Liver Transcription Research Articles

Developmental regulation and tissue distribution of the liver transcription factor LFB1 (HNF1) in Xenopus laevis.

The transcription factor LFB1 (HNF1) was initially identified as a regulator of liver-specific gene expression in mammals. It interacts with the promoter element HP1, which is functionally conserved between mammals and amphibians, suggesting that a homologous factor, XLFB1, also exists in Xenopus laevis. To study the role of LFB1 in early development, we isolated two groups of cDNAs coding for this factor from a Xenopus liver cDNA library by using a rat LFB1 cDNA probe. A comparison of the primary structures of the Xenopus and mammalian proteins shows that the myosin-like dimerization helix, the POU-A-related domain, the homeo-domain-related region, and the serine/threonine-rich activation domain are conserved between X. laevis and mammals, suggesting that all these features typical for LFB1 are essential for function. Using monoclonal antibodies, we demonstrate that XLFB1 is present not only in the liver but also in the stomach, intestine, colon, and kidney. In an analysis of the expression of XLFB1 in the developing Xenopus embryo, XLFB1 transcripts appear at the gastrula stage. The XLFB1 protein can be identified in regions of the embryo in which the liver diverticulum, stomach, gut, and pronephros are localized. The early appearance of XLFB1 expression during embryogenesis suggests that the tissue-specific transcription factor XLFB1 is involved in the determination and/or differentiation of specific cell types during organogenesis.

ADH1, 2 and 3: genes whose times have come.

The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF–1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer–binding protein α (C/EBPα), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D–element–binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF–1 or C/EBPα, and a significant synergism between C/EBPα and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBPα and LAP homo– and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBPα (i.e., —160, — 120, — 40, and — 20 bp), but DBP bound strongly only to the site located at —40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the —40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF–1 and C/EBPα help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high–level ADH gene family transcription in postnatal liver.

Effects of STZ-induced diabetes and fasting on insulin receptor mRNA expression and insulin receptor gene transcription in rat liver.

Insulinopenic states in rodents are known to cause an increase in the number of hepatic insulin receptors. To determine if this change is related to an abnormality in insulin receptor gene expression, insulin receptor binding, insulin receptor mRNA levels, and insulin receptor gene transcription rates have been measured in livers from rats rendered hypoinsulinemic by STZ administration (65 mg/kg) or fasting. In the two groups of experimental rats, insulin binding to liver plasma membranes was increased (approximately 40 and 25%, respectively) relative to control, normoinsulinemic animals. Northern blot analysis of either total or poly (A)+ RNA from livers of hypo- and normoinsulinemic rats revealed two major insulin receptor mRNA species of 9.5 and 7.5 kbs. In hypoinsulinemic rats, insulin receptor mRNA levels were increased > or = 10-fold, with similar effects on the two mRNA species. The effects of STZ administration and fasting on insulin receptor binding and insulin receptor mRNA levels were fully reversed by insulin treatment or refeeding, respectively. Injection of ACT D, an inhibitor of gene transcription, decreased insulin receptor mRNA levels by > or = 80% in control and diabetic rats and suppressed the overexpression of mRNA seen in diabetic rats. In vitro nuclear transcription assays showed that the rate of transcription of the insulin receptor gene was increased 2-fold in STZ-induced diabetic rats and fasted rats relative to control animals. Taken together, these results suggest that the upregulation of the insulin receptor induced by chronic insulinopenia results, at least in part, from an increase in insulin receptor gene transcription.

Hormonal control of insulin-like growth factor-II gene expression in the rat liver.

IGF-II in the rat is an embryonic/fetal growth factor. Transcription of its gene falls abruptly in the liver at about 18-20 days postnatally. In an attempt to elucidate the mechanisms controlling this phenomenon, we used Northern analysis to investigate the effect of corticosterone and thyroid hormones (tri-iodothyronine and tetra-iodothyronine) on hepatic IGF-II mRNA levels. The administration of either corticosterone or tri-iodothyronine to 8-day-old pups resulted in a significant decrease in IGF-II mRNA when the animals were examined on day 12 of life. Adrenalectomy, thyroidectomy or adrenalectomy combined with thyroidectomy were, however, without effect. Our results indicate that glucocorticoid and thyroid hormones are not the exclusive regulators of IGF-II gene transcription in the rat liver, but participate in this process, which appears to involve multifactorial interactions.

