Browsing by Subject "Basic Helix-Loop-Helix Transcription Factors"
Now showing 1 - 11 of 11
Results Per Page
Sort Options
Item The bHLH/PAS Transcription Factor SIM1 is a Novel Obesity Gene(2005-05-03) Holder, Jimmy Lloyd, Jr.; Zinn, Andrew R.Obesity is epidemic in the United States and other developed countries. Obesity is a major risk factor for type II diabetes, hypertension, hyperlipidemia and osteoarthritis. I report a unique girl with early-onset obesity (47.5 kg, +9.3 s.d. above mean at age 67 months) and a de novo balanced translocation between chromosomes 1 and 6. She has normal energy expenditure and a voracious appetite. I show that her translocation disrupts a transcription factor gene, SIM1, on chromosome 6q16.2. I also present data that Sim1 haploinsufficiency causes obesity in mice. Animals heterozygous for a Sim1 null allele fed a standard chow diet (4% fat) developed obesity around the time of sexual maturity, were 33-45% heavier than wild-type littermates by 5 months of age, and had increased adiposity by DEXA scans. In contrast, the human subject developed obesity by two years of age, well before puberty. To investigate whether differences in dietary fat consumption might explain this discrepancy in human and mouse phenotypes, I fed mutant mice and wild type littermates a "Westernized" diet (35% fat). Heterozygous Sim1 mice fed this diet became obese prior to 6 weeks of age. The obesity was also more severe, especially in females, who by 8 weeks of age weighed 72% more than controls compared with 13% on a low fat diet. Heterozygous Sim1 mice maintained on a 4% fat diet ate more than controls over a 5 day period (delta kcals 12-14%), and became even more hyperphagic when acutely challenged with increased dietary fat (delta kcals 46-68% over 5 days). This altered behavior was evident within the first day of exposure to the high fat diet: during this time, heterozygous Sim1 mice failed to significantly change the mass of food consumed, whereas wild-type littermates decreased their food consumption by >15%. These data suggest that Sim1 is critical for the acute and chronic homeostatic response to elevated dietary fat. This data demonstrates that normal Sim1 gene dosage is critical for proper regulation of feeding behavior and body weight regulation.Item Control of Cardiac and Limb Development by the bHLH Transcription Factors DHAND and eHAND(2004-05-04) McFadden, David Glenn; Srivastava, DeepakMembers of the basic helix-loop-helix (bHLH) transcription factor family regulate the specification and differentiation of multiple cell lineages during embryonic development. The bHLH proteins dHAND and eHAND are expressed in complimentary and overlapping patterns during embryogenesis, and gene knockout studies have demonstrated that dHAND and eHAND null embryos die from defects in right ventricular and placental development, respectively. Therefore, we have investigated three aspects of HAND gene biology. Firstly, in order to determine the mechanisms that establish chamber identity during cardiac development, we have identified a transcriptional enhancer that controls dHAND expression in the embryonic right ventricle, and demonstrated that activity of this element depends on paired binding sites for the GATA family of zinc-finger transcription factors. Secondly, we have generated floxed alleles of murine eHAND in order to investigate the role of eHAND during heart formation. These studies have identified a novel role for eHAND during cardiac valve formation, and demonstrated genetic redundancy of HAND genes during mammalian cardiac morhpogenesis. Finally, we have utilized tissue culture assays and transgenic mice to investigate the mechanisms by which dHAND regulates transcription of downstream target genes. These results suggest that dHAND and eHAND may regulate gene expression independently of direct DNA binding and transcriptional activation.Item Defining a Novel Role for Hypoxia Inducible Factor-2 Alpha (HIF-2a)/EPAS1 : Maintenance of Mitochondrial and Redox Homeostasis(2005-12-20) Oktay, Yavuz; Garcia, Joseph A.The Epas1 gene encodes HIF-2alpha , a member of the Hypoxia Inducible Factor family of transcriptional regulators. The biological role for HIF-2alpha has been elusive due to embryonic lethality of the initial Epas1-/- mouse strains. Our lab reported the generation of the first viable Epas1-/- mice using a genetic breeding strategy. Adult Epas1-/- mice exhibit gross, histological, biochemical, and molecular evidence consistent with mitochondrial dysfunction. Similarities between Epas1 and Sod2 deficient strains suggest a biochemical etiology, increased oxidative stress, as well as a molecular etiology, decreased Sod2 gene expression, for the mitochondrial dysfunction in Epas1-/- mice. Consistent with this hypothesis, Sod2 gene expression is reduced in Epas1-/- mice whereas HIF-2a induces Sod2 gene promoter in transient transfection studies. Further studies revealed