Binding Of Upstream Stimulatory Factor (USF)-1 To An E-box Activates GATA5 Gene Expression

Author(s):  
Bohao Chen ◽  
Rona Hsu ◽  
Paul Kogut ◽  
Nanyue Chen ◽  
Julian Solway
2012 ◽  
Vol 446 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Bohao Chen ◽  
Rona Hsu ◽  
Zhenping Li ◽  
Paul C. Kogut ◽  
Qingxia Du ◽  
...  

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.


1998 ◽  
Vol 12 (5) ◽  
pp. 714-726 ◽  
Author(s):  
Adrienne N. Harris ◽  
Pamela L. Mellon

Abstract Tissue-specific expression of the mammalian FTZ-F1 gene is essential for adrenal and gonadal development and sexual differentiation. The FTZ-F1 gene encodes an orphan nuclear receptor, termed SF-1 (steroidogenic factor-1) or Ad4BP, which is a primary transcriptional regulator of several hormone and steroidogenic enzyme genes that are critical for normal physiological function of the hypothalamic-pituitary-gonadal axis in reproduction. The objective of the current study is to understand the molecular mechanisms underlying transcriptional regulation of SF-1 gene expression in the pituitary. We have studied a series of deletion and point mutations in the SF-1 promoter region for transcriptional activity in αT3–1 and LβT2 (pituitary gonadotrope), CV-1, JEG-3, and Y1 (adrenocortical) cell lines. Our results indicate that maximal expression of the SF-1 promoter in all cell types requires an E box element at −82/−77. This E box sequence (CACGTG) is identical to the binding element for USF (upstream stimulatory factor), a member of the helix-loop-helix family of transcription factors. Studies of the SF-1 gene E box element using gel mobility shift and antibody supershift assays indicate that USF may be a key transcriptional regulator of SF-1 gene expression.


2003 ◽  
Vol 371 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Cyrus C. MARTIN ◽  
Christina A. SVITEK ◽  
James K. OESER ◽  
Eva HENDERSON ◽  
Roland STEIN ◽  
...  

Islet-specific glucose-6-phosphatase (G6Pase) catalytic-subunit-related protein (IGRP) is a homologue of the catalytic subunit of G6Pase, the enzyme that catalyses the final step of the gluconeogenic pathway. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion-gene expression through transient transfection of islet-derived βTC-3 cells revealed that multiple promoter regions, located between −306 and −97, are required for maximal IGRP-CAT fusion-gene expression. These regions correlated with trans-acting factor-binding sites in the IGRP promoter that were identified in βTC-3 cells in situ using the ligation-mediated PCR (LMPCR) footprinting technique. However, the LMPCR data also revealed additional trans-acting factor-binding sites located between −97 and +1 that overlap two E-box motifs, even though this region by itself conferred minimal fusion-gene expression. The data presented here show that these E-box motifs are important for IGRP promoter activity, but that their action is only manifest in the presence of distal promoter elements. Thus mutation of either E-box motif in the context of the −306 to +3 IGRP promoter region reduces fusion-gene expression. These two E-box motifs have distinct sequences and preferentially bind NeuroD/BETA2 neurogenic differentiation/β-cell E box transactivator 2 and upstream stimulatory factor (USF) in vitro, consistent with the binding of both factors to the IGRP promoter in situ, as determined using the chromatin-immunoprecipitation (ChIP) assay. Based on experiments using mutated IGRP promoter constructs, we propose a model to explain how the ubiquitously expressed USF could contribute to islet-specific IGRP gene expression.


1999 ◽  
Vol 341 (2) ◽  
pp. 315-322 ◽  
Author(s):  
Jin QIAN ◽  
Elizabeth N. KAYTOR ◽  
Howard C. TOWLE ◽  
L. Karl OLSON

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.


Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 492-500 ◽  
Author(s):  
Naotetsu Kanamoto ◽  
Tetsuya Tagami ◽  
Yoriko Ueda-Sakane ◽  
Masakatsu Sone ◽  
Masako Miura ◽  
...  

Type 1 iodothyronine deiodinase (D1), a selenoenzyme that catalyzes the bioactivation of thyroid hormone, is expressed mainly in the liver. Its expression and activity are modulated by several factors, but the precise mechanism of its transcriptional regulation remains unclear. In the present study, we have analyzed the promoter of human D1 gene (hDIO1) to identify factors that prevalently increase D1 activity in the human liver. Deletion and mutation analyses demonstrated that a forkhead box (FOX)A binding site and an E-box site within the region between nucleotides −187 and −132 are important for hDIO1 promoter activity in the liver. EMSA demonstrated that FOXA1 and FOXA2 specifically bind to the FOXA binding site and that upstream stimulatory factor (USF) specifically binds to the E-box element. Overexpression of FOXA2 decreased hDIO1 promoter activity, and short interfering RNA-mediated knockdown of FOXA2 increased the expression of hDIO1 mRNA. In contrast, overexpression of USF1/2 increased hDIO1 promoter activity. Short interfering RNA-mediated knockdown of FOXA1 decreased the expression of hDIO1 mRNA, but knockdown of both FOXA1 and FOXA2 restored it. The response of the hDIO1 promoter to USF was greatly attenuated in the absence of FOXA1. Taken together, these results indicate that a balance of FOXA1 and FOXA2 expression modulates hDIO1 expression in the liver.


2003 ◽  
Vol 369 (3) ◽  
pp. 549-561 ◽  
Author(s):  
Judy M. COULSON ◽  
Jodie L. EDGSON ◽  
Zoe V. MARSHALL-JONES ◽  
Robert MULGREW ◽  
John P. QUINN ◽  
...  

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.


2010 ◽  
Vol 111 (3) ◽  
pp. 720-726 ◽  
Author(s):  
Nélida C. Olave ◽  
Maximiliano H. Grenett ◽  
Martin Cadeiras ◽  
Hernan E. Grenett ◽  
Paul J. Higgins

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