HNF4 Binding Site Research Articles

Differentiation-dependent activation of the human intestinal alkaline phosphatase promoter by HNF-4 in intestinal cells

The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the enterocytes, we have conducted a computer-assisted cis-element search of the proximal human ALPI promoter sequence. A putative recognition site for the transcription factor hepatocyte nuclear factor (HNF)-4 was predicted at the positions from -94 to -82 in relation to the translational start site. The ability of HNF-4alpha to stimulate the expression from the ALPI promoter was investigated in the nonintestinal Hela cell line. Cotransfection with an HNF-4alpha expression vector demonstrated a direct activation of the ALPI promoter through this -94 to -82 element. EMSA showed that HNF-4alpha from nuclear extracts of differentiated intestinal epithelial cells (Caco-2) bound with high affinity to the predicted HNF-4 binding site. A 521 bp promoter fragment containing the HNF-4 binding site demonstrated a differentiation-dependent increase in promoter activity in Caco-2 cells. The presence of the HNF-4 binding site was necessary for this increase to occur.

Binding of HNF-3α in nuclear extracts of pancreatic islet α-cell line to the novel HNF-3 site-A in the glucagon gene

The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the hepatocyte nuclear factor-3 (HNF-3, also called Foxa) family are expressed in endoderm-derived tissues such as liver and pancreatic islets and, as part of their metabolic control, are required for cell-specific activation of the glucagon gene in pancreatic islet α-cells. However, their action at the glucagon gene is poorly understood. We have shown previously that the well characterized HNF-3 binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. However, a novel HNF-3 site (called site-A) has been identified that is conserved in rat, mouse, and humans and that mediates an activation of the glucagon gene by HNF-3 proteins expressed in an heterologous cell line. In the present study, a molecular characterization using protein-DNA binding and transient transfection assays revealed that the novel HNF-3 site-A confers cell-specific promoter activity in glucagon-producing pancreatic islet α-cell lines (InR1G9, αTC2). In contrast to other HNF-3 binding sites in the glucagon promoter that bind nuclear HNF-3β, the novel HNF-3 site-A was found to bind preferentially HNF-3α in nuclear extracts of a glucagon-producing pancreatic islet α-cell line. Mice with a targeted disruption of the HNF-3α gene have been shown to develop normally but exhibit neonatal growth retardation and die postnatally prior to 4 weeks of age, exhibiting hypoglycemia associated with inappropriately low levels of circulating glucagon. The results of the present study offer a mechanism that explains the decrease in glucagon gene expression in HNF-3α-deficient mice. The novel HNF-3 site-A is located just upstream of the TATA box (between -30 and -50) suggesting a role for HNF-3 proteins in addition to direct transcriptional activation

Mutation analysis of HNF-4 binding sites in the human glucose-6-phosphatase promoter

HNF-4, a member of the nuclear receptor superfamily, binds to HNF-4 response elements (HRE), consisting of a direct repeat of the hexameric half-sites spaced by 1 nt (direct repeat 1) and activates a number of genes, which play central roles in fatty acids and glucose metabolism. Glucose-6-phosphatase (G6Pase) catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. A previous study has shown that HNF-4 binds to two DR1s in the regions A (located between -266 and -234) and B (located between -306 and -274) on the human G6Pase promoter. We found that the region B contains the one more DR1 element, composed of the two half-sites, designated half-sites a and b, the latter of which overlaps with the previously identified DR1 consisting of two half-sites, designated half-sites b and c. In this study, electrophoretic mobility shift assay (EMSA) using point mutations in each half-site a, b, or c indicated that HNF-4 binds to the combination of half-sites a and b, but not to half-sites b and c. Furthermore, mutational analysis demonstrated that, in the context of the human G6Pase promoter, the half-sites a and b, but not the half-sites b and c, are required for the stimulatory effect of HNF-4. These results suggested that the DR1 element containing the half-sites a and b is a functional HRE that mediates the induction of hG6Pase promoter activity by HNF-4.

