HNF1 Binding Research Articles

Expression of the α7 Isoform of Hepatocyte Nuclear Factor (HNF) 4 Is Activated by HNF6/OC-2 and HNF1 and Repressed by HNF4α1 in the Liver

The hepatocyte nuclear factor (HNF) 4alpha gene possesses two promoters, proximal P1 and distal P2, whose use results in HNF4alpha1 and HNF4alpha7 transcripts, respectively. Both isoforms are expressed in the embryonic liver, whereas HNF4alpha1 is almost exclusively in the adult liver. A 516-bp fragment, encompassing a DNase I-hypersensitive site associated with P2 activity that is still retained in adult liver, contains functional HNF1 and HNF6 binding sites and confers full promoter activity in transient transfections. We demonstrate a critical role of the Onecut factors in P2 regulation using site-directed mutagenesis and embryos doubly deficient for HNF6 and OC-2 that show reduced hepatic HNF4alpha7 transcript levels. Transient transgenesis showed that a 4-kb promoter region is sufficient to drive expression of a reporter gene in the stomach, intestine, and pancreas, but not the liver, for which additional activating sequences may be required. Quantitative PCR analysis revealed that throughout liver development HNF4alpha7 transcripts are lower than those of HNF4alpha1. HNF4alpha1 represses P2 activity in transfection assays and as deduced from an increase in P2-derived transcript levels in recombinant mice in which HNF4alpha1 has been deleted and replaced by HNF4alpha7. We conclude that although HNF6/OC-2 and perhaps HNF1 activate the P2 promoter in the embryo, increasing HNF4alpha1 expression throughout development causes a switch to essentially exclusive P1 promoter activity in the adult liver.

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4.

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.

Integrase-specific enhancement and suppression of retroviral DNA integration by compacted chromatin structure in vitro.

Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1. In contrast, integration by avian sarcoma virus (ASV) integrase was more efficient after compaction by either histone H1 or a high salt concentration, suggesting that the compacted structure enhances this reaction. Furthermore, although site-specific binding of transcription factors HNF3 and GATA4 blocked ASV DNA integration in extended nucleosome arrays, local opening of H1-compacted chromatin by HNF3 had no detectable effect on integration, underscoring the preference of ASV for compacted chromatin. Our results indicate that chromatin structure affects integration site selection of the HIV-1 and ASV integrases in opposite ways. These distinct properties of integrases may also affect target site selection in vivo, resulting in an important bias against or in favor of integration into actively transcribed host DNA.

Analysis of gene structures and promoter activities of the chipmunk alpha(1)-antitrypsin-like genes.

The chipmunk hibernation-specific protein HP-55 is a component of a 140-kDa complex whose levels are drastically decreased in the blood during hibernation. It is highly homologous to alpha(1)-antitrypsin (AT). In the chipmunk, several alpha(1)-AT-like genes in addition to HP-55 (or CM55-ML) are expressed in the liver and have distinct patterns of regulation during hibernation: in hibernating chipmunks, the level of CM55-ML gene expression is greatly reduced, that of the CM55-MS gene is slightly increased, and the expression of the CM55-MM gene is hardly affected. As a first step towards understanding the hibernation-associated gene regulation of these chipmunk alpha(1)-AT-like genes, we isolated genomic clones for the CM55-ML, CM55-MM, and CM55-MS genes, and analyzed their promoter activities. These alpha(1)-AT-like genes are composed of five exons, and show a similar gene structure to that of the human alpha(1)-AT gene, suggesting that they were generated by the duplication of an ancestral alpha(1)-AT gene. Transient transfection studies using HepG2 and COS-7 cells revealed that for all three alpha(1)-AT-like genes, approximately 150-bp 5' flanking sequences were sufficient for the liver-specific promoter activity, and that the binding of HNF-1 to the promoter region could transactivate transcription. In addition, analysis of the activity of chimeric promoters composed of CM55-ML and CM55-MS gene sequences indicated that the lack of a TATA box-like sequence in the CM55-MS gene is responsible for its weak promoter activity.

