Transcription factors and liver-specific genes.
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Animals↗
Biomedical subjects
R Cortese
Publications and source records attributed to R Cortese.
A POU-A related region dictates DNA binding specificity of LFB1/HNF1 by orienting the two XL-homeodomains in the dimer.
LFB1/HNF1 regulates the hepatocyte-specific transcription of several genes, binding as a dimer to cis-acting elements that match the inverted palindrome GTTAATNATTAAC. The DNA binding domain of LFB1/HNF1 is characterized by a unique tripartite structure that includes an unusually long homeodomain (domain C), a region related to the POU-specific A-box (domain B) and a short N-terminal dimerization domain (domain A). We report that a recombinant peptide corresponding to the isolated homeodomain of LFB1/HNF1 binds as a monomer to a half-palindrome binding site, but shows diminished sequence specificity. Domain B, in addition to the homeodomain, is required and sufficient for proper recognition of LFB1/HNF1-responsive sites. A protein consisting of only these latter two domains is a monomer in solution, but forms dimers upon DNA binding. The protein-protein contacts established within the bound dimer restrain the orientation of the two homeodomains with respect to one another, thus contributing in a critical fashion to the recognition of the dyad symmetry-related LFB1/HNF1 sites. The DNA-independent dimerization domain (domain A) is required to increase the affinity of DNA binding, but does not influence the dimer geometry.
Amino Acid Sequence↗
Chromosomal localization in man and rat of the genes encoding the liver-enriched transcription factors C/EBP, DBP, and HNF1/LFB-1 (CEBP, DBP, and transcription factor 1, TCF1, respectively) and of the hepatocyte growth factor/scatter factor gene (HGF).
By means of somatic cell hybrids segregating either human or rat chromosomes, we determined the chromosome localization of three genes encoding transcription factors expressed in hepatocytes, namely, C/EBP (CCAAT/enhancer binding protein), DBP (D site of albumin promoter binding protein), and HNF1/LFB-1 (designated transcription factor 1, gene symbol: TCF1), and of the hepatocyte growth factor gene, which is identical to the mitogenic and chemotactic factor designated scatter factor (gene symbol:HGF). The CEBP and DBP genes, encoding two related transcription factors, were found to be syntenic both on human chromosome 19 and on rat chromosome 1. These results provide further evidence for conservation of synteny on these two chromosomes (and on mouse chromosome 7). The TCF1 gene was found to be located on chromosome 12 in both man and rat, thereby defining a new segment of homology between these two species (and a segment of mouse chromosome 5). The HGF gene was mapped to rat chromosome 4, confirming homology between this chromosome and human chromosome 7, which carries the human HGF gene.
Animals↗
The dimerization domain of LFB1/HNF1 related transcription factors: a hidden four helix bundle?
LFB1/HNF1 alpha and LFB3/HNF1 beta bind DNA as dimers and form heterodimers together in vivo and in vitro. The dimerization domain has been located in both proteins in the 32 N-terminal residues. In previous papers we have described the conformational stability as determined by CD and the secondary structure by NMR studies of a peptide with the amino acid sequence of the dimerization domain of LFB1/HNF1 alpha. This study presents a more complete characterization of similar synthetic peptides spanning the LFB3/HNF1 beta dimerization domain and the alpha/beta heterodimer. The HNF1 peptides represent an example of structures which cannot be determined by NOE data alone because they are not sufficient to define one unique solution. An approach is presented which combines NMR data, the protein structure database and structural analyses according to known principles of protein structure. On this basis we are able to determine possible solutions and to identify a four helix bundle as the structure most consistent with the experimental evidence.
DNA-Binding Proteins↗
Extinction of alpha 1-antitrypsin gene expression in somatic cell hybrids: evidence for multiple controls.
Expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene is extinguished in hepatoma/fibroblast hybrids. To define the mechanism of extinction, we identified DNA sequences involved in this process by transiently transfecting mutant alpha 1AT promoters into parental and hybrid cells. The wild-type alpha 1AT promoter (-554 to +44 bp) was highly expressed in rat hepatoma cells, but activity was 100-fold less in fibroblasts or cell hybrids. Mutations in this region failed to activate alpha 1AT expression in nonhepatic cells, but mutations in the binding site for liver factor B1 (LF-B1) reduced hepatic-specific expression greater than 100-fold. Furthermore, the hybrid cells failed to express LF-B1-binding activity and mRNA. This suggested that alpha 1AT extinction in hybrids might be an indirect, lack-of-activation phenotype mediated primarily through repression of LF-B1. To test this possibility, we stably transfected an LF-B1 expression cassette into parental and hybrid cells and monitored expression of transfected and endogenous alpha 1AT genes. Surprisingly, although constitutive LF-B1 expression could activate alpha 1AT-CAT transgenes in these cells, it neither prevented nor reversed extinction of the chromosomal alpha 1AT genes. We conclude that although extinction of the LF-B1 trans-activator accompanies alpha 1AT extinction in cell hybrids, it does not play a causal role in this process.
Animals↗
Myocardial mechanical, biochemical, and structural alterations induced by chronic ethanol ingestion in rats.
To determine the effects of moderate ethanol consumption on the mechanical, biochemical, and structural characteristics of the heart, myocardial mechanical performance, contractile protein enzyme activity, and the number and size of myocytes were measured in male Fischer 344 rats after the ingestion of 30% oral ethanol. Papillary muscles removed from the left ventricle were greater in length, weight, and cross-sectional area than the corresponding muscles from the right side. However, no differences were found between control and ethanol-treated myocardium when either the left or right side was compared separately. Chronic ethanol ingestion resulted in an increase in resting tension in left ventricular muscles, with no alteration in peak developed tension. Moreover, time to peak tension was significantly prolonged, whereas a depression was observed in the peak rate of isometric tension development. Isotonically, left muscles from ethanol-treated rats revealed a prolongation of time to peak shortening and a marked depression in the velocity of shortening at physiological loads. No changes were noted in muscles from the right ventricle. Contractile protein enzyme activity revealed no differences in myofibrillar Mg(2+)-ATPase activity in right and left ventricular myocardium between control and ethanol-treated rats in the presence of EGTA. However, at physiological activating levels of calcium, an upward shift of the myofibrillar Mg(2+)-ATPase activity-calcium curve occurred in left myocardium, whereas a depression in this relation was seen in the right ventricle. As a result of chronic ethanol intake, a decrease was noted in the volume percent of myocardium occupied by myocytes, and that myocyte cell volume per nucleus was found to remain essentially constant throughout the various layers of the ventricular wall. Importantly, a 14% significant decrease in the total number of myocyte nuclei was demonstrated in the left ventricular myocardium of rats on chronic ethanol consumption. Thus, chronic but moderate alcohol ingestion resulted in depressed contractile performance, alterations in myofibrillar Mg(2+)-ATPase activity, and myocyte loss. These events may serve to function as preliminary indicators of the onset of heart failure of alcoholic origin in this animal model.
Adenosine Triphosphatases↗
LFB1 and LFB3 homeoproteins are sequentially expressed during kidney development.
LFB1 (HNF-1/HNF-1 alpha/APF) and LFB3 (vHNF-1/HNF-1 beta) are two homeoproteins involved in the transcriptional regulation of several liver-specific genes. Both genes are expressed in the polarized epithelia of a wide range of tissues, including liver, the digestive tract and kidney. We have analyzed the expression pattern of LFB1 and LFB3 in the developing rat kidney by in situ hybridization. Our results show that LFB3 transcripts can be detected in mesoderm-derived cells as soon as they are induced to differentiate into a polarized epithelium, while LFB1 transcripts appear only at a later stage when the three different segments of the nephron become apparent. LFB1 transcripts are restricted to the proximal and distal tubules, whereas LFB3 is also detected in the collecting ducts. Neither LFB1 nor LFB3 are expressed in the glomeruli or in the transition epithelia of the ureters and of the urinary bladder, none of which are involved in active transport mechanisms. The sequential activation of these two genes is also observed in transfilter organ cultures of nephrogenic mesenchyme at different stages after induction. This expression pattern suggests that LFB3 and LFB1 play a role in two critical stages of the developmentally regulated conversion of the nephric mesenchyme into a polarized epithelium: the early inductory phase (LFB3) and the postinductory phase (LFB1+LFB3).
Animals↗
The different tissue transcription patterns of genes for HNF-1, C/EBP, HNF-3, and HNF-4, protein factors that govern liver-specific transcription.
