HNF-1 Alpha Research Articles

Transcriptional Regulation of the Phosphoenolpyruvate Carboxykinase Gene by Cooperation Between Hepatic Nuclear Factors

To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-1c1c7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the alpha-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1 alpha (HNF-1 alpha) and HNF-4 and the late-expressed factor C/EBP alpha, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1 alpha but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1 alpha resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.

Tissue-specific regulation of mouse hepatocyte nuclear factor 4 expression.

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

Tissue-specific regulation of mouse hepatocyte nuclear factor 4 expression.

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

Apolipoprotein(a) gene transcription is regulated by liver-enriched trans-acting factor hepatocyte nuclear factor 1 alpha.

Elevated levels of lipoprotein(a) (Lp(a)) in the plasma are a risk factor for coronary artery disease and stroke. Plasma Lp(a) concentrations are highly heritable and predominantly determined by the liver-specific apolipoprotein(a) (apo(a)) gene. In this report we show by deletion analysis that sequences from -98 to +130 of the apo(a) gene are sufficient to direct liver-specific transcription. DNase I protection analysis of this region using HepG2 nuclear extracts revealed six major protein-binding sites, designated A to F. A mutation within footprint C, situated in the 5'-untranslated region of the gene, resulted in a marked reduction of luciferase expression from a reporter construct to 12% of wild type. This was not due to a decrease in mRNA stability. Gel mobility shift assays demonstrated that site C binds hepatocyte nuclear factor 1 alpha (HNF-1 alpha), and overexpression of HNF-1 alpha in HepG2 cells resulted in a significant stimulation of transcription from this promoter fragment. Mutation of footprint B resulted in a 2-fold enhancement of transcription. These results show that positive regulation of transcription of the apo(a) gene is dependent on the binding of HNF-1 alpha to a regulatory element situated downstream of the mRNA start site, and suggest that an as yet unidentified protein may negatively regulate apo(a) transcription by binding to a discrete sequence within the 5'-untranslated region.

Expression of hepatic transcription factors during liver development and oval cell differentiation.

The oval cells are thought to be the progeny of a liver stem cell compartment and strong evidence now exists indicating that these cells can participate in liver regeneration by differentiating into different hepatic lineages. To better understand the regulation of this process we have studied the expression of liver-enriched transcriptional factors (HNF1 alpha and HNF1 beta, HNF3 alpha, HNF3 beta, and HNF3 gamma, HNF4, C/EBP, C/EBP beta, and DBP) in an experimental model of oval cell proliferation and differentiation and compared the expression of these factors to that observed during late stages of hepatic ontogenesis. The steady-state mRNA levels of four (HNF1 alpha, HNF3 alpha, HNF4, and C/EBP beta) "liver-enriched" transcriptional factors gradually decrease during the late period of embryonic liver development while three factors (HNF1 beta, HNF3 beta, and DBP) increase. In the normal adult rat liver the expression of all the transcription factors are restricted to the hepatocytes. However, during early stages of oval cell proliferation both small and large bile ducts start to express HNF1 alpha and HNF1 beta, HNF3 gamma, C/EBP, and DBP but not HNF4. At the later stages all of these factors are also highly expressed in the proliferating oval cells. Expression of HNF4 is first observed when the oval cells differentiate morphologically and functionally into hepatocytes and form basophilic foci. At that time the expression of some of the other factors is also further increased. Based on these data we suggest that the upregulation of the "establishment" factors (HNF1 and -3) may be an important step in oval cell activation. The high levels of these factors in the oval cells and embryonic hepatoblasts further substantiates the similarity between the two cell compartments. Furthermore, the data suggest that HNF4 may be responsible for the final commitment of a small portion of the oval cells to differentiate into hepatocytes which form the basophilic foci and eventually regenerate the liver parenchyma.

Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta regulate transcription via two elements in an intestine-specific promoter.

Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta are transcription factors that contain a divergent homeodomain, bind to DNA as homo- or heterodimers, and act to regulate transcription of many genes that are expressed primarily in liver. We show that HNF-1 alpha and HNF-1 beta act as regulatory factors for transcription of sucrase-isomaltase (SI), the gene for which is expressed exclusively in absorptive enterocytes of the small intestine. HNF-1 alpha and HNF-1 beta bind to two evolutionarily conserved sites in the SI promoter, previously named SIF2 and SIF3, with different relative affinities. HNF-1 alpha binds to the SIF3 element with greater affinity than to the SIF2 element, whereas HNF-1 beta binds to both elements equally. Co-transfection experiments demonstrated that HNF-1 alpha is able to increase transcriptional initiation from a minimal SI intestine-specific promoter. In contrast, HNF-1 beta has no functional effect on transcription of the SI promoter. The functional and DNA binding differences of HNF-1 alpha and HNF-1 beta on elements in the SI promoter suggest that these transcription factors may play a role in the complex spatial patterns of SI gene expression in the intestine.

Effect of amino acid limitation on the expression of 19 genes in rat hepatoma cells

We showed previously that the abundance of serum albumin mRNA is decreased in H4-II-E rat hepatoma cells limited for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan). To define the specificity of this phenomenon, we examined the effect of amino acid limitation on the abundance of mRNAs for 19 genes in the H4-II-E cells. These genes included six genes whose expression is either completely liver-specific or highly enriched in the liver compared with other tissues [albumin, transthyretin (TTR), transferrin, carbamyl phosphate synthetase-I, urate oxidase, class I alcohol dehydrogenase], as well as a number of ubiquitously expressed "housekeeping" genes. The results indicated that the 19 genes could be divided into three classes based on their response to amino acid limitation. Class I genes (the six liver-specific genes and alpha-tubulin) exhibit decreased expression in response to amino acid limitation. The expression of class II genes [beta 2-microglobulin, hypoxanthine-guanine phosphoribosyl transferase (HPRT), H-ferritin, ubiquitin (UbB), insulin-like growth factor binding protein-4, HNF-1 alpha] is not significantly affected by amino acid limitation. Class III genes [gadd153, beta-actin, ubiquitin (UbC), phosphoglycerate kinase-1, C/EBP alpha, C/EBP beta] exhibit increased expression in response to amino acid limitation. Thus, specific inductive as well as repressive effects on gene expression are quite common in amino acid-limited cells. The observation that all six genes whose expression is liver-specific exhibited decreased expression in amino acid-limited cells suggests a common mode of regulation of these genes by amino acid availability. The strong induction by amino acid limitation of the C/EBP inhibitor gadd153 is of interest in this regard, as increased levels of gadd153 could interfere with C/EBP, which is required for high expression of most liver-specific genes. To investigate further the molecular mechanism for the decrease in albumin mRNA abundance, albumin nuclear transcript levels were quantified in control and tryptophan-limited cells. Tryptophan limitation caused a decrease in albumin nuclear transcript abundance, and this decrease preceded the decrease in albumin mRNA, suggesting that the decrease in albumin mRNA was caused at least partly by a decrease in albumin gene transcription. Additional experiments with actinomycin D indicated that albumin mRNA was also destabilized in the tryptophan-limited cells. Thus, the overall results indicate that the decrease in albumin mRNA in the tryptophan-limited cells is caused by a specific decrease in albumin nuclear transcript abundance and destabilization of albumin mRNA.

Multiple, functional DBP sites on the promoter of the cholesterol 7 alpha-hydroxylase P450 gene, CYP7. Proposed role in diurnal regulation of liver gene expression.

Hepatic cytochrome P450 cholesterol 7 alpha-hydroxylase, CYP7, is regulated in vivo at the protein and the mRNA level in response to multiple physiological factors, including liver cholesterol synthesis, bile acid feedback inhibition, and diurnal rhythm. In the present study we investigated whether the liver transcription factor DBP (albumin promoter D-site binding protein), which undergoes a striking diurnal rhythm in rat liver (DBP levels during evening/morning approximately 100:1), contributes to the diurnal regulation of CYP7 gene expression. DNase I footprinting analysis using bacterially expressed DBP and a cloned 5'-flanking DNA segment of the rat CYP7 gene revealed five distinct DBP-binding sites, designated A-E, distributed between nucleotides (nts) -41 and -295 relative to the CYP7 transcription start site. CYP7-directed gene transcription in HepG2 cells transfected with a 5'-CYP7 promoter-chloramphenicol acetyl-transferase reporter was activated up to 12-fold upon cotransfection of a DBP expression vector, whereas an HNF-1 alpha expression vector did not stimulate CYP7 gene activity. 5'-Deletion analyses and site-specific mutagenesis revealed that this stimulating effect of DBP can in part be ascribed to its functional interaction with DBP binding sites B (nts -115/-125), C (nts -172/-195), and D (nts -214/-230). C/EBP beta (LAP), another liver-enriched basic-leucine zipper transcription factor, bound to these same sites but effected a more modest increase in CYP7-directed gene transcription (up to 3-4-fold) when expressed in HepG2 cells. Competition for CYP7 promoter-binding sites between C/EBP, which undergoes < or = 2-fold diurnal change in rat liver, and the diurnally regulated DBP is proposed to determine the relative rates of basal versus diurnally regulated CYP7 gene transcription and thus may be a primary mechanism for setting the 3-6-fold amplitude that characterizes the circadian rhythm of liver CYP7 expression. Moreover, since DBP is first expressed in rat liver 3-4 weeks after birth, these findings may account for both the enhanced expression and the onset of the diurnal pattern of CYP7 enzyme levels at this stage of development.