Factors influencing therapeutic efficacy and the host immune response to helper‐dependent adenoviral gene therapy in hemophilia A mice

Adenoviral-based methods of gene therapy have been ineffective at providing sustained factor (F)VIII expression in outbred populations of large animal hemophilic models primarily due to the immunogenicity of these vectors. Improvements have been made in vector design leading to the development of the helper-dependent adenoviral (HD) system. Unfortunately, it remains unclear whether these modifications are sufficient to circumvent the induction of inhibitor formation associated with adenoviral gene transfer. To develop an HD vector capable of mediating sustained FVIII expression and to determine the variables that influence inhibitor development. HD vectors were constructed encoding the canine FVIII B-domain deleted transgene under the control of either the cytomegalovirus (CMV) promoter or a tissue-restricted hybrid element consisting of five HNF-1 binding sites, located upstream of the human FVIII proximal promoter. Inbred and outbred populations of hemophilic mice were treated, and monitored for vector-induced toxicity, therapeutic efficacy, and inhibitor formation. When HD vectors utilizing the CMV promoter were administered, all hemophilic mice developed high levels of FVIII inhibitors. In contrast, vectors under the control of the HNF/FVIII element were capable of achieving sustained elevations of FVIII for over 6 months. Strain-specific differences were also observed, with outbred animals showing a greater propensity towards inhibitor development in response to treatment. HD vectors can be used to provide long-term FVIII expression in hemophilic animals, but treatment outcome and the induction of inhibitors is dependent on a number of variables including the transgene promoter, the vector dose, and the genetic background of the host.

In vivo regulation of murine CYP7A1 by HNF-6: a novel mechanism for diminished CYP7A1 expression in biliary obstruction.

Disruption of the enterohepatic bile acid circulation during biliary tract obstruction leads to profound perturbation of the cholesterol and bile acid metabolic pathways. Several families of nuclear receptor proteins have been shown to modulate this critical process by regulating hepatic cholesterol catabolism and bile acid synthesis through the transcriptional control of cholesterol 7-alpha hydroxylase (CYP7A1). Hepatocyte nuclear factor (HNF) 6 (also known as OC-1) is a member of the ONECUT family of transcription factors that activate numerous hepatic target genes essential to liver function. We have previously shown that hepatic expression of mouse HNF-6 messenger RNA (mRNA) and protein significantly decrease following bile duct ligation. Because CYP7A1 contains potential HNF-6 binding sites in its promoter region, we tested the hypothesis that HNF-6 transcriptionally regulates CYP7A1. Following bile duct ligation, we demonstrated that diminished HNF-6 mRNA levels correlate with a reduction in CYP7A1 mRNA expression. Increasing hepatic levels of HNF-6 either by infection with recombinant adenovirus vector expressing HNF-6 cDNA by growth hormone treatment leads to an induction of CYP7A1 mRNA. To directly evaluate if HNF-6 is a transcriptional activator for CYP7A1, we used deletional and mutational analyses of CYP7A1 promoter sequences and defined sequences -206/-194 to be critical for CYP7A1 transcriptional stimulation by HNF-6 in cotransfection assays. In conclusion, the HNF-6 protein is a component of the complex network of hepatic transcription factors that regulates the expression of hepatic genes essential for bile acid homeostasis and cholesterol/lipid metabolism in normal and pathological conditions.

Regulation of apolipoprotein M gene expression by MODY3 gene hepatocyte nuclear factor-1alpha: haploinsufficiency is associated with reduced serum apolipoprotein M levels.

