Hepatocyte nuclear factor-4 regulates intestinal expression of the guanylin/heat-stable toxin receptor

1999 ◽  
Vol 276 (3) ◽  
pp. G728-G736 ◽  
Author(s):  
E. Scott Swenson ◽  
Elizabeth A. Mann ◽  
M. Lynn Jump ◽  
Ralph A. Giannella
Keyword(s):  
Binding Site ◽  
Expression Vector ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Surface Epithelium ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
T84 Cells ◽  
Mobility Shift ◽  
Hepatocyte Nuclear Factor 4

We have investigated the regulation of gene transcription in the intestine using the guanylyl cyclase C (GCC) gene as a model. GCC is expressed in crypts and villi in the small intestine and in crypts and surface epithelium of the colon. DNase I footprint, electrophoretic mobility shift assay (EMSA), transient transfection assays, and mutagenesis experiments demonstrated that GCC transcription is regulated by a critical hepatocyte nuclear factor-4 (HNF-4) binding site between bp −46 and −29 and that bp −38 to −36 were essential for binding. Binding of HNF-4 to the GCC promoter was confirmed by competition EMSA and by supershift EMSA. In Caco-2 and T84 cells, which express both GCC and HNF-4, the activity of GCC promoter and/or luciferase reporter plasmids containing 128 or 1973 bp of 5′-flanking sequence was dependent on the HNF-4 binding site in the proximal promoter. In COLO-DM cells, which express neither GCC nor HNF-4, cotransfection of GCC promoter/luciferase reporter plasmids with an HNF-4 expression vector resulted in 23-fold stimulation of the GCC promoter. Mutation of the HNF-4 binding site abolished this transactivation. Transfection of COLO-DM cells with the HNF-4 expression vector stimulated transcription of the endogenous GCC gene as well. These results indicate that HNF-4 is a key regulator of GCC expression in the intestine.

scholarly journals Regulation of Cyp2a5 transcription in mouse primary hepatocytes: roles of hepatocyte nuclear factor 4 and nuclear factor I

2004 ◽  
Vol 381 (3) ◽  
pp. 887-894 ◽  
Author(s):  
Johanna ULVILA ◽  
Satu ARPIAINEN ◽  
Olavi PELKONEN ◽  
Kaoru AIDA ◽  
Tatsuya SUEYOSHI ◽  
...  
Keyword(s):  
Binding Site ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Primary Hepatocytes ◽  
Double Mutation ◽  
Factor I ◽  
Nuclear Factor I ◽  
Hepatocyte Nuclear Factor 4

The cytochrome P4502a5 (Cyp2a5) gene is expressed principally in liver and olfactory mucosa. In the present study, the transcriptional mechanisms of hepatocyte-specific expression of Cyp2a5 were studied in mouse primary hepatocytes. The Cyp2a5 5′-flanking region −3033 to +10 was cloned in front of a luciferase reporter gene and transfected into hepatocytes. Deletion analysis revealed two major activating promoter regions localized at proximal 271 bp and at a more distal area from −3033 to −2014 bp. The proximal activation region was characterized further by DNase I footprinting, and a single clear footprint was detected in the studied area centred over a sequence similar to the NF-I (nuclear factor I)-binding site. The binding of NF-I was confirmed using an EMSA (electrophoretic mobility-shift assay). A putative HNF-4 (hepatocyte nuclear factor 4)-binding site was localized at the proximal promoter by computer analysis of the sequence, and HNF-4α was shown to interact with the site using an EMSA. The functional significance of HNF-4 and NF-I binding to the Cyp2a5 promoter was evaluated by site-directed mutagenesis of the binding motifs in reporter constructs. Both mutations strongly decreased transcriptional activation by the Cyp2a5 promoter in primary hepatocytes, and double mutation almost completely abolished transcriptional activity. Also, the functionality of the distal activation region was found to be dependent on the intact HNF-4 and NF-I sites at the proximal promoter. In conclusion, these results indicate that HNF-4 and NF-I play major roles in the constitutive regulation of hepatic expression of Cyp2a5.

scholarly journals Severe Factor VII Deficiency Due to a Mutation Disrupting a Hepatocyte Nuclear Factor 4 Binding Site in the Factor VII Promoter

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 176-182 ◽  
Author(s):  
Arnaldo A. Arbini ◽  
Eleanor S. Pollak ◽  
Janet K. Bayleran ◽  
Katherine A. High ◽  
Kenneth A. Bauer
Keyword(s):  
Binding Site ◽  
Factor Vii ◽  
Severe Bleeding ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Wild Type ◽  
Mobility Shift ◽  
Factor Vii Deficiency ◽  
Hepatocyte Nuclear Factor 4 ◽  
Mutant Promoter

AbstractAlthough small deletions, splice site abnormalities, missense, and nonsense mutations have been identified in patients with factor VII deficiency, there have been no reports of mutations in the factor VII promoter. We investigated a girl with factor VII levels that were less than 1% of normal in association with a severe bleeding diathesis. The patient is homozygous for a T to G transversion that occurs 61 bp before the translation start site. This nucleotide is in a sequence that is an hepatocyte nuclear factor 4 (HNF-4) binding site within the factor VII promoter (ACTTTG Æ → ACGTTG). Using gel mobility shift assays, we show that the mutation disrupts the binding of HNF-4 to its cognate binding site. In growth hormone reporter gene assays, the activity of a plasmid containing the mutant promoter was 6.7% of the wild-type promoter plasmid. Although HNF-4 was able to transactivate the wild-type factor VII promoter 5.4-fold in HeLa cells, no transactivation could be shown with the mutant promoter. These findings indicate that HNF-4 exerts a major positive regulatory effect on factor VII expression and provides in vivo evidence that binding of this transcription factor is critical for normal factor VII expression.

scholarly journals Severe Factor VII Deficiency Due to a Mutation Disrupting a Hepatocyte Nuclear Factor 4 Binding Site in the Factor VII Promoter

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 176-182 ◽  
Author(s):  
Arnaldo A. Arbini ◽  
Eleanor S. Pollak ◽  
Janet K. Bayleran ◽  
Katherine A. High ◽  
Kenneth A. Bauer
Keyword(s):  
Binding Site ◽  
Factor Vii ◽  
Severe Bleeding ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Wild Type ◽  
Mobility Shift ◽  
Factor Vii Deficiency ◽  
Hepatocyte Nuclear Factor 4 ◽  
Mutant Promoter

Although small deletions, splice site abnormalities, missense, and nonsense mutations have been identified in patients with factor VII deficiency, there have been no reports of mutations in the factor VII promoter. We investigated a girl with factor VII levels that were less than 1% of normal in association with a severe bleeding diathesis. The patient is homozygous for a T to G transversion that occurs 61 bp before the translation start site. This nucleotide is in a sequence that is an hepatocyte nuclear factor 4 (HNF-4) binding site within the factor VII promoter (ACTTTG Æ → ACGTTG). Using gel mobility shift assays, we show that the mutation disrupts the binding of HNF-4 to its cognate binding site. In growth hormone reporter gene assays, the activity of a plasmid containing the mutant promoter was 6.7% of the wild-type promoter plasmid. Although HNF-4 was able to transactivate the wild-type factor VII promoter 5.4-fold in HeLa cells, no transactivation could be shown with the mutant promoter. These findings indicate that HNF-4 exerts a major positive regulatory effect on factor VII expression and provides in vivo evidence that binding of this transcription factor is critical for normal factor VII expression.

scholarly journals Hemophilia B Leyden: substitution of thymine for guanine at position - 21 results in a disruption of a hepatocyte nuclear factor 4 binding site in the factor IX promoter

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 151-158 ◽  
Author(s):  
MJ Reijnen ◽  
K Peerlinck ◽  
D Maasdam ◽  
RM Bertina ◽  
PH Reitsma
Keyword(s):  
Androgen Receptor ◽  
Binding Site ◽  
Promoter Region ◽  
Factor Ix ◽  
Hemophilia B ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Hepatocyte Nuclear Factor 4 ◽  
Responsive Element

Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by an altered developmental expression of blood coagulation factor IX. This form of hemophilia B has been found to be associated with a variety of single point mutations in the factor IX promoter region. We now describe a novel point mutation, T-->G at position -21, in two related patients with the hemophilia B Leyden phenotype. This mutation lies within the factor IX promoter region (-40 to -9) that contains overlapping binding sites for hepatocyte nuclear factor 4 (HNF-4) and androgen receptor. Transient transfection assays in HepG2 cells show that the -21 mutation causes a significant reduction in factor IX promoter activity. Gel mobility shift assays and transient cotransfection experiments revealed that the HNF-4-binding site but not the androgen-responsive element is disrupted by the -21 mutation. A comparison of the -21 mutation with the previously described -20 T-->A mutation (associated with the hemophilia B Leyden phenotype) and -26 G-->C mutation (associated with severe hemophilia B throughout life) was made. It shows that the -21 mutation reduced HNF-4 binding and transactivation to a similar level as the -20 mutation, whereas the -26 mutation completely abolished HNF-4 binding and transactivation. Mobility shift experiments indicate that there was no significant difference in binding affinity of recombinant androgen receptor protein for oligonucleotides containing wild-type and -21 or - 20 mutated DNA. The binding affinity for the oligonucleotide containing the -26 mutation was twofold lower. The results indicate that the disruption of the HNF-4-binding site by the -21 T-->G mutation is the cause of the bleeding disorder in these two patients. This study adds further support for the notion that the recovery from hemophilia at puberty may not only be related to an intact androgen-responsive element but also to the degree of disruption of the HNF-4-binding site.

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.

scholarly journals Involvement of Hepatocyte Nuclear Factor-4 in the Expression of the Growth Hormone Receptor 1A Messenger Ribonucleic Acid in Bovine Liver

2001 ◽  
Vol 15 (6) ◽  
pp. 1023-1034 ◽  
Author(s):  
Honglin Jiang ◽  
Matthew C. Lucy
Keyword(s):  
Binding Site ◽  
Cell Lines ◽  
Regulatory Region ◽  
Bovine Liver ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Specific Expression ◽  
Ghr Gene ◽  
Hepatocyte Nuclear Factor 4 ◽  
Flanking Region

Abstract The GH receptor 1A mRNA (GHR 1A mRNA) is one of the major GHR mRNA variants that differ in the 5′-untranslated region. The GHR 1A mRNA is unique because it is exclusively expressed in liver. The objective of the present study was to understand the mechanism for the liver-specific expression of the GHR 1A mRNA in the bovine. Twenty-six kilobases of 5′-flanking region of the bovine GHR gene was cloned and sequenced. The first exon (exon 1A) that corresponded to the 5′-untranslated region of the GHR 1A mRNA was 15,250 bp upstream from exon 2 in the GHR gene. The major transcription start site for the GHR 1A mRNA was 19 bp downstream from a putative TATA box. Transient transfection analyses of the 5′-flanking region of exon 1A in liver cell lines vs. nonliver cell lines did not reveal a positively regulatory region responsible for the liver-specific expression of the GHR 1A mRNA perhaps because the liver cell lines do not recapitulate the in vivo hepatic environment. A putative regulatory region was then found by deoxyribonuclease I footprinting analyses of the proximal 5′-flanking region of exon 1A with nuclear extracts from bovine liver tissue. This regulatory region contained a putative binding site for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4). Binding of HNF-4 in bovine liver to this putative HNF-4 binding site was confirmed by electrophoretic mobility shift assays. Overexpression of HNF-4 enhanced the transcriptional activity of the 5′-proximal region of exon 1A in various cell lines. Mutation of the HNF-4 binding site abolished the transactivation. In addition, the HNF-4 mRNA was found to be primarily expressed in liver and absent in most nonhepatic tissues in the bovine. Collectively, these observations suggest that the liver-enriched transcription factor HNF-4 plays a role in the expression of GHR 1A mRNA in bovine liver.

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.

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