Molecular mechanisms of hereditary persistence of α-fetoprotein (AFP) in two Japanese familiesA hepatocyte nuclear factor-1 site mutation leads to induction of the AFP gene expression in adult livers

alpha-Fetoprotein (AFP) is produced abundantly in fetal liver but is hardly detectable in adults. In this study, we investigated two unrelated Japanese families with hereditary persistence of AFP. A g-->a substitution at nucleotide -119 (-119g-->a) in the hepatocyte nuclear factor (HNF)-1 binding site of the AFP promoter was identified in both families. The activity of the wild- or variant-type human AFP promoter was evaluated by in vitro and in vivo transfection experiments. This substitution in the AFP promoter significantly stimulated its transcriptional activity in human hepatoma cells, regardless of their prior AFP production. The variant-type AFP promoter was also active in adult mouse livers in vivo. Additionally, overexpression of HNF-1alpha stimulated the activity of both the wild- and variant-type AFP promoters in hepatoma cells. HNF-1alpha expression also activated both AFP promoters even in nonhepatoma cells, and this activation was suppressed by nuclear factor (NF)-I overexpression. These results indicate that an HNF-1 binding site mutation leads to induction of the AFP gene expression in adult liver, and suggest that competition between HNF-1 and NF-I in this region is involved in transcriptional regulation of the AFP gene during hepatic development.

Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1beta in human papillary thyroid cancer cells.

We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2-7) and lower (e.g. BHP 14-9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2-7 and BHP 14-9 cells. In BHP 2-7 cells, promoter activity was dependent on an upstream sequence. In BHP 14-9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1beta (HNF-1beta) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1beta was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1beta-positive BHP 2-7 cells, but not in the HNF-1beta-negative BHP 14-9 cells. HNF-1beta bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1beta-specific antibody. Cotransfection of a HNF-1beta expression plasmid increased NNMT promoter activity significantly in both HNF-1beta-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1beta in BHP 14-9 cells increased endogenous NNMT protein levels. In summary, HNF-1beta functions as a transcription activator for NNMT gene expression in some papillary thyroid cancer cells.

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.

Regulation of hepatitis B virus core promoter by transcription factors HNF1 and HNF4 and the viral X protein.

Hepatitis B virus (HBV) core promoter contains a binding site for nuclear receptors. A natural double mutation in this binding site, which changes nucleotide (nt) 1765 from A to T and nt 1767 from G to A, selectively abolishes the binding of several nuclear receptors without affecting that of HNF4. This double mutation also creates a binding site for the transcription factor HNF1 and changes two amino acids in the overlapping X protein sequence. In this study, we have examined the roles of HNF1, HNF4, and the X protein in the regulation of the core promoter activities in Huh7 hepatoma cells. Our results indicate that HNF4 could stimulate the expression of the precore RNA and the core RNA from the core promoter of both the wild-type (WT) HBV and the double mutant, although its effect on the former was more prominent. In contrast, HNF1, which did not affect the WT core promoter, suppressed the precore RNA expression of the double mutant. Further analysis using HBV genomic constructs, with and without the ability to express the X protein, indicates that the X protein did not affect the HNF4 activity on the core promoter and affected the HNF1 activity on the core promoter of only the double mutant. Thus, our results indicate that the phenotypic differences of HBV WT and double-mutant core promoters are at least partially due to the differential activities of HNF1, HNF4, and the X protein on these two promoters.

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.

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.

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.

Functional characterization of transcription factor binding sites for HNF1-alpha, HNF3-beta (FOXA2), HNF4-alpha, Sp1 and Sp3 in the human prothrombin gene enhancer

Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.

Differential regulation of alternate UDP-glucuronosyltransferase 1A6 gene promoters by hepatic nuclear factor-1