impaired mitochondrial respiration, sensitized mitochondrial permeability transition pore opening, increased electron transport chain activity and reduced mitochondrial aconitase activity. Given that it is the most sensitive enzymatic marker for oxidative stress, aconitase inhibition may explain impaired respiration. Also, redox balance in Epas1-/- liver is disturbed: the reduced cytoplasmic environment, and a relative oxidized environment for mitochondria from Epas1-/- liver implies a role for HIF-2a in maintenance of cellular redox balance. All these data suggest that HIF-2a is essential for maintenance of mitochondrial function, reactive oxygen species detoxification, and redox balance.Item HIF-2: Standing Guard at the Crossroads of Stress and Aging(2009-06-15) Dioum, El Hadji Mamadou; Garcia, Joseph A.The capacity of mammalian organisms to cope with hypoxic or ischemic stress is in part mediated by stress-induced transcription factors. Hypoxia-induced mediators include transcription factors, such as the α (alpha) subunit of Hypoxia inducible factors (HIF-1alpha and HIF-2 alpha). HIF-1 alpha and HIF-2 alpha have similar structural organization, and after forming an obligate heterodimer with the common partner ARNT/HIF-1 alpha, bind to the same recognition element located in target gene promoter or enhancer regions. However, despite these similarities, HIF-1 alpha and HIF-2 alpha regulate distinct target genes. In previous studies from the Garcia laboratory using mouse knockout studies, we demonstrated the importance of HIF-2 alpha in the in vivo regulation of genes involved in the cellular response to hypoxic and oxidative stress. These genes include Erythropoietin (epo), vascular endothelial cell growth factor (Vegf), superoxide dismutase 2 (Sod2) and other genes encoding major antioxidant enzymes (AOE). Novel roles for HIF-2 alpha have been found not only in hematopoiesis, but also in the control of reactive oxygen species and mitochondrial homeostasis. The molecular mechanism by which HIF-2 alpha selectively regulates its target genes remains an exciting area of research. In the first part of my thesis, I identified a novel molecular mechanism regulating activity of the enhancer region in the Epo gene. First, by using bioinformatics to perform an unbiased sequence comparison of several mammalian 3 prime Epo enhancer region, we identified a previously unrecognized evolutionary conserved region. Second, we determined the functional significance of these conserved sequences using transient transfection and mutation analyses in cell culture studies and determined that these sequences contribute to HIF-2 alpha selectivity. Finally, using a candidate factor strategy, we determined that members of the early growth response (Egr) transcription factor family bind to these elements and act synergistically with HIF-2 alpha to augment Epo gene expression. In the second part of my thesis, we demonstrate that the redox-sensing, NAD+ dependent deacetylase enzyme Sirtuin 1, also known as Sirt1 or silent mating type information regulator 2 (Sir2) homolog 1, selectively stimulates HIF-2 alpha signaling during hypoxia. In lower organisms and cell culture models, the FoxO family of transcription factor regulates the transcription of SOD2 and other major AOE. During oxidative stress, Sirt1 modulates FoxO transcriptional activity, promoting the protective cellular response to oxidative stress. We hypothesized that Sirt1 would be activated by redox changes induced by hypoxia and that activated Sirt1 would in turn modulate HIF-2 signaling. We determined that HIF-2 alpha signaling is indeed increased by Sirt1 in transfection assays. Sirt1/HIF-2 alpha signaling does not involve previously described oxygen-dependent HIF-2 alpha modifications. Sirt1 augmentation of HIF-2 alpha transcriptional activity involves direct binding to and deacetylation of HIF-2 alpha. In cultured cells and in mice models, interventions that decrease or increase Sirt1 activity affect expression of the HIF-2 alpha target gene epo accordingly. Thus, Sirt1 is a molecular switch that promotes HIF-2 signaling during hypoxia and likely other environmental stresses.Item The Mammalian Hypoxia Response Pathway: Regulation of HIf and HIF Prolyl Hydroxylases(2007-05-22) Ozer, Abdullah; Bruick, Richard K.Cells exposed to hypoxia -limited oxygen availability- initiate an adaptive response orchestrated by a transcription factor called Hypoxia Inducible Factor (HIF). HIF is composed of an oxygen-sensitive alpha -, and an oxygen-insensitive beta -subunit (ARNT). The stability and transcriptional activity of HIF alpha are controlled by two different Fe(II)- and 2- oxoglutarate-dependent dioxygenases that utilize molecular oxygen during hydroxylation of HIF alpha -subunit. When oxygen levels are sufficient (normoxia), HIF Prolyl Hydroxylases (HPH-1, -2, and -3) hydroxylate the Oxygen-dependent Degradation Domain (ODD) of HIF-alpha targeting