Temporal Expression of the Human Alcohol Dehydrogenase Gene Family during Liver Development Correlates with Differential Promoter Activation by Hepatocyte Nuclear Factor 1, CCAAT/Enhancer-Binding Protein α, Liver Activator Protein, and D-Element-Binding Protein

The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver.

Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.

The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver.

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.

Extracellular matrix regulation of hepatic transcription factors is dependent on cell structure.

The extracellular matrix (ECM) promotes tissue morphogenesis, cell migration, and the differentiation of a variety of cell types. However, the mechanisms by which ECM causes differentiated gene expression have been unknown. In this report, we show that culturing the hepatocyte–derived cell line H2.35 on an ECM gel changes cell morphology and selectively stimulates the transcription of a subset of liver–specific genes, including serum albumin. Transcriptional activation by ECM also occurs with transfected plasmids bearing the transcriptional enhancer of the albumin gene. ECM substrates of different composition activated the albumin enhancer only when the ECM promoted a cuboidal, differentiated cell morphology. Enhancer activation by the ECM was mediated by two liver transcription factors, HNF3α and eH–TF, which appear to be regulated differently by matrix. Specifically, we found that a collagen gel substratum caused a selective increase in the factor HNF3α at the levels of mRNA accumulation and DNA–binding activity in nuclear extracts, both in H2.35 cells and in the hepatoma cell line HepG2. We conclude that the ECM can stimulate cell differentiation by selectively activating transcriptional regulatory factors and that such regulation occurs coordinately with ECM–promoted changes in cell shape.

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.

Developmental expression of hexokinase 1 in the rat

Hexokinase 1 (HK1) activity varies in a developmental stage- and tissue-specific manner and is a key step in glucose homeostasis and energy metabolism. We conducted studies to determine if HK1 expression is regulated at the transcriptional level. Expression of HK1 was examined in selected pre- and postnatal rat tissues using Northern blot analyses and RNAase protection assays. We found that brain and kidney exhibited significantly higher expression than heart, lung, spleen and skeletal muscle. Brain HK1 expression was constant prenatally, peaked at 7 days of age and reached a constant level after weaning. In kidney, HK1 expression was essentially constant or perhaps gradually decreased after weaning. HK1 transcription in heart, skeletal muscle and liver was higher during fetal stages than postnatally. Lung expression was essentially constant except in the adult. HK1 mRNA levels were compared to phosphoglycerate kinase (PGK) mRNA. PGK steady state mRNA levels were relatively constant in all tissues and developmental stages, except in skeletal muscle where there was a postnatal rise. The developmental profiles of HK1 and PGK mRNA expression are not consistent with enzyme activity measurements in all the tissues examined. We conclude that regulation of HK1 expression involves both transcriptional and posttranscriptional mechanisms that are tissue and stage specific.

The Restricted Promoter Activity of the Liver Transcription Factor Hepatocyte Nuclear Factor 3β Involves a Cell-Specific Factor and Positive Autoactivation

The transcription factor hepatocyte nuclear factor 3 (HNF-3) is involved in the coordinate expression of several liver genes. HNF-3 DNA binding activity is composed of three different liver proteins which recognize the same DNA site. The HNF-3 proteins (designated alpha, beta, and gamma) possess homology in the DNA binding domain and in several additional regions. To understand the cell-type-specific expression of HNF-3 beta, we have defined the regulatory sequences that elicit hepatoma-specific expression. Promoter activity requires -134 bp of HNF-3 beta proximal sequences and binds four nuclear proteins, including two ubiquitous factors. One of these promoter sites interacts with a novel cell-specific factor, LF-H3 beta, whose binding activity correlates with the HNF-3 beta tissue expression pattern. Furthermore, there is a binding site for the HNF-3 protein within its own promoter, suggesting that an autoactivation mechanism is involved in the establishment of HNF-3 beta expression. We propose that both the LF-H3 beta and HNF-3 sites play an important role in the cell-type-specific expression of the HNF-3 beta transcription factor.