The transcription factors that act in hepatocyte-specific gene expression include proteins that are present mainly in liver cells (HNF-1/LFB1, C/EBP, HNF-3, HNF-4) (HNF, hepatocyte nuclear factor; C/EBP, rat enhancer binding protein) and proteins that are widely distributed (AP-1, NF-1, NF-Y/ACF). We show here that the genes encoding each of these liver-enriched factors exhibit different patterns of transcriptional control in different tissues. In addition, there were several instances in which transcription was detected (e.g., for HNF-1) when no mRNA or specific DNA binding protein was found, suggesting the importance of posttranscriptional control in some instances for these factors. These experiments identify C/EBP, HNF-3, and HNF-4, and perhaps also HNF-1, as targets for the study of cascades of transcriptionally controlled transcription factors in differentiated cells.
Base Sequence↗
The transcription factor LF-A1 interacts with a bipartite recognition sequence in the promoter regions of several liver-specific genes.
The transcription factors LF-A1 and LF-B1 are required for the cell-specific expression of the human alpha 1-antitrypsin gene in hepatocytes. We report here the purification and preliminary characterization of LF-A1. This protein, purified to homogeneity from calf liver nuclei by site-specific DNA affinity chromatography and reverse-phase HPLC, has a molecular mass of 40 kDa. Binding sites of LF-A1 are present in the promoter regions of several genes expressed in the liver (alpha 1-antitrypsin, apolipoproteins A1, B1, A4 and pyruvate kinase). Interestingly, the binding site of LF-A1 is bipartite and consists of two short sequence motifs (consensus: TGGACT/CT/C and TGGCCC) separated by a variable 'spacer' region. Insertion or deletion of 1-4 nucleotides in the 'spacer' region of the site in the alpha 1-antitrypsin promoter does not abolish DNA binding.
Animals↗
Isolation of two cDNAs encoding zinc finger proteins which bind to the alpha 1-antitrypsin promoter and to the major histocompatibility complex class I enhancer.
Two partial cDNAs coding for DNA-binding proteins (AT-BP1 and AT-BP2) have been isolated. Both proteins, when prepared from lambda gt11 lysogens, bind to the B-domain of the alpha 1-antitrypsin promoter, an element which is important for the liver-specific expression of alpha 1-antitrypsin. Analysis of the cDNA sequences encoding these proteins reveals that both contain two zinc fingers of the Cys2-His2 type followed by a highly acidic stretch of 20 amino acids. AT-BP1 contains a second putative DNA-binding domain consisting of an 8-fold repeat of a SPKK (Ser-Pro-Lys/Arg-Lys/Arg) motif. Both proteins bind to the NF-kappa B recognition site in the MHC gene enhancer with significantly higher affinity than to the kappa immunoglobulin gene enhancer, or to the B-domain of the alpha 1-antitrypsin gene promoter. Analysis of mRNA expression shows that AT-BP1 and AT-BP2 are expressed in all the tissues examined. While the physiological roles of AT-BP1 and AT-BP2 remain to be elucidated, their predicted amino acid sequence and their DNA-binding characteristics suggest a role as transcriptional regulators.
Amino Acid Sequence↗
Circular dichroism study on the conformational stability of the dimerization domain of transcription factor LFB1.
LFB1, a dimeric DNA binding protein, is a major determinant of hepatocyte-specific transcription. The thermal and chemical equilibrium unfolding of a 32-residue alpha-helical peptide comprising its dimerization domain (B1-Dim) was monitored by circular dichroism spectroscopy. The conformational stability of this peptide is shown to be concentration dependent, and the unfolding reaction is described as a two-state transition between folded dimers and unfolded monomers. The thermodynamic parameters associated with the unfolding reaction were determined under the two-state assumption by the van't Hoff procedure. The enthalpy of unfolding increases linearly with temperature, and the corresponding value of delta Cp, the difference in heat capacity between the unfolded and the folded forms of the peptide, is estimated to be ca. 0.7 kcal mol-1 K-1. The dimeric folded structure of the peptide is stabilized, at 25 degrees C, by a delta G of about 11.5 kcal mol-1, which is equivalent to a dimerization constant greater than 10(8) mol-1. These results indicate that the dimerization domain of LFB1 can fold and dimerize independently of the rest of the protein, with a thermodynamic stability comparable to that of a small globular protein.
Amino Acid Sequence↗
1H resonance assignment and secondary structure determination of the dimerization domain of transcription factor LFB1.
