bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mapping the Domains of the Interaction of the Vitamin D Receptor and Steroid Receptor Coactivator-1
Authors: Gill Rajbir K. , Atkins Loretta M. , Hollis Bruce W. , Bell Norman H. 
Published: January 2014

AbstractThe vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) and mediates the effects of the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3[ 1,25-(OH)2D3], on gene expression. The VDR binds to the VDRE as a heterodimeric complex with retinoid X receptor. In the present study, we have used a yeast two-hybrid system to clone complementary DNA that codes for VDR-interacting protein(s). We found that the human steroid receptor coactivator-1 (SRC-1) interacts with the VDR in a ligand-dependent manner, as demonstrated by β-galactosidase production. The interaction of the VDR and the SRC-1 takes place at physiological concentrations of 1,25(OH)2D3. A 48.2-fold stimulation of β-galactosidase activity was observed in the presence of 10−10m 1,25-(OH)2D3. In addition, a direct interaction between the ligand-activated glutathione-S-transferase-VDR and 35S-labeled SRC-1 was observed in vitro. Deletion-mutation analysis of the VDR established that the ligand-dependent activation domain (AF-2) of the VDR is required for the interaction with SRC-1. One deletion mutant, pGVDR-(1–418), bound the ligand but failed to interact with the SRC-1, whereas another deletion mutant, pGVDR-(1–423), bound the ligand and interacted with the SRC-1. We demonstrated that all the deletion mutants were expressed as analyzed by a Gal4 DNA-binding domain antibody. Deletion mutation analysis of the SRC-1 demonstrated that 27 amino acids (DPCNTNPTPMTKATPEEIKLEAQS-QFT) of the SRC-1 are essential for interaction with the AF-2 motif of the VDR.
Endothelin-1 Is a Potent Survival Factor for c-Myc-Dependent Apoptosis
Authors: Shichiri Masayoshi , Sedivy John M. , Marumo Fumiaki , Hirata Yukio 
Published: January 2014

AbstractMany vertebrate cells are resistant to apoptotic stimuli, whose variety and the mechanisms involved are not fully understood. Endothelin-1 is an endothelium-derived vasoactive peptide that mediates many physiological functions, such as vasoconstriction and cell proliferation. Deregulated expression of c-Myc induces apoptosis in serum-deprived fibroblasts. Using a panel of isogenic fibroblast cell lines with differential c-myc expression levels, we demonstrate that low doses of endothelin-1 protect fibroblasts against serum deprivation-induced apoptosis, which occurs through a c-Myc-dependent process. The endothelin-1-induced cell survival was mediated by the ETA receptor and was not linked to the ability of endothelin-1 to induce cell proliferation. The survival function of endothelin-1 was abrogated by inhibiting the mitogen-activated protein kinase pathway. These results demonstrate a hitherto unappreciated role of endothelin-1 as a potent survival factor for c-Myc-dependent apoptosis, a process mediated by the ETA receptor and the mitogen-activated protein kinase pathway.
Androgen Responsiveness of Mouse Kidney β-Glucuronidase Requires 5′-Flanking and Intragenic Gus-s Sequences
Authors: Thornton S. , Thomas D. W. , Gallagher P. M. , Ganschow R. E. 
Published: January 2014

AbstractGenetics studies of natural variants of the androgen response of mouse β-glucuronidase (GUS) reveal a cis-active element closely linked to the GUS structural gene (Gus-s) that is necessary for this kidney-specific response. Results of our previous studies suggested sequences within or near an androgen-inducible deoxyribonuclease I-hypersensitive site (DH site) located in the ninth intron of Gus-s are associated with the androgen response of GUS. Using transgenic mice, we now demonstrate that at least two regions of sequence within Gus-s are involved in regulating the androgen response of GUS. The first, located within 3.8 kb of Gus-s 5′-flanking sequence, directs the response and its tissue specificity, while the second, located within a 6.4-kb fragment of Gus-s extending from the third through the ninth intron of Gus-s, protects the androgen responsiveness of the transgene from repressive influences of the insertion site.
Truncated SNAP-25 (1–197), Like Botulinum Neurotoxin A, Can Inhibit Insulin Secretion from HIT-T15 Insulinoma Cells
Authors: Huang Xiaohang , Wheeler Michael B. , Kang You-hou , Sheu Laura , Lukacs Gergely L. , Trimble William S. , Gaisano Herbert Y. 
Published: January 2014

