scholarly journals Upstream stimulatory factor activates the vasopressin promoter via multiple motifs, including a non-canonical E-box

2003 ◽  
Vol 369 (3) ◽  
pp. 549-561 ◽  
Author(s):  
Judy M. COULSON ◽  
Jodie L. EDGSON ◽  
Zoe V. MARSHALL-JONES ◽  
Robert MULGREW ◽  
John P. QUINN ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Upstream Stimulatory Factor ◽  
Mobility Shift ◽  
Transcriptional Initiation ◽  
E Box ◽  
E Boxes ◽  
Stimulatory Factor

We have described previously a complex E-box enhancer (-147) of the vasopressin promoter in small-cell lung cancer (SCLC) extracts [Coulson, Fiskerstrand, Woll and Quinn, (1999) Biochem. J. 344, 961—970]. Upstream stimulatory factor (USF) heterodimers were one of the complexes binding to this site in vitro. We now report that USF overexpression in non-SCLC (NSCLC) cells can functionally activate vasopressin promoter-driven reporters that are otherwise inactive in this type of lung cancer cell. Site-directed mutagenesis and electrophoretic mobility-shift analysis demonstrate that although the −147 E-box contributes, none of the previously predicted E-boxes (-147, −135, −34) wholly account for this USF-mediated activation in NSCLC. 5′ Deletion showed the key promoter region as −52 to +42; however, USF-2 binding was not reliant on the −34 E-box, but on a novel adjacent CACGGG non-canonical E-box at −42 (motif E). This mediated USF binding in both SCLC and USF-2-transfected NSCLC cells. Mutation of motif E or the non-canonical TATA box abolished activity, implying both are required for transcriptional initiation on overexpression of USF-2. Co-transfected dominant negative USF confirmed that binding was required through motif E for function, but that the classical activation domain of USF was not essential. USF-2 bound motif E with 10-fold lower affinity than the −147 E-box. In NSCLC, endogenous USF-2 expression is low, and this basal level appears to be insufficient to activate transcription of arginine vasopressin (AVP). In summary, we have demonstrated a novel mechanism for USF activation, which contributes to differential vasopressin expression in lung cancer.

scholarly journals Upstream stimulatory factor 1 activates GATA5 expression through an E-box motif

2012 ◽  
Vol 446 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Bohao Chen ◽  
Rona Hsu ◽  
Zhenping Li ◽  
Paul C. Kogut ◽  
Qingxia Du ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Hypermethylation ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Upstream Stimulatory Factor ◽  
Mobility Shift ◽  
Reverse Transcription Pcr ◽  
E Box ◽  
Upstream Stimulatory Factor 1 ◽  
Stimulatory Factor

Silencing of GATA5 gene expression as a result of promoter hypermethylation has been observed in lung, gastrointestinal and ovarian cancers. However, the regulation of GATA5 gene expression has been poorly understood. In the present study, we have demonstrated that an E (enhancer)-box in the GATA5 promoter (bp −118 to −113 in mice; bp −164 to −159 in humans) positively regulates GATA5 transcription by binding USF1 (upstream stimulatory factor 1). Using site-directed mutagenesis, EMSA (electrophoretic mobility-shift analysis) and affinity chromatography, we found that USF1 specifically binds to the E-box sequence (5′-CACGTG-3′), but not to a mutated E-box. CpG methylation of this E-box significantly diminished its binding of transcription factors. Mutation of the E-box within a GATA5 promoter fragment significantly decreased promoter activity in a luciferase reporter assay. Chromatin immunoprecipitation identified that USF1 physiologically interacts with the GATA5 promoter E-box in mouse intestinal mucosa, which has the highest GATA5 gene expression in mouse. Co-transfection with a USF1 expression plasmid significantly increased GATA5 promoter-driven luciferase transcription. Furthermore, real-time and RT (reverse transcription)–PCR analyses confirmed that overexpression of USF1 activates endogenous GATA5 gene expression in human bronchial epithelial cells. The present study provides the first evidence that USF1 activates GATA5 gene expression through the E-box motif and suggests a potential mechanism (disruption of the E-box) by which GATA5 promoter methylation reduces GATA5 expression in cancer.

scholarly journals The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites.

