Purification of intercalator-released p67, a polypeptide that interacts specifically with the c-fos serum response element.

Incubation of intact nuclei in buffers containing the DNA intercalating drug chloroquine leads to release of proteins that interact with DNA. We demonstrate here that a protein which binds to a motif within the human c-fos promoter, identified as the serum response element (SRE), is quantitatively released from HeLa nuclei, whereas nuclear factor 1 (NF 1) is not. Purification of the SRE binding protein by affinity chromatography to greater than 95% homogeneity allowed us to identify it as a polypeptide of approximately 67,000 daltons. The DNA contacts made by p67, as identified by methylation interference experiments, are indistinguishable from those of the serum response factor described previously.     

CCAAT/enhancer binding protein beta mediates the activation of the murine alpha1(I) collagen promoter by acetaldehyde

Acetaldehyde was previously shown to activate the alpha1(I) and alpha2(I) collagen promoters and to increase collagen production in activated stellate cells. Also, CCAAT/enhancer binding protein beta (C/EBPbeta) binds and activates the mouse alpha1(I) collagen promoter. This study investigates the role of C/EBