Upstream stimulatory factors stimulate transcription through E-box motifs in the PF4 gene in megakaryocytes

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 2027-2034 ◽  
Author(s):  
Yoshiaki Okada ◽  
Eri Matsuura ◽  
Zenzaburo Tozuka ◽  
Ryohei Nagai ◽  
Ayako Watanabe ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Regulatory Element ◽  
The Novel ◽  
Upstream Stimulatory Factor ◽  
Platelet Factor ◽  
Cd34 Cells ◽  
E Box ◽  
Hel Cells ◽  
B Cell Leukemia ◽  
Stimulatory Factor

Abstract Platelet factor 4 (PF4) is expressed during megakaryocytic differentiation. We previously demonstrated that the homeodomain proteins (myeloid ecotropic integra tion site 1 [MEIS1], Pbx-regulating protein 1 [PREP1], and pre-B-cell leukemia transcription factors [PBXs]) bind to the novel regulatory element tandem repeat of MEIS1 binding element [TME] and transactivate the rat PF4 promoter. In the present study, we investigated and identified other TME binding proteins in megakaryocytic HEL cells using mass spectrometry. Among identified proteins, we focused on upstream stimulatory factor (USF1) and USF2 and investigated their effects on the PF4 promoter. USF1 and 2 bound to the E-box motif in the TME and strongly transactivated the PF4 promoter. Furthermore, physiologic bindings of USF1 and 2 to the TME in rat megakaryocytes were demonstrated by the chromatin immunoprecipitation (ChIP) assay. Interestingly, the E-box motif in the TME was conserved in TME-like sequences of both the human and mouse PF4 promoters. USF1 and 2 also bound to the human TME-like sequence and transactivated the human PF4 promoter. Expressions of USF1 and 2 were detected by reverse-transcriptase–polymerase chain reaction (RT-PCR) in the human megakaryocytes derived from CD34+ cells. Thus, these studies demonstrate that the novel TME binding transcription factors, USF1 and 2, transactivate rat and human PF4 promoters and may play an important role in megakaryocytic gene expression.

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

2002 ◽  
Vol 30 (6) ◽  
pp. 1070-1072 ◽  
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.

scholarly journals Roles of USF, Ikaros and Sp proteins in the transcriptional regulation of the human reduced folate carrier B promoter

2004 ◽  
Vol 383 (2) ◽  
pp. 249-257 ◽  
Author(s):  
Mingjun LIU ◽  
Johnathan R. WHETSTINE ◽  
Scott G. PAYTON ◽  
Yubin GE ◽  
Robin M. FLATLEY ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dominant Negative ◽  
Upstream Sequence ◽  
Reduced Folate Carrier ◽  
Upstream Stimulatory Factor ◽  
Important Region ◽  
Ht1080 Cells ◽  
E Box ◽  
Gc Box ◽  
High Level

The hRFC (human reduced folate carrier) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to seven non-coding regions (A1, A2, A, B, C, D and E) and at least four promoters. For the hRFC-B basal promoter, regulation involves binding of Sp (specificity protein) transcription factors to a critical GC-box. By transiently transfecting HT1080 cells with 5′- and 3′-deletion constructs spanning 1057 bp of upstream sequence, a transcriptionally important region was localized to 158 bp flanking the transcriptional start sites. By gel shift and chromatin immunoprecipitation assays, USF (upstream stimulatory factor), Sp1 and Ikaros-related proteins were bound to consensus elements (one E-box, two GC-box and three Ikaros) within this region. The functional importance of these elements was confirmed by transient tranfections of HT1080 cells with hRFC-B reporter constructs in which they were mutated, and by co-transfections of Drosophila Mel-2 cells with wild-type hRFC-B promoter and expression constructs for USF1, USF2a, Sp1 and Ikaros 2 and 8. Both USF1 and Sp1 proteins transactivated the hRFC-B promoter. Sp1 combined with USF1 resulted in a synergistic transactivation. Identical results were obtained with USF2a. Ikaros 2 was a repressor of hRFC-B promoter activity whose effects were partly reversed by the dominant-negative Ikaros 8. In HT1080 cells, transfection with Ikaros 2 decreased endogenous hRFC-B transcripts, whereas USF1 and Sp1 increased transcript levels. Ikaros 2 also decreased reporter gene activity and levels of acetylated chromatin associated with the endogenous promoter. Collectively, these results identify transcriptionally important regions in the hRFC-B promoter that include multiple GC-box, Ikaros and E-box elements. Our results also suggest that co-operative interactions between transcription factors Sp1 and USF are essential for high-level hRFC-B transactivation and imply that these effects are modulated by the family of Ikaros proteins and by histone acetylation.

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

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 492-500 ◽  
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.

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.

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

2001 ◽  
Vol 357 (1) ◽  
pp. 97-105 ◽  
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.

Cis and trans regulation of hepcidin expression by upstream stimulatory factor

Blood ◽  
2006 ◽  
Vol 108 (13) ◽  
pp. 4237-4245 ◽  
Author(s):  
Henry K. Bayele ◽  
Harry McArdle ◽  
Surjit K.S. Srai
Keyword(s):  
Transcription Factors ◽  
Iron Metabolism ◽  
Iron Status ◽  
Transcriptional Regulators ◽  
Negative Regulator ◽  
Gene Promoters ◽  
Upstream Stimulatory Factor ◽  
Hepcidin Expression ◽  
Zip Family ◽  
Stimulatory Factor

Abstract Hepcidin is the presumed negative regulator of systemic iron levels; its expression is induced in iron overload, infection, and inflammation, and by cytokines, but is suppressed in hypoxia and anemia. Although the gene is exquisitely sensitive to changes in iron status in vivo, its mRNA is devoid of prototypical iron-response elements, and it is therefore not obvious how it may be regulated by iron flux. The multiplicity of effectors of its expression also suggests that the transcriptional circuitry controlling the gene may be very complex indeed. In delineating enhancer elements within both the human and mouse hepcidin gene promoters, we show here that members of the basic helix-loop-helix leucine zipper (bHLH-ZIP) family of transcriptional regulators control hepcidin expression. The upstream stimulatory factor 2 (USF2), previously linked to hepcidin through gene ablation in inbred mice, appears to exert a polar or cis-acting effect, while USF1 may act in trans to control hepcidin expression. In mice, we found variation in expression of both hepcidin genes, driven by these transcription factors. In addition, c-Myc and Max synergize to control the expression of this hormone, supporting previous findings for the role of this couple in regulating iron metabolism. Transcriptional activation by both USF1/USF2 and c-Myc/Max heterodimers occurs through E-boxes within the promoter. Site-directed mutagenesis of these elements rendered the promoter unresponsive to USF1/USF2 or c-Myc/Max. Dominant-negative mutants of USF1 and USF2 reciprocally attenuated promoter transactivation by both wild-type USF1 and USF2. Promoter occupancy by the transcription factors was confirmed by DNA-binding and chromatin immunoprecipitation assays. Taken together, it would appear that synergy between these members of the bHLH-ZIP family of transcriptional regulators may subserve an important role in iron metabolism as well as other pathways in which hepcidin may be involved.

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