it to ubiquitin-mediated proteosomal degradation. Factor Inhibiting HIF 1 (FIH-1, an asparaginyl hydroxylase), on the other hand, hydroxylates C-terminal Transactivation Domain (CTAD) thereby abolishing recruitment of transcriptional co-activators by HIF alpha. However, under hypoxic conditions, both hydroxylations are diminished allowing HIF alpha to escape degradation and induce transcription by associating with co-activators. Because of its critical role as an oxygen sensor, we studied HIF Prolyl Hydroxylase 2 (HPH-2) and focused on protein-protein interactions expecting that some of the interacting proteins might regulate its function. We characterized the function of a HPH-2 interacting protein identified in yeast two-hybrid screen; Inhibitor of Growth 4 (ING4) -a candidate tumor suppressor protein-, and showed that ING4 represses HIF transcriptional activity under hypoxia in a chromatindependent manner. Recruitment of ING4 to alter HIF transcriptional activity represents a novel function of HPH-2. To shed some light on the mechanism of this transcriptional repression, we purified ING4 containing co-repressor complex containing MYST2 and JADE3. Furthermore, we showed that ING4 and MYST2 targets not only HIF but also NF- κB transcription factor, a previously identified target of ING4, perhaps misregulation of which in the absence of functional ING4 protein contributes to tumor progression. Moreover, we identified additional HPH-2 interacting proteins and found that HPH enzymes can be modified by Protein Arginine Methyltransferase 1 (PRMT1) in vitro. Inhibition of methyltransferases in vivo further stabilized and activated HIF-1alpha suggesting a role for methyltransferases in regulation of HIF that might be mediated through HPH enzymes. Methylation of HPH enzymes, the first identified post-translational modification of these enzymes, adds another layer of complexity to the regulation of HIF alpha and it may serve as an interface between the hypoxia response pathway and other signaling pathways.Item Myogenic BHLH Transcription Factors: Their Overlapping Functions and Direct Regulation of MEF2C Provide a Regulatory Network for the Maintenance and Amplification of Vertebrate Myogenesis(2003-04-01) Valdez, Melissa Renee; Mangelsdorf, DavidThe myogenic basic helix-loop-helix (bHLH) genes - Myf5, MyoD, myogenin and MRF4 - exhibit distinct, but overlapping expression patterns during vertebrate myogenesis. Loss-of-function mutations in these genes have defined an in vivo model for myogenesis in which MyoD and Myf5 have redundant functions in myoblast specification, whereas myogenin acts to control myoblast differentiation. A role for MRF4 in differentiation has been suggested by various studies, but not defined. Through the analysis of MyoD-/-MRF4-/- and myogenin-/-MRF4-/- mutants, we show that MRF4 plays a role in differentiation which it shares with MyoD, but not myogenin, thereby defining a novel myogenin-independent differentiation pathway. The functional redundancy of the myogenic bHLH factors demonstrated in these and other studies led us to investigate the ability of a single factor to direct the myogenic program in the absence of the other myogenic bHLH proteins. Analysis of myogenin-/-MyoD-/-MRF4-/- mutant animals showed that alone, Myf 5 was unable to bring about differentiation, although specification of myoblasts was not affected. These results suggest that these myogenic factors possess specialized functions. However, the remarkably low level of Myf5 available in triple mutant neonatal muscle leaves open the possibility that it is the total level of myogenic bHLH transcription factors that is critical to the completion of muscle differentiation. The auto- and cross-regulation that the myogenic bHLH factors provide for one another, combined with their functional redundancy, comprises a mechanism whereby myogenesis is induced and maintained. Members of the MEF2 family of transcription factors cooperate with the myogenic bHLH factors to control the expression of muscle specific genes, thereby contributing to the maintenance and amplification of muscle development. To determine the mechanisms that regulate the expression of MEF2C, the earliest of the MEF2 factors expressed in the myogenic lineage, the mouse MEF2C gene was analyzed for cis-regulatory elements that direct its expression in the skeletal muscle lineage in vivo. As described herein, such a control region was identified, characterized and shown to be a direct transcriptional target of myogenic bHLH and MEF2 proteins. These results further define the regulatory circuit that induces, amplifies and maintains myogenesis in vivo.Item Neurogenesis and Gliogenesis from Ascl1 (Mash1) Expressing Progenitors in the Central Nervous System(2010-05-14) Kim, Euiseok Joshua; Johnson, JaneFor the functional architecture of the central nervous system, a small population of neural stem cells generates the correct numbers and types