Expression of mRNAs for alpha-fetoprotein (AFP) and albumin and incorporation of AFP and docosahexaenoic acid in baboon fetuses.

alpha-Fetoprotein and albumin, two members of a multigene family, reversibly bind fatty acids with high affinity. The origin of alpha-fetoprotein (AFP) and albumin present in fetal tissues other than the liver and yolk sac is a subject of controversy. In this work, we have searched for the presence of the albumin and AFP mRNA molecules in different fetal organs of the baboon (Papio cinocephalus), using a highly sensitive gel-blot hybridization assay with human albumin and AFP cDNA probes. Large amounts of albumin and AFP mRNA molecules were found in the fetal liver; significant quantities were also present in the gastrointestinal tract and in the kidney. No detectable levels were found in the other tissues examined (brain, skin, spleen, pancreas, muscle, heart, thymus, placenta, and amnion). After injection of radiolabeled AFP into pregnant baboons, all fetal tissues took up the protein. White adipose tissue, kidney, intestine, lung, liver, and cerebral cortex showed a great uptake of exogenous AFP. [14C]Docosahexaenoic acid (22:6, n-3), injected at the same time, was actively transferred from the maternal compartment across the placenta and incorporated into cellular lipids by all fetal tissues and particularly by liver (around 70% of total incorporation). The levels of [14C]docosahexaenoic acid per gram of tissue increased in the order: maternal blood less than placenta less than fetal liver, indicating a selective accumulation of this fatty acid by the fetus. These results indicate that intracellular AFP in non-hepatic tissues of the developing baboon is, for the most part, of plasma origin.

Binding of a liver-specific factor to the human albumin gene promoter and enhancer.

A segment of 1,022 base pairs (bp) of the 5'-flanking region of the human albumin gene, fused to a reporter gene, directs hepatoma-specific transcription. Three functionally distinct regions have been defined by deletion analysis: (i) a negative element located between bp -673 and -486, (ii) an enhancer essential for efficient albumin transcription located between bp -486 and -221, and (iii) a promoter spanning a region highly conserved throughout evolution. Protein-binding studies have demonstrated that a liver trans-acting factor which interacts with the enhancer region is the well-characterized transcription factor LF-B1, which binds to promoters of several liver-specific genes. A synthetic oligodeoxynucleotide containing the LF-B1-binding site is sufficient to act as a tissue-specific transcriptional enhancer when placed in front of the albumin promoter. The fact that the same binding site functions in both an enhancer and a promoter suggests that these two elements influence the initiation of transcription through similar mechanisms.

The organization of repetitive sequences in the albumin and alpha-fetoprotein gene loci in the rat.

The distribution of middle repetitive sequences in the genic and extragenic regions of the rat albumin and alpha-fetoprotein genes was analyzed. Their presence was determined by probing Southern blots of restriction fragments of albumin and alpha-fetoprotein genomic subclones with 32P-labeled total rat DNA. Repetitive sequences were detected in both genes. They were classified as weak, moderate and intense hybridizing elements according to the intensity of hybridization. Weak repetitive sequences were characterized as dG.dT repeats by using 32P-labeled poly-(dG.dT)(dC.dA) oligomer probe. They occurred in 5' and 3' extragenic regions of the two genes and in introns 4 and 5 of the albumin gene. The moderate repetitive sequence present in intron 6 of the albumin gene was identified as the rat SINES element. 4D12. The intense repetitive sequence, localized in the 3' non-coding region of the albumin gene, corresponded to the terminal segment of a rat high repeat long interspersed DNA family, L1Rn. 4D12 and L1Rn sequences were also scattered throughout the alpha-fetoprotein locus as moderate and intense repetitive elements, respectively, but their distribution was different from that of the albumin genomic region. These results indicate that repetitive sequences invaded the two loci in a non-conservative manner.

Albumin and alpha-fetoprotein gene expression in various nonhepatic rat tissues.

