Dedifferentiated Hepatoma Cells Research Articles

458 Inhibition of TGFβ2 production in mouse dedifferentiated hepatoma cells leads to decrease of their tumourigenic and metastatic potential

458 Inhibition of TGFβ2 production in mouse dedifferentiated hepatoma cells leads to decrease of their tumourigenic and metastatic potential

Apoptosis of Dedifferentiated Hepatoma Cells is Independent of NF-κB Activation in Response to LPS

Dedifferentiated hepatoma cells, in contrast to most other cell types including hepatoma cells, undergo apoptosis when treated with lipopolysaccharide (LPS) plus the protein synthesis inhibitor cycloheximide (CHx). We recently reported that the dedifferentiated hepatoma cells also exhibit a strong and prolonged NF-kappaB induction phenotype upon exposure to LPS, suggesting that NF-kappaB signaling may play a pro-survival role, as reported in several other cell systems. To test the role of NF-kappaB in preventing LPS-mediated apoptosis, we examined the dedifferentiated cell line M38. Results show that antioxidants strongly inhibited LPS + CHx-mediated cell death in the M38 cells, yet only modestly inhibited NF-kappaB induction. In addition, inhibition of NF-kappaB translocation by infection of the M38 cells with an adenoviral vector expressing an IkappaBalpha super-repressor did not result in LPS-mediated cell death. These results suggest that unlike TNFalpha induction, the cell survival pathway activated in response to LPS is independent of NF-kappaB translocation in the dedifferentiated cells. Addition of inhibitors of JNK, p38 and ERK pathways also failed to elicit LPS-mediated apoptosis similar to that observed when protein synthesis is prevented. Thus, cell survival pathways other than those involving NF-kappaB inducible gene expression or other well-known pathways appear to be involved in protecting the dedifferentiated hepatoma variant cells from LPS-mediated apoptosis. Importantly, this pro-apoptotic function of LPS appears to be a function of loss of hepatic gene expression, as the parental hepatoma cells resist LPS-mediated apoptosis in the presence of protein synthesis inhibitors.

Tissue-specificity of apoptosis in hepatoma-derived cell lines

Apoptosis is known to play a critical role in development and homeostasis in metazoans. Although apoptotic responses vary widely among cell types, the underlying mechanisms responsible for these differences are not known. In order to understand the molecular basis for these differences, we have studied a cell culture model comparing hepatoma cells to dedifferentiated cell lines derived from them. We recently reported evidence suggesting that a common regulatory locus affects both liver-specific function and sensitivity to lipopolysaccharide (LPS)-mediated apoptosis. Here, we show that dedifferentiated hepatoma cells undergo apoptosis in response to multiple compounds, including sorbitol (to induce hyperosmotic shock), TNF alpha and the microtubule damaging agent vinblastin. In contrast, the hepatoma parental cells fail to undergo apoptosis in response to any of the compounds tested. Further analysis of LPS-mediated cell death found that antioxidants N-acetylcysteine and alpha-tocopherol partially prevented apoptosis. Lastly, evidence is presented showing that LPS-mediated cell death of the hepatoma variant cell lines is caspase-dependent. These results suggest that pathways dictating hepatic phenotype also affect general cellular survival mechanisms in response to multiple agents. The dedifferentiated cells provide a model to examine the influence of cell-type specific expression on apoptotic signaling.

