Browsing by Subject "Basic Helix-Loop-Helix Transcription Factors"
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Item Analysis of ASCL1 in Neuroendocrine Lung Cancer(2015-08-20) He, Min; Buszczak, Michael; Cobb, Melanie H.; White, Michael A.; Liu, QinghuaSmall cell lung cancer (SCLC) is an understudied tumor subset with aggressive neuroendocrine carcinoma features. Previous studies have determined that the basic helix-loop-helix (bHLH) transcription factor achaete-scute homolog 1 (ASCL1) is essential for the survival and progression of many pulmonary neuroendocrine (NE) cancer cells, which include both SCLC and some non-small cell lung cancer (NSCLC). To understand how ASCL1 initiates tumorigenesis in pulmonary neuroendocrine cancer and identify the transcriptional targets of ASCL1, whole-genome RNA-sequencing (RNA-seq) analysis combined with chromatin immunoprecipitation-sequencing (ChIP-seq) were performed with a series of lung cancer cell lines. We discovered the gene SCNN1A, which encodes the alpha subunit of the epithelial sodium channel (αENaC), is highly correlated with ASCL1 expression in SCLC cells. We confirmed that SCNN1A is under the transcriptional control of ASCL1, indicating that SCNN1A represents a newly recognized ASCL1 target. The product of the SCNN1A gene ENaC can be pharmacologically inhibited by amiloride, a drug that has been used clinically for nearly 50 years. Amiloride-treated ASCL1-dependent tumor cells stopped cell growth in vitro. Analysis of downstream targets of ASCL1 broadens our understanding how ASCL1 functions, and further provides a step forward in the development of drug-targeted therapy for pulmonary neuroendocrine cancer. We also discovered that ASCL1 may negatively regulate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway through a negative feedback mechanism. Finally, we found that expression of ASCL1 in certain NSCLC could induce neuroendocrine features which are reminiscent of SCLC.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 Characterizing ASCL1-Dependent Neuroendocrine Non-Small Cell Lung Cancers(2013-06-27) Augustyn, Alexander; Amatruda, James F.; Minna, John D.; Johnson, Jane E.; White, Michael A.In order to achieve personalized medicine for the treatment of lung cancer, it is important to accurately classify tumors using a combination of factors, including patho-physiological features, tumor gene expression profiles, response to therapy, and oncogene/tumor suppressor mutation status. Gene expression analyses, including immunohistochemistry, single mRNA transcript analyses, and genome-wide mRNA expression profiling, performed over the course of the last three decades suggest that distinct, poorly performing neuroendocrine tumors occur in about 10% of otherwise pathologically indistinguishable non-small cell lung cancers. A complete molecular characterization of these tumors is lacking because no pre-clinical model exists. Utilizing genome-wide mRNA expression profiling from lung cancer cell lines established from a variety of patients, it was discovered that a rare subgroup of non-small cell lung cancer (NSCLC) lines demonstrated similar gene expression compared to a known neuroendocrine tumor, small cell lung cancer (SCLC). Validation of transcript analysis verified this data, and demonstrated that a particular transcription factor, ASCL1, required during development for the formation of pulmonary neuroendocrine cells, is dramatically upregulated in the subgroup of non-small cell lung cancer with neuroendocrine features (NE-NSCLC). Other cancer models have demonstrated addiction of tumors to developmental transcription factors and termed these genes “lineage oncogenes.” By showing that NE-NSCLC cell lines are addicted to ASCL1 expression and function, it was established that ASCL1 is also a lineage oncogene. Transcription factors of the basic helix-loop-helix category are historically difficult to target with small molecules, so a downstream target analysis was performed in order to understand the ASCL1 transcriptome. ChIP-Seq analysis demonstrated that ASCL1 regulates many genes, including several that are inherently druggable. Further studies proved that ASCL1 directly regulates the transcription of the anti-apoptotic regulator BCL2. Inhibition of BCL2 in vitro and in vivo led to induction of apoptosis and tumor xenograft regression suggesting that BCL2 is a potential therapeutic target in ASCL1-dependent NE-NSCLCs. Analysis of the upstream regulation