AbstractWe and others have previously shown that insulin-secreting cells of the pancreas express high levels of SNAP-25 (synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) implicated in synaptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin. To determine whether the inhibition of release was due to the depletion of functional SNAP-25 or the accumulation of proteolytic by-products, we transfected cells with SNAP-25 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin. This modified SNAP-25 (amino acids 1–197) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K+-mediated plasma membrane depolarization. Electron microscopy revealed that cells transfected with either BoNT/A or truncated SNAP-25 contained significantly higher numbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhibitory action of BoNT/A toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.
Regulation of the Rat Thyrotropin Receptor Gene by the Methylation-Sensitive Transcription Factor GA-Binding Protein
Authors: Yokomori Norihiko , Tawata Masato , Saito Tukasa , Shimura Hiroki , Onaya Toshimasa 
Published: January 2014

AbstractThe GA-binding protein (GABP), a transcription factor with a widespread tissue distribution, consists of two subunits,α and β1, and acts as a potent positive regulator of various genes. The effect of GABP on transcription of the TSH receptor (TSHR) gene in rat FRTL-5 thyroid cells has now been investigated. Both deoxyribonuclease I footprint analysis and gel mobility-shift assays indicated that bacterially expressed glutathione S-transferase fusion proteins of GABP subunits bind to a region spanning nucleotides (nt) −116 to −80 of the TSHR gene. In gel mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells yielded several specific bands with a probe comprising nt −116 to− 80. Supershift assays with antibodies to GABPα and to GABPβ1 showed that GABP was a component of the probe complexes formed by the nuclear extracts. Immunoblot analysis confirmed the presence of both GABP subunits in the nuclear extracts. A reporter gene construct containing the TSHR gene promoter was activated, in a dose-dependent manner, in FRTL-5 cells by cotransfection with constructs encoding both GABPα and GABPβ1. Both GABP binding to and activation of the TSHR gene promoter were prevented by methylation of CpG sites at nt −93 and− 85.These CpG sites were highly methylated (&gt;82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.
The Adrenomedullin Gene Is a Target for Negative Regulation by the Myc Transcription Complex
Authors: Wang Xueyan , Peters Mette A. , Utama Fransiscus E. , Wang Yuzhen , Taparowsky Elizabeth J. 
Published: January 2014

AbstractThe Myc family of transcription factors plays a central role in vertebrate growth and development although relatively few genetic targets of the Myc transcription complex have been identified. In this study, we used mRNA differential display to investigate gene expression changes induced by the overexpression of the MC29 v-Myc oncoprotein in C3H10T1/2 mouse fibroblasts. We identified the transcript of the adrenomedullin gene (AM) as an mRNA that is specifically down-regulated in v-Myc overexpressing C3H10T1/2 cell lines as well as in a Rat 1a cell line inducible for c-Myc. Nucleotide sequence analysis of the mouse AM promoter reveals the presence of consensus CAAT and TATA boxes as well as an initiator element (INR) with significant sequence similarity to the INR responsible for Myc-mediated repression of the adenovirus major late promoter (AdMLP). Reporter gene assays confirm that the region of the AM promoter containing the INR is the target of Myc-mediated repression. Exogenous application of AM peptide to quiescent C3H10T1/2 cultures does not stimulate growth, and constitutive expression of AM mRNA in C3H10T1/2 cells correlates with a reduced potential of the cells to be cotransformed by v-Myc and oncogenic Ras p21. Additional studies showing that AM mRNA is underrepresented in C3H10T1/2 cell lines stably transformed by Ras p21 or adenovirus E1A suggest that AM gene expression is incompatible with deregulated growth in this cell line. We propose a model in which the repression of AM gene expression by Myc is important to the role of this oncoprotein as a potentiator of cellular transformation in C3H10T1/2 and perhaps other cell lines.
An Explanation for Observed Estrogen Receptor Binding to Single-Stranded Estrogen-Responsive Element DNA
Authors: Driscoll Mark D. , Sathya Ganesan , Saidi Layla F. , DeMott Michael S. , Hilf Russell , Bambara Robert A. 
Published: January 2014

Abstract
Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.
Activator Protein-2 Mediates Transcriptional Activation of the CYP11A1 Gene by Interaction with Sp1 Rather than Binding to DNA
Authors: Pena Pilar , Reutens Anne T. , Albanese Chris , D’Amico Mark , Watanabe Genichi , Donner Amy , Shu I-Wei , Williams Trevor , Pestell Richard G. 
Published: January 2014