The restricted promoter activity of the liver transcription factor hepatocyte nuclear factor 3 beta involves a cell-specific factor and positive autoactivation.

The transcription factor hepatocyte nuclear factor 3 (HNF-3) is involved in the coordinate expression of several liver genes. HNF-3 DNA binding activity is composed of three different liver proteins which recognize the same DNA site. The HNF-3 proteins (designated alpha, beta, and gamma) possess homology in the DNA binding domain and in several additional regions. To understand the cell-type-specific expression of HNF-3 beta, we have defined the regulatory sequences that elicit hepatoma-specific expression. Promoter activity requires -134 bp of HNF-3 beta proximal sequences and binds four nuclear proteins, including two ubiquitous factors. One of these promoter sites interacts with a novel cell-specific factor, LF-H3 beta, whose binding activity correlates with the HNF-3 beta tissue expression pattern. Furthermore, there is a binding site for the HNF-3 protein within its own promoter, suggesting that an autoactivation mechanism is involved in the establishment of HNF-3 beta expression. We propose that both the LF-H3 beta and HNF-3 sites play an important role in the cell-type-specific expression of the HNF-3 beta transcription factor.

Identification and functional characterization of acis-acting positive DNA element regulating CYP 2B1/B2 gene transcription in rat liver

A positive cis-acting DNA element in the near 5'-upstream region of the CYP2B1/B2 genes in rat liver was found to play an important role in the transcription of these genes. An oligonucleotide covering -69 to -98 nt mimicked the gel mobility shift pattern given by the fragment -179 to +29 nt, which was earlier found adequate to confer the regulatory features of this gene. Two major complexes were seen, of which the slower and faster moving complexes became intense under uninduced and Phenobarbitone-induced conditions respectively. Minigene cloned DNA plasmid covering -179 to +181 nt in pUC 19 and Bal 31 mutants derived from this parent were transcribed in whole nuclei and cell free transcription extracts and mutants containing only upto -75 nt of the upstream were poorly transcribed. Transcription extracts from phenobarbitone-injected rat liver nuclei were significantly more active than extracts from uninduced rats in transcribing the minigene constructs. Addition of the oligonucleotide (-69 to -98nt) specifically inhibited the transcription of the minigene construct (-179 to +181 nt) in the cell free transcription system. It is therefore, concluded that the region -69 to -98 nt acts as a positive cis-acting element in the transcription of the CYP2B1/B2 genes and in mediating the inductive effects of phenobarbitone.

Hormonal Regulation of Serine Dehydratase Gene Expression in Liver and Kidney of the Adrenalectomized Rat

We have previously demonstrated that glucagon but not dexamethasone could induce serine dehydratase (SDH: EC.4.2.1.13) in liver, and either glucagon or dexamethasone could induce the enzyme in kidney of normal rats. The mechanism(s) of the hormonal regulation of SDH gene expression in liver and kidney was further studied using adrenalectomized rats. Simultaneous administration of glucagon and dexamethasone induced the activity, rate of SDH synthesis, and accumulation of SDH mRNA in both liver and kidney of the rat. The increased SDH activity was reflected by changes in the amount of enzyme protein and in the rate of SDH protein synthesis, both parameters closely paralleling the changes in the levels of SDH mRNA. The rates of transcription of the SDH gene as measured in run-on experiments with isolated nuclei were also increased by the administration of these hormones. These results indicate that the expression of the SDH gene was regulated primarily at the transcriptional level under these conditions. When glucagon or dexamethasone was injected separately into adrenalectomized rats, significant increases in the levels of SDH mRNA and the rate of SDH gene transcription were observed in liver. Although glucagon was more effective than dexamethasone, both hormones were required for the maximal induction of SDH gene transcription in liver. In contrast, dexamethasone alone effectively increased the rate of SDH gene transcription in kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Disruption of the LF-A1 and LF-B1 binding sites in the human alpha-1-antitrypsin gene has a differential effect during development in transgenic mice.

