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.

Tissue-specific regulation of mouse hepatocyte nuclear factor 4 expression

1994 ◽  
Vol 14 (11) ◽  
pp. 7276-7284
Author(s):  
W Zhong ◽  
J Mirkovitch ◽  
J E Darnell
Keyword(s):  
Transcription Factor ◽  
Transgenic Mice ◽  
Transient Transfection ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Distal Enhancer ◽  
Specific Expression ◽  
Tissue Specific ◽  
Hypersensitive Sites ◽  
Hepatocyte Nuclear Factor 4

Hepatocyte nuclear factor 4 (HNF-4) is a liver-enriched transcription factor and a member of the steroid hormone receptor superfamily. HNF-4 is required for the hepatoma-specific expression of HNF-1 alpha, another liver-enriched transcription factor, suggesting the early participation of HNF-4 in development. To prepare for further study of HNF-4 in development, the tissue-specific expression of the mouse HNF-4 gene was studied by analyzing the promoter region for required DNA elements. DNase-hypersensitive sites in the gene in liver and kidney tissues were found in regions both distal and proximal to the RNA start that were absent in tissues in which HNF-4 expression did not occur. By use of reporter constructs in transient-transfection assays and with transgenic mice, a region sufficient to drive liver-specific expression of HNF-4 was identified. While an HNF-1 binding site between bp -98 and -68 played an important role in the hepatoma-specific promoter activity of HNF-4 in transient-transfection assays, it was not sufficient for the liver-specific expression of a reporter gene in transgenic mice. Distal enhancer elements indicated by the presence of DNase I-hypersensitive sites at kb -5.5 and -6.5, while not functional in transient-transfection assays, were required for the correct expression of the mouse HNF-4 gene in animals.

scholarly journals Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.

1997 ◽  
Vol 17 (10) ◽  
pp. 6002-6013 ◽  
Author(s):  
K L Wu ◽  
M Gannon ◽  
M Peshavaria ◽  
M F Offield ◽  
E Henderson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Beta Cell ◽  
Beta Cells ◽  
Cell Lines ◽  
Pancreatic Beta Cell ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Specific Expression ◽  
Islet Beta Cells ◽  
Beta Galactosidase

The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas.

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 Serum transferrin as a biomarker of hepatocyte nuclear factor 4 alpha activity and hepatocyte function in liver diseases

BMC Medicine ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Nurdan Guldiken ◽  
Josepmaria Argemi ◽  
Berivan Gurbuz ◽  
Stephen R. Atkinson ◽  
Martin Oliverius ◽  
...  
Keyword(s):  
Liver Disease ◽  
Liver Failure ◽  
Regulatory Region ◽  
Serum Transferrin ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Primary Hepatocytes ◽  
Advanced Liver Disease ◽  
Hepatocyte Nuclear Factor 4

Abstract Background Serum transferrin levels represent an independent predictor of mortality in patients with liver failure. Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of hepatocyte functions. The aim of this study was to explore whether serum transferrin reflects HNF4α activity. Methods Factors regulating transferrin expression in alcoholic hepatitis (AH) were assessed via transcriptomic/methylomic analysis as well as chromatin immunoprecipitation coupled to DNA sequencing. The findings were corroborated in primary hepatocytes. Serum and liver samples from 40 patients with advanced liver disease of multiple etiologies were also studied. Results In patients with advanced liver disease, serum transferrin levels correlated with hepatic transferrin expression (r = 0.51, p = 0.01). Immunohistochemical and biochemical tests confirmed reduced HNF4α and transferrin protein levels in individuals with cirrhosis. In AH, hepatic gene-gene correlation analysis in liver transcriptome revealed an enrichment of HNF4α signature in transferrin-correlated transcriptome while transforming growth factor beta 1 (TGFβ1), tumor necrosis factor α (TNFα), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) negatively associated with transferrin signature. A key regulatory region in transferrin promoter was hypermethylated in patients with AH. In primary hepatocytes, treatment with TGFβ1 or the HNF4α inhibitor BI6015 suppressed transferrin production, while exposure to TNFα, IL-1β, and IL-6 had no effect. The correlation between hepatic HNF4A and transferrin mRNA levels was also seen in advanced liver disease. Conclusions Serum transferrin levels constitute a prognostic and mechanistic biomarker. Consequently, they may serve as a surrogate of impaired hepatic HNF4α signaling and liver failure.

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