Comparative genomic analysis of the HNF-4α transcription factor gene

Hepatocyte nuclear factor-4α (HNF-4α), the gene for the maturity-onset diabetes of the young type 1 (MODY1) form of type 2 diabetes mellitus (T2DM), is within the T2DM-linked region on chromosome 20q12-q13.1 and consequently, is a positional candidate gene for T2DM. Mutations in the coding region of HNF-4α are rare in diabetes affected subjects. Altered regulation of HNF-4α gene expression, controlled by distant enhancer sequences, may contribute to the development of type 2 diabetes. Comparative sequence analysis was performed between 13 kb of genomic DNA 5 ′ to the P1 promoter sequences of the human, mouse, and rat HNF-4α coding sequences. Three regions, located at −10.5 kb (295 bp in length), −6.25 kb (421 bp in length), and −5.36 kb (263 bp in length), have significant sequence identity between the species. These three regions were functionally characterized using the chloramphenicol acetyltransferase (CAT) reporter assay, in which the conserved 5 ′ regions of mouse HNF-4α were cloned in front of the herpes simplex virus thymidine kinase promoter driving transcription of the CAT gene. A fragment containing the 421 bp conserved region significantly increased CAT activity in differentiated rat hepatoma cells (13.7- ± 1.9-fold control), while only a modest increase in CAT activity was observed in pancreatic cells (2.5- ± 0.9-fold control; 1.6- ± 0.1-fold control) and dedifferentiated hepatoma cells (1.7- ± 0.4-fold control). The remaining two conserved regions increased CAT activity minimally in pancreatic (1.1- ± 0.1-fold control to 1.9- ± 0.1-fold control) and hepatic (1.6- ± 0.5-fold control to 2.3- ± 0.4-fold control) cell lines. Denaturing high-performance liquid chromatography (DHPLC) was used to search for sequence variants in DNA from 259 T2DM individuals. Two single nucleotide polymorphisms (SNPs) were identified, both of which increased CAT activity in the insulinoma cell lines in the CAT reporter assay (1.4-fold increase over wild-type; 1.7-fold increase over wild-type). These results suggest that comparative sequence analysis can efficiently identify regulatory elements and that sequence variants in regulatory elements of HNF-4α can contribute to altered HNF-4α gene expression.

An enhancer element 6 kb upstream of the mouse HNF4alpha1 promoter is activated by glucocorticoids and liver-enriched transcription factors.

We have characterized a 700 bp enhancer element around -6 kb relative to the HNF4alpha1 transcription start. This element increases activity and confers glucocorticoid induction to a heterologous as well as the homologous promoters in differentiated hepatoma cells and is transactivated by HNF4alpha1, HNF4alpha7, HNF1alpha and HNF1beta in dedifferentiated hepatoma cells. A 240 bp sub-region conserves basal and hormone-induced enhancer activity. It contains HNF1, HNF4, HNF3 and C/EBP binding sites as shown by DNase I footprinting and electrophoretic mobility shift assays using nuclear extracts and/or recombinant HNF1alpha and HNF4alpha1. Mutation analyses showed that the HNF1 site is essential for HNF1alpha transactivation and is required for full basal enhancer activity, as is the C/EBP site. Glucocorticoid response element consensus sites which overlap the C/EBP, HNF4 and HNF3 sites are crucial for optimal hormonal induction. We present a model that accounts for weak expression of HNF4alpha1 in the embryonic liver and strong expression in the newborn/adult liver via the binding sites identified in the enhancer.

Multiple elements for negative regulation of the rat catalase gene expression in dedifferentiated hepatoma cells.

Like such hepatic genes as those for albumin and aldolase B, the rat catalase gene shows markedly reduced expression in carcinogenesis of hepatocytes. Strong silencer activity has been widely observed in the 5'-flanking region of the gene, downstream from the G-rich sequence identified in a previous study. In this study, we identified and characterized multiple elements involved in negative regulation of catalase gene expression by reporter assay and gel shift assay. One of the silencer elements is located 3 kb upstream of the gene and has GATATCCCGATATC as core sequence. The observation that protein binding to the element is abundantly expressed in dedifferentiated hepatoma cell lines, but scarcely in well-differentiated cell lines suggests that this element is involved in negative regulation of the catalase gene expression in hepatocarcinogenesis. This element was targeted by a novel 20-kDa nuclear protein, which is designated HNRF (hepatocarcinogenesis-related negative regulatory factor).

Defective NF-kappaB signaling in dedifferentiated hepatoma cells.