Hepatocyte nuclear factor-1a (HNF-1alpha) is a transcription factor that plays an important role in regulation of gene expression in pancreatic beta-cells, intestine, kidney, and liver. Heterozygous mutations in the HNF-1alpha gene are responsible for maturity-onset diabetes of the young (MODY3), which is characterized by pancreatic beta-cell-deficient insulin secretion. HNF-1alpha is a major transcriptional regulator of many genes expressed in the liver. However, no liver defect has been identified in individuals with HNF-1alpha mutations. In this study, we show that Hnf-1alpha is a potent transcriptional activator of the gene encoding apolipoprotein M (apoM), a lipoprotein that is associated with the HDL particle. Mutant Hnf-1alpha(-/-) mice completely lack expression of apoM in the liver and the kidney. Serum apoM levels in Hnf-1alpha(+/-) mice are reduced approximately 50% compared with wild-type animals and are absent in the HDL and HDLc fractions of Hnf-1alpha(-/-). We analyzed the apoM promoter and identified a conserved HNF-1 binding site. We show that Hnf-1alpha is a potent activator of the apoM promoter, that a specific mutation in the HNF-1 binding site abolished transcriptional activation of the apoM gene, and that Hnf-1alpha protein can bind to the Hnf-1 binding site of the apoM promoter in vitro. To investigate whether patients with mutations in HNF-1alpha mutations (MODY3) have reduced serum apoM levels, we measured apoM levels in the serum of nine HNF-1alpha/MODY3 patients, nine normal matched control subjects (HNF-1alpha(+/+)), and nine HNF-4alpha/MODY1 subjects. Serum levels of apoM were decreased in HNF-1alpha/MODY3 subjects when compared with control subjects (P < 0.02) as well as with HNF-4alpha/MODY1 subjects, indicating that HNF-1alpha haploinsufficiency rather than hyperglycemia is the primary cause of decreased serum apoM protein concentrations. This study demonstrates that HNF-1alpha is required for apoM expression in vivo and that heterozygous HNF-1alpha mutations lead to an HNF-1alpha-dependent impairment of apoM expression. ApoM levels may be a useful serum marker for the identification of MODY3 patients.

Characterization of six base pair deletion in the putative HNF1-binding site of human PXR promoter.

Pregnane X receptor (PXR) regulates transcription of drug metabolism genes such as CYP3A4 and MDR1. Several species of PXR transcripts have been reported, including hPAR-2 with an extended amino-terminus. Database search identified a 6-bp deletion at the putative HNF1 binding element on the proximal region flanking to the hPAR-2 transcription start site. Aspirin-induced asthma (AIA) is a typical drug-induced phenotype due to aspirin or nonsteroidal antiinflammatory drugs, and these drugs are metabolized by CYP2C9 and UGT1A6, which are regulated by PXR. We examined a possible association between the 6-bp deletion variant and AIA; 129 AIA patients and 117 controls were genotyped, and no allelic association was observed. Characterization of the hPAR-2 promoter revealed that the proximal region of 1.5-kb from the transcription start site conferred a promoter activity and that the 6-bp deletion diminished the activity. These results suggest that the putative HNF1 binding element is essential for the transcriptional activity of hPAR-2 and also, that substantial numbers of Japanese are in a deficient state. Because of the biological significance of the 6-bp deletion of PXR, the variant might potentially associate with as yet unknown phenotype.

Hepatocyte nuclear factor HNF-6 regulates hepatic cholesterol 7 alpha-hydroxylase (CYP7A1) during liver injury from biliary obstruction

