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 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.

Mutation in the factor VII hepatocyte nuclear factor 4α-binding site contributes to factor VII deficiency

2011 ◽  
pp. 1 ◽  
Author(s):  
Xing-Wu Zheng ◽  
Rama Kudaravalli ◽  
Theresa T. Russell ◽  
Donna M. DiMichele ◽  
Constance Gibb ◽  
...  
Keyword(s):  
Binding Site ◽  
Factor Vii ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Factor Vii Deficiency ◽  
Hepatocyte Nuclear Factor 4Α

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 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.

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 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.

scholarly journals Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.

1997 ◽  
Vol 17 (8) ◽  
pp. 4208-4219 ◽  
Author(s):  
B Viollet ◽  
A Kahn ◽  
M Raymondjean
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Hepatocyte Nuclear Factor 4 ◽  
Kinase A

Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers.

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