Previous work in transfected cell lines and in nuclear extracts has led to the identification of two cis-acting elements important for transcription of the human alpha-1-antitrypsin (A1AT) gene, which bind to two liver specific trans-acting factors, LF-A1 and LF-B1. Mutations EM3 and PM1, which abolish the binding of LF-A1 and LF-B1 respectively, drastically reduce transcription activity of the A1AT gene in vitro and in cell culture. The same mutants have now been introduced in a larger DNA context and their effect has been tested in transgenic mice. A stretch of DNA was constructed which carries two transcriptional units: 18 kb of the human retinol binding protein (RBP) gene, driving the expression of the bacterial chloramphenicol acetyl transferase, linked to 17.5 kb containing the entire A1AT coding sequence with additional 5' and 3' flanking sequences. Transcription from the RBP promoter was shown to predominate in liver, and could be used as an internal marker of 'active copy number'. Mutations in the A1AT gene promoter were introduced by homologous recombination in bacterial cells. The results show that base pair substitutions in the binding site for LF-A1 and LF-B1 drastically reduce transcription in non-hepatic adult tissues, yolk sac, and fetal liver, whereas only LF-B1 binding site mutations have a marked, albeit variable, effect in adult liver.

Involvement of heme in the transcriptional activation of CYPIIB1/B2 gene by phenobarbitone in rat liver—Studies with succinylacetone

Earlier studies in this laboratory had implicated heme to function as a positive modulator of phenobarbitonemediated activation of CYPIIB1/B2 gene transcription in rat liver. However, recent reports have indicated that succinylacetone, a specific inhibitor of δ-aminolevulinate dehydrase, does not affect this process. The present studies indicate that succinylacetone does inhibit the phenobarbitone-mediated increase in CYPIIB1/B2 mRNAs and their transcription in rat liver at early time points (45 min to 3 h), but the inhibition is not pronounced at later time points (16 h). Succinylacetone is a weaker inhibitor of heme biosynthesis than CoCl 2, 3-amino-1,2,4-triazole, or thioacetamide used earlier in this laboratory. Succinylacetone induces δ-aminolevulinate synthase, whereas the other compounds depress the levels of the enzyme. There is a good correlation between the amount of freshly synthesized nuclear heme pool and the activation of CYPIIB1/B2 transcription by phenobarbitone. A model implicating a nuclear heme pool regulating the transcription of δ-aminolevulinate synthase, CYPIIB1/ B2, and heme oxygenase genes is proposed.

The Extracellular Matrix Coordinately Modulates Liver Transcription Factors and Hepatocyte Morphology

The extracellular matrix (ECM) promotes tissue morphogenesis, cell migration, and the differentiation of a variety of cell types. However, the mechanisms by which ECM causes differentiated gene expression have been unknown. In this report, we show that culturing the hepatocyte-derived cell line H2.35 on an ECM gel changes cell morphology and selectively stimulates the transcription of a subset of liver-specific genes, including serum albumin. Transcriptional activation by ECM also occurs with transfected plasmids bearing the transcriptional enhancer of the albumin gene. ECM substrates of different composition activated the albumin enhancer only when the ECM promoted a cuboidal, differentiated cell morphology. Enhancer activation by the ECM was mediated by two liver transcription factors, HNF3 alpha and eH-TF, which appear to be regulated differently by matrix. Specifically, we found that a collagen gel substratum caused a selective increase in the factor HNF3 alpha at the levels of mRNA accumulation and DNA-binding activity in nuclear extracts, both in H2.35 cells and in the hepatoma cell line HepG2. We conclude that the ECM can stimulate cell differentiation by selectively activating transcriptional regulatory factors and that such regulation occurs coordinately with ECM-promoted changes in cell shape.

The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.