of ASCL1 showed that it depends on a paradoxical activation of the RAS/RAF/MEK/ERK pathway. Small molecule agonists of this pathway were utilized to demonstrate reduction of ASCL1 levels and induction of apoptosis. The combination of ERK activation with BCL2 inhibition was then shown to be a viable therapeutic strategy for ASCL1-dependent tumors in vitro.Item Chemistry to Biology and Back Again: Small Molecule Regulation of HIF Transcription Factors and the Development of a Platform for the Discovery of New Biocatalyzed Organic Transformations(2013-04-08) Rogers, Jamie Lee 1985-; Tambar, Uttam; MacMillan, John; Ready, Joseph M.; Gardner, Kevin H.This work is divided into two parts. The first is the description of the regulation of the hypoxic response pathway via small molecule inhibitors. The hypoxia response pathway is a way in which cells sense and regulate oxygen levels in cells. Specifically, when oxygen levels in the cells are low, a family of transcription factors known as hypoxia inducible factors (HIFs) is up regulated. Importantly, the hypoxic response pathway is often mis–regulated in cancer. As a result, the regulation of this pathway offers a promising target for cancer treatment. Previous work on HIF identified a large internal cavity, which provided an opportunity for allosteric binding and regulation of HIF. After identification of a small molecule inhibitor of HIF through a high–throughput screening campaign, an SAR analysis was performed on the lead molecule. This led to a greater understanding of the structural requirements to strong binding with HIF. In addition, a sterol natural product was identified through the screen that also inhibits HIF. This molecule led to the search for the endogenous ligand of the HIF transcription factor and has developed a better understanding of the natural regulation of the hypoxic response pathway. The second part of this work describes the development of a new discovery platform for the identification of new, biocatalyzed organic transformations. Biocatalyzed organic transformations have been used by organic chemists for decades as these reactions offer many benefits to the synthetic chemist. For example, reactions catalyzed by biological enzymes tend to be very stereo- and regiospecific. In addition, biocatalyzed transformation can occur on unfunctionalized organic substrates. However, research into new biocatalyzed reactions has been limited due to the challenges in searching for these new reactions. We have developed a discovery platform designed to screen a vast library of bacteria for new reactivity by introducing 13C labeled substrates to cultures. This platform is illustrated with the discovery of a tunable indole oxidation reaction.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 The Developmental Transcription Factor Neurogenic Differentiation 1 in Migration and Survival of Neuroendocrine Carcinomas(2013-03-12) Osborne, Jihan K.; Minna, John D.; White, Michael A.; Johnson, Jane E.; Cobb, Melanie H.Differentiation and determination of cell fate during embryogenesis is decided by a collection of transcription factors, including the large family of basic-helix-loop-helix (bHLH) transcription factors. Neurogenic differentiation 1 (NeuroD1) is a bHLH transcription factor responsible for neuronal and neuroendocrine islet differentiation during development of the central and peripheral nervous systems and the pancreas respectively. NeuroD1 has also been shown to be anomalously expressed in a subset of aggressive neuroendocrine tumors. Initial examination of microarray data revealed that subsets of aggressive small cell lung cancers (SCLC) and certain neuroendocrine non-small cell lung cancers (NSCLC-NE) have high expression of NeuroD1 as compared to human bronchial epithelial cells (HBEC) and other non-small cell lung cancers (NSCLC). In several neuroendocrine carcinomas, including subsets of neuroendocrine lung cancers, melanoma and some undifferentiated prostate cell lines, NeuroD1 directly induces the expression of signaling pathways that support survival and migration. Loss-of-function/gain-of-function studies in cell lines from each of these cancer types reveled that NeuroD1 regulates both survival and the migration potential of neuroendocrine carcinomas that have lost or mutated p53. Subsequently, loss of p53 has been shown to up-regulate NeuroD1 expression in non-transformed HBECs and cancer cells with neuroendocrine features. The actions of NeuroD1 are