Albumin and alpha-fetoprotein (AFP), two major serum proteins, are synthesized predominantly in the liver and yolk sac of mammals. In the present paper we report on the developmental expression of the corresponding genes in nonhepatic rat tissues. Significant quantities of mature albumin and AFP mRNAs were revealed in kidney, pancreas, heart, and lung of fetal and/or newborn rats using dot blot and Northern blot assays. Very low levels of these mRNA sequences were also detected in adult kidney and pancreas using sensitive RNA-cDNA solution hybridization assays. In situ hybridization analysis revealed that the albumin and AFP gene transcripts are present in the tubular cells of the 20-day-old fetal kidney. In order to elucidate further the mechanisms governing this expression, we studied the chromatin structure and methylation pattern in the 5'-end of these two genes. A faint band, corresponding to a specific DNase I-hypersensitive site upstream from the albumin gene, was detected in the fetal and neonatal kidney nuclei but not in adult kidney. For both genes, a site CG, demethylation of which is correlated with expression in liver and hepatoma cell lines, is highly methylated in fetal kidney even though AFP and albumin genes are expressed. Taken together, these results show the presence of a cell population in the rat kidney that actively transcribes both the albumin and AFP genes. The expression of these genes may be mediated by mechanisms differing in at least some steps from those exerted in the liver.

Differences in methylation patterns of the alpha-fetoprotein and albumin genes in hepatic and non hepatic developing rat tissues.

By use of different restriction enzymes sensitive to internal cytosine methylation (HpaII, AvaI, HhaI) we have analysed the methylation patterns of albumin and AFP genes in tissues and cell lines with high (liver, yolk sac, hepatoma cell lines), low (fetal and neonatal kidney) or undetectable (spleen, JF1 fibroblasts) expression of either gene. We show that expression of the AFP gene is associated to the demethylation of a whole region or domain extending from -4 to +3 Kb. Moreover, demethylation of a site located at the upstream limit of this domain appears to be correlated with the commitment of the cell type to synthesize AFP. As concerns the albumin gene, we show that the domain in which demethylation is correlated with active gene transcription in hepatoma cell lines has different borders than in tissue. This difference might be related to the different amounts of mRNA synthesized or to an alteration in gene regulation in tumor cells. Finally, we show that low expression of albumin and AFP genes in fetal and neonatal kidney is not correlated with domain demethylation, suggesting that the regulatory mechanisms of expression of these genes are different in kidney as compared with liver.

The rat alpha-fetoprotein and albumin genes. Transcriptional control and comparison of the sequence organization and promoter region.

Functional and structural approaches were used to characterize the transcription units of the rat alpha-feto-protein (AFP) and albumin genes. A cell-free nuclear transcription assay and several genomic clones were used to show that: 1) the rate of transcription of these genes is closely related to the levels of corresponding mRNAs in the yolk sac and during rat liver development, indicating that the expression of the albumin and AFP genes is mainly regulated at the transcriptional level in the rat, and 2) the in vivo 5' end boundaries of the rat AFP and albumin transcription domains were mapped near the respective first exons. Due to the presence of repeated sequences, the 3' end boundary of both genes could not be accurately defined in the same manner. 3) No transcription could be detected until 7 kilobases upstream from the cap site of these genes. In addition, the organization of the rat AFP gene was analyzed by restriction endonuclease mapping, S1 nuclease mapping, and nucleotide sequencing. Our results indicate that: 1) the rat AFP gene is 20 kilobase pairs long and is split into 15 exons by 14 intervening sequences; 2) the transcription initiation site of the rat AFP gene is heterogenous; 3) the 5'-flanking region upstream from the rat AFP gene exhibits 60-90% similarity with the mouse and human AFP genes while no major nucleotide identity is found with the rat albumin gene; 4) a 90-base pair sequence present as one copy upstream from the rat and mouse AFP genes is present as two copies in the human genome; 5) several inverted repeats are mapped in the 5'-flanking region indicating potential stem-loop structures. One highly conserved structure encompasses an enhancer-like core sequence and the sequence recognized by the TGGCA-binding protein.

Differential stability of the higher order structure of chromatin associated with genes having different transcriptional activity.

We have investigated the stability of the higher order structure of chromatin associated to genes which display a different transcriptional activity in adult rat live. Nuclei were digested with micrococcal nuclease and chromatin was fractionated by sedimentation in sucrose gradients. Specific DNA sequences were revealed by dot-blotting. In conditions of physiological ionic strength the distribution of the inactive gamma-casein gene sequences is similar than the bulk of chromatin. In the same conditions the relative content of the albumin gene, highly expressed in adult rat liver, revealed an enhanced instability of the chromatin superstructure. The distribution of the potentially active but silent alpha-fetoprotein sequences in adult liver showed an intermediate unfolding of its chromatin superstructure. These distinct behavior was not observed in non-physiological ionic strength conditions. Our results suggest that distinct folding of the local higher order structure of chromatin actually occurs in the region of active, potentially active and inactive genes.

