In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors

This is the first attempt to identify regulatory elements that are involved in early choriogenesis of the silkworm Bombyx mori. A new cis element in the promoter region of five early chorion genes was identified. The consensus sequence of this element matches the consensus of the C/EBP DNA binding site. Moreover, this sequence interacts with a 70 kD protein (pX2) present in follicular nuclear extracts and complex formation exhibits early developmental specificity. There is strong evidence that this factor belongs to the C/EBP family. Surprisingly, the same protein binds with the same developmental specificity to a similar sequence of a late chorion gene promoter, which has been previously defined as the binding site for a putative late specific factor, BCFII. The possibility that pX2 and BCFII are isoforms or modifications of the same protein factor, which is presumably able to bind to the highly similar sequence elements of both early and late genes, is discussed. A hypothesis involving protein-protein interactions between C/EBP (pX2/BCFII) and GATA during choriogenesis is presented to explain the temporal specificity of chorion genes.     

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.     

Multiple elements within the glucocorticoid regulatory unit of the rat alpha 1-acid glycoprotein gene are recognition sites for C/EBP.

Sequences between -106 and -42, located immediately downstream of the glucocorticoid response element, are essential for efficient glucocorticoid-stimulated expression of the alpha 1-acid glycoprotein (AGP) gene. We have used mobility shift assays with oligonucleotides bearing wild type and mutated sequences from segmented portions of this region to characterize the specific interaction of similar binding factors from rat liver and HTC rat hepatoma cell nuclear extracts. One of these factors, AGP nuclear factor 2 (ANF-2), appears capable of dual interaction with the homologous recognition sites, HA (-133 to -104) and HB (-81 to -72), which overlap and are located downstream of the glucocorticoid response element, respectively. Using an affinity matrix containing the HB sequence we have isolated ANF-2 from rat liver nuclear extracts. On the basis of immunological evidence rat liver ANF-2 was confirmed to be highly related and probably identical to CCAAT/enhancer-binding protein (C/EBP). Methylation protection analyses with partially purified, rat liver ANF-2 confirmed HA and HB as recognition sites for C/EBP-related factors and are consistent with the location of a third interaction site for these transactivating proteins at HX (-102 to -93). We propose that the sequences HA, HX, and HB, spanning residues -113 to -72 of the AGP promoter, might serve as recognition sites for a family of C/EBP-like nuclear factors that coordinate the glucocorticoid-mediated induction of the AGP gene.     

Effect of the CCAAT/enhancer binding protein on expression of the gene for chicken malic enzyme

The gene for malic enzyme is expressed at a high level in chick embryo-hepatocytes (CEH) treated with triiodothyronine (T3) and at a low level in the absence of T3. In chick-embryo fibroblasts (CEF), expression of the malic enzyme gene is low and not regulated by T3. Specific nuclear proteins from both CEH and CEF bound to a consensus CCAAT/enhancer binding protein (C/EBP) site at -335 to -327 bp of the malic enzyme gene. The level of binding was much higher in extracts from CEH than in extracts of CEF, and the complexes formed had different mobilities. C/EBPalpha was present in the complex that bound to the C/EBP site in nuclear extracts from CEH but not in those from CEF. The C/EBP element was necessary and sufficient to bestow full T3 responsiveness to 5800 bp of 5'-flanking DNA of the malic enzyme gene in CEH. C/EBPalpha was not detectable in wild-type CEF, and deletion of the C/EBP binding site had no effect on expression of transgenes containing 5800 bp of 5'-flanking DNA of the malic enzyme gene. In CEF, overexpression of C/EBPalpha stimulated promoter activity of constructs that contained the C/EBP site linked to the malic enzyme promoter or a heterologous reporter. The results suggest that C/EBPalpha or a closely related isoform is involved in the tissue-specific expression of the malic enzyme gene.