Dedifferentiated rat hepatoma cells contain defects that result in the loss of hepatic gene expression, including the liver-enriched HNF4/HNF1alpha pathway. We examined induction of NF-kappaB, a key mediator of the inflammatory response, in hepatoma and dedifferentiated hepatoma cells. We show that exposure of dedifferentiated hepatoma cells, but not rat and human hepatoma cell lines, to proinflammatory cytokines or lipopolysaccharide resulted in rapid and sustained NF-kappaB induction. IkappaB-beta levels, but not NF-kappaB subunit p65 or IkappaB-alpha levels, were elevated compared with those for parental hepatoma cells. Interestingly, LPS-mediated activation of NF-kappaB was found to be independent of degradation of IkappaB-alpha or IkappaB-beta. Thus, these results suggest that loci responsible for maintaining hepatic gene expression also influence cellular responses to inflammatory agents.

Hepatocyte nuclear factor 4 provokes expression of epithelial marker genes, acting as a morphogen in dedifferentiated hepatoma cells.

We have recently shown that stable expression of an epitope-tagged cDNA of the hepatocyte- enriched transcription factor, hepatocyte nuclear factor (HNF)4, in dedifferentiated rat hepatoma H5 cells is sufficient to provoke reexpression of a set of hepatocyte marker genes. Here, we demonstrate that the effects of HNF4 expression extend to the reestablishment of differentiated epithelial cell morphology and simple epithelial polarity. The acquisition of epithelial morphology occurs in two steps. First, expression of HNF4 results in reexpression of cytokeratin proteins and partial reestablishment of E-cadherin production. Only the transfectants are competent to respond to the synthetic glucocorticoid dexamethasone, which induces the second step of morphogenesis, including formation of the junctional complex and expression of a polarized cell phenotype. Cell fusion experiments revealed that the transfectant cells, which show only partial restoration of E-cadherin expression, produce an extinguisher that is capable of acting in trans to downregulate the E-cadherin gene of well-differentiated hepatoma cells. Bypass of this repression by stable expression of E-cadherin in H5 cells is sufficient to establish some epithelial cell characteristics, implying that the morphogenic potential of HNF4 in hepatic cells acts via activation of the E-cadherin gene. Thus, HNF4 seems to integrate the genetic programs of liver-specific gene expression and epithelial morphogenesis.

Hepatocyte nuclear factor 4 expression overcomes repression of the hepatic phenotype in dedifferentiated hepatoma cells.

The capacity of the liver-enriched transcription factor hepatocyte nuclear factor 4 (HNF4) to direct redifferentiation of dedifferentiated rat hepatoma cells was investigated by stable transfection of epitope-tagged HNF4 cDNA into H5 variant cells. HNF4-producing cells expressed the previously silent HNF1 gene and showed activation of some hepatic functions, including alpha1-antitrypsin, beta-fibrinogen, and transthyretin, but not of the endogenous HNF4 gene. Expression of the other hepatocyte-enriched transcription factors was not modified. Treatment of the HNF4tag-expressing cells with dexamethasone induced expression of the transgene by 10-fold, resulting in enhanced expression of target genes of both glucocorticoid hormones and HNF4. The set of activated hepatic genes was extended by treatment of cells with the demethylating agent 5-azacytidine followed by selection in dexamethasone-containing glucose-free medium. Some of the colonies that developed reexpressed the entire set of hepatic functions tested. Fusion of HNF4tag-producing H5 cells with well-differentiated Fao cells showed that only those hybrids which maintained expression of HNF4tag were protected from complete extinction, including that of the Fao HNF4 gene. Thus, H5 cells must produce an extinguisher of the HNF4 gene. In addition, this result implies that HNF4 itself, or its target HNF1, is a positive regulator of HNF4. In conclusion, HNF4tag expression overcomes repression of the hepatic phenotype of the H5 cell without abolishing its potential to extinguish an active genome. Taken together, these results predict that expression of HNF4 should be sufficient to establish heritable expression of many parameters of the hepatic differentiated state.

Members of the nuclear receptor superfamily regulate transcription from the hepatitis B virus nucleocapsid promoter.

The role of members of the nuclear receptor superfamily of transcription factors in regulating hepatitis B virus (HBV) transcription was investigated. Hepatocyte nuclear factor 4 (HNF4), the retinoid X receptor (RXR), and the peroxisome proliferator-activated receptor (PPAR) were examined for their capacity to modulate the level of transcriptional activity from the four HBV promoters by transient-transfection analysis in the dedifferentiated hepatoma cell line, HepG2.1. It was found that the nucleocapsid and large surface antigen promoters were transactivated in the presence of HNF4 whereas the enhancer I/X gene, nucleocapsid, and large surface antigen promoters were transactivated in the presence of RXR and PPAR. Characterization of the nuclear receptors interacting with the nucleocapsid promoter region demonstrated that HNF4 is the primary transcription factor binding to the regulatory region spanning nucleotides -127 to -102 whereas HNF4, RXR-PPAR heterodimers, COUPTF1, and ARP1 bind the regulatory region spanning nucleotides -34 to -7. Transcriptional transactivation from the nucleocapsid promoter by HNF4 appears to be mediated through the two HNF4 binding sites in the promoter, whereas modulation of the level of transcription from the nucleocapsid promoter by RXR-PPAR appears to be regulated by the regulatory sequence element spanning nucleotides -34 to -7 and the HBV enhancer 1 region. These observations indicate that HBV transcription, and pregenomic RNA synthesis in particular, is regulated by ligand-dependent nuclear receptors. Agonists and antagonists capable of regulating the activity of these nuclear receptors may permit the modulation of HBV transcription and consequently replication during viral infection.

Regulation of transcription from the hepatitis B virus large surface antigen promoter by hepatocyte nuclear factor 3

The influence of hepatocyte nuclear factor 3 (HNF3) on the level of transcriptional activity from the four hepatitis B virus promoters was investigated by transient-transfection analysis in the dedifferentiated hepatoma cell line, HepG2.1. It was found that the large surface antigen promoter and, to a much lesser extent, the nucleocapsid promoter were transactivated in the presence of HNF3. DNase I footprinting analysis demonstrated that purified recombinant HNF3 alpha protects one region of the large surface antigen promoter. Gel retardation analysis showed that a double-stranded oligonucleotide containing this HNF3-binding site formed a specific complex with DNA-binding proteins in the differentiated hepatoma cell lines, Huh7 and HepG2. The complex formed with Huh7 cell extract comigrated with exogenously expressed HNF3 beta in HeLa S3 extracts and was specifically inhibited from forming by the addition of HNF3 beta antiserum. The promoter element which appears to mediate the HNF3 transactivation was functionally mapped by mutational analysis to a region between nucleotides -65 and -54 relative to the transcriptional start site. This regulatory sequence is within the region protected from DNase I digestion by HNF3 alpha and contains 10 of 12 nucleotides homologous to the HNF3-binding-site consensus sequence. A synthetic promoter construct containing this HNF3-binding site was able to mediate transactivation by HNF3 beta. These and previous results suggest that the hepatitis B virus large surface antigen promoter is regulated by at least two liver-enriched transcription factors, HNF1 and HNF3, which together may contribute to the differentiated liver cell type specificity of this promoter.

Analysis of the Inductive Effect of the Genomic Equivalent of HALF1 Sequence in the Reversion of Rat Dedifferentiated Hepatoma Cells

HALF1 (human activator of liver function 1) is a closed DNA duplex implicated in reversion of rat dedifferentiated C2 hepatoma cells to a well-differentiated state. A copy of HALF1 is found in high-molecular-weight DNA in the human genome. The genomic equivalent of HALF1 and its flanking sequences [gH(5′-3′) fragment] have previously been cloned and sequenced. To analyze the ability of the gH(5′-3′) fragment to induce reversion process of C2 cells, two series of transfections were performed: (1) cotransfection of gH(5′-3′) and plasmid pSVneo1 and (2) transfection of gH(5′-3′) inserted into pSVneo1. The frequency of reversion was enhanced in transfected cells from the first experiment whereas no revertants were obtained from transfected cells in the second one. DNA analysis of the revertant clones revealed that reversion is associated with the transient presence of nonintegrated gH(5′-3′) molecules. C2 cells were also transfected with gH(5′-3′) cloned in pSV2dhfr. In the products of this transfection, the genesis of revertants correlated with amplification process of the gH(5′-3′) sequence. We conclude that the presence of integrated copies of gH(5′-3′), even in high copy number, is not sufficient to induce the reversion process. We propose that extrachromosomal forms of gH(5′-3′), either given to cells or formed during amplification cycles, are involved in the reversion process of C2 cells.

