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 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.

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.

Lineage Specific Erythropoietin Receptor Expression Regulation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4779-4779
Author(s):  
Li Wang ◽  
Heather Marie Rogers ◽  
Constance Tom Noguchi
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Erythroid Differentiation ◽  
C2c12 Cells ◽  
Proximal Promoter ◽  
Erythroid Cells ◽  
E Box ◽  
High Level ◽  
Epor Expression ◽  
In Erythroid Cells

Abstract Abstract 4779 Erythropoietin receptor (EpoR) is a member of the cytokine receptor superfamily. During erythroid differentiation of hematopoietic stem cells, Epo acts through binding of EpoR on the surface of early erythroid progenitor cells to promote cell survival, proliferation and differentiation down the erythroid lineage. The extent of Epo response is dependent on the level of EpoR expression. The EpoR proximal promoter contains an inverted GATA-1 binding site (TTATCT) located at position -179 from the first codon of the human EpoR (hEpoR) gene. Three E boxes sites (CAGCTG) are also present in the 5′ untranslated transcribed region (UTR) of the EpoR gene and appear to be evolutionarily conserved in mammals. The expression of EpoR is not restricted to the erythroid lineage and can be found in several non-hematopoietic cells including endothelial, neural, muscle, cardiovascular and renal tissues. We previously found that EpoR is also expressed in primary satellite cells from skeletal muscle and in myoblast C2C12 cells but not in terminally differentiated myotubes. Epo stimulates proliferative and/or anti-apoptotic activities in myoblasts. Here we observed that erythroid MEL cells and C2C12 cells in the undifferentiated state express EpoR at a similar order of magnitude, but while EpoR expression increases with MEL cell differentiation, expression decreases with C2C12 differentiation. To gain insight on Epo activity during lineage specific differentiation, we compared EpoR expression during erythroid differentiation of MEL cells with myoblast differentiation of C2C12 cells. The conserved E-box region acts as a binding site for tissue specific basic-helix-loop-helix transcription factors, TAL1 in erythroid cells and the MyoD transcription factor family member in myoblasts. Deletion or mutation of the E-box motifs resulted in down regulated transcriptional activity of the EPO-R proximal promoter in erythroid and myoblast cells. These data suggest that the E-box region contributes to high activation of EPO-R transcription in both cell types. We also found that EPO-R expression is also dependent on the GATA-motif in the proximal promoter in myoblasts. While GATA-1, required for high level EpoR expression in erythroid cells is not expressed in myoblasts, we determined other GATA proteins in C2C12 cells associate with the GATA-1 binding site to provide transcription activity. However, unlike erythroid cells that exhibit high level induction of GATA-1 with erythroid differentiation and thus, high level EpoR expression, GATA proteins are down regulated with myoblast differentiation and EpoR expression is down regulated. Overall, during differentiation we observed an increase in histone acetylation and H3-K4 dimethylation in chromatin associated with the EpoR promoter in erythroid cells in contrast to a decrease in differentiating C2C12 cells, suggesting that the chromatin structure of EpoR in myoblast is less accessible than in erythroid cells and reflects the lower EpoR expression in non-hematopoietic cells. While DNA binding motifs for GATA and basic-helix-loop-helix transcription factors regulate EpoR expression in both erythroid and myoblast cells, important differences during lineage specific differentiation in EpoR chromatin accessibility and induction of the corresponding transcription factors that regulate EpoR expression explain in part the high induction of EpoR during erythroid differentiation in comparison to the low level expression in non-hematopoietic tissues. Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals Arabidopsis heat shock transcription factor HSFA7b positively mediates salt stress tolerance by binding to an E-box-like motif to regulate gene expression

2019 ◽  
Vol 70 (19) ◽  
pp. 5355-5374 ◽  
Author(s):  
Dandan Zang ◽  
Jingxin Wang ◽  
Xin Zhang ◽  
Zhujun Liu ◽  
Yucheng Wang
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Heat Shock ◽  
Salt Tolerance ◽  
Stress Responses ◽  
Ion Homeostasis ◽  
Cellulose Synthase ◽  
Regulate Gene Expression ◽  
E Box ◽  
Regulate Gene