AbstractThe ovine P450 side chain cleavage (CYP11A1) enzyme gene, which catalyzes the initial enzymatic step in steroid hormone biosynthesis is transcriptionally regulated in cultured steroidogenic human trophoblastic JEG-3 cells. The ovine CYP11A1 promoter contains two GC-rich footprinted regions referred to as ovine footprints 5 (OF5) and OF3, which are well conserved among the CYP11A1 promoters of different species. These GC-rich sequences resemble activator protein-2 (AP-2)/Sp1 binding sites and were previously implicated in basal and cAMP-regulated activity of the bovine and ovine CYP11A1 promoters. In the current studies, AP-2 induced the ovine CYP11A1 promoter 4.5-fold in JEG-3 cells with full induction requiring the previously defined cAMP-responsive elements. Point mutation of OF3 abolished induction by AP-2, and OF3 was sufficient for induction by AP-2 when linked to a heterologous promoter. AP-2 induction of the CYP11A1 promoter required the basic region (N165-N278) and the carboxy terminus of AP-2 (N413-N437). In the course of investigating the mechanisms by which OF5 and OF3 regulated CYP11A1 transcription, we found that OF5 and OF3 bound Sp1 and Sp3 in JEG-3 cells. AP-2 did not bind OF5 or OF3 directly but rather formed a multiprotein complex with Sp1 in JEG-3 cells. AP-2 associated directly with Sp1 in vitro requiring the AP-2 basic region and the Sp1 carboxy terminus. AP-2 induced Sp1/Sp3 activity independently of AP-2 binding to DNA using a GAL4 paradigm. The Sp1 and Sp3 transactivation domains were linked to the DNA-binding domain of GAL4, and their activity was assessed using a luciferase reporter gene containing only the GAL4 DNA-binding sites linked to the minimal TATA site. AP-2 induced Sp1/Sp3-GAL4 activity 3- to 4-fold, requiring both the amino and extreme carboxy terminus of AP-2. We conclude that AP-2 can bind to and stimulate Sp1 activity and induces the ovine CYP11A1 promoter through conserved Sp1/Sp3-binding sites in JEG-3 cells. The induction of Sp1 activity by AP-2 may contribute to the induction of other genes that bind Sp1.
A Synthetic Triterpenoid, 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO), Is a Ligand for the Peroxisome Proliferator-Activated Receptor γ
Authors: Wang Yongping , Porter Weston W. , Suh Nanjoo , Honda Tadashi , Gribble Gordon W. , Leesnitzer Lisa M. , Plunket Kelli D. , Mangelsdorf David J. , Blanchard Steven G. , Willson Timothy M. , Sporn Michael B. 
Published: January 2014

AbstractA novel synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), previously reported to have potent differentiating, antiproliferative, and antiinflammatory activities, has been identified as a ligand for the peroxisome proliferator-activated receptor γ (PPARγ). CDDO induces adipocytic differentiation in 3T3-L1 cells, although it is not as potent as the full agonist of PPARγ, rosiglitazone. Binding studies of CDDO to PPARγ using a scintillation proximity assay give a Ki between 10−8 to 10−7m. In transactivation assays, CDDO is a partial agonist for PPARγ. The methyl ester of CDDO, CDDO-Me, binds to PPARγ with similar affinity, but is an antagonist. Like other PPARγ ligands, CDDO synergizes with a retinoid X receptor (RXR)-specific ligand to induce 3T3-L1 differentiation, while CDDO-Me is an antagonist in this assay. The partial agonism of CDDO and the antagonism of CDDO-Me reflect the differences in their capacity to recruit or displace cofactors of transcriptional regulation; CDDO and rosiglitazone both release the nuclear receptor corepressor, NCoR, from PPARγ, while CDDO-Me does not. The differences between CDDO and rosiglitazone as either partial or full agonists, respectively, are seen in the weaker ability of CDDO to recruit the coactivator CREB-binding protein, CBP, to PPARγ. Our results establish the triterpenoid CDDO as a member of a new class of PPARγ ligands.
GCN5 and ADA Adaptor Proteins Regulate Triiodothyronine/GRIP1 and SRC-1 Coactivator-Dependent Gene Activation by the Human Thyroid Hormone Receptor
Authors: Anafi Mordecai , Yang Yong-Fan , Barlev Nick A. , Govindan Manjapra V. , Berger Shelley L. , Butt Tauseef R. , Walfish Paul G. 
Published: January 2014

AbstractWe have used yeast genetics and in vitro protein-protein interaction experiments to explore the possibility that GCN5 (general control nonrepressed protein 5) and several other ADA (alteration/deficiency in activation) adaptor proteins of the multimeric SAGA complex can regulate T3/GRIP1 (glucocorticoid receptor interacting protein 1) and SRC-1 (steroid receptor coactivator-1) coactivator-dependent activation of transcription by the human T3 receptor β1 (hTRβ1). Here, we show that in vivo activation of a T3/GRIP1 or SRC-1 coactivator-dependent T3 hormone response element by hTRβ1 is dependent upon the presence of yeast GCN5, ADA2, ADA1, or ADA3 adaptor proteins and that the histone acetyltransferase (HAT) domains and bromodomain (BrD) of yGCN5 must be intact for maximal activation of transcription. We also observed that hTRβ1 can bind directly to yeast or human GCN5 as well as hADA2, and that the hGCN5387−837 sequence could bind directly to either GRIP1 or SRC-1 coactivator. Importantly, the T3-dependent binding of hTRβ1to hGCN5387−837 could be markedly increased by the presence of GRIP1 or SRC1. Mutagenesis of GRIP1 nuclear receptor (NR) Box II and III LXXLL motifs also substantially decreased both in vivo activation of transcription and in vitro T3-dependent binding of hTRβ1 to hGCN5. Taken together, these experiments support a multistep model of transcriptional initiation wherein the binding of T3 to hTRβ1 initiates the recruitment of p160 coactivators and GCN5 to form a trimeric transcriptional complex that activates target genes through interactions with ADA/SAGA adaptor proteins and nucleosomal histones.
β-Cell Differentiation from a Human Pancreatic Cell Line in Vitro and in Vivo
Authors: Dufayet de la Tour Dominique , Halvorsen Tanya , Demeterco Carla , Tyrberg Björn , Itkin-Ansari Pamela , Loy Mary , Yoo Soon-Jib , Hao Ergeng , Bossie Stuart , Levine Fred 
Published: January 2014