Differentiation-Specific Transcriptional Regulation of the Hepatitis B Virus Nucleocapsid Gene in Human Hepatoma Cell Lines

Transcription from the hepatitis B virus (HBV) nucleocapsid promoter is regulated in a cell-type-specific manner and can be modulated by the HBV enhancer 1 element. Mutagenesis analysis of the nucleocapsid promoter demonstrated that the two Sp1-binding sites (CpB and CpC) in the minimal promoter were the major determinants of transcriptional activity in the dedifferentiated hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3. In contrast, binding sites for transcription factors located immediately upstream (CpE) and downstream (CpF) of the two Sp1-binding sites were shown to be important determinants of nucleocapsid promoter activity in the differentiated hepatoma cell lines, Huh7 and HepG2. The role of these elements in the regulation of the nucleocapsid promoter activity correlated with the formation of specific DNA-protein complexes between Huh7 and HepG2 nuclear extracts and the CpE and CpF region sequences. Characterization of the influence of the nucleocapsid promoter mutations in the presence or absence of the enhancer 1 demonstrated that modification of individual transcription factor binding sites does not prevent enhancer-mediated activation of transcription from the nucleocapsid promoter. These results indicate that differentiated-hepatoma-specific transcription factors plus the Sp1 transcription factor interacting with the nucleocapsid promoter and the enhancer 1 regulatory region contribute to the level of transcription from this HBV promoter.

The transcription factor HNF1 acts with C/EBP alpha to synergistically activate the human albumin promoter through a novel domain.

HNF1 and C/EBP alpha are transcription factors that bind to and trans-activate the human albumin gene proximal promoter. Various 5' deletions of the human albumin promoter were coupled to a luciferase reporter gene (alb-luc constructs) and co-electroporated with HNF1 and/or C/EBP alpha expression vectors into HeLa cells. Luciferase activities from co-electroporation of the HNF1 and C/EBP alpha expression vectors with the alb-luc constructs were approximately 10-fold greater than the sum of the activities achieved with HNF1 and C/EBP alpha alone. Analysis of COOH-terminal or internal deletions of the HNF1 expression vector revealed that the domain important for collaborative interaction with C/EBP alpha could be localized to a 157 amino acid region not previously described. This domain is proline and glutamine-rich and is highly homologous (66%) to a portion of vHNF1, an evolutionarily related gene first identified in dedifferentiated hepatoma cells. A construct linking the negatively charged activation domain of herpes simplex virus protein VP16 to the DNA-binding domain of HNF1 showed that it could also synergize with C/EBP alpha to trans-activate the human albumin gene promoter. Our studies delineate a domain in HNF1 important for synergistic activation with C/EBP alpha.

Chimeric liver transcription factors LFB1 (HNF1) containing the acidic activation domain of VP16 act as positive dominant interfering mutants.

The transcription factor LFB1 (HNF1) involved in the expression of liver-specific genes is characterized by a serine/threonine-rich activation domain whose transactivation potential differs between mammals and Xenopus. Exchanging the activation domain between the Xenopus and rat LFB1, we produced chimeric transactivators whose activities are primarily determined by the origin of the activation domain. By replacing the serine/threonine-rich activation domain of LFB1 with the acidic activation domain of VP16, we generated transcription factors that act as dominant positive interfering mutants on endogenous LFB1 in differentiated hepatoma cells. As these LFB1/VP16 chimeras show no self-squelching as observed with wild-type LFB1 and increase the activity of saturating LFB1, we postulate that acidic and serine/threonine-rich activation domains use different targets of the basal transcription machinery. Stable transfection of various LFB1 derivatives, including those containing the VP16 transactivation domain, into the dedifferentiated C2 hepatoma cell resulted in cell clones stably expressing LFB1 function. However, as in none of these clones the chromosomal albumin genes are activated, we conclude that the presence of functional LFB1 may not be sufficient to reactivate liver-specific functions lost in dedifferentiated hepatoma cells.

