The CCAAT/enhancer binding proteins (C/EBP) alpha and beta of the bZIP family of transcription factors each occur as multiple forms due to translation initiation at different in-frame AUG codons from the same messenger RNA. The C/EBP alpha mRNAs of chicken, rat and Xenopus all contain a small 5' open reading frame (5'ORF) whose size (18 nucleotides) and distance (seven nucleotides) to the C/EBP alpha cistron has been conserved in vertebrate evolution. The present studies shows that the small 5'ORF is crucial to the leaky scanning mechanism of ribosomes causing a fraction of them to ignore the first C/EBP alpha AUG codon and to start at internal AUGs. Our data challenge the view that translational start site multiplicity is mainly governed by the sequence context of the potential initiation codons. Western analysis showed that the two major chicken C/EBP alpha translation products, the full-length cC/EBP alpha-42 which acts a trans-activator in liver and the N-terminally truncated cC/EBP alpha-29 which lacks transcription activation potential, occur in a fixed ratio which is similar in different expressing tissues, like liver, lung and small intestine. The presence of a similar, thusfar unnoticed, small ORF 5' to the major initiation codon of C/EBP beta mRNA suggests that start site multiplicity from this mRNA may be governed by the same mechanism.

Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed.

DNAseI-hypersensitive sites were localized in apoVLDL II chromatin from chicken. In the liver two sites at 1.75 and 1.0 kb upstream from the cap-site are present before the gene is activated. After induction by estradiol a number of additional sites appear, three in the promotor region of the gene, one within the coding region and two behind the poly-A signal. These sites disappear when the expression of the gene is shut off upon estradiol withdrawal. All sites appear to be tissue-specific in that they are not found in other tissues of the rooster. However, in oviduct of the laying hen we find a hypersensitive site at 1.6 kb in front of the gene.

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The transcription factors CCAAT/enhancer-binding proteins a and / 3 (CEBPa and CIEBPP) are highly expressed in liver and are believed to function in maintaining the differentiated state of the hepatocytes. CIEBPa appears to be a critical regulator of genes involved in metabolic processes. We are interested in the roles of C/EBP in the expression of the very-low-density apolipoprotein I1 (apoVLDL 11) gene. This gene encodes an avian yolk protein, is induced by estrogens and is only expressed in liver. To examine the role of CEBP in apoVLDL I1 expression, footprinting and electromobility-shift analysis were performed. For three of the protein-binding sites in the apoVLDL I T promoter region, C/EBPa and CIEBPP were identified as the major DNA-binding activities. For one of the C/EBP genes, CIEBPa, the effect of the gene products on apoVLDL I1 transcription was examined. From transfection experiments we conclude that maximal estrogen-dependent activity of the apoVLDL I1 promoter requires the dual action of the estrogen receptor and CEBP. The level of activity is different depending on the nature of the CEBPa translational isoform transfected, the full-length CEBPn polypeptide being the most active isoform and the N-terminally truncated isoform being moderately active. The present results suggest a role of CEBPa translational isoform ratio in the modulation of expression of C/EBP target genes, such as those involved in metabolic processes.