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.     

Developmental changes in the methylation of the rat albumin and alpha-fetoprotein genes

We have analyzed methylation of the rat albumin and alpha-fetoprotein (AFP) genes by hydridizing labeled cDNA clones to HpaII and MspI digests of DNA from different stages of development. These CCGG-cutting enzymes distinguish 5-methylcystosine in mCCGG (sensitive to HpaII) and CmCGG (sensitive to MspI). In the liver, the albumin gene is heavily methylated at 18 days gestation and uniformly demethylated in the adult. The AFP gene is also heavily methylated at 18 days gestation, and develops demethylated regions at the 3' half of the gene in the adult. These methylation changes are not observed in other embryonic or adult tissues. We also evaluated expression of these genes by measuring their corresponding mRNAs. The albumin gene is actively transcribed in 18-day fetal liver, when it is heavily methylated, as well as in adult liver, when it is unmethylated. In contrast, the AFP gene is transcribed only in fetal liver, even though it is less methylated in adult liver. These findings suggest that specific methylation changes are associated with changes in gene expression, but that this association is not adequately described by the simple hypothesis that methylation turns genes off.     

DNaseI sensitivity of the rat albumin and alpha-fetoprotein genes.

We have analyzed the DNaseI sensitivity of chromatin from the rat albumin and alpha-fetoprotein genes in the fetal liver (which synthesizes albumin and alpha-fetoprotein), adult liver (which synthesizes albumin), fetal yolk sac (which synthesizes alpha-fetoprotein), and adult kidney (which synthesizes neither). Active genes were much more sensitive than their kidney counterparts, and the adult liver alpha-fetoprotein and fetal yolk sac albumin genes showed intermediate levels of sensitivity. Sensitivity was analyzed as a function of the extent of DNaseI digestion. Rate constants were calculated for the degradation of individual DNA hybridization bands and normalized to the intrinsic rate constants of the same bands degraded in purified DNA. This enabled us to eliminate the inconsistencies that otherwise result from comparing chromatin sensitivity of different DNA sequences, or chromatin sensitivity in different nuclear environments.     

Localization of DNA protein-binding sites in the proximal and distal promoter regions of the mouse alpha-fetoprotein gene.

DNase I footprinting assays were performed to identify the binding sites for putative trans-acting factors involved in the control of alpha-fetoprotein (AFP) gene expression using mouse AFP promoter fragments (-839 to +56) and nuclear protein extracts from fetal, newborn, and adult livers and from brain and kidney. Our studies have shown that with nuclear protein from adult mouse liver, there are 14 protected regions in the AFP promoter up to -839 base pairs (bp). Region I (-82 to -43) was protected by at least three different factors, one of which is CCAAT-binding/enhancer-binding protein. This region is highly conserved in the mouse, rat, and human AFP genes and has been shown previously to be essential for the regulation of tissue-specific expression in mouse. Differences in DNase I protection with fetal, newborn, and adult nuclear proteins have been observed in the proximal promoter region (up to -202 bp) and in regions further upstream (up to -839 bp). Significant differences among liver, kidney, and brain nuclear protein-binding sites have also been observed. In these studies, we have mapped the fetal and adult nuclear protein-binding sites of the cis-acting DNA sequences of the mouse AFP proximal promoter (up to -200) and have identified specific protein-binding sites in the distal promoter (-200 to -839). We have also identified the sites of the AFP promoter which bind nuclear proteins from highly differentiated tissues in which AFP is not expressed.     

Regulators of fetal liver differentiation in vitro

Seventeen-day-old fetal rat hepatocytes were employed to examine factors required to promote differentiation in vitro. In the absence of effectors, primary fetal hepatocytes dedifferentiated, as characterized by the rapid decline in synthesis of fetal alpha-fetoprotein (AFP), albumin, and transferrin. On the other hand, cells maintained in the presence of glucocorticoid hormone produced high levels of albumin and transferrin. Glucocorticoid could not prevent the decline in fetal AFP synthesis, but induced synthesis of the 65K variant AFP--the major AFP species produced by adult rat liver. Fetal hepatocytes maintained in the presence of 8-bromo-cAMP (8-BrcAMP), or methyl isobutyl xanthine (MIX), an agent that increases intracellular cAMP levels, synthesized high levels of fetal AFP and albumin but reduced levels of transferrin. Both glucocorticoid and 8-BrcAMP or MIX induced expression of adult liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), suggesting that these fetal hepatocytes have matured. Cells maintained in the presence of glucocorticoid hormone and MIX (or 8-BrcAMP) contained more albumin, TAT, and PEPCK mRNAs and synthesized increased amounts of the 65K variant AFP than those with either agent alone. However, the glucocorticoid/MIX cells produced intermediate levels of the fetal AFP and transferrin. Our data indicate that both glucocorticoid hormone and cAMP are necessary for optimal differentiation of fetal hepatocytes in vitro.     

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.     

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.     

核蛋白成分参与胚胎肝细胞AFP基因的活化

利用体外转录分析方法,在大鼠胚胎肝细胞核蛋白中,检测到促进AFP基因转录的蛋白因子,用同样的方法在成年大鼠肝细胞核蛋白中没有检测到促进AFP基因转录的任何成分。DNA结合分析找到了可能为蛋白因子的核蛋白成分,它们的大小分别为89、50、46和36kDa。