carried out by downstream targets which include the signaling molecules, the tyrosine kinase, tropomyosin-related kinase B (TrkB), and the adhesion molecule, neural cell adhesion molecule (NCAM), and the ion channels, the nicotinic acetylcholine receptor subunit cluster of α3, α5, and β4 (nAChR), to name a few. Impaired expression of each of these downstream targets mirrors the various phenotypes associated with loss of NeuroD1. These findings ultimately have implications for the potential of NeuroD1 acting as a lineage-dependent oncogene in neuroendocrine carcinomas.Item Dual Mechanisms Regulating Alpha Subunit-Specific Activity in Hypoxia-Inducible Factor Signaling(2015-12-02) Nagati, Jason Sharif; Terada, Lance; Liu, Zhi-Ping; Yanagisawa, Hiromi; Munshi, Nikhil; Garcia, Joseph A.The ability to adapt to and protect from environmental stresses is essential to survival and has played a major role in fitness selection during evolution. As oxygen is essential to most life, many organisms have developed a response to conditions of low oxygen availability. Throughout the animal kingdom, hypoxia-inducible factor has emerged as a master regulator of this response. These bHLH transcription factors enhance transcription of a variety of genes that work to maintain oxygen homeostasis and allow adaptation to decreased oxygen availability. Two homologues, HIF-1α and HIF-2α, have been extensively studied in this field. Though they have similar domain structures and amino acid sequences, display overlap in some gene targets, and share regulatory mechanisms, they also perform distinct roles. They differ in tissue expression patterns, both temporally during development and spatially, hypoxia-driven expression kinetics, target genes, and fold induction. To elucidate mechanisms of this differential behavior, I investigated two aspects of HIF-2α-specific regulation. Firstly, I explored the contribution of early growth response transcription factors, EGRs, to HIF-2α-directed erythropoietin expression. Through reporter assays and chromatin immunoprecipitation, these factors were determined to occupy the erythropoietin enhancer adjacent to the HIF-2α binding site. Overexpression analysis showed they could amplify HIF-2α transactivation of erythropoietin, while knockdown experiments showed they were necessary for full, endogenous expression. And co-immunoprecipitation studies revealed a physical interaction between EGRs and HIF-2α that was necessary for cooperative activity. Secondly, I investigated the mechanism by which modulation of HIF-2α activity by CBP/SIRT1-dependent acetylation was signaled. Our studies revealed ACSS2, an acetyl CoA synthetase, as the source of acetyl CoA required for HIF-2α complex formation with the acetyltransferase CBP, subsequent HIF-2α acetylation, and target gene activation. The ACSS2 substrate acetate is produced during hypoxia, and exogenous acetate supplementation to cell culture media induced this pathway independent of hypoxia. Acetate administration in mice also augmented the HIF-2α-influenced pathways of red blood cell production and tumor growth in an ACSS2-dependent manner. Thus, EGRs represent novel HIF-2α cofactors in erythropoietin induction, while acetate, through ACSS2, regulates HIF-2α acetylation-dependent activity.Item Genome-Wide Analysis of Transcription Factors Ascl1 and Ptf1a in Development and Cancer(2013-11-26) Borromeo, Mark Dominic; Yu, Gang; Lu, Q. Richard; Kim, Tae-Kyung; Johnson, Jane E.Cell fate specification in the developing embryo relies on combinations of transcription factors to regulate tissue specific gene programs. Many of the same transcription factors can be found in multiple tissue types and are crucial for their development, and at other times these same factors can be misused in disease states. The basic helix-loop-helix (bHLH) factors Ascl1 and Ptf1a are examples of factors that give rise to and function in multiple tissues. Ascl1 and Ptf1a are essential for generating the correct number and sub-type of neurons in multiple regions of the nervous system. In addition, Ptf1a is required in the developing pancreas for both its formation and maturation, while Ascl1 is crucial for tumor growth in malignant small cell lung carcinoma (SCLC). It is unknown if Ascl1 and Ptf1a directly regulate different genes programs in these disparate tissues. Furthermore, Ptf1a and Ascl1 are members of same transcription factor family, which recognize and bind a similar DNA sequence. How these