1989 ◽  
Vol 9 (9) ◽  
pp. 4079-4082 ◽  
Author(s):  
T Deng ◽  
Y Li ◽  
K Jolliff ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Promoter Region ◽  
Promoter Activity ◽  
Initiation Site ◽  
Essential Elements ◽  
Site Directed Mutagenesis ◽  
Upstream Stimulatory Factor ◽  
Transcriptional Initiation ◽  
Close Proximity ◽  
Stimulatory Factor

The promoter region of the mouse thymidylate synthase gene was analyzed by deletion and site-directed mutagenesis. Elimination of an upstream Sp1 element reduced expression threefold, whereas elimination of an adenovirus upstream stimulatory factor element had little effect. All of the upstream elements that are essential for promoter activity are located within 22 nucleotides of the first transcriptional initiation site.

scholarly journals Identification of upstream stimulatory factor as transcriptional activator of the liver promoter of the glucokinase gene

1998 ◽  
Vol 333 (3) ◽  
pp. 705-712 ◽  
Author(s):  
Patrick B. IYNEDJIAN
Keyword(s):  
Rat Liver ◽  
Transient Transfection ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Nuclear Proteins ◽  
Dominant Negative Effect ◽  
Upstream Stimulatory Factor ◽  
Glucokinase Gene ◽  
Expression Plasmid ◽  
Stimulatory Factor

A functionally important cis-acting element termed P2 was identified in the liver promoter of the glucokinase gene. Element P2 was delineated by footprinting in vitro with nuclear proteins from rat liver and spleen. Its core sequence in the rat gene is a canonical CACGTG E-box. In the electrophoretic mobility-shift assay with nuclear proteins from rat liver, hepatocytes and hepatoma cells, an oligonucleotide with P2 in the context of the glucokinase promoter sequence gave rise to a DNA–protein complex shown to contain the upstream stimulatory factor (USF) by specific competition experiments and by reactivity with anti-USF antibodies. Transient transfection of hepatoma HepG2 cells, combined with site-directed mutagenesis, demonstrated that the P2 element was important for liver glucokinase promoter activity. Co-transfection of an expression plasmid coding for USF1 activated reporter gene expression in a manner dependent on an intact P2 element, whereas an expression plasmid for c-Myc was ineffective. Expression of a truncated form of USF1 lacking the transcription activation domain and the basic region decreased reporter activity by a dominant-negative effect. The functional significance of the P2 element was also demonstrated in transient transfection of primary hepatocytes.

The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites

1989 ◽  
Vol 9 (9) ◽  
pp. 4079-4082
Author(s):  
T Deng ◽  
Y Li ◽  
K Jolliff ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Promoter Region ◽  
Promoter Activity ◽  
Initiation Site ◽  
Essential Elements ◽  
Site Directed Mutagenesis ◽  
Upstream Stimulatory Factor ◽  
Transcriptional Initiation ◽  
Close Proximity ◽  
Stimulatory Factor

The promoter region of the mouse thymidylate synthase gene was analyzed by deletion and site-directed mutagenesis. Elimination of an upstream Sp1 element reduced expression threefold, whereas elimination of an adenovirus upstream stimulatory factor element had little effect. All of the upstream elements that are essential for promoter activity are located within 22 nucleotides of the first transcriptional initiation site.

scholarly journals Characterization of the human liver fructose-1,6-bisphosphatase gene promoter

2000 ◽  
Vol 351 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Birger HERZOG ◽  
Mary WALTNER-LAW ◽  
Donald K. SCOTT ◽  
Klaus ESCHRICH ◽  
Daryl K. GRANNER
Keyword(s):  
Hormonal Regulation ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Nuclear Factor Κb ◽  
Site Directed Mutagenesis ◽  
Upstream Stimulatory Factor ◽  
Mobility Shift ◽  
Fructose 1,6 Bisphosphatase