The extracellular matrix (ECM) promotes tissue morphogenesis, cell migration, and the differentiation of a variety of cell types. However, the mechanisms by which ECM causes differentiated gene expression have been unknown. In this report, we show that culturing the hepatocyte-derived cell line H2.35 on an ECM gel changes cell morphology and selectively stimulates the transcription of a subset of liver-specific genes, including serum albumin. Transcriptional activation by ECM also occurs with transfected plasmids bearing the transcriptional enhancer of the albumin gene. ECM substrates of different composition activated the albumin enhancer only when the ECM promoted a cuboidal, differentiated cell morphology. Enhancer activation by the ECM was mediated by two liver transcription factors, HNF3 alpha and eH-TF, which appear to be regulated differently by matrix. Specifically, we found that a collagen gel substratum caused a selective increase in the factor HNF3 alpha at the levels of mRNA accumulation and DNA-binding activity in nuclear extracts, both in H2.35 cells and in the hepatoma cell line HepG2. We conclude that the ECM can stimulate cell differentiation by selectively activating transcriptional regulatory factors and that such regulation occurs coordinately with ECM-promoted changes in cell shape.

Tissue-specific interactions between nuclear proteins and the aminopeptidase N promoter.

Aminopeptidase N/CD13 is a metallopeptidase found in many tissues. Aminopeptidase N activity is high in the small intestinal mucosa, moderate in the liver, and low in the spleen. Using DNase I footprinting and electrophoretic mobility shift assays with nuclear extracts from these tissues, three cis elements (DF, LF-B1, UF) were identified in the aminopeptidase N promoter. The DF region (-53 to -30) interacts with the ubiquitously expressed transcription factor Sp1. The LF-B1 region (-85 to -58) interacts with the liver transcription factor LF-B1 (HNF-1) which was detected as well in nuclei from small intestinal mucosa. The UF region (-112 to -90) interacts with nuclear factors which seem to be expressed differentially in the liver and the small intestine. Transfection of promoter deletions into HepG2 cells showed that the LF-B1 region is necessary for high expression of the aminopeptidase N gene in liver cells. LF-B1 could not be detected in spleen nuclei. In accordance with this, RNA analysis demonstrated that the aminopeptidase N promoter operating in the small intestine and in the liver is inactive in the spleen. In this tissue initiation of transcription from the aminopeptidase N gene occurs from an upstream promoter.

Structure of the gene coding for the alpha polypeptide chain of the human complement component C4b-binding protein.

The human gene coding for the 70-kD polypeptide of the complement regulatory component C4b-binding protein (C4BP alpha) spans over 40 kb of DNA and is composed of twelve exons. Upon transcription in liver, or in Hep-G2 cells, this gene produces a single transcript of 2,262 nucleotides, excepting the poly A tail, that presents an unusually long 5' untranslated region (5' UTR) of 223 nucleotides. The C4BP alpha gene is organized as follows: the first exon codes for the first 198 nucleotides of the 5' UTR. It is separated by a large intron from the second exon including the remaining of the 5' UTR and the coding region for the signal peptide. Each of the eight 60-amino acid repeats (short consensus repeats [SCRs]) that compose the C4BP alpha polypeptide chain is encoded by a single exon, except for the second SCR, which is split in two exons. At the 3' end of the C4BP alpha gene, the twelfth exon codes for the COOH-terminal 57 amino acids of the mature protein, which have no similarities to the SCRs, and the 245 nucleotides of the 3' UTR. Examination of the nucleotide sequence of the first exon revealed an interesting characteristic, strongly suggesting that this exon may specify a functional domain of the C4BP alpha transcript. It includes two in-phase ATG codons, in a different frame respect to that coding the C4BP alpha polypeptide, followed by an in-frame termination codon, also within the first exon. Comparison between mouse and human C4BP alpha transcripts indicates conservation of this structure within the 5' UTR. C4BP is expressed in the liver and is an acute phase protein. A computer search of the genomic sequences upstream the transcription start site demonstrates the presence of potential cis-acting regulatory elements similar to those found in the promoters of other liver-expressed and/or acute phase genes.