A role for CCAAT/enhancer-binding protein in hepatic expression of thrombin-activable fibrinolysis inhibitor

Thrombin-activable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase B-like zymogen that upon activation by thrombin, thrombin-thrombomodulin, or plasmin attenuates fibrin clot lysis by inhibiting positive feedback in the fibrinolytic cascade. The concentration of TAFI in plasma varies in the human population and thus may constitute a risk factor for thrombotic disorders. In addition, TAFI has been reported to be a positive acute phase reactant in mice. We have initiated molecular analysis of the human TAFI promoter to understand the mechanisms underlying regulation of TAFI gene expression. We identified a putative C/EBP-binding site between -53 and -40 of the promoter. Mutations in this site that abolish C/EBP binding decrease TAFI promoter activity in human hepatoma (HepG2) cells by approximately 80%. Gel mobility shift analyses indicated that C/EBP-beta present in HepG2 nuclear extracts and C/EBP-alpha and -beta present in adult rat liver nuclear extracts bind to the C/EBP site. C/EBP-alpha, -beta, and -delta isoforms are all capable of binding to the C/EBP site and activating the TAFI promoter. The identification of a functional C/EBP-binding site in the human TAFI promoter may have important implications for the regulation of expression of this gene during development and in response to inflammatory stimuli.     

Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene. Identification of an element, upstream from the promoter, required for efficient expression of phoE protein

The phoE gene of Escherichia coli codes for an outer membrane pore protein whose expression is induced under phosphate limitation. The promoter of this gene contains a 17 base-pair fragment, designated a pho box, which is present also in other phosphate-controlled promoters. The mRNA start site was determined and found to be located downstream from the pho box, such that this element is located in the -35 region of the phoE promoter. A set of promoter deletions was generated in vitro and analysis of these deletions revealed that sequences upstream from the pho box are required for the efficient expression of phoE. The required upstream region is located (in part) between positions -106 and -121 relative to the mRNA start site, and contains sequences homologous to a pho box and a correctly spaced Pribnow box, but in the reversed orientation relative to the regular -35 and -10 regions. A proper spacing between this upstream region and the -35 region appears to be important, since an oligonucleotide insertion in the intervening region interferes with phoE expression. By cloning the upstream region in a lacZ operon fusion vector, a weak phosphate limitation-inducible promoter activity could be detected.     

Sequences in the promoter region of the chicken lysozyme gene required for steroid regulation and receptor binding

We have constructed a series of deletion mutants in the lysozyme promoter region fused to the SV40 T-antigen coding region. Regulated expression was tested after microinjection of the lysozyme deletion mutants into primary cultures of chicken oviduct cells using fluorescent antibodies against T antigen. Deletion of lysozyme gene sequences upstream of position - 164 was accompanied by loss of both progesterone- and glucocorticoid-induced expression. Using the rat liver glucocorticoid receptor for binding studies, two separate binding sites have been identified: a strong binding site that is destroyed by deletion of lysozyme sequences between positions -74 and -39 and a weaker binding site contained between positions -208 and -161 upstream of the lysozyme cap site.     

DNA methylation pattern and restriction endonuclease accessibility in chromatin of a germ-line specific gene, the rainbow trout protamine gene.

The chromatin structure of a germ-line specific gene, the TPG-3 gene, one of the rainbow trout protamine genes was analyzed in various tissues. The protamine genes are expressed in early stage testis but not in late stage testis, liver or erythrocyte. Five potential CpG methylation sites in the coding and flanking regions of the TPG-3 protamine gene were monitored in early and late stage testis, nucleoprotamine, liver and erythrocyte. In all cases the patterns of methylation were identical with only one CpG site at position -740 being methylated. Thus, the methylation pattern of this protamine gene remained the same independently of the expression of the gene. Two Msp I sites at positions -293 and/or -275 and +155 were accessible to the enzyme in the TPG-3 chromatin of early stage testis. Since the Msp I site at position -293 and/or -275 was also present in the TPG-3 chromatin of liver, only the site at position +155 within the transcribed region correlated with the expression of the protamine gene.     

Functional characterization of the rat gamma-glutamyl transpeptidase promoter that is expressed in transformed rat liver epithelial cells

In the rat the gamma-glutamyl transpeptidase (GGT) gene codes for at least four different messenger RNAs (mRNA I to mRNA IV) which differ only in their 5' untranslated regions and are transcribed from a single copy gene in a tissue-specific manner. In the liver GGT expression is up-regulated in transformed cells. To understand the induction mechanisms of GGT activity by transformation, we previously cloned the 5' region of the rat GGT gene which contains the 5' untranslated leader sequence for mRNA I. In the present study, using transfection and reporter gene assays, I have demonstrated that (1) the sequence from positions -369 to +226 drives a relatively strong promoter activity in C5 and AKG cell lines, transformed rat liver epithelial cells, but a very weak one in RLE-228 cells, a normal rat liver epithelial cell line; and (2) removal of the region between -418 and -369 increases CAT activity more than 10-fold in RLE-228, C5 and AKG cells, and the DNA fragment spanning nucleotides -761 to -292 significantly reduces CAT activity driven by the adenine phosphoribosyl transferase (APRT) promoter in RLE-228 cells.