Abstract Plant heat shock transcription factors (HSFs) are involved in heat and other abiotic stress responses. However, their functions in salt tolerance are little known. In this study, we characterized the function of a HSF from Arabidopsis, AtHSFA7b, in salt tolerance. AtHSFA7b is a nuclear protein with transactivation activity. ChIP-seq combined with an RNA-seq assay indicated that AtHSFA7b preferentially binds to a novel cis-acting element, termed the E-box-like motif, to regulate gene expression; it also binds to the heat shock element motif. Under salt conditions, AtHSFA7b regulates its target genes to mediate serial physiological changes, including maintaining cellular ion homeostasis, reducing water loss rate, decreasing reactive oxygen species accumulation, and adjusting osmotic potential, which ultimately leads to improved salt tolerance. Additionally, most cellulose synthase-like (CSL) and cellulose synthase (CESA) family genes were inhibited by AtHSFA7b; some of them were randomly selected for salt tolerance characterization, and they were mainly found to negatively modulate salt tolerance. By contrast, some transcription factors (TFs) were induced by AtHSFA7b; among them, we randomly identified six TFs that positively regulate salt tolerance. Thus, AtHSFA7b serves as a transactivator that positively mediates salinity tolerance mainly through binding to the E-box-like motif to regulate gene expression.

scholarly journals Angiotensin II-Induced Protein Kinase D Activates the ATF/CREB Family of Transcription Factors and Promotes StAR mRNA Expression

Endocrinology ◽  
2014 ◽  
Vol 155 (7) ◽  
pp. 2524-2533 ◽  
Author(s):  
Lawrence O. Olala ◽  
Vivek Choudhary ◽  
Maribeth H. Johnson ◽  
Wendy B. Bollag
Keyword(s):  
Transcription Factors ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Mrna Expression ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Dependent Manner ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Constitutively Active

Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

scholarly journals Formaldehyde-Responsive Proteins TtmR and EfgA Reveal a Trade-off between Formaldehyde Resistance and Efficient Transition to Methylotrophy in Methylorubrum extorquens

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Jannell V. Bazurto ◽  
Eric L. Bruger ◽  
Jessica A. Lee ◽  
Leah B. Lambert ◽  
Christopher J. Marx
Keyword(s):  
Transcription Factors ◽  
Sole Source ◽  
Growth Delay ◽  
Enzymatic Reactions ◽  
Environmental Niche ◽  
Carbon Utilization ◽  
Trade Off ◽  
Content Type ◽  
Formaldehyde Sensing ◽  
High Level

ABSTRACT For bacteria to thrive, they must be well adapted to their environmental niche, which may involve specialized metabolism, timely adaptation to shifting environments, and/or the ability to mitigate numerous stressors. These attributes are highly dependent on cellular machinery that can sense both the external and intracellular environment. Methylorubrum extorquens is an extensively studied facultative methylotroph, an organism that can use single-carbon compounds as its sole source of carbon and energy. In methylotrophic metabolism, carbon flows through formaldehyde as a central metabolite; thus, formaldehyde is both an obligate metabolite and a metabolic stressor. Via the one-carbon dissimilation pathway, free formaldehyde is rapidly incorporated by formaldehyde activating enzyme (Fae), which is constitutively expressed at high levels. In the presence of elevated formaldehyde levels, a recently identified formaldehyde-sensing protein, EfgA, induces growth arrest. Here, we describe TtmR, a formaldehyde-responsive transcription factor that, like EfgA, modulates formaldehyde resistance. TtmR is a member of the MarR family of transcription factors and impacts the expression of 75 genes distributed throughout the genome, of which many encode transcription factors and/or are involved in stress response, including efgA. Notably, when M. extorquens is adapting its metabolic network during the transition to methylotrophy, efgA and ttmR mutants experience an imbalance in formaldehyde production and a notable growth delay. Although methylotrophy necessitates that M. extorquens maintains a relatively high level of formaldehyde tolerance, this work reveals a trade-off between formaldehyde resistance and the efficient transition to methylotrophic growth and suggests that TtmR and EfgA play a pivotal role in maintaining this balance. IMPORTANCE All organisms produce formaldehyde as a by-product of enzymatic reactions and as a degradation product of metabolites. The ubiquity of formaldehyde in cellular biology suggests that all organisms have evolved mechanisms of mitigating formaldehyde toxicity. However, formaldehyde sensing is poorly described, and the prevention of formaldehyde-induced damage is understood primarily in the context of detoxification. Here, we used an organism that is regularly exposed to elevated intracellular formaldehyde concentrations through high-flux one-carbon utilization pathways to gain insight into the role of formaldehyde-responsive proteins that modulate formaldehyde resistance. Using a combination of genetic and transcriptomic analyses, we identified dozens of genes putatively involved in formaldehyde resistance, determined the relationship between two different formaldehyde response systems, and identified an inherent trade-off between formaldehyde resistance and optimal transition to methylotrophic metabolism.

Sign in / Sign up

Export Citation Format

Share Document