scholarly journals Cloning and characterization of the human beta2-glycoprotein I (beta2-GPI) gene promoter: roles of the atypical TATA box and hepatic nuclear factor-1alpha in regulating beta2-GPI promoter activity

2004 ◽  
Vol 380 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Hsueh-Hsiao WANG ◽  
An-Na CHIANG
Keyword(s):  
Gene Expression ◽  
Hela Cells ◽  
Promoter Activity ◽  
Hepatoma Cell ◽  
Site Directed Mutagenesis ◽  
Tata Box ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Glycoprotein I

β2-Glycoprotein I (β2-GPI) is a plasma glycoprotein primarily synthesized in the liver. The interindividual variability of β2-GPI expression in subjects with various metabolic syndromes and disease states suggests that it may have clinical importance. However, the regulation of β2-GPI gene expression has not been clarified. To gain more insight into the control of β2-GPI gene expression, we cloned the 4.1-kb 5´-flanking region and characterized the proximal promoter of the β2-GPI gene in this study. Cis-acting elements required for β2-GPI promoter activity were identified with transient transfection assays in the hepatoma cell lines HepG2 and Huh7 and in non-hepatic HeLa cells. Serial deletion analyses of the β2-GPI 5´-flanking sequence revealed that the region from −197 to +7 had strong promoter activity in hepatoma cells but not in HeLa cells. Truncation and site-directed mutagenesis of putative cis-elements within this region showing an atypical TATA box and a HNF-1 (hepatic nuclear factor-1) element were both essential for the β2-GPI promoter activity. Subsequent gel mobility shift assays confirmed the interaction of HNF-1α with the HNF-1 site residing downstream of the TATA box. Co-transfection of β2-GPI promoter-luciferase vector with HNF-1α expression vector in Huh7 and HNF-1-deficient HeLa cells demonstrated the transactivation effect of HNF-1α on β2-GPI promoter activity. In addition, overexpression of HNF-1α enhanced the endogenous β2-GPI expression. These results suggest that the atypical TATA box and HNF-1 cis-element are critical for β2-GPI transcription and HNF-1α may play an important role in cell-specific regulation of β2-GPI gene expression.

Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene

1989 ◽  
Vol 9 (6) ◽  
pp. 2627-2640
Author(s):  
J N Buskin ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Muscle Creatine Kinase ◽  
Mouse Muscle ◽  
Nuclear Extracts ◽  
Muscle Creatine

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

scholarly journals Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.

1989 ◽  
Vol 9 (6) ◽  
pp. 2627-2640 ◽  
Author(s):  
J N Buskin ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Muscle Creatine Kinase ◽  
Mouse Muscle ◽  
Nuclear Extracts ◽  
Muscle Creatine

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

scholarly journals GATA augments GNRH-mediated increases in Adcyap1 gene expression in pituitary gonadotrope cells

2013 ◽  
Vol 51 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Robin L Thomas ◽  
Natalie M Crawford ◽  
Constance M Grafer ◽  
Weiming Zheng ◽  
Lisa M Halvorson
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Proximal Promoter ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Mobility Shift ◽  
Subunit Expression ◽  
Mobility Shift Assay

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine–paracrine factor in this tissue. GNRH has been shown to increase pituitaryAdcyap1gene expression through the interaction of CREB and jun/fos with CRE/AP1cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognatecis-elements which regulateAdcyap1gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulateAdcyap1promoter activity via a GATAcis-element located at position −191 in the ratAdcyap1gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation ofAdcyap1gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.

Transcriptional regulation of the KEL gene and Kell protein expression in erythroid and non-erythroid cells

2001 ◽  
Vol 356 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Valérie CAMARA-CLAYETTE ◽  
Cécile RAHUEL ◽  
Claude LOPEZ ◽  
Claude HATTAB ◽  
Virginie VERKARRE ◽  
...  
Keyword(s):  
Hela Cells ◽  
Blot Analysis ◽  
Promoter Activity ◽  
K562 Cells ◽  
Site Directed Mutagenesis ◽  
Transcriptional Analysis ◽  
Lymphoid Tissues ◽  
Dot Blot ◽  
Mobility Shift ◽  
Exon 1

The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only. Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5′ distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete. KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis. DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue. In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues. Moreover, most tissues analysed exhibited low levels of Kell transcripts. The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells. Altogether, the results indicated that KEL expression is not restricted to erythroid tissue.

scholarly journals Different Sp1 family members differentially affect transcription from the human elongation factor 1 A-1 gene promoter

1998 ◽  
Vol 333 (3) ◽  
pp. 511-517 ◽  
Author(s):  
S⊘ren J. NIELSEN ◽  
Morten PRÆSTEGAARD ◽  
Helle F. J. ØRGENSEN ◽  
Brian F. C. CLARK
Keyword(s):  
Hela Cells ◽  
Promoter Activity ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Elongation Factor ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Gc Box ◽  
Elongation Factor 1 ◽  
Human Elongation Factor

The GC box is an important transcriptional regulatory element present in the promoters of many mammalian genes. In the present study we examine the effect of known GC-box-binding proteins on the promoter of the human elongation factor 1 A-1 (hEF1A-1) gene in human HeLa cells and Drosophila SL2 cells. In HeLa cells co-transfection with the GC-box-binding protein BTEB resulted in a 4–10-fold increase in hEF1A-1 promoter activity. This stimulation was dependent on a single GC box located between positions -69 and -50 of the promoter. Little or no effect was observed of other GC-box-binding proteins including Sp1, Sp3, Sp4 and BTEB2. In SL2 cells stimulation by Sp1 and Sp3 through the single GC box of the proximal promoter led to 13-fold and 21-fold increases respectively in promoter activity. Inclusion of further upstream sequences resulted in high levels of expression when Sp1 or Sp3 was co-transfected with the reporter plasmid. In this setting Sp1 stimulated transcription by 750-fold, whereas Sp3 was even more potent, yielding a 1150-fold stimulation. Mobility-shift assays performed with the promoter-proximal GC box demonstrated the binding of Sp1, Sp3 and Sp4 to this sequence. To our knowledge, the present study represents the first comparison of all known GC-box-binding proteins on a natural promoter.

Down-regulation of hepatic nuclear factor 4α on expression of human hepatic stimulator substance via its action on the proximal promoter in HepG2 cells

2008 ◽  
Vol 415 (1) ◽  
pp. 111-121 ◽  
Author(s):  
Duo Guo ◽  
Ling-yue Dong ◽  
Yuan Wu ◽  
Lin Yang ◽  
Wei An
Keyword(s):  
Hepg2 Cells ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Mrna Level ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Hepatic Stimulator Substance

hHSS (human hepatic stimulator substance) stimulates hepatocyte growth. To understand the mechanism controlling hHSS expression, we analysed the proximal promoter activity and identified two regulatory regions (−212/−192 and −152/−132) that were important for transcription in HepG2 cells. Using the luciferase reporter assay, gel-shift experiments and ChIP (chromatin immunoprecipitation), we found that the transcription factors HNF4α (hepatocyte nuclear factor 4α) and Sp1 (stimulating protein-1) were essential for hHSS promoter activity and could directly bind to regions −209/−204 and −152/−145 respectively. We also confirmed that activation and repression of hHSS transcription induced by Sp1 and HNF4α resulted from binding of these factors to these two cis-elements respectively. Overexpression of HNF4α led to a dramatic repression of the promoter activity and, in contrast, the activity was markedly elevated by overexpression of Sp1. Furthermore, overexpression of HNF4α1, one of the HNF4α isoforms, resulted in a dramatic suppression of the promoter activity. Moreover, repression of HNF4α expression by siRNA (small interfering RNA) remarkably enhanced the hHSS mRNA level. It has been reported previously that expression of HNF4α is functionally regulated by dexamethasone. To further confirm the transcriptional control of HNF4α on hHSS, we tested the effect of dexamethasone on hHSS transcription in HepG2 cells. In the present study we have demonstrated that the expression of the hHSS gene was down-regulated at the transcriptional level by dexamethasone in HepG2 cells. A deletion and decoy assay revealed that binding of HNF4α to nucleotides −209/−204 was responsible for the suppression of hHSS promoter activity by dexamethasone. Increases in the HNF4α-binding activity and expression were simultaneously observed in an electrophoretic mobility-shift assay and Western blot analysis. These results suggested that Sp1 activates hHSS basal expression, but HNF4α inhibits hHSS gene expression.

5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)

2001 ◽  
Vol 353 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Luciana E. GIONO ◽  
Cecilia L. VARONE ◽  
Eduardo T. CÁNEPA
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Putative Transcription Factor

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

scholarly journals Thyroid transcription factor 1 and Pax8 synergistically activate the promoter of the human thyroglobulin gene

2001 ◽  
Vol 27 (1) ◽  
pp. 59-67 ◽  
Author(s):  
CR Espinoza ◽  
TL Schmitt ◽  
U Loos
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Transcriptional Start Site ◽  
Promoter Sequence ◽  
Specific Protein ◽  
Site Directed Mutagenesis ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Thyroid Transcription Factor 1 ◽  
Thyroid Hormone Biosynthesis

Thyroglobulin (Tg) is an essential thyroid-specific protein, which serves as the matrix for thyroid hormone biosynthesis. To obtain new insights in the regulation of Tg gene expression, we investigated the interaction of the human Tg promoter with the thyroid-specific transcription factors TTF-1 and Pax8. A reporter gene, containing a 202 bp fragment from the human Tg 5'-flanking region including the promoter sequence and the transcriptional start site, and expression vectors containing the cDNAs for human TTF-1 and Pax8 were used in cotransfection experiments, in the non-thyroidal cell lines COS-7 and HeLa. Pax8 increased the specific transcriptional activity of the Tg promoter about threefold, whereas cotransfection with the homeodomain-containing protein TTF-1 stimulated promoter activity from six- to tenfold. The simultaneous expression of both factors stimulated the Tg promoter activity in a multiplicative manner up to 25-fold. TTF-1 binding sites could be localized precisely by lectron mobility shift assay. The two binding elements corresponded to sites A and C in the rat Tg promoter. Site-directed mutagenesis of three nucleotides in each binding element inhibited binding of TTF-1 to the two oligonucleotides. In cotransfection experiments, the mutant site C decreased TTF-1 transactivation to 26% of the wild-type, whereas an additional mutation in the site A reduced this value to almost zero, thus proving the physiological relevance of these sites. The present results demonstrate that the activity of the human Tg promoter is closely dependent on the function of TTF-1 and Pax8, opening the field for further investigations of pathological alterations of Tg gene expression.

scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

scholarly journals Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

2018 ◽  
Vol 314 (1) ◽  
pp. G14-G21 ◽  
Author(s):  
Saminathan Muthusamy ◽  
Jong Jin Jeong ◽  
Ming Cheng ◽  
Jessica A. Bonzo ◽  
Anoop Kumar ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Core Promoter ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Protein Levels ◽  
Hepatocyte Nuclear Factor 4Α

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

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