of neurons, oligodendrocytes and astrocytes in a precisely coordinated manner. Basic helix-loop-helix (bHLH) transcription factors play central roles in determining distinct neural cell fates and thus contribute to mechanisms controlling neural cell type diversity during the embryogenesis. Fundamental to understanding nervous system formation is to uncover links between early cell type specification mechanisms, the developmental dynamics of each lineage, and the contributions of specific molecules to these processes to form the mature nervous system structures. Ascl1 (previously Mash1) is a bHLH transcription factor essential for neuronal differentiation and neural sub-type specification. Ascl1 is present in proliferating progenitor cells but these cells are actively differentiating as evidenced by their rapid migration out of germinal zones. Although it has been studied for its role in several neural lineages, the full complement of lineages arising from Ascl1 progenitor cells and the molecular mechanism of Ascl1’s functions are not completely understood. Using an inducible Cre-flox genetic fate-mapping strategy, Ascl1 lineages were determined in both the embryonic and adult central nervous system. In chapter two, the fate of Ascl1+ progenitor cells throughout the brain was described. Depending on the temporal and spatial context during embryogenesis, Ascl1+ cells contribute to distinct neuronal and glial cells in each major brain division. In chapter three, by labeling Ascl1+ progenitor cells at distinct phases of their development, I delineated the temporal lineage relationship of distinct subtypes of neurons and glia in the developing spinal cord. Two spatially and temporally distinct Ascl1+ progenitor populations contribute differentially to inhibitory dILA and excitatory dILB neurons in the dorsal spinal cord. At later stages of embryogenesis, Ascl1+ progenitors are restricted to glial lineages giving rise to both astrocytes and oligodendrocytes. Analysis of conditional mutants of Ascl1 demonstrated that Ascl1 is required for only one division of each lineage. Loss of Ascl1 results in a reduction of inhibitory dILA neurons and oligodendrocytes, but not excitatory dILB neurons and astrocytes. In chapter four, the physiological functions of Nicastrin in gliogenesis were investigated. Nicastrin is a requisite subunit of the !-secretase complex essential for activating Notch signaling pathway. Conditional mutant of Nicastrin leads to the increased level of oligodendrocytes lineage markers in the neural tube, the opposite phenotype of that for Ascl1. Thus, I propose that Notch signaling in constraining levels of Ascl1 is required in oligodendrogenesis. In chapter five, I revealed that Ascl1 is a common molecular marker of early progenitors of both neurons and oligodendrocytes in the adult brain, and these Ascl1 defined progenitors mature with distinct dynamics in different brain regions. In this thesis, I define Ascl1 as a neural differentiation factor crucial for neural cell type diversification, playing important roles in cell differentiation and subtype specification at several different nodes of cell fate decisions throughout neurodevelopment.Item RBP-L and RBP-J Have Critical Roles in the Functioning of Two Forms of the Pancreas Transcription Factor Complex PTF1(2005-05-04) Beres, Thomas Matthew; MacDonald, Raymond J.The decision of pancreatic precursor cells to differentiate into acinar or endocrine cells is regulated by a complex network of signaling and transcription factor pathways. P48 is a tissue-specific basic-helix-loop-helix (bHLH) transcription factor, which is essential for pancreas development and function. Mice lacking both p48 alleles lack an exocrine pancreas and have a greatly reduced endocrine pancreas. The active form of P48 is the heterotrimeric complex, PTF1. This complex binds and regulates the transcription of genes encoding digestive enzymes within the exocrine pancreas. The PTF1 complex forms on the rat elastase 1 (ELA1) promoter by synergistically binding to a 21 base-pair site comprising an E-box (CANNTG) and a TC-box separated by one helical turn. P48 binds the E-box as a heterodimer with class A bHLH proteins, while the third member of the complex contacts the TC-box, but cannot stably bind without the P48 heterodimer. PTF1 activates the genes encoding the digestive enzymes specifically in the acinar cells of the pancreas, but no developmentally relevent target genes for P48 have been identified. Human mutations that truncate P48 are associated with permanent neonatal diabetes mellitus (PNDM), a genetic disorder characterized by pancreatic and cerebellar agenesis. DNA binding and transcriptional activity of PTF1 is dependent on the interaction of P48 with either RBP-J, or its paralogue, RBP-L. The exclusive form of PTF1 in mature pancreatic acinar cells is a potent transcriptional activator containing RBP-L; however, RBP-J can form a similar, but