two factors achieve specificity of DNA binding and gene target selection in vivo is unknown. These questions have long been unanswered due in part to the lack of known direct transcriptional targets. Thus, to understand how Ascl1 and Ptf1a function in these processes, the direct transcriptional targets were identified genome-wide in the multiple tissues using ChIP-Seq and RNA-Seq. Overwhelmingly, Ascl1 and Ptf1a directly regulate different gene programs important for each tissue. Within a given tissue, the specificity of Ascl1 and Ptf1a function is partly explained by their differences in E-box sequence preferences. Ptf1a and Ascl1 are co-expressed in a subset of cells in the dorsal neural tube, and comparative analysis of their binding sites show that they bind a common E-box. However, Ptf1a can also bind a distinct E-box, which is found enriched in binding sites unique to Ptf1a. However, analysis of Ascl1 binding in SCLC and Ptf1a binding in the developing pancreas, shows that their E-box preferences change and does not reflect the same type of E-box they bind in the neural tube. Mechanisms in addition to E-box specificity are likely in use because tissue specific binding coincides with tissue-specific chromatin accessibility and enrichment of lineage-specific transcription factor binding motifs. Thus, Ascl1 and Ptf1a make use of different tissue-specific co-factors to regulate tissue-specific genes. This study provides insights into how a single factor can regulate the transcription of different genes in different tissue types, and how two related E-box binding proteins regulate distinct genes.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 Inhibition of Karyopherin-β1-Mediated Nuclear Import of Lineage-Defining Transcription Factors in Small Cell Lung Cancer(2021-05-01T05:00:00.000Z) Kelenis, Demetra Patrica; Minna, John D.; Johnson, Jane E.; Cobb, Melanie H.; McFadden, David G.Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor that accounts for approximately 16% of lung cancer diagnoses. Recent genomic studies support the classification of this disease into four different subtypes based on the expression of the lineage-defining transcription factors ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P), and YAP1 (SCLC-Y). Together, the SCLC-A and SCLC-N subtypes account for a majority of SCLC. ASCL1 and NEUROD1 are Class II bHLH transcription factors that command the expression of SCLC oncogenes and are known to drive distinct transcriptional programs, conferring SCLC-A and SCLC-N with different molecular and physiological features. ASCL1 has also been shown to be required for tumor formation in SCLC mouse models, and, where tested, both ASCL1 and NEUROD1 play key roles in maintaining growth of SCLC-A and SCLC-N cell lines. Together, these findings suggest that strategies to inhibit ASCL1 and NEUROD1 activity may represent an attractive SCLC therapy, and provide new insight into the underlying plasticity of the major SCLC subtypes. ASCL1 and NEUROD1 are translated in the cytoplasm and must be transported into the nucleus in order to regulate gene expression. Interestingly, it has been shown that NEUROD1 is selectively imported into the nucleus via the nuclear transport receptor Karyopherin-β1, or KPNB1 (also known as Importin-β1) in several mouse and human cell lines. Additionally, a muscle-specific bHLH transcription factor, MYOD, has also been shown to be imported into the nucleus by KPNB1, suggesting a common nuclear import mechanism for Class II bHLH transcription factors. However, whether ASCL1 is also imported into the nucleus via the same mechanism, and whether the nuclear import of NEUROD1 is mediated by KPNB1 in the setting of SCLC, had not been tested. Here, I 1. identified KPNB1 as a nuclear import receptor for ASCL1 and NEUROD1 in SCLC, 2. showed that KPNB1 inhibition disrupts SCLC- A patient derived xenograft (PDX) growth in vivo, and preferentially disrupts the growth of ASCL1/NEUROD1+ SCLC in vitro, and 3. compared the changes in gene expression following the inhibition of ASCL1 or KPNB1 activity across in vitro and in vivo models of SCLC.Item Intrinsic Specificity of Binding and Regulatory Function of Class II bHLH Transcription Factors(2016-11-28) Casey, Bradford Harris; Krämer, Helmut; Johnson, Jane E.; Konopka, Genevieve; MacDonald, Raymond J.Embryonic development begins with a single cell, and gives rise to the many diverse cells which comprise the complex structures of the adult