Introduction: The liver enriched transcription factor HNF-6 (OC-1) is expressed in the embryonic and mature hepatocytes and bile duct epithelia, and has potential DNA binding sites for numerous hepatic target genes involved in critical liver function. The relevance of HNF-6 in liver development and function is illustrated in the HNF-6 knock out mice where the extra and intrahepatic bile ducts are abnormally differentiated and animals developed non obstructive cholestatic injury. Hypothesis: HNF-6 regulates in vivo hepatic genes involved in bile acid metabolism. Methods: CD1 mice underwent common bile duct ligation (BDL) (n = 4) or sham operation (SH) (n = 2) for 24 hours and the effects of biliary obstruction on the expression of hepatic HNF-6 and its in vivo target genes involved in bile acid transport and synthesis are examined. Whole liver total RNA was extracted for RNAse protection analyses. Data were collected on the Phosphoimager and analyzed by the ImageQuant program. Differences in mRNA signals between groups were evaluated by the student t test. Results: A 4 fold reduction in HNF-6 mRNA expression in BDL samples relative to SH samples (p = 0.005) is associated with a 3 fold (p = 0.001) and a 10 fold (p = 0.005) downregulation in the sodium dependent taurocholate bile acid transporter NTCP and the bile acid synthesis enzyme Cholesterol 7α hydroxylase (CYP7A1) respectively, but a 7 fold upregulation in the apical bile acid transporter protein (ABAT). Overexpression of HNF-6 mRNA and protein in liver following 48 hours of recombinant adenovirus expressing HNF-6 cDNA (AdH6 n = 3) or control LacZ (n = 2) by tail vein injection showed a 4 fold upregulation of Cyp7A1 (p = 0.03) but no effect on NTCP, or ASBT expression in AdH6 infected liver. Electromobility shift assay using liver nuclear protein extracts, HNF-6 antibody and oligonucleotide of the CYP7A1 promoter bearing the DNA consensus sequences for HNF-6 binding confirmed that the mouse Cyp7A1 promoter at nt -1442/–1429 contains a binding site for HNF-6. Conclusions: CYP7A1 is a likely in vivo target gene for HNF-6. The downregulation of HNF-6 transcription factor expression in association with a reduction in Cyp7A1 during biliary obstruction is a likely protective mechanism to further limit the production and therefore the excess of intracellular toxic bile acids accumulated during cholestasis.

The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes.

Androgens and mesenchymal factors are essential extracellular signals for the development as well as the functional activity of the prostate epithelium. Little is known of the intraepithelial determinants that are involved in prostatic differentiation. Here we found that hepatocyte nuclear factor-3 alpha (HNF-3 alpha), an endoderm developmental factor, is essential for androgen receptor (AR)-mediated prostatic gene activation. Two HNF-3 cis-regulatory elements were identified in the rat probasin (PB) gene promoter, each immediately adjacent to an androgen response element. Remarkably, similar organization of HNF-3 and AR binding sites was observed in the prostate-specific antigen (PSA) gene core enhancer, suggesting a common functional mechanism. Mutations that disrupt these HNF-3 motifs significantly abolished the maximal androgen induction of PB and PSA activities. Overexpressing a mutant HNF-3 alpha deleted in the C-terminal region inhibited the androgen-induced promoter activity in LNCaP cells where endogenous HNF-3 alpha is expressed. Chromatin immunoprecipitation revealed in vivo that the occupancy of HNF-3 alpha on PSA enhancer can occur in an androgen-depleted condition, and before the recruitment of ligand-bound AR. A physical interaction of HNF-3 alpha and AR was detected through immunoprecipitation and confirmed by glutathione-S-transferase pull-down. This interaction is directly mediated through the DNA-binding domain/hinge region of AR and the forkhead domain of HNF-3 alpha. In addition, strong HNF-3 alpha expression, but not HNF-3 beta or HNF-3 gamma, is detected in both human and mouse prostatic epithelial cells where markers (PSA and PB) of differentiation are expressed. Taken together, these data support a model in which regulatory cues from the cell lineage and the extracellular environment coordinately establish the prostatic differentiated response.

