scholarly journals Human hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes.

1996 ◽  
Vol 16 (3) ◽  
pp. 925-931 ◽  
Author(s):  
T Drewes ◽  
S Senkel ◽  
B Holewa ◽  
G U Ryffel
Keyword(s):  
Splice Variant ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Transactivation Potential ◽  
Genes Encoding ◽  
Hepatocyte Nuclear Factor 4 ◽  
Hnf4 Alpha

Hepatocyte nuclear factor 4 (HNF4) was first identified as a DNA binding activity in rat liver nuclear extracts. Protein purification had then led to the cDNA cloning of rat HNF4, which was found to be an orphan member of the nuclear receptor superfamily. Binding sites for this factor were identified in many tissue-specifically expressed genes, and the protein was found to be essential for early embryonic development in the mouse. We have now isolated cDNAs encoding the human homolog of the rat and mouse HNF4 splice variant HNF4 alpha 2, as well as a previously unknown splice variant of this protein, which we called HNF alpha 4. More importantly, we also cloned a novel HNF4 subtype (HNF4 gamma) derived from a different gene and showed that the genes encoding HNF 4 alpha and HNF4 gamma are located on human chromosomes 20 and 8, respectively. Northern (RNA) blot analysis revealed that HNF4 GAMMA is expressed in the kidney, pancreas, small intestine, testis, and colon but not in the liver, while HNF4 alpha RNA was found in all of these tissues. By cotransfection experiments in C2 and HeLa cells, we showed that HNF4 gamma is significantly less active than HNF4 alpha 2 and that the novel HNF4 alpha splice variant HNF4 alpha 4 has no detectable transactivation potential. Therefore, the differential expression of distinct HNF4 proteins may play a key role in the differential transcriptional regulation of HNF4-dependent genes.

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.

scholarly journals Hepatocyte nuclear factor 4 alpha P2 promoter variants associate with insulin resistance.

2011 ◽  
Vol 58 (2) ◽  
Author(s):  
Riyadh Saif-Ali ◽  
Roslan Harun ◽  
S Al-Jassabi ◽  
Wan Zurinah Wan Ngah
Keyword(s):  
Insulin Resistance ◽  
Metabolic Syndrome ◽  
Fasting Blood Glucose ◽  
Nuclear Factor ◽  
Nucleotide Polymorphisms ◽  
Hepatocyte Nuclear Factor ◽  
Coding Region ◽  
P2 Promoter ◽  
Hepatocyte Nuclear Factor 4 ◽  
Hnf4 Alpha

This study aimed to investigate the associations of hepatocyte nuclear factor 4 (HNF4) alpha single nucleotide polymorphisms (SNPs) and haplotype with insulin resistance and metabolic syndrome parameters. Nine SNPs spanning the HNF4 alpha P2 promoter (rs4810424, rs1884613 and rs1884614) and coding region (rs2144908, rs6031551, rs6031552, rs1885088, rs1028583 and rs3818247) were genotyped in 160 subjects without diabetes or metabolic syndrome. The HNF4 alpha P2 promoter SNPs rs4810424, rs1884613 and rs1884614 were associated with insulin resistance (p = 0.017; 0.037; 0.024) and body mass index (BMI) (p = 0.03; 0.035; 0.039). The intron 1D SNP rs2144908 was associated with high-density lipoprotein cholesterol (HDLc) (p = 0.020) and the intron 9 SNP rs3818247 showed association with systolic (p = 0.02) and diastolic (p = 0.034) blood pressure. HNF4 alpha common haplotype CCCGTC associated with higher insulin resistance (p = 0.022), fasting blood glucose (FBG) (p = 0.035) and lower HDLc (p = 0.001). In conclusion, subjects with HNF4 alpha P2 variants and haplotypes have been shown to have a higher insulin resistance and are therefore at a higher risk for developing type 2 diabetes mellitus.

Activity of hepatocyte nuclear factor 1α and hepatocyte nuclear factor 1β isoforms is differently affected by the inhibition of protein phosphatases 1/2A

2001 ◽  
Vol 354 (2) ◽  
pp. 301-308 ◽  
Author(s):  
Véronique CARRIÈRE ◽  
Michel LACASA ◽  
Monique ROUSSET
Keyword(s):  
Dna Binding ◽  
Binding Capacity ◽  
Protein Phosphatases ◽  
Physiological Role ◽  
Binding Activity ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Gene Encoding ◽  
Dna Binding Activity

Phosphorylation/dephosphorylation processes are known to control the activity of several transcription factors. The nutrition-dependent expression of sucrase–isomaltase and Na+/glucose co-transporter 1, two proteins implicated in the intestinal absorption of glucose, has been shown to be closely related to modifications of hepatocyte nuclear factor 1 (HNF1) activity. This study was conducted to determine whether phosphorylation/dephosphorylation processes could control HNF1 activity. We show that expression of the gene encoding sucrase–isomaltase is inhibited in the enterocytic Caco-2 clone TC7 by okadaic acid at a concentration that is known to inhibit protein phosphatases 1/2A and that does not affect cell viability. At the same concentration, phosphorylation of the HNF1α and HNF1β isoforms is greatly enhanced and their DNA-binding capacity is decreased. The phosphorylation state of HNF1β isoforms directly affects their DNA-binding capacity. In contrast, the decreased DNA-binding activity of the HNF1α isoforms, which was observed after the inhibition of protein phosphatases 1/2A, is due to a net decrease in their total cellular and nuclear amounts. Such an effect results from a decrease in both the HNF1α mRNA levels and the half-life of the protein. This is the first evidence for the implication of protein phosphatases 1/2A in the control of the activity of HNF1 isoforms. Moreover, these results emphasize a physiological role for the balance between phosphatases and kinases in the nutrition-dependent regulation of HNF1-controlled genes.

Immunocytochemical study for hepatocyte nuclear factor 4^|^alpha; (HNF4^|^alpha;) in body cavity fluids

2014 ◽  
Vol 53 (3) ◽  
pp. 176-181
Author(s):  
Mika SUGAI ◽  
Makoto NAITO ◽  
Kanae TAKAHASHI ◽  
Chikashi IKEGAME ◽  
Chiaki SAKASHITA ◽  
...  
Keyword(s):  
Body Cavity ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Immunocytochemical Study ◽  
Hepatocyte Nuclear Factor 4 ◽  
Hnf4 Alpha

scholarly journals Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay.

1986 ◽  
Vol 6 (5) ◽  
pp. 1363-1373 ◽  
Author(s):  
J F Diffley ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Assay ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Factor I ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Nuclear Factor I ◽  
Dna Fragment

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.

Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay

1986 ◽  
Vol 6 (5) ◽  
pp. 1363-1373
Author(s):  
J F Diffley ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Assay ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Factor I ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Nuclear Factor I ◽  
Dna Fragment

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.

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