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 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 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.

The role of NeuroD1 in the upregulation of SMYD3 by HBx.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 183-183
Author(s):  
Junyao Xu ◽  
Qingqi Hong ◽  
Chuanchao He ◽  
Jie Wang
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Promoter Region ◽  
Gene Transfection ◽  
Histone Methyltransferase ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Cis Element ◽  
E Box

183 Background: SET and MYND Domain-Containing Protein 3 (SMYD3) is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis B virus x protein (HBx) is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Methods: Immunohistochemical staining was used to detect the expression of HBx and SMYD3 in HCC tumor tissues. HBx gene transfection, RNAi, and histone methyltransferase(H3-K4) activity assay were performed to reveal the transcrpitionally activation of HBx on functional SMYD3 gene expression. Chromatin immunoprecipitation (ChIP), Co-immunoprecipitation (Co-IP), Electrophoretic mobility shift assay (EMSA) were applied to investigate the underlying mechanism. Dual-luciferase reporter assay was used to search for the HBx responsive cis-element of SMYD3 gene. Results: Immunohistochemistry identified the positive correlation between HBx and SMYD3 expression in 42 HCC tissues. Up-regulation of HBx on SMYD3 expression was validated through experiments involving overexpression or knock-down of HBx in different HCC cell lines. And up-regulated SMYD3 is functionally active as histone methyltransferase. Next we found that HBx transcriptionally regulated SMYD3 gene expression by interacting with RNA polymerase IIand altering its binding site to a proximal promoter region(SD2) from a distant promoter region(SD6) of SMYD3. Truncated and mutant reporter assays revealed that the cis-element mapped in -178~-203bp in SMYD3 promotor is responsive for HBx-transactivation. And this 25bp cis-element contains a E-box 3 unit, which is a binding site for the transcriptional factor Neurogenic differentiation 1(NeuroD1). EMSA and Chip showed that HBx increased NeuroD1 binding to SMYD3 proximal promotor, however transcient expression of antisense NeuroD1 abolished HBx-induced SMYD3 expression. Conclusions: HBx transcriptionally up-regulates SMYD3 and that this process is mediated by NeuroD1 through binding to the E-box 3 site of SMYD3 promotor.

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.

Promoter I of the ovine acetyl-CoA carboxylase-α gene: an E-box motif at −114 in the proximal promoter binds upstream stimulatory factor (USF)-1 and USF-2 and acts as an insulin-response sequence in differentiating adipocytes

2001 ◽  
Vol 359 (2) ◽  
pp. 273-284 ◽  
Author(s):  
Maureen T. TRAVERS ◽  
Amanda J. VALLANCE ◽  
Helen T. GOURLAY ◽  
Clare A. GILL ◽  
Izabella KLEIN ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Binding Protein ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Acetyl Coa Carboxylase ◽  
Upstream Stimulatory Factor ◽  
Acetyl Coa ◽  
Rnase Protection ◽  
E Box ◽  
Stimulatory Factor

Acetyl-CoA carboxylase-α (ACC-α) plays a central role in co-ordinating de novo fatty acid synthesis in animal tissues. We have characterized the regulatory region of the ovine ACC-α gene. Three promoters, PI, PII and PIII, are dispersed throughout 50kb of genomic DNA. Expression from PI is limited to adipose tissue and liver. Sequence comparison of the proximal promoters of ovine and mouse PIs demonstrates high nucleotide identity and that they are characterized by a TATA box at −29, C/EBP (CCAAT enhancer-binding protein)-binding motifs and multiple E-box motifs. A 4.3kb ovine PI-luciferase reporter construct is insulin-responsive when transfected into differentiated ovine adipocytes, whereas when this construct is transfected into ovine preadipocytes and HepG2 cells the construct is inactive and is not inducible by insulin. By contrast, transfection of a construct corresponding to 132bp of the proximal promoter linked to a luciferase reporter is active and inducible by insulin in all three cell systems. Insulin signalling to the −132bp construct in differentiated ovine adipocytes involves, in part, an E-box motif at −114. Upstream stimulatory factor (USF)-1 and USF-2, but not sterol regulatory element-binding protein 1 (SREBP-1), are major components of protein complexes that bind this E-box motif. Activation of the 4.3kb PI construct in differentiated ovine adipocytes is associated with endogenous expression of PI transcripts throughout differentiation; PI transcripts are not detectable by RNase-protection assay in ovine preadipocytes, HepG2 cells or 3T3-F442A adipocytes. These data indicate the presence of repressor motifs in PI that are required to be de-repressed during adipocyte differentiation to allow induction of the promoter by insulin.

scholarly journals The Basic Helix-Loop-Helix, Leucine Zipper Transcription Factor, USF (Upstream Stimulatory Factor), Is a Key Regulator of SF-1 (Steroidogenic Factor-1) Gene Expression in Pituitary Gonadotrope and Steroidogenic Cells

1998 ◽  
Vol 12 (5) ◽  
pp. 714-726 ◽  
Author(s):  
Adrienne N. Harris ◽  
Pamela L. Mellon
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Transcriptional Regulator ◽  
Orphan Nuclear Receptor ◽  
Upstream Stimulatory Factor ◽  
Specific Expression ◽  
Steroidogenic Factor 1 ◽  
Helix Loop Helix ◽  
E Box ◽  
Stimulatory Factor

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.

scholarly journals Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression

2003 ◽  
Vol 371 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Cyrus C. MARTIN ◽  
Christina A. SVITEK ◽  
James K. OESER ◽  
Eva HENDERSON ◽  
Roland STEIN ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Fusion Gene ◽  
Catalytic Subunit ◽  
Related Protein ◽  
Upstream Stimulatory Factor ◽  
Factor Binding ◽  
E Box ◽  
Stimulatory Factor

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.

Regulation of human β2-microglobulin transactivation in hematopoietic cells

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3058-3064 ◽  
Author(s):  
Sam J. P. Gobin ◽  
Paula Biesta ◽  
Peter J. Van den Elsen
Keyword(s):  
Hematopoietic Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Upstream Stimulatory Factor ◽  
Β2 Microglobulin ◽  
E Box ◽  
Infection And Inflammation ◽  
Upstream Stimulatory Factor 1 ◽  
Stimulatory Factor

Abstract β2-Microglobulin (β2m) is a chaperone of major histocompatibility complex (MHC) class I (–like) molecules that play a central role in antigen presentation, immunoglobulin transport, and iron metabolism. It is therefore of importance that β2m is adequately expressed in cells that perform these functions, such as hematopoietic cells. In this study, we investigated the transcriptional regulation of β2m in lymphoid and myeloid cell lines through a promoter containing a putative E box, Ets/interferon-stimulated response element (ISRE), and κB site. Here we show that upstream stimulatory factor 1 (USF1) and USF2 bind to the E box and regulate β2m transactivation. The nuclear factor κB (NF-κB) subunits p50 and p65 bind to the κB box and p65 transactivates β2m. Interferon regulatory factor 1 (IRF1), IRF2, IRF4, and IRF8, but not PU.1, bind to the Ets/ISRE, and IRF1 and IRF3 are strong transactivators of β2m. Together, all 3 boxes are important for the constitutive and cytokine-induced levels of β2m expression in lymphoid and myeloid cell types. As such, β2m transactivation is under the control of important transcriptional pathways, which are activated during injury, infection, and inflammation.

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