animal. Distinct cell fates require precise regulation to develop and maintain their functional characteristics. Transcription factors provide a mechanism to select tissue-specific programs of gene expression from the shared genome. ASCL1, ASCL2, and MYOD are class II basic Helix-Loop-Helix (bHLH) transcription factors which play crucial roles in lineage specification in the developing embryo. In vivo, these factors bind to distinct genomic sites, and regulate distinct transcriptional programs. The mechanisms by which they select their cognate binding sites remain poorly defined. Here, we utilize an inducible system to express these master regulatory factors in embryonic stem cells to characterize early events in bHLH factor binding and function in a common cellular context, removed from their role as endogenous master regulators of lineage specification. Using genome-wide sequencing approaches, we demonstrate that these factors maintain distinct binding when ectopically expressed in a common context. We observe that they initiate distinct transcriptional programs, which include key regulators in lineage specification. By comparing chromatin accessibility of bHLH binding sites, we reveal a shared ability for these factors to bind nucleosome-occupied sites, and meet the criteria which define pioneer transcription factors. We further characterize epigenetic features of the empirically observed genome-wide binding sites of these factors, and compare these findings to the conventional understanding of bHLH factor function. This work represents the first comprehensive approach to direct comparison of early events in the binding and transcriptional profiles of ASCL1, ASCL2, and MYOD.Item Investigating the Transcriptional Regulation of PTF1A and the Function of PRDM13 to Specify Dorsal Spinal Interneurons(2019-03-28) Mona, Bishakha; Zhang, Chun-Li; Johnson, Jane E.; Wu, Jiang I.; Meeks, Julian P.Transcriptional regulation is a fundamental process required for cell fate specification. During neural development, complex interactions of diverse transcription activators and repressors regulate lineage specific programs. The multitudes of neuronal subtypes generated during development is dependent on spatio-temporally controlled expression of these transcription factors such as bHLH factors, required for cell fate specification. PTF1A is one such factor whose expression is required for specification of the inhibitory fate while silencing the excitatory fate in the neural tube. Previous studies from our lab determined that PTF1A directly activates the inhibitory neuron lineage specific genes to direct neural progenitors towards an inhibitory neuronal fate and initiates a repressive program to suppress the excitatory neuron gene expression program through PRDM13. My thesis work uncovered additional functions of PRDM13 including suppression of ventral neural tube specification genes in the dorsal neural tube through repressing the activity of multiple bHLH transcriptional activators. Additionally, PRDM13 feedback inhibits PTF1A through a Ptf1a auto-regulatory enhancer. Given the indispensable requirement of PTF1A in neural specification, I continued to explore the regulatory network in place for Ptf1a expression. An enhancer with activity specifically in the dorsal neural tube specific was identified for Ptf1a using reporter says in mice and chick. The function the auto-regulatory enhancer and the dorsal neural tube specific enhancer was tested in situ. Mutations in the auto-regulatory enhancer, but not the dorsal neural tube enhancer, resulted in reduced levels of PTF1A, decreased the number of inhibitory neurons generated during dorsal spinal cord development, and exhibit a spontaneous scratching phenotype. The increased sensitivity to itch can be attributed at least in part to loss of BHLHB5;PAX2+ inhibitory neurons, known to play a role in itch. The absence of altered sensitivities in other somatosensory modalities whose information is known to be modulated in dorsal spinal cord circuits suggests itch circuitry is more dependent on PTF1 levels than the other sensory pathways. Together, these findings examine the role of two specific factors PTF1A and PRDM13 in neuronal specification during development.