We have started the structure determination of the dimerization domain of LFB1 in solution by nuclear magnetic resonance in order to elucidate the way that the LFB1 protein dimerizes and then interacts with DNA. A 32 amino acid peptide was synthesized, and full assignment of the NMR resonances in acidic solution was achieved. The secondary structure determination is presented here. Three structurally distinct regions can be distinguished. The N-terminal region from residues 1 to 6 is extended. Two helical regions span from residues 7 to 18 and from 23 to 32. The absence of dipolar effects involving residues more than four positions apart in the sequence excludes the possibilities both of a four-helix bundle formed by two hairpins and of an antiparallel dimer; the domain must therefore be arranged as a parallel dimer formed by kinked monomers. This structural solution presents important differences from the leucine zipper-type structure observed in other transcriptional activators. Although further studies are still necessary to determine the 3D structure of the peptide, we can exclude the possibility of a coiled-coil structure.
Amino Acid Sequence↗
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.
Aging↗
LFB3, a heterodimer-forming homeoprotein of the LFB1 family, is expressed in specialized epithelia.
We have cloned and characterized a mouse cDNA coding for LFB3, a DNA binding protein containing an extra-large homeodomain. The first 315 amino acids of LFB3 are highly homologous to the DNA binding domain of LFB1, a regulatory protein involved in the expression of several liver-specific genes. LFB3 is a transcriptional activator which binds to DNA as a dimer and forms heterodimers with LFB1 both in vitro and in vivo. However, LFB3 expression seems not to be directly correlated with the liver-specific phenotype, since it is detected in dedifferentiated hepatoma cell lines which express neither LFB1 nor several liver-specific genes. LFB3 expression starts before that of LFB1 during mouse and rat development, and is strongly increased upon retinoic acid induced differentiation of F9 embryonic carcinoma cells. LFB3 and LFB1 are expressed in the epithelial component of many organs of endodermal and mesodermal origin, suggesting that they may play a more general role associated with the differentiation of specialized epithelia.
Amino Acid Sequence↗
The Interleukin-6-dependent DNA-binding protein gene (transcription factor 5: TCF5) maps to human chromosome 20 and rat chromosome 3, the IL6 receptor locus (IL6R) to human chromosome 1 and rat chromosome 2, and the rat IL6 gene to rat chromosome 4.
Using two panels of somatic cell hybrids segregating either human or rat chromosomes, the gene encoding the interleukin-6-dependent DNA-binding protein, also called liver activator protein (designated transcription factor 5: TCF5), was assigned to human chromosome 20 and to rat chromosome 3. The TCF5 gene might be identical with the NF-IL6 gene. The locus encoding the IL6 receptor gene (IL6R) was localized to human chromosome 1 and rat chromosome 2. An IL6R-like (IL6RL) locus was also assigned to human chromosome 9. In addition, the rat interleukin-6 (IL6) gene was assigned to rat chromosome 4. These mapping data allow one to extend comparison between the rat, mouse, and human gene maps.
Animals↗
Transcriptional regulation of liver-specific gene expression.
Liver-specific gene expression is regulated by four families of transcriptional activatory proteins that are not liver-specific, but are still restricted to a subset of tissues. The current opinion is that liver-specificity is a consequence of the combinatorial action of these factors, which could represent 'functional compartments' coexisting in the hepatocyte.
Animals↗
Biologically active recombinant prothrombin and antithrombin III expressed in a human hepatoma/vaccinia virus system.
Several systems have been devised that are based on viral promoters suitable for gene expression in eukaryotic cells. One such system, vaccinia virus, has been successfully used to express a variety of heterologous proteins following the construction of a recombinant virus. Indeed, because of the ability of vaccinia virus to infect a wide range of host cells, the expression system can be custom-designed so that a cell line can be instrumental in ensuring the correct processing of the recombinant polypeptide. We show that recombinant vaccinia virus and human hepatoma cells in culture are an efficient expression system with which to produce correctly modified and biologically active human prothrombin (15 micrograms/10(7) cells) and antithrombin III (40 micrograms/10(7) cells).
Animals↗
Generation of small mutation in large genomic fragments by homologous recombination: description of the technique and examples of its use.
We have developed a technique of homologous recombination in bacteria which allows the mutagenesis of large genomic fragments cloned in cosmids. The desired mutation is first introduced into a plasmid clone and is then transferred to the appropriate cosmid clone by the means of double antibiotic selection coupled with phenotypic selection. We describe three different types of construct made by this technique.
Cloning, Molecular↗