low activity, complex. The P48-RBP interaction is primarily through two conserved peptides that resemble the RBP-J-interacting motif of the Notch intracellular domain (NotchIC). However, the NotchIC is excluded from PTF1 because it lacks affinity for RBP-L, and P48 occupies its docking site on RBP-J. PNDM associated mutations delete one or both critical peptides, indicating the requirement of a PTF1 complex for proper embryonic development. The inability of the NotchIC to integrate into PTF1 complexes demonstrates a Notch-independent role for mammalian Suppressor of Hairless (RBP-J) and its paralogue RBP-L.Item Regulation and Lineage Analysis of Neurog1 in the Developing Spinal Cord(2007-05-23) Quinones-Figueroa, Herson Isaac; Johnson, Jane E.The bHLH transcription factor Neurog1 is involved in neuronal differentiation and cell-type specification in distinct regions of the developing nervous system. I developed mouse models that efficiently drive expression of GFP or Cre recombinase in all Neurog1 (Ngn1, NeuroD3) domains. Deleting highly conserved sequences from a BAC containing 113kb 5' and 71kb 3' genomic sequence surrounding the Neurog1 coding region allowed the identification of enhancer elements required to drive Neurog1 expression. I show that a 3.8 kb fragment located 4.2 kb 5' of Neurog1 is required for efficient reporter expression in all Neurog1 domains. This sequence contains previously identified enhancer elements for midbrain, hindbrain and dorsal neural tube, and has two sequences conserved from human to fish. A 16kb fragment containing 8.9 kb 5' and 5.2 kb 3' of the Neurog1 coding sequence was not sufficient to drive expression in all domains. Reporter expression was observed in the dorsal neural tube, the midbrain, hindbrain and trigeminal ganglia, but was missing in the olfactory epithelium, dorsal root ganglia, dorsal telencephalon, and ventral neural tube. A 2.3 kb enhancer element located 8 kb 5' of the Neurog1 coding region was identified that is necessary to direct expression in the ventral neural tube. In addition, these mouse models allowed both short-term and long-term lineage analyses. I show that derivatives of Neurog1-expressing progenitor cells in the neural tube largely comprise the interneuron populations dI2, dI6, V0, V1, and V2, and to a lesser extent motorneurons. This is seen in the co-expression of GFP driven by Neurog1 regulatory sequences with the neuronal identity markers Brn3a, Islet1/2, Lhx1/5, Lhx3, Pax2, and Chx10. Genetic fate mapping in vivo using Cre recombinase reveals that although Neurog1-expressing cells primarily give rise to neurons, minor populations of oligodendrocytes and astrocytes are also identified in the lineage by adult stages in the spinal cord. Adding temporal control to the fate mapping strategy demonstrates that the neurons are generated from Neurog1-expressing cells prior to E13, and glial cells after E13, placing Neurog1 in lineage restricted precursor cells during embryogenesis.Item Role of Mash1-E Protein Heterodimers in Mash1 Function in the Developing Neural Tube(2003-05-01) Collisson, Tandi Louise; Lu, Qing RichardNeural-specific Class II bHLH transcription factors heterodimerize with ubiquitous Class I bHLH E proteins to form complexes required for neural differentiation. There are four known E proteins, HEB, E12, E47 and E2.2, in the mammalian nervous system, which potentially form heterodimers with Mash1 in the neural tube. To test the relevance of particular Mash1-E protein heterodimer combinations in vivo, I constructed tethered Mash1-E protein heterodimers for over-expression in the chick neural tube. By comparing overexpression of Mash1 with over-expression of these Mash1-E protein heterodimers, their abilities to effect neural differentiation and cell-type specification were analyzed. Mash1-E protein heterodimers are interchangeable in the function of driving neurogenesis in the chick neural tube. The effects of Mash1-E protein heterodimers on cell-type specificity were different, suggesting non-redundant functions in effecting dorsal interneuron populations. Furthermore, additional Mash1 heterodimer partners may be required for the cell-type specification function of Mash1.Item Transcriptional Regulation of Cardio-pulmonary Development(2004-01-14) Aiyer, Aparna R.; Srivastava, DeepakOrganogenesis is a complex process, disruption of which results in developmental anomalies. In recent years, genetic dissection of the pathways involved in cardiogenesis have shown a striking similarity in molecular mechanisms across species. One conserved protein is dHAND, a basic helix-loop-helix (bHLH) transcription factor that is required for normal development of the right ventricle, the pharyngeal arches and limb buds. Loss of dHAND leads to apoptosis in the aforementioned tissues and to embryonic lethality at E10.0. A differential display analysis was performed to identify genes dysregulated in dHAND-/- hearts.