Abstract
Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human β-cells, inducibly transformed pancreatic β-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of β-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional β-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human β-cells is a major step toward developing a cell transplantation therapy for diabetes.
Isoform-Specific Transcriptional Regulation by Thyroid Hormone Receptors: Hormone-Independent Activation Operates through a Steroid Receptor Mode of Coactivator Interaction
Authors: Yang Zhihong , Privalsky Martin L. 
Published: January 2014

Abstract
Thyroid hormone receptors (T3Rs) are hormone-regulated transcription factors that play important roles in vertebrate homeostasis, differentiation, and development. T3Rs are synthesized as multiple isoforms that display tissue-specific expression patterns and distinct transcriptional properties. Most T3R isoforms associate with coactivator proteins and mediate transcriptional activation only in the presence of thyroid hormone. The pituitary-specific T3Rβ-2 isoform departs from this general rule and is able to interact with p160 coactivators, and to mediate transcriptional activation in both the absence and presence of hormone. We report here that this hormone-independent activation is mediated by contacts between the unique N terminus of T3Rβ-2 and an internal interaction domain in the SRC-1 (steroid receptor coactivator-1) and GRIP-1 (glucocorticoid receptor interacting protein 1) coactivators. These hormone-independent contacts between T3Rβ-2 and the p160 coactivators are distinct in sequence and function from the LXXLL motifs that mediate hormone-dependent transcriptional activation and resemble instead a mode of coactivator recruitment previously observed only for the steroid hormone receptors and only in the presence of steroid hormone. Our results suggest that the transcriptional properties of the different T3R isoforms represent a combinatorial mixture of repression, antirepression, and hormone-independent and hormone-dependent activation functions that operate in conjunction to determine the ultimate transcriptional outcome.
Reduction of Coactivator Expression by Antisense Oligodeoxynucleotides Inhibits ERα Transcriptional Activity and MCF-7 Proliferation
Authors: Cavarretta Ilaria T. R. , Mukopadhyay Ratna , Lonard David M. , Cowsert Lex M. , Bennett C. Frank , O’Malley Bert W. , Smith Carolyn L. 
Published: January 2014

AbstractSteroid receptor RNA activator (SRA) is a novel coactivator for steroid receptors that acts as an RNA molecule, whereas steroid receptor coactivator (SRC) family members, such as steroid receptor coactivator-1 (SRC-1) and transcriptional intermediary factor 2 (TIF2) exert their biological effects as proteins. Individual overexpression of each of these coactivators, which can form multimeric complexes in vivo, results in stimulated ERα transcriptional activity in transient transfection assays. However there is no information on the consequences of reducing SRC-1, TIF2, or SRA expression, singly or in combination, on ERα transcriptional activity. We therefore developed antisense oligodeoxynucleotides (asODNs) to SRA, SRC-1, and TIF2 mRNAs, which rapidly and specifically reduced the expression of each of these coactivators. ERα-dependent gene expression was reduced in a dose-dependent fashion by up to 80% in cells transfected with these oligonucleotides. Furthermore, treatment of cells with combinations of SRA, SRC-1, and TIF2 asODNs reduced ERα transcriptional activity to an extent greater than individual asODN treatment alone, suggesting that these coactivators cooperate, in at least an additive fashion, to activate ERα-dependent target gene expression. Finally, treatment of MCF-7 cells with asODN against SRC-1 and TIF2 revealed a requirement of these coactivators, but not SRA, for hormone-dependent DNA synthesis and induction of estrogen-dependent pS2 gene expression, indicating that SRA and SRC family coactivators can fulfill specific functional roles. Taken together, we have developed a rapid method to reduce endogenous coactivator expression that enables an assessment of the in vivo role of specific coactivators on ERα biological action and avoids potential artifacts arising from overexpression of coactivators in transient transfection assays.