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.

Specific sets of DNase I-hypersensitive sites are associated with the potential and overt expression of the rat albumin and alpha-fetoprotein genes.

We have examined the chromatin structure of the 5'-flanking region of the albumin and alpha-fetoprotein (Afp) genes in different developing rat tissues and cloned cell lines that display various functional states of these genes. Nuclease-hypersensitive sites were probed with DNase I, using an indirect end-labeling technique. In albumin-producing rat cells two major DNase I-hypersensitive sites were found near the promoter region and one additional site was located approximately 3 kilobases (kb) upstream. Similarly, in Afp-producing rat tissues and cell lines we mapped one DNase I-hypersensitive region close to the promoter region and two cleavage sites further upstream at approximately 2.2 and approximately 3.8 kb from the cap site. The DNase I-hypersensitive sites of both genes were absent in nonhepatic rat cells and therefore appear to be tissue specific. Loss of specific sets of DNase I-hypersensitive sites accompanies the cessation of transcription for the Afp gene in adult rat liver and in a "dedifferentiated" hepatoma cell line. Likewise, specific sets of DNase I-hypersensitive sites disappear during the inactivation of the albumin gene in hepatoma cells. The distal upstream sites of the Afp and albumin genes display the same DNase I sensitivity in expressing and potentially expressible states. These findings suggest that reversible changes in short chromatin regions may be involved in the actual transcription of the albumin and Afp genes, while more permanent tissue-specific changes at other sites correlate with the capacity of these genes to be expressed during hepatic differentiation and neoplasia.

Phenobarbital-mediated modulation of gene expression in rat liver. Analysis of cDNA clones.

Phenobarbital evokes a pleiotypic response in the liver characterized by cell hypertrophy and mono-oxygenase induction. These phenomena arise through complex modulation mechanisms changing the pattern of protein synthesis, distinct from those triggered by other well known inducers, like steroid hormones or polycyclic hydrocarbons. To investigate the mechanisms involved in regulating the expression of the phenobarbital-inducible tissue-specific genes, we constructed two libraries of recombinant bacterial plasmids pBR322 in Escherichia coli. Each library contains cDNA copies of polysomal poly(A)-rich RNA obtained from control and 16-h phenobarbital-induced rat liver. A thousand cloned sequences from each library were screened by double-cross colony hybridization using [32P]cDNA prepared from homologous and heterologous poly(A)-rich RNAs as the probes. The statistical analysis of the results revealed that phenobarbital treatment significantly changes the relative abundance of different polysomal mRNA classes in rat liver. Clones corresponding to mRNAs clearly induced following phenobarbital treatment have been further selected by plasmid DNA dot hybridization, and used as probes for measuring the changes in each mRNA concentration in the whole cell and in the polysomal RNAs from rat livers, at different times after phenobarbital treatment. The fact that changes in the concentration of each specific mRNA in the polysomes does not parallel the variation of its total concentration in the cell indicates that the induced modulation of protein synthesis in the liver is brought about by mechanisms involving both transcriptional and translational regulation, since besides the increases in whole cellular mRNA concentration a marked mobilization of mRNA into active polysomes could be demonstrated during the onset of the adaptive response to phenobarbital.

Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period.

To analyze at the cellular level the decrease in alpha-fetoprotein (AFP) gene expression during the early postnatal growth, we searched for AFP gene transcripts by in situ hybridization using a specific cDNA probe, and for the corresponding protein by immunocytochemistry, on rat liver sections at various times of the perinatal period. The relative number of mRNA sequences was evaluated by Northern blot analysis. Albumin (ALB) gene expression was studied simultaneously with the same techniques. In 17-19-d-old fetuses all hepatocytes express simultaneously, for both genes, the mRNAs and the corresponding proteins. During the first postnatal weeks, at a time when the global number of AFP mRNA molecules decreases, all hepatocytes still contain cytoplasmic transcripts and protein. A zonal heterogeneity in the level of AFP gene expression develops around the first week, a higher number of gene products being detected in perivenous than in periportal hepatocytes. This heterogeneity persists until the fourth week when AFP mRNA sequences and protein are barely detectable. All hepatocytes express the ALB gene after birth, but at around the second week, a periportal intensification of the in situ hybridization signal and immunostaining becomes apparent. Our data indicate that co-expression of the AFP and ALB genes by all hepatocytes is a normal step in liver ontogeny; the diminution of AFP gene expression after birth is not the result of the disappearance of specialized cell clones; and zonal quantitative differences in the level of AFP and ALB gene expression are observed within the maturing liver lobule.

