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.

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 Identification of DNA binding-site preferences for nuclear factor I-A

FEBS Letters ◽  
1996 ◽  
Vol 390 (1) ◽  
pp. 44-46 ◽  
Author(s):  
Shigehiro Osada ◽  
Shoko Daimon ◽  
Tsutomu Nishihara ◽  
Masayoshi Imagawa
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Nuclear Factor ◽  
Factor I ◽  
Site Preferences ◽  
Dna Binding Site ◽  
Nuclear Factor I

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.

In vivo stimulation of a chimeric promoter by binding sites for nuclear factor I

1991 ◽  
Vol 11 (6) ◽  
pp. 2946-2951
Author(s):  
J J Knox ◽  
P J Rebstein ◽  
A Manoukian ◽  
R M Gronostajski
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Tata Box ◽  
Nuclear Factor ◽  
Chimeric Promoter ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor I (NFI) is composed of a family of site-specific DNA-binding proteins which recognize a DNA-binding site with the consensus sequence TGGC/A(N)5GCCAA. Binding sites for NFI have previously been shown to stimulate mRNA synthesis in vitro when present upstream of the TATA box of the adenovirus major late promoter (AdMLP). We have examined the effect of NFI-binding sites on transcription in vivo in transiently transfected HeLa and COS cells. An NFI-binding site isolated from the human genome activated expression from the minimal AdMLP in vivo in both the absence and presence of the simian virus 40 enhancer. A point mutation that decreased NFI binding affinity for the site in vitro reduced expression to near the basal level of the AdMLP. Several NFI-binding sites which differed in their spacer and flanking sequences were tested for their ability to activate expression in vivo. The ability of these sites to activate expression correlated with the strength of NFI binding in vitro. An NFI-binding site stimulated expression equally well when placed from 33 to 65 bp upstream of the TATA box. However, expression dropped to basal levels when the site was located from 71 to 77 bp upstream of the TATA box. These studies indicate that an NFI-binding site in this chimeric promoter activates expression in vivo only if located within a critical distance of the TATA box.

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 Identification of a Nuclear Factor-I Family Protein-binding Site in the Silencer Region of the Cartilage Matrix Protein Gene

1995 ◽  
Vol 270 (17) ◽  
pp. 10212-10221 ◽  
Author(s):  
Piroska Szabó ◽  
Jaideep Moitra ◽  
Altanchimeg Rencendorj ◽  
Gábor Rákhely ◽  
Tibor Rauch ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Matrix Protein ◽  
Protein Gene ◽  
Protein Binding Site ◽  
Nuclear Factor ◽  
Matrix Protein Gene ◽  
Factor I ◽  
Family Protein ◽  
Nuclear Factor I

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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