Distinct Gene Expression Programs Function in Progenitor and Mature Islet Cells

Homeodomain transcription factor Nkx2.2 is required for the final differentiation of the beta-cells in the pancreas and for the production of insulin. Nkx2.2 is expressed in islet cell precursors during pancreatic development and persists in a subset of mature islet cells including all beta-cells. To understand the mechanisms regulating the expression of Nkx2.2 in these different cell populations, we outlined the structure of the mouse nkx2.2 gene and identified regions that direct cell type-specific expression. The nkx2.2 gene has two noncoding alternative first exons (exons 1a and 1b). In transgenic mice, sequences upstream from exon 1a directed expression predominantly in mature islet cells. Within this exon 1a promoter, cooperative interactions between HNF3 and basic helix-loop-helix factors neurogenin-3 or NeuroD1 binding to adjacent sites played key roles in its islet cell-specific expression. In contrast, sequences upstream from exon 1b restricted expression specifically to islet cell precursors. These studies reveal distinct mechanisms for directing the expression of a key differentiation factor in precursors versus mature islet cells.

Increased expression of hepatocyte nuclear factor 3β (HNF3β) in hepatocellular carcinoma is associated with repression of the human organic anion transporting polypeptide 8 (OATP8) gene

Organic anion transporting poly~ptides (OATPs) mediate the uptake ofxenobiotics, peptides and organic anions across the basolateral (sinusoidal) hepatocyte membrane. OATP-C (SLC21A6) and OATP8 (gLC21A8) are highly expressed in human liver It is unknown, however, whether expression levels are altered in hepatocellular carcinoma (HCC). The aim of this study, therefore, was to quantify the expression of OATP-C and OATP8 in eight surgically resected HCCs and to analyze the mechanism of OATP regulation in HCC. Using real*time PCR, OATP8 expression was thund to be decreased 5-50Dld in 6 out of 8 HCCs compared to surrounding non-minor liver tissue from the same patients Expression of the liver-enriched transcription factor hepatocyte nuclear factor 313 (HNF3~) was 2-50fold higher in these 6 HCCs than in non-tumor liver tissue. In one HCC, expression of HNF3[8 was decreased compared to non-tumor liver tissue and this was associated wnh increased expression of OATP8. In cnutrast to OATP8, expression of OATP-C was not significantly different in mnmr compared to non-tumor liver tissue. Sequence analysts of the 5'-flanking sequence of the OATP-C and OATP8 genes revealed consensus binding motifs for HNF31~ in both geoe promoters. Reporter gene luciterase assays in transt?cted Huh7 cells showed 70% inhibition of OATP8 promoter activity in the presence of cotransfected HNF3[3 Transfection of deletional OATP8 promoter constructs indicated that repression was mediated by an HNF3~ motif located at nt -23 to -39 t~lative to the transcription initiation site. Binding of HNF3[3 to this motif was cc, nfirmed in electrophomtic mobility Shit~ assays (EMSA). The OATP.C gene promoter was not inhibited by HNF3[3 in reporter gene assays, even though HNF3~ ,,','as shown by EMSA to bind to a proximal motif. In conclusion, HNF3~ represses tratkscription of the OATP8 but not the OATP-C gene Increased HNF3]?, expression in HCC explains reduced expression ot OATP8. On the basis of the substrate specificities of OATP-C and OATP8, these findings could permit the development of novel diagnostic and therapeutic strategies for the managemem of HCC.

Nitric oxide downregulates factor VII gene by inhibiting the binding of SP1 and HNF-4

Nitric oxide downregulates factor VII gene by inhibiting the binding of SP1 and HNF-4

Hepatocyte Nuclear Factor-4 Is a Novel Downstream Target of Insulin via FKHR as a Signal-regulated Transcriptional Inhibitor