Activation of alpha-fetoprotein synthesis in rat hepatoma cells with reduced sensitivity to dexamethasone.

The Faza 967 'differentiated', dexamethasone (DEX)-sensitive cell line of Reuber rat hepatoma cells does not synthesize detectable amounts of alpha-fetoprotein (AFP), whereas it does produce albumin. AFP production was activated in 'differentiated' variants of Faza 967 cells with reduced glucocorticoid sensitivity upon culture for several months in the presence of high concentrations of dexamethasone. The stability of AFP production differed among the variants, while albumin synthesis did not change, thus indicating that the regulation of these two genes is not co-ordinated. Using molecular hybridization techniques, we found that the AFP message could not be detected in the non-AFP-producing cells, suggesting that the lack of AFP synthesis most probably originates from a transcriptional block of the AFP gene. AFP-producing and non-AFP-producing variants of Faza 967 cells constitute a valuable model system for studying the regulatory mechanisms involved in the activation and inactivation of the gene coding for the oncodevelopmental protein, AFP.

Human inter-alpha-trypsin-inhibitor: characterization and partial nucleotide sequencing of a light chain-encoding cDNA.

A synthetic Inter-alpha-Trypsin-Inhibitor (ITI) -specific oligonucleotide probe was used to isolate a clone from a human liver cDNA library. The amino-acid sequence deduced from partial nucleotide sequencing of the corresponding cDNA insert perfectly matched a known ITI sequence, apart from an as yet unreported C-terminal dipeptide. Hybridization on Northern blots evidenced that this cDNA insert originated from an ITI light chain-encoding mRNA whose size was estimated to be 1 300 bases.

A lethal deletion on mouse chromosome 7 affects regulation of liver-cell-specific functions: posttranscriptional control of serum protein and transcriptional control of aldolase B synthesis.

Steady-state levels of mRNAs were determined for the serum proteins albumin, alpha-fetoprotein (AFP), and transferrin, as well as for aldolase B in livers of newborn mice homozygous for a radiation-induced lethal deletion (c14CoS) in chromosome 7. Deficiencies in synthesis and secretion of the serum proteins as well as in activities of certain liver-specific enzymes characterize these homozygotes. The results of RNA dot and gel-blot hybridizations with the respective cloned cDNA probes showed a decrease to one-fourth of aldolase B mRNA levels in homozygous mutant livers compared to normal littermates, in contrast to normal levels of mRNA sequences for the three serum proteins in the mutants. Furthermore, the mRNA sequences were shown to be present as mature mRNA molecules in both mutant and normal littermate livers. We suggest that the deficiencies of liver-specific serum proteins and those of the enzymes caused by the lethal deletions around the albino locus on chromosome 7 of the mouse are due to different causes. In the case of the liver-specific enzyme examined here--i.e., aldolase B--control at the level of transcription or of message stability is affected in the homozygous deletion mutants, whereas the deficiencies of serum proteins are not reflected on the mRNA level and owe their origin to an effect on a posttranscriptional or translational level. These results lend further support to the assumption that the deleted portion of the genome includes genes concerned with the control and regulation of liver cell differentiation.

Structural basis for restriction-site polymorphism at the albumin locus in inbred strains of rats.

Two types of variant EcoRI restriction enzyme patterns of albumin-gene DNA fragments are found in different inbred strains of rats and reflect allelic polymorphism. The structural basis of the two allelic forms has been analyzed by mapping the EcoRI fragments using cloned albumin cDNA probes corresponding to the 5' or 3' end of the rat albumin mRNA and different genomic subclones. Additional restriction fragment length polymorphism has been detected using the restriction endonucleases HindIII and MspI. The results suggest that the two allelic variants differ from each other by multiple cleavage-site variations (base-pair substitutions) and by an insertion or deletion of DNA sequences. An extensive DNA sequence variation appears to exist between the two forms of the albumin gene; we have estimated that as much as 4% of the nucleotides in this region varied between the two alleles. All of this genetic variation is found in the intervening sequences of the gene and has no phenotypic manifestation.