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 Modulation of Excitatory and Inhibitory Neuronal Balance Through Regulation of Ptf1a by Factors Binding to Zinc Finger and POU Motifs(2014-05-01) Avila, John Manuel, Jr.; MacDonald, Raymond J.; Wu, Jiang I.; Hsieh, Jenny; Johnson, Jane E.The proper function of the nervous system depends on a delicate balance between excitatory and inhibitory neurons. Transcription factors of the basic helix-loop-helix (bHLH) family have been shown to be particularly important in generating the correct numbers of these neurons during development. One of these, Ptf1a, is required in the specification of inhibitory neurons in multiple regions of the nervous system including the dorsal spinal cord. The absence of Ptf1a in null mice disrupts the balance of excitatory and inhibitory neurons, as Ptf1a is required for generating inhibitory neurons while suppressing the excitatory phenotype. Therefore, discovering the regulators of Ptf1a expression will identify mechanisms controlling the generation of a balanced neural network required for processing somatosensory information. Using sequence conservation between divergent vertebrate species, a 1.2 kb enhancer that directs expression of a reporter gene to Ptf1a expressing domains in transgenic mice was identified approximately 11 kb 3’ of the coding region. A series of mutations across the 1.2 kb enhancer were generated to identify sequences required for activity of this enhancer. The activity of the enhancer in directing expression specifically to the developing dorsal neural tube requires at least two distinct motifs: a putative POU motif required for activity, and a zinc finger which represses activity in non-Ptf1a-expressing populations within the neural tube. The activities of these two motifs were tested by in chick and transgenic mice. Coupled with a bioinformatics approach, several candidates for the upstream transcription factors have been identified and were tested for their role in regulating the temporal and spatial specific-activity of the Ptf1a-enhancer. One factor, Zic1 was shown to repress expression of Ptf1a. Thus, a combination of transcriptional activators and repressors are required to control Ptf1a expression, which regulates the subsequent balanced generation of inhibitory and excitatory neurons in the dorsal spinal cord.Item Molecular Dissection of Hand2 During the Formation of Pacemaker-Like Myocytes During Direct Reprogramming(2019-03-06) Fernandez-Perez, Antonio; Zhang, Chun-Li; Cleaver, Ondine; Olson, Eric N.; Munshi, NikhilDirect reprogramming of one cell type into another has great promise for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocytes (iCM) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes. Recent evidence has shown that iCM formation and diversity can be enhanced by the addition of Hand2 to GMT (GHMT). These four transcription factors give rise to a heterogenous CM population, consisting of atrial (iAM), ventricular (iVM), and pacemaker myocytes (iPM). However, the molecular mechanisms that drive this plastic fate conversion remain poorly understood. Although chromatin and single-cell studies in GMT-iCM have shown the existence of a set of temporal steps that orchestrate iCM formation, little is known about how Hand2 enhances this process. In the present study, we seek to characterize these Hand2-dependent mechanisms. We hypothesize that Hand2 regulates a discrete pacemaker regulatory network that becomes active during GHMT-iCM reprogramming. To test this, we compared the transcriptional and genomic profiles of fibroblasts, GMT, GHMT, and endogenous mouse Pacemaker cells. We observe similar chromatin landscape and gene expression profiles between Hand2-iPM and endogenous sinoatrial node (SAN), however several known key PM pathways are not active. Activation of these networks further enhances iCM-iPM fo Moreover, we show that Hand2 enhances chromatin accessibility in regions related to sarcomere function and electrical coupling, as well as promoting the closing of regions related to alternative fates. Utilizing integrative genomics between ATAC-seq and RNA-seq datasets, we identify the desmosome machinery as an important feature of iPM formation. In parallel, we define a novel Hand2 domain region that regulates cardiac subtype diversity. Taken together, our results showcase Hand2-dependent mechanisms for iPM formation and gives insight into the improvement of future iPM engineering.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, David J.The 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, Jane E.For 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) Quiñones-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.