Localization of the gene for DNA polymerase epsilon (POLE) to human chromosome 12q24.3 and rat chromosome 12 by somatic cell hybrid panels and fluorescence in situ hybridization.

DNA polymerase epsilon [DNA pol epsilon (EC 2.7.7.7)] is one of the four nuclear DNA polymerases in eukaryotic cells. The mammalian enzyme is involved in DNA repair and possibly also in replication of chromosomal DNA. The gene encoding pol epsilon (POLE) was assigned to human and rat chromosomes 12 by Southern blot analysis of genomic DNA from mouse-human and mouse-rat somatic cell hybrid panels using human cDNA probe. The human gene was then regionally localized to band 12q24.3 by fluorescence in situ hybridization of metaphase spreads of chromosomes from human lymphocytes using genomic DNA probe. POLE is closely linked to HNF1A, a gene encoding a liver-enriched transcription factor, HNF1 alpha. The two genes thus define a new synteny group retained on human and rat chromosomes 12. Another gene mapping to the same or close-by region as human POLE is a gene for inherited disorder tuberous sclerosis 3, TSC3.

Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.

Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.

A Novel cis-Acting Element Controlling the Rat CYP2D5 Gene and Requiring Cooperativity Between C/EBPβ and an Sp1 Factor

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

Homeostatic regulation of hepatocyte nuclear transcription factor 1 expression in cultured hepatoma cells.

Serum colloid osmotic pressure is believed to control the hepatic output of plasma proteins, including albumin. The present study was aimed at identifying the molecular basis for feedback control of albumin gene expression in highly differentiated hepatoma cells. The steady-state level of albumin mRNA and the activity of a 282-bp albumin promoter-chloramphenicol acetyltransferase reporter gene in cells incubated in the presence of increasing amounts of serum albumin or dextran were significantly and selectively decreased. When nuclear extracts from cells exposed to 5% (wt/vol) serum albumin were tested in a gel-retardation assay with six oligonucleotide probes containing DNA elements of the albumin promoter, only the element B-retarded band, which contains the nucleotide recognition sequence for hepatocyte nuclear transcription factor 1 alpha (HNF-1 alpha), was consistently decreased as compared to nuclear extract from cells not exposed to serum albumin. Moreover, the activity of a reporter gene with a basal TATA-promoter driven by multiple HNF-1 alpha recognition elements was selectively inhibited in cells incubated in the presence of 5% serum albumin. A reduction of HNF-1 alpha mRNA appears to be responsible for this response to a change in the level of macromolecules in the incubation medium. These results indicate that activity of a dominant liver transcription factor, HNF-1 alpha, controlling the transcription of several liver-specific genes, is modulated by a fluctuation in the level of oncotically active macromolecules.

HNF-1 alpha and HNF-1 beta expression in mouse intestinal crypts.