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major UGT contributing to the glucuronidation of small phenolic compounds. The gene for rat 1A6 is expressed using two promoters, a distal promoter P1 and a proximal promoter P2. Transcripts from P2 are high in liver, gastrointestinal tract, and kidney, whereas P1 transcripts predominate in other tissues. Here we report evidence for primary control of the P2 promoter by hepatic nuclear factor 1 (HNF1). Transient transfection of a P2 reporter plasmid, p(−1354/+65) 1A6P2-luc, resulted in enhanced luciferase activity in HepG2 but not Hepa1 cells compared to cells transfected with pGL3-Basic control vector. A truncated reporter under the control of −224 to +65 exhibited comparable activity. Footprint analysis of the −224/+65 fragment revealed specific binding by rat liver nuclear protein to a region between bases −60 and −37. The binding activity was also observed with HepG2 cell but not Hepa1 cell extract. Electrophoretic mobility shift assays were consistent with the presence of HNF1 in the binding complexes. The functionality of an HNF1-binding site at −51/−37 is also supported by (1) marked decreases in the activity of P2 reporter plasmids containing a three-base substitution in the proposed HNF1 binding site and (2) the enhancement of P2 reporter activity following cotransfection of an HNF1α expression plasmid. The UGT1A6 P1 promoter lacks an HNF1 binding site in the analogous position and showed little response to HNF1 overexpression. Although these data do not strictly rule out an interaction between the P1 promoter and HNF1 bound to −51/−37 of P2, the results suggest a mechanism for the more abundant expression of P2-derived UGT1A6 transcripts in liver and other HNF1-enriched tissues.

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.

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.

The role of the HNF4α enhancer in type 2 diabetes : variants of the HNF4α enhancer are not a common cause of susceptibility to type 2 diabetes

The genetic causes of type 2 diabetes are not well understood. The disease has been linked to chromosome 20q12–q13.1 a region which harbors the transcription factor HNF4 α. Mutations in the coding region of HNF4 α cause maturity onset diabetes of the young, an autosomal dominant form of diabetes, but do not account for the linkage to this region. An enhancer element has recently been characterized 6 kb 5 ′ of the HNF4α P1 promoter containing binding sites for the transcription factors HNF1, HNF4, HNF3, and C/EBP, which are overlapped by glucocorticoid consensus sites. We hypothesized that variation in the enhancer element disrupts HNF4 α expression in the liver and increases susceptibility to type 2 diabetes. We screened for variants of the enhancer element in 39 white UK young onset diabetic subjects, giving >95% power to identify variants with minor allele frequencies of >5%. No variants of the enhancer element were found in this population. We conclude that variation in the HNF4α enhancer element is not a common cause of susceptibility to type 2 diabetes.

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.

HNF-3β Is a Critical Factor for the Expression of the Jaagsiekte Sheep Retrovirus Long Terminal Repeat in Type II Pneumocytes but Not in Clara Cells

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a sheep lung cancer that resembles human lung adenocarcinoma or bronchioloaveolar carcinoma (BAC). JSRV is the only retrovirus that shows lung tropism and induces pulmonary carcinoma. Several lines of evidence suggest that the lung tropism for JSRV is mainly determined by the viral long terminal repeats (LTR). In a previous study, we showed that HNF-3alpha and -3beta were able to transactivate the JSRV LTR when cotransfected into 3T3 cells. The JSRV LTR contains two putative HNF-3 binding sites; to investigate the contribution of each HNF-3 binding site to transcription, we generated reporter constructs with deletions or nucleotide substitutions in one or both of the putative HNF-3 binding sites. In murine MLE-15 cells (derived from type II pneumocytes), mutations within the upstream site (minus sign147 to minus sign128 bp) resulted in a 72% reduction of the LTR activity, while mutation of the downstream site had little effect. In contrast, transactivation of the JSRV LTR was greatly reduced in 3T3 cells cotransfected with an HNF-3alpha or -3beta expression plasmid when the downstream site was eliminated. Electrophoretic mobility shift assays (EMSA) revealed that nuclear extracts from MLE-15 cells, but not 3T3 cells, were able to form a retarded complex with oligonucleotides encompassing either the upstream or the downstream sites. Anti-HNF-3beta antiserum, but not anti-HNF-3alpha antiserum, supershifted both protein-DNA complexes. These results indicate that the JSRV LTR is activated by the lung-specific transcription factor HNF-3beta and that the upstream HNF-3 binding site is essential for expression in MLE-15 cells. In contrast, transactivation by HNF-3beta in 3T3 cells is mediated through the downstream HNF-3 site. On the other hand, JSRV LTR expression in a mouse lung Clara cell-derived line (mtCC1-2) did not appear to be strongly dependent on either HNF-3 binding site. These results support the notion that JSRV lung tropism is determined by the transcriptional specificity of the JSRV LTR, which is governed by interactions with lung-specific transcription factors.