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), an important gluconeogenic enzyme, catalyses the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and Pi. Enzyme activity is mainly regulated by the allosteric inhibitors fructose 2,6-bisphosphate and AMP. Although some observations about hormonal regulation of the enzyme have been published, the FBPase promoter has not been studied in detail. Here we report an in vitro characterization of the FBPase promoter with respect to the elements that are required for basal promoter activity. Transient transfection of H4IIE rat hepatoma cells, combined with site-directed mutagenesis, demonstrated that an enhancer box, three GC-boxes and a nuclear factor κB (NF-κB)-binding element are important for hepatic FBPase promoter activity. These elements are found in the region located between -405 to +25bp relative to the transcription start site. Electrophoretic-mobility-shift assays and supershift analysis confirmed that upstream stimulatory factor 1 (USF1)/USF2, specificity protein 1 (Sp1)/Sp3 and NF-κB respectively bind to these sites. The present study provides the basis for a more comprehensive screening for mutations in FBPase-deficient patients and for further studies of the transcriptional regulation of this gene.

scholarly journals Upstream stimulatory factor regulates Pdx-1 gene expression in differentiated pancreatic β-cells

1999 ◽  
Vol 341 (2) ◽  
pp. 315-322 ◽  
Author(s):  
Jin QIAN ◽  
Elizabeth N. KAYTOR ◽  
Howard C. TOWLE ◽  
L. Karl OLSON
Keyword(s):  
Gene Expression ◽  
Gene Promoter ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Upstream Stimulatory Factor ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Protein Levels ◽  
E Box ◽  
Stimulatory Factor

The homeobox gene Pdx-1 plays a key role in the development of the pancreas. In the adult, however, expression of the Pdx-1 gene is restricted to pancreatic β-cells and endocrine cells of duodenal epithelium. Recently, the transcription factor, upstream stimulatory factor (USF), has been shown to bind invitro to a mutationally sensitive E-box motif within the 5′-flanking region of the Pdx-1 gene [Sharma, Leonard, Lee, Chapman, Leiter and Montminy (1996) J. Biol. Chem. 271, 2294-2299]. In the present study, we show that USF not only binds to the Pdx-1 gene promoter but also functionally regulates the expression of the Pdx-1 gene in differentiated pancreatic β-cells. Adenovirus-mediated overexpression of a dominant negative form of USF2 decreased binding of endogenous USF to the E-box element by ~ 90%. This reduction in endogenous USF binding led to a greater than 50% decrease in Pdx-1 gene promoter activity, which, in turn, resulted in marked reductions in Pdx-1 mRNA and protein levels. Importantly, the lower Pdx-1 protein levels led to a greater than 50% reduction in Pdx-1 binding activity to the A3 element on the insulin gene promoter, and a significant reduction in insulin mRNA levels. Overall, our results show that USF functionally regulates Pdx-1 gene expression in differentiated pancreatic β-cells and provide the first functional data for a role of USF in the regulation of a normal cellular gene.

The role of upstream stimulatory factor in expression of the vasopressin gene in lung cancer

Lung Cancer ◽  
2000 ◽  
Vol 29 (1) ◽  
pp. 204-205 ◽  
Author(s):  
J.M Coulson ◽  
J.L Edgson ◽  
R.J Mulgrew ◽  
J.P Quinn ◽  
P.J Woll
Keyword(s):  
Lung Cancer ◽  
Upstream Stimulatory Factor ◽  
Stimulatory Factor ◽  
Vasopressin Gene

scholarly journals DEC1 Modulates the Circadian Phase of Clock Gene Expression

2008 ◽  
Vol 28 (12) ◽  
pp. 4080-4092 ◽  
Author(s):  
Ayumu Nakashima ◽  
Takeshi Kawamoto ◽  
Kiyomasa K. Honda ◽  
Taichi Ueshima ◽  
Mitsuhide Noshiro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clock Genes ◽  
Luciferase Reporter ◽  
Constant Darkness ◽  
Mobility Shift ◽  
Behavioral Rhythms ◽  
Circadian Phase ◽  
Clock Gene Expression ◽  
E Box ◽  
E Boxes

ABSTRACT DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E′ boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E′-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E′ box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1 −/− mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.

Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK

1999 ◽  
Vol 276 (2) ◽  
pp. G322-G330 ◽  
Author(s):  
Brian K. Dieckgraefe ◽  
Danielle M. Weems
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Erk Activation ◽  
Kinase C

The signaling pathways activated in response to gastrointestinal injury remain poorly understood. Previous work has implicated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase as a mediator of wound-signal transduction and a possible regulator of epithelial restitution. Monolayer injury resulted in rapid activation of p42 and p44 ERK. Injury-induced ERK activation was blocked by protein kinase C inhibition or by disruption of the cell cytoskeleton. Significant increases in Fos and early growth response (Egr)-1 mRNA levels were stimulated by injury, peaking by 20 min. ERK activation and the induction of Egr-1 mRNA were inhibited in a dose-dependent fashion with PD-98059. Fos mRNA expression was partially blocked by PD-98059. Western blot analysis demonstrated strong expression and nuclear localization of Fos and Egr after wounding. Electrophoretic mobility shift assays demonstrated that nuclear extracts contained a protein that specifically bound double-stranded oligonucleotides containing the Egr consensus binding element. Gel supershift assays demonstrated that the protein-DNA complexes were recognized by anti-Egr antibody. Inhibition of injury-induced ERK activation by PD-98059 or direct interference with Egr by expression of a dominant negative mutant led to significantly reduced in vitro monolayer restitution.

scholarly journals Adenovirus RIDα regulates endosome maturation by mimicking GTP-Rab7

2007 ◽  
Vol 179 (5) ◽  
pp. 965-980 ◽  
Author(s):  
Ankur H. Shah ◽  
Nicholas L. Cianciola ◽  
Jeffrey L. Mills ◽  
Frank D. Sönnichsen ◽  
Cathleen Carlin
Keyword(s):  
Growth Factor Receptor ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Site Directed Mutagenesis ◽  
Oxysterol Binding Protein ◽  
Early Endosomes ◽  
Catalytically Active ◽  
Guanosine Triphosphatase ◽  
Endocytic Vesicles

The small guanosine triphosphatase Rab7 regulates late endocytic trafficking. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are guanosine triphosphate (GTP)–Rab7 effectors that instigate minus end–directed microtubule transport. We demonstrate that RILP and ORP1L both interact with the group C adenovirus protein known as receptor internalization and degradation α (RIDα), which was previously shown to clear the cell surface of several membrane proteins, including the epidermal growth factor receptor and Fas (Carlin, C.R., A.E. Tollefson, H.A. Brady, B.L. Hoffman, and W.S. Wold. 1989. Cell. 57:135–144; Shisler, J., C. Yang, B. Walter, C.F. Ware, and L.R. Gooding. 1997. J. Virol. 71:8299–8306). RIDα localizes to endocytic vesicles but is not homologous to Rab7 and is not catalytically active. We show that RIDα compensates for reduced Rab7 or dominant-negative (DN) Rab7(T22N) expression. In vitro, Cu2+ binding to RIDα residues His75 and His76 facilitates the RILP interaction. Site-directed mutagenesis of these His residues results in the loss of RIDα–RILP interaction and RIDα activity in cells. Additionally, expression of the RILP DN C-terminal region hinders RIDα activity during an acute adenovirus infection. We conclude that RIDα coordinates recruitment of these GTP-Rab7 effectors to compartments that would ordinarily be perceived as early endosomes, thereby promoting the degradation of selected cargo.

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