Hepatocyte nuclear factors 1 alpha and 1 beta (HNF-1 alpha and HNF-1 beta) are homologous homeodomain-containing transcription factors that form homodimers or heterodimers that bind the same consensus sequence in the promoters of several genes expressed in liver. Many of these genes, including the mouse alpha-fetoprotein (AFP) gene, are also expressed in the intestinal epithelium. AFP expression is restricted to the enteroendocrine cell lineage in the adult small intestine, and we have investigated the distribution of HNF-1 alpha and HNF-1 beta mRNA in the mouse intestine to determine whether expression of either of these regulatory factors colocalizes with AFP. We found that transcripts encoding both factors are expressed at the highest levels in crypts of small and large intestine. Quantitation shows that the relative ratio of HNF-1 alpha to HNF-1 beta mRNAs appears lower in the colon, where AFP is not expressed. Alterations in the ratio of HNF-1 alpha to HNF-1 beta along the length of the intestine may influence HNF-1 dimer formation and expression of target genes. Although HNF-1 is necessary for transcription of the AFP gene, other factors must be involved in eliciting the cell type-specific pattern of AFP expression in the intestine.

Functional interaction of nuclear factors EF-C, HNF-4, and RXR alpha with hepatitis B virus enhancer I

Hepatitis B virus (HBV) enhancer I contains cis-acting elements that are both sufficient and essential for liver-specific enhancer function. The EF-C binding site was previously shown to be a key element in enhancer I. EF-C binding activity is evident in hepatic and nonhepatic cells. Although the EF-C binding site is required for efficient HBV enhancer I function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. We have defined a novel region in HBV enhancer I, termed the GB element, that is adjacent to and functions in conjunction with the EF-C binding site. The GB element and EF-C site confer interdependent liver-specific enhancer activity in the absence of flanking HBV enhancer sequences. The nucleotide sequence of the GB element is similar to sequences of the DNA binding sites for members of the steroid receptor superfamily. Among these proteins, we demonstrate that HNF-4, RXR (retinoid X receptor), and COUP-TF bind to the GB element in vitro. HNF-4 transactivates a promoter linked to a multimerized GB/EF-C domain via the GB element in vivo in a manner that is dependent on the integrity of the adjacent EF-C binding site. RXR alpha also transactivates promoter expression via the GB element in vivo in response to retinoic acid but in a largely EF-C-independent manner. Finally, we show that COUP-TF antagonizes the activity of the GB element in human liver cells.

Hepatic nuclear factor 1 alpha (HNF-1 alpha) is expressed in the oviduct of hens and interacts with regulatory elements of the lysozyme gene.

HNF-1 alpha is a nuclear transcriptional regulatory protein required for the expression of a variety of liver-specific genes. This factor was previously considered liver-specific but later shown to be expressed also in a few other mammalian tissues. Here we report on the occurrence of HNF-1 alpha in the avian oviduct. This finding is of particular interest because HNF-1 alpha is not expressed in female reproductive organs of mammals. The avian oviduct is the site of assembly for the avian egg and the site of tissue-specific synthesis of the major egg white proteins, such as lysozyme. We also demonstrate that the chicken lysozyme gene contains HNF-1 recognition sites within two of its important upstream regulatory elements. The presence of HNF-1 recognition elements in functionally significant regulatory sites of the lysozyme gene and high levels of HNF-1 alpha in the oviduct is a strong indication for the involvement of HNF-1 alpha in the control of the lysozyme gene and possibly other egg white protein genes in the chicken oviduct. Apparently, HNF-1 alpha performs functions in the avian oviduct that were lost upon development from birds to mammals.

Genomic structure of the POU-related hepatic transcription factor HNF-1 alpha.

Hepatocyte Nuclear Factor-1 alpha (HNF-1 alpha) is a vertebrate transcriptional regulatory protein which plays a prominent role in the activation of a large family of hepatocyte-specific genes. It is the prototype of a new subfamily of POU-related proteins important in embryogenesis and cell-type specification. Here we report on the genomic structure and DNA sequence of HNF-1 alpha of the hen. Functional domains are non-uniformly conserved between hen and mammals consistent with the notion of functions in the hen oviduct that were lost in mammals after avian mammalian separation. The unusual homeo-related DNA-binding domain is well preserved between birds and mammals. The exon/intron pattern of the gene changed during evolution. The rat gene consists of 9 exons, the chicken gene of 10. In mammals one intron was eliminated that in hens subdivides the serine-rich transactivation domain. Comparison of the sequences at this junction indicates the involvement of elements of the V(D)J recombination system in the process of intron elimination.

Role of the liver-enriched transcription factor DBP in expression of the cytochrome P450 CYP2C6 gene.