Previous studies have shown that FKHR, a member of the forkhead family of transcription factors, acts as a DNA binding-independent cofactor of nuclear receptors, including estrogen, retinoid, and thyroid hormone receptors, in addition to the original function as a DNA binding transcription factor that redistributes from the nucleus to the cytoplasm by insulin-induced phosphorylation. Here, we demonstrated the physical interaction of FKHR with hepatocyte nuclear factor (HNF)-4, a member of steroid/thyroid nuclear receptor superfamily, and the repression of HNF-4 transactivation by FKHR. FKHR interacted with the DNA binding domain of HNF-4 and inhibited HNF-4 binding to the cognate DNA. Furthermore, the binding affinity of HNF-4 with phosphorylated FKHR significantly decreased in comparison to that with unphosphorylated FKHR. Therefore, a phosphorylation of FKHR by insulin followed by its dissociation from HNF-4 and the redistribution of FKHR from the nucleus to the cytoplasm would expect to induce the transcriptional activation of HNF-4 by facilitating to the access of HNF-4 to its DNA element. Indeed, most intriguingly, insulin stimulation reversed the repression of HNF-4 transcriptional activity by phosphorylation-sensitive (wild-type) FKHR, but not by phosphorylation-deficient FKHR. These results suggest that insulin regulates the transcriptional activity of HNF-4 via FKHR as a signal-regulated transcriptional inhibitor.

In silico searching of human and mouse genome data identifies known and unknown HNF1 binding sites upstream of β-cell genes

HNF1-α is a transcription factor present in β-cells. Mutations in the HNF1-α gene cause maturity-onset diabetes of the young (MODY), but the exact mechanism is not known. Several studies have highlighted genes down-regulated in β-cells lacking this gene, but it is not clear if these are directly regulated by HNF1-α. To better understand this, we used human and mouse genome data to examine 29 genes expressed in the β-cell. Using an in silico approach (with software available at www.BindGene.org) we examined 2 kb upstream of each gene for possible HNF1 binding sequences. In five genes we also examined 100 kb upstream of each gene, but only the portions strongly conserved between humans and mice. We identified nine putative HNF1 binding sites upstream of seven genes ( p<0.1 and good alignment between species or p<0.05). Six of these nine sites had some experimental corroboratory evidence and included the recently identified sites 6 and 45 kb upstream of HNF4-α. Three novel sites were identified. These were 92 bp upstream of SLC3A1, 52 bp upstream of PCBD (DCOH), and 42202 bp upstream of TCF2(HNF1-β). In conclusion, our computer search identified some known HNF1 sites, and suggested three novel sites indicating these genes are very likely to be directly activated by HNF1. This should help in designing experiments to discover the mechanisms of β-cell dysfunction due to HNF1 disruption.

In Vivo Functional Characterization of the Aldolase B Gene Enhancer

A 400-bp intronic enhancer fragment in conjunction with the proximal promoter of the aldolase B gene provided correct tissue-specific expression in transgenic mice together with hormonal regulation in the liver. We investigated in vivo and in cultured cells the contribution of the intronic regulatory sequences and their interaction with the promoter elements in controlling aldolase B gene expression. Transgene activity was completely abolished by disruption of the two hepatocyte nuclear factor 1 (HNF1) binding sites in the enhancer, whereas mutation of one HNF1 site had no effect in the liver but strongly decreased activity in the kidney. Our data show that the HNF1 binding site(s) in the enhancer were key regulators of aldolase B transgene expression both in the liver and kidney. Deletion of the CCAAT/enhancer-binding protein site in the promoter completely abolished the enhancer function in HepG2 cells. These results suggest that expression of the aldolase B gene in the liver requires cooperative interactions between CCAAT/enhancer-binding protein and HNF1. Deletion of the HNF4 binding site in the enhancer suppressed expression in both liver and kidney in half of the transgenic lines, suggesting that this element might play a role in chromatin opening at the insertion site. We firmly establish that the endogenous aldolase B gene's first response to glucagon or cyclic AMP exposure was a transient increase in the expression in the liver, followed by a secondary decline in the transcription, as previously reported. This response was reproduced by all transgenes studied, indicating that neither HNF1 nor HNF4 binding sites in the enhancer were involved in this biphasic cyclic AMP response.

Activation of glucokinase gene expression by hepatic nuclear factor 4alpha in primary hepatocytes.