The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors.

Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.

The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors.

Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.

Characterization of the hepatitis B virus- and nucleocapsid gene transcriptional regulatory elements

The regulatory DNA sequence elements that control the expression of the hepatitis B virus X- and nucleocapsid genes in the differentiated human hepatoma cell lines, Huh7, Hep3B, PLC/PRF/5, and HepG2, the dedifferentiated human hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3, were analyzed using transient transfection assays. In this system, the hepatitis B virus enhancer I located between coordinates 1071 (−239) and 1238 (−72) increases transcription from the X-gene promoter located between coordinates 1239 (−71) and 1376 (+67) more than 30-fold in the differentiated hepatoma and the HeLa S3 cell lines. In the dedifferentiated hepatoma cell line, HepG2.1, the enhancer I sequence increases the level of transcription from the X-gene promoter approximately 10-fold. The enhancer I subregion between coordinates 1117 (−193) and 1204 (−106) appears to be important for enhancer function only in the differentiated hepatoma cell lines, whereas the enhancer I subregion between coordinates 1222 (−88) and 1238 (−72) is required for enhancer activity in each of the cell lines examined. In all of the cell lines, the X-gene minimal promoter element was within a 138-nucleotide sequence located between coordinates 1239 (−71) and 1376 (+67). The enhancer I sequence increases transcription from the nucleocapsid promoter approximately 3- to 10-fold in the Huh7, Hep3B, PLC/PRF/5, and HeLa S3 cell lines, whereas it had little influence on the level of transcription from this promoter in HepG2 and HepG2.1 cells. The minimal nucleocapsid promoter element was within a 105 nucleotide sequence located between coordinates 1700 (-85) and 1804 (+20). This indicates that the levels of transcription from the X- and nucleocapsid gene promoters are determined in a cell-type-specific manner, in part, by the hepatitis B virus enhancer I and the corresponding minimal promoter sequence.

VHNF1 is expressed in epithelial cells of distinct embryonic origin during development and precedes HNF1 expression

HNF1 (Hepatic Nuclear Factor 1) and vHNF1 are transcriptional regulators containing a highly divergent homeodomain. The first was initially found in liver nuclear extracts and is crucial for the transcription of albumin and many other hepatocyte specific genes, while the second was found in dedifferentiated hepatoma cells. Both recognize the same DNA binding site and can form homo and heterodimers in vitro and in vivo. In situ hybridization analyses have been performed to delineate the spatial and temporal pattern of expression of vHNF1 relative to HNF1 during mouse embryogenesis. The results show that accumulation of vHNF1 mRNAs expression is detected in several tissues of the embryo of both endodermal and mesodermal origin. Expression occurs in the yolk sac, the primitive gut, the liver primordium, and at different stages of kidney development in polarized epithelial structures and usually precedes that of HNF1. vHNF1 expression seems particularly prevalent with morphogenetic events in the kidney and may be a marker for certain polarized epithelium.

VHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1.

vHNF1 and HNF1 are two nuclear proteins that bind to an essential element in the promoter proximal sequences of albumin and of many other liver-specific genes. HNF1 predominates in hepatocytes but is absent in dedifferentiated hepatoma cells. These cells contain vHNF1 but fail to express most of the liver traits. In the present work we have isolated cDNA clones for vHNF1 and found that it is a homeoprotein homologous to HNF1 in regions important for DNA binding. Unexpectedly, vHNF1 transactivated the albumin promoter in transfection experiments. Like the HNF1 mRNA, the vHNF1 message was found in kidney, liver and intestine although in different proportions. The fact that vHNF1 and HNF1 readily form heterodimers in vitro and the biochemical characterization of vHNF1/HNF1 heterodimers in nuclear extracts of kidney, liver and several cell lines, strongly argue that such heterodimers exist in vivo. Our results raise the possibility that heterodimerization between homeoproteins could be a common phenomenon in higher eukaryotes, which may have implications in the regulatory network sustained between these factors.