The CYP2C6 gene becomes maximally transcriptionally activated in livers of postpubertal rats. We examined the role of upstream DNA and liver-specific transcription factors in regulation of this promoter by use of transient transfection of heterologous chloramphenicol acetyltransferase gene constructs and vectors containing cDNAs encoding the liver-enriched transcription factors HNF-1 alpha, C/EBP, and DBP. Only DBP was able to activate the CYP2C6 promoter in HepG2 cells. Transactivation was not observed in one mouse and two human nonhepatic origin cell lines tested. Analysis of various constructs in which CYP2C6 upstream DNA was deleted revealed that DNA between -38 to -103 was involved in DBP-mediated activation. A partially purified preparation of DBP produced a footprint between -43 and -64 bp upstream of the transcription start site. A 32P-labeled double-stranded oligonucleotide, containing sequence information corresponding to -40 to -65, bound to both partially pure DBP and extracts from livers of rats as young as 1 week and as old as 25 weeks of age, as assessed by gel mobility shift analysis. This binding was eliminated by coincubation with excess unlabeled -40/-65 double-stranded oligonucleotide and by an oligonucleotide corresponding to the D site of the rat albumin gene. A gel mobility shift-Western immunoblot analysis revealed that the -40/-65 sequence bound to DBP only in liver nuclear extracts from rats older than 3 weeks; maximal binding was observed by 7 weeks of age, and no binding was detected from 1-week-old rat liver extracts. Interestingly, the DBP-binding regions of both CYP2C6 and albumin bind to C/EBP, but this factor is capable of transactivating only the latter gene. Although the DBP-binding regions in these two genes share no obvious sequence similarities, the CYP2C6 region contains consensus palindromic half sites for DBP-related binding proteins and affinity for recombinant DBP of 17-fold greater than that of the D site of albumin. This difference in affinity is probably responsible for the markedly lower amounts of DBP required for half-maximal activation of the CYP2C6 promoter, as compared with the albumin promoter, in transactivation transfection assays. These data indicate that the CYP2C6 gene may be regulated, at least in part, by DBP, a liver transcription factor produced when rats reach puberty that may also be involved in maintenance of albumin gene transcription.

Role of the liver-enriched transcription factor DBP in expression of the cytochrome P450 CYP2C6 gene.

The CYP2C6 gene becomes maximally transcriptionally activated in livers of postpubertal rats. We examined the role of upstream DNA and liver-specific transcription factors in regulation of this promoter by use of transient transfection of heterologous chloramphenicol acetyltransferase gene constructs and vectors containing cDNAs encoding the liver-enriched transcription factors HNF-1 alpha, C/EBP, and DBP. Only DBP was able to activate the CYP2C6 promoter in HepG2 cells. Transactivation was not observed in one mouse and two human nonhepatic origin cell lines tested. Analysis of various constructs in which CYP2C6 upstream DNA was deleted revealed that DNA between -38 to -103 was involved in DBP-mediated activation. A partially purified preparation of DBP produced a footprint between -43 and -64 bp upstream of the transcription start site. A 32P-labeled double-stranded oligonucleotide, containing sequence information corresponding to -40 to -65, bound to both partially pure DBP and extracts from livers of rats as young as 1 week and as old as 25 weeks of age, as assessed by gel mobility shift analysis. This binding was eliminated by coincubation with excess unlabeled -40/-65 double-stranded oligonucleotide and by an oligonucleotide corresponding to the D site of the rat albumin gene. A gel mobility shift-Western immunoblot analysis revealed that the -40/-65 sequence bound to DBP only in liver nuclear extracts from rats older than 3 weeks; maximal binding was observed by 7 weeks of age, and no binding was detected from 1-week-old rat liver extracts. Interestingly, the DBP-binding regions of both CYP2C6 and albumin bind to C/EBP, but this factor is capable of transactivating only the latter gene. Although the DBP-binding regions in these two genes share no obvious sequence similarities, the CYP2C6 region contains consensus palindromic half sites for DBP-related binding proteins and affinity for recombinant DBP of 17-fold greater than that of the D site of albumin. This difference in affinity is probably responsible for the markedly lower amounts of DBP required for half-maximal activation of the CYP2C6 promoter, as compared with the albumin promoter, in transactivation transfection assays. These data indicate that the CYP2C6 gene may be regulated, at least in part, by DBP, a liver transcription factor produced when rats reach puberty that may also be involved in maintenance of albumin gene transcription.