Glucokinase (GK) is a key enzyme for glucose utilization in liver and shows a higher expression in the perivenous zone. In primary rat hepatocytes, the GK gene expression was activated by HNF (hepatic nuclear factor)-4alpha via the sequence -52/-39 of the GK promoter. Venous pO2 enhanced HNF-4 levels and HNF-4 binding to the GK-HNF-4 element. Thus, HNF-4alpha could play the role of a regulator for zonated GK expression.

Structure, upstream promoter region, and functional domains of a mouse and human Mix paired-like homeobox gene

Mix/Bix proteins represent a vertebrate subgroup of paired-like homeodomain proteins which are known to function around the time of gastrulation. Here we report the structures of the genomic and upstream promoter regions of a mouse and human Mix-like gene. Both genes map to syntenic regions of chromosome 1 and contain two coding exons, with the paired-type homeodomain split between the exons within helix 3. Differentiating mouse embryonic stem cells transcribe a messenger RNA of ∼2.6 kb. The first exon encodes the translation initiation codon and a 5′ untranslated region of ∼90 bp. Sequence analysis of the 960 bp upstream of the transcription start site of the mouse Mix gene revealed the presence of a putative initiator region and TATA box as well as potential Smad, FoxH1/FAST, T-box, COUP-TF, C/EBP, GATA, HNF3 binding sites and retinoic acid response elements. A number of these sites are conserved in the human Mix promoter. We find that most paired-related homeodomain proteins, including mouse and human Mix, contain a proline-rich region within their amino termini which may interact with other proteins. Mouse and human Mix proteins contain highly conserved carboxy-terminal polar/acidic regions with the potential to form an amphipathic helix and the ability to activate transcription in yeast. Mouse Mix expressed in COS cells or in vitro binds a DNA consensus sequence identified previously for paired class homeodomain proteins. These studies suggest that a number of features of paired-like protein structure and function are conserved across diverse species and provide a useful framework for studying the function and regulation of the mouse Mix gene.

TGF-beta down regulates factor VII gene promoter by inhibiting the binding of HNF-4

TGF-beta down regulates factor VII gene promoter by inhibiting the binding of HNF-4

Two Initiator-like Elements Are Required for the Combined Activation of the Human Apolipoprotein C-III Promoter by Upstream Stimulatory Factor and Hepatic Nuclear Factor-4

Human apoC-III (-890/+24) promoter activity is strongly activated by hepatic nuclear factor (HNF)-4 through its binding to the proximal (-87/-72) element B. This site overlaps the binding site for an activity that we identified as the ubiquitously expressed upstream stimulatory factor (USF) (Ribeiro, A., Pastier, D., Kardassis, D., Chambaz, J., and Cardot, P. (1999) J. Biol. Chem. 274, 1216-1225). In the present study, we characterized the relationship between USF and HNF-4 in the activation of human apoC-III transcription. Although USF and HNF-4 binding to element B is mutually exclusive, co-transfection experiments in HepG2 cells surprisingly showed a combined effect of USF and HNF-4 in the transactivation of the (-890/+24) apoC-III promoter. This effect only requires the proximal region (-99/+24) of the apoC-III promoter and depends neither on USF binding to its cognate site in element B nor on a USF-dependent facilitation of HNF-4 binding to its site. By contrast, we found by electrophoretic mobility shift assay and footprinting analysis two USF low affinity binding sites, located within the proximal promoter at positions -58/-31 (element II) and -19/-4 (element I), which are homologous to initiator-like element sequence. Co-transfection experiments in HepG2 cells show that a mutation in element II reduces 2-fold the USF transactivation effect on the proximal promoter of apoC-III and that a mutation in element I inhibits the combined effect of USF and HNF-4. In conclusion, these initiator-like elements are directly involved in the transactivation of the apoC-III promoter by USF and are necessary to the combined effect between USF and HNF-4 for the apoC-III transcription.