Organization of the albumin and alpha-fetoprotein genes in fetal and adult rat tissues, and rat hepatomas.

We compared the organization of the albumin and alpha-fetoprotein (AFP) genes in chromosomal DNA from different fetal and adult rat tissues as well as from two rat hepatomas. These two genes are expressed at widely different levels in the tissues and hepatomas analysed. Southern blots of DNAs digested with the restriction endonucleases EcoRI, HindIII or MspI were hybridized to albumin and AFP complementary DNA (cDNA) and genomic probes. No significant difference was observed in the hybridization patterns obtained for the DNAs from the different tissues, except for some interstrain variation between the chromosomal DNAs isolated from Sprague-Dawley and Buffalo rats, which was due to allelic polymorphism. We cannot rule out the possibility of changes in chromosomal gene organization which would result either in small alterations of restriction fragment size or in translocations of large blocks of DNA containing whole sets of restriction enzyme fragments within the chromosome; however, our results indicate that the gross organization of the albumin and AFP genes remains constant throughout the regulatory processes involved in the tissue- and time-specific transcription of these genes.

DNA restriction polymorphism of alpha-fetoprotein gene after digestion by MspI endonuclease.

Hybridization of alpha-fetoprotein clones cDNA with human DNAs digested by seven restriction endonucleases reveals one polymorphism. This polymorphism, detected after restriction by MspI, is at low frequency (f = 0.02) and is validated by family analysis. It corresponds to an intronic sequence, for a methylated site external to the probes utilized.

Differential DNase I sensitivity of the albumin and alpha-fetoprotein genes in chromatin from rat tissues and cell lines.

We have examined the DNase I sensitivity of the albumin and alpha-fetoprotein (AFP) genes in different rat tissues (adult liver and kidney) and cloned cell lines (hepatoma 7777-C8, JF1 fibroblasts), which show drastic differences in the level of expression of these two genes. This was done by studying the disappearance of defined restriction endonuclease fragments of these genes as a function of limited DNase I digestion. The sensitivity of these genes was compared to that of a gene not expressed in the hepatic cells and to that of a ubiquitously expressed gene. In nuclei from adult rat liver the albumin and AFP genes were preferentially degraded by the nucleolytic action of DNase I, whereas they were not in rat kidney nuclei. In the hepatoma cells the AFP gene was much more sensitive to DNase I digestion than the albumin gene; both genes were very resistant to DNase I action in fibroblastic nuclei. When analyzed in relation to the level of gene expression our results indicate that alterations in the chromatin structure of the albumin and AFP genes might be involved in the early establishment of the tissue-specific potential of overt gene expression; such alterations reflected in an altered DNase I sensitivity do not appear to be responsible for the changes in gene activity occurring during the terminal differentiation of the hepatocyte; and modifications in the chromatin structure of these genes might occur during oncogenic events; these structural modifications could be related to the changes in gene expression observed in hepatocarcinogenic processes.

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat.

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.

Selective detection of rat and mouse specific albumin and alpha-fetoprotein mRNA molecules under highly stringent hybridization conditions.

The molecular analysis of some important interactions observed between the parental genomes in interspecific cell hybrids requires the availability of highly specific hybridization assays to selectively quantitate mRNA sequences coding for the same protein but transcribed from the two different genomes. Specific hybridization techniques which should permit the selective detection of rat and mouse albumin and alpha-fetoprotein (AFP) mRNA molecules in a mixture of the two types of mRNAs are presented here. The high degree of homology existing between the AFP mRNA sequences coding for mouse and rat AFP, and, presumably, albumin, results in extensive cross-hybridization with the cDNA probes under standard hybridization conditions. No size differences could be detected between the two types of mRNA molecules from the two species. A Tm difference of 7 degrees C between the intra- and interspecific mRNA:rat cDNA hybrids allowed the establishment of highly stringent solution hybridization conditions necessary to measure separately the contents of rat albumin and AFP mRNAs. Mouse albumin and AFP cDNA clones were then isolated from mouse liver and yolk sac cDNA libraries, and used to show the usefulness of highly stringent washing conditions to discriminate between rat and mouse albumin and AFP mRNA molecules in conventional "Northern blotting" techniques. In combination with the solution hybridization assay, these filter hybridization techniques can be used to specifically quantitate the content of rat and mouse albumin and AFP mRNA molecules in interspecific cell hybrids.