting signals may not be limited to the proline-tyrosine nuclear localization signal.Item Regulation and Function of PTF1a in the Developing Nervous System(2012-08-15) Meredith, David Miles; Johnson, Jane E.Basic helix-loop-helix transcription factors serve many roles in development, including regulation of neurogenesis. Many of these factors are activated in naive neural progenitors and function to promote neuronal differentiation and cell-type specification. Ptf1a is a basic helix-loop-helix protein that is required for proper inhibitory neuron formation in several regions of the developing nervous system, including the spinal cord, cerebellum, retina, and hypothalamus. In addition, Ptf1a is essential for proper pancreas formation and exocrine function. In both the nervous system and pancreas, Ptf1a functions as a switch in cell fate determination. In the absence of Ptf1a, inhibitory neurons are lost and those cells instead adopt an excitatory identity. Similarly, endodermal progenitors will assume duodenal characteristics in place of a pancreatic identity when Ptf1a is lost. Like most other tissue-specific basic-helix-loop-helix factors, Ptf1a dimerizes with E-proteins and binds a degenerate hexameric E-box motif (CANNTG). Ptf1a is unique, however, in that it also requires the presence of Rbpj(l) to form an active transcription complex, PTF1. This interaction is central to Ptf1a function, as disruption of Ptf1a?s ability to bind Rbpj in vivo phenocopies the Ptf1a null in the nervous system and pancreas. Similarly, all targets described thus far for Ptf1a require an intact PTF1 binding site, which includes both an E-box and Rbpj binding site. In order to understand how a factor such as Ptf1a is capable of giving rise to such disparate organs, I wanted to place it in context of a larger regulatory network that directs a multipotent progenitor into a mature inhibitory neuron. Thus, I examine two regulatory schemes controlling Ptf1a expression during development in Chapters two and three. I then investigate direct Ptf1a targets in a genome-wide fashion using massively parallel sequencing technology in Chapters four and five. These efforts uncovered that Ptf1a employs several mechanisms to achieve proper cell-type specification, including initiation of transcription factor cascades, direct activation of inhibitory neuron machinery, and direct suppression of the excitatory neuron program. Furthermore, I identify novel binding modes and potential co-regulatory factors that could impart tissue-specific function. [Keywords: Ptf1a; neural tube; pancreas; Rbpj; bHLH; ChlP-Seq; RNA-Seq; transcription; development; GABAergic]Item Regulation of Accessibility of the Variable Gene Segments of the Mouse Immunoglobulin Kappa Light Chain Gene Locus(2005-08-11) Brekke, Katherine Meyers; Garrard, WilliamUnderstanding the development of the expression of antibody heavy and light chain molecules is required to fully explain the adaptive immune response. This involves understanding the mechanisms underlying the selection of individual Vκ genes for V-J joining. It has been assumed that this selection involves alterations in the chromatin structure surrounding Vκ genes. Therefore, the focus of my work has centered on understanding the factors influencing the selection of a particular Vκ gene as well as the accompanying alterations in chromatin structure. I have assembled the complete nucleotide sequence of the 3.2 Mb Mus musculus Igκ gene locus and mapped all of the functional and pseudo-Vκ genes onto the sequence. I have also determined the patterns of potential transcription factor binding sites surrounding these Vκ genes. These analyses revealed statistically significant patterns of transcription factor motifs that are clustered either upstream of the transcription start site, in the intronic region, or downstream of the recombination signal sequence (RSS). The conservation of these sites has been tied to the presence of DNase I hypersensitive sites at the pre-B cell stage of development. These hypersensitive sites appear just prior to the V-J joining step and disappear after the Igκ locus has completed the rearrangement process. These sites are present at or near the promoter of the particular Vκ gene and are sometimes present near the RSS as well. I also looked for the presence of transcripts from the promoters of the Vκ genes that had HSP and HSRSS. I found that the presence of transcripts and the presence of HS was uncoupled. There were instances where both HS and transcripts were present, neither was present, or only one phenomenon was observed. This led me to conclude that these events were separable. The presence of HSRSS as well as the corresponding binding sites for transcription factors in the downstream region, suggests a model for generating accessibility of Igκ V genes for V-J joining.Item The Regulation of TCF POP-1 Asymmetry and its Function in Early Cell Fate Specification in Caenorhabditis Elegans(2010-01-12T18:53:02Z) Huang, Shu-Yi; Lin, RueylingCell-cell signaling pervades all aspects of development. Signaling pathways need to be highly robust to ensure reproducible outcomes. In the cases of several major pathways, including the Wnt signaling pathway, default repression and signal-dependent activation are both mediated by the same response elements and transcription factors. My work focuses on how the C. elegans TCF protein POP-1 is converted from a repressor into an activator upon Wnt and MAPK signaling. C. elegans embryos exhibit a nearly invariant cell lineage composed primarily of a stepwise binary diversification of anteroposterior blastomere identities. The nuclear level of TCF/POP-1 is lowered in all posterior cells in the A-P cell divisions. I showed that the ?-catenin homolog, SYS-1, exhibits reiterated asymmetry which is reciprocal to that of POP-1. SYS-1 functions as a limiting coactivator for POP-1. The SYS-1-to-POP-1 ratio is critical for both anterior and posterior cell fates. A high ratio drives the posterior cell fate whereas a low ratio drives the anterior cell fate in multiple A-P divisions throughout development. I showed genetically that while POP-1 nuclear asymmetry is mainly regulated by the MAPK pathway, SYS-1 asymmetry is regulated solely by the Wnt pathway. Besides the quantitative differences between A-P nuclei, the Wnt and MAPK pathways also modify POP-1 qualitatively, affecting the transcriptional activity of POP-1. I showed that LIT-1/WRM-1-dependent phosphorylation of POP-1 reduces its binding to the coactivator SYS-1. Deleting the PCA domain also weakens the interaction between POP-1 and SYS-1 independent of LIT-1/WRM-1 phosphorylation. The PCA domain is required for POP-1 nuclear asymmetry and all LIT-1/WRM-1-dependent phosphorylation of POP-1. Within the PCA domain, mutating POP-1 threonine 426 to aspartic acid, but not alanine, shows similar effects to deleting the PCA domain. This study shows that Wnt and MAPK signaling pathways regulate the TCF protein POP-1 both quantitatively for its nuclear asymmetry, and qualitatively for its transcriptional activity. The two pathways not only change the levels of POP-1 and SYS-1 but also modify the strength of binding, strongly favoring the formation of POP-1/SYS-1 activation complex in the posterior cell, thereby driving A-P differential cell fates.