Upstream Stimulatory Factor Binding to the E-box at −65 Is Required for Insulin Regulation of the Fatty Acid Synthase Promoter

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.

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Transcription factors acting on the promoter of the rat fatty acid synthase gene

Biochemical Society Transactions ◽

10.1042/bst0301070 ◽

2002 ◽

Vol 30 (6) ◽

pp. 1070-1072 ◽

Cited By ~ 24

Author(s):

M. Schweizer ◽

K. Roder ◽

L. Zhang ◽

S. S. Wolf

Keyword(s):

Transcription Factors ◽

Fatty Acid ◽

Binding Sites ◽

Fatty Acid Synthase ◽

Regulatory Element ◽

Upstream Stimulatory Factor ◽

Element Binding Protein ◽

Sterol Regulatory Element ◽

Responsive Element ◽

Stimulatory Factor

Fatty acid synthase (FAS), one of the main lipogenic enzymes, converts dietary calories into a storage form of energy. The transcription factors, stimulatory proteins 1 and 3 (Sp1 and Sp3), nuclear factor Y (NF-Y), upstream stimulatory factor (USF) and sterol regulatory element binding protein-1 (SREBP-1) have cognate binding sites on the promoter of the FAS gene. It was shown that Sp1 and NF-Y interact co-operatively at the diet-induced DNase I-hypersensitive site at position —500. Adjacent binding sites for NF-Y and Sp1 have also been found between —71 and —52, and —91 and —83. cAMP regulation is mediated via the inverted CAAT element (ICE) at —99 to —92, which binds NF-Y. The FAS insulin-responsive element 3 (FIRE3)-binding site at —71 to —52 is capable of binding NF-Y, USF and SREBP-1, and is required for the sterol response in conjunction with the co-activator NF-Y around —100. Surprisingly, both FIRE3 and ICE are also necessary for the response to retinoic acid that plays a role in development and is an essential component of the diet.

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Forkhead Box A1 (FOXA1) and A2 (FOXA2) Oppositely Regulate Human Type 1 Iodothyronine Deiodinase Gene in Liver

Endocrinology ◽

10.1210/en.2011-1310 ◽

2012 ◽

Vol 153 (1) ◽

pp. 492-500 ◽

Cited By ~ 11

Author(s):

Naotetsu Kanamoto ◽

Tetsuya Tagami ◽

Yoriko Ueda-Sakane ◽

Masakatsu Sone ◽

Masako Miura ◽

...

Keyword(s):

Binding Site ◽

Promoter Activity ◽

Short Interfering Rna ◽

Upstream Stimulatory Factor ◽

Iodothyronine Deiodinase ◽

Forkhead Box ◽

E Box ◽

Stimulatory Factor ◽

Interfering Rna

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.

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

Biochemical Journal ◽

10.1042/bj20021176 ◽

2003 ◽

Vol 369 (3) ◽

pp. 549-561 ◽

Cited By ~ 22

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.

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Occupancy and Function of the −150 Sterol Regulatory Element and −65 E-Box in Nutritional Regulation of the Fatty Acid Synthase Gene in Living Animals

Molecular and Cellular Biology ◽

10.1128/mcb.23.16.5896-5907.2003 ◽

2003 ◽

Vol 23 (16) ◽

pp. 5896-5907 ◽

Cited By ~ 78

Author(s):

Maria-Jesus Latasa ◽

Michael J. Griffin ◽

Yang Soo Moon ◽

Chulho Kang ◽

Hei Sook Sul

Keyword(s):

Fatty Acid ◽

Binding Sites ◽

Fatty Acid Synthase ◽

Regulatory Element ◽

Nutritional Regulation ◽

Fed State ◽

E Box ◽

Sterol Regulatory Element ◽

E Boxes

ABSTRACT Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5′ deletions as well as site-specific mutations of the FAS promoter. We show that the −332 and −65 E-boxes are bound by USF in both fasted and refed mice, while the −150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the −150 SRE or the −65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the −150 SRE but, unexpectedly, of the −65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the −150 SRE and that USF binding to the −65 E-box is also required for SREBP binding and activation of the FAS promoter.

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Interaction of upstream stimulatory factor proteins with an E-box located within the human CYP1A2 5′-flanking gene contributes to basal transcriptional gene activation

Biochemical Pharmacology ◽

10.1016/s0006-2952(03)00037-6 ◽

2003 ◽

Vol 65 (7) ◽

pp. 1087-1096 ◽

Cited By ~ 10

Author(s):

George V. Pickwell ◽

Hsueh Shih ◽

Linda C. Quattrochi

Keyword(s):

Gene Activation ◽

Upstream Stimulatory Factor ◽

E Box ◽

Stimulatory Factor ◽

Human Cyp1a2

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Transcriptional regulation of the human cystathionine β-synthase −1b basal promoter: synergistic transactivation by transcription factors NF-Y and Sp1/Sp3

Biochemical Journal ◽

10.1042/bj3570097 ◽

2001 ◽

Vol 357 (1) ◽

pp. 97-105 ◽

Cited By ~ 32

Author(s):

Yubin GE ◽

Mark A. KONRAD ◽

Larry H. MATHERLY ◽

Jeffrey W. TAUB

Keyword(s):

Reporter Gene ◽

Intermediate Step ◽

Nuclear Factor ◽

Upstream Stimulatory Factor ◽

E Box ◽

Gc Box ◽

Specific Regulation ◽

Specificity Protein ◽

Upstream Stimulatory Factor 1 ◽

Stimulatory Factor

Cystathionine β-synthase (CBS) catalyses the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. Human CBS encodes five distinct 5′ non-coding exons, the most frequent termed CBS −1a and CBS −1b, each transcribed from its own unique GC-rich TATA-less promoter. The minimal transcriptional region (−3792 to −3667) of the CBS −1b promoter was defined by 5′- and 3′-deletions, and transient transfections of reporter gene constructs in HepG2 cells, characterized by CBS transcription exclusively from the −1b promoter. Included in this 125bp region are 3 GC-boxes (termed GC-a, GC-b and GC-c), an inverted CAAT-box and an E-box. By gel-shift and supershift assays, binding of specificity protein (Sp)1 and Sp3 to the GC-box elements, upstream stimulatory factor 1 (USF-1) to the E-box, and both nuclear factor (NF)-Y and an NF-1-like factor to the CAAT box could be demonstrated. By transient trans fections and reporter gene assays in HepG2 and Drosophila SL2 cells, a functional interplay was indicated between NF-Y binding to the CAAT-box, or between USF-1 binding to the E-box, and Sp1/Sp3 binding to the GC-box elements. In SL2 cells, NF-Y and Sp1/Sp3 were synergistic. Furthermore, both Sp1 and the long Sp3 isoform transactivated the CBS −1b minimal promoter; however, the short Sp3 isoforms were potent repressors. These results may explain the cell- or tissue-specific regulation of CBS transcription, and clarify the bases for alterations in CBS gene expression in human disease and Down's syndrome.

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Upstream stimulatory factor regulates E box-dependent PAI-1 transcription in human epidermal keratinocytes

Journal of Cellular Physiology ◽

10.1002/jcp.20211 ◽

2005 ◽

Vol 203 (1) ◽

pp. 156-165 ◽

Cited By ~ 29

Author(s):

Rosalie R. Allen ◽

Li Qi ◽

Paul J. Higgins

Keyword(s):

Epidermal Keratinocytes ◽

Upstream Stimulatory Factor ◽

Human Epidermal Keratinocytes ◽

E Box ◽

Stimulatory Factor ◽

Pai 1

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Follicle-Stimulating Hormone (FSH) Transiently Blocks FSH Receptor Transcription by Increasing Inhibitor of Deoxyribonucleic Acid Binding/Differentiation-2 and Decreasing Upstream Stimulatory Factor Expression in Rat Sertoli Cells

Endocrinology ◽

10.1210/en.2008-1261 ◽

2009 ◽

Vol 150 (8) ◽

pp. 3783-3791 ◽

Cited By ~ 15

Author(s):

Pushpa Viswanathan ◽

Michelle A. Wood ◽

William H. Walker

Keyword(s):

Protein Binding ◽

Sertoli Cells ◽

Fsh Receptor ◽

Upstream Stimulatory Factor ◽

Binding Studies ◽

Nuclear Extracts ◽

E Box ◽

Stimulatory Factor ◽

Stimulation Of

FSH acts through the FSH receptor (FSHR) to modulate cell processes that are required to support developing spermatozoa. Within the testis, only Sertoli cells possess receptors for FSH and are the major targets for this regulator of spermatogenesis. FSH stimulation of Sertoli cells for 24–48 h is known to induce Fshr mRNA expression through an E-box motif (CACGTG) located 25 bp upstream of the transcription start site. In contrast, FSH stimulation for 8 h inhibits Fshr transcription. DNA-protein binding studies performed using nuclear extracts from Sertoli cells show that protein binding to the Fshr promoter E-box was reduced 68% after 6 h of FSH stimulation but increased 191% over basal levels after 48 h of stimulation. The proteins binding to the Fshr E-box were identified as upstream stimulatory factor (USF)-1 and -2. FSH stimulation transiently decreased USF1 levels and increased the expression of the inhibitor of DNA binding/differentiation (ID)-2 repressor protein with the same kinetics as the decreased USF/E-box interactions. Overexpression of ID2 resulted in a dose-dependent decrease in USF-driven Fshr promoter activity in the MSC-1 Sertoli cell line, and ID2 inhibited USF binding to the Fshr E-box. Together, these studies suggest that stimulation of Sertoli cells with FSH transiently decreases expression of the USF1 activator and induces accumulation of the ID2 repressor, to block USF binding to the Fshr promoter and delay activation of Fshr transcription. This FSH-regulated mechanism may explain the cyclical changes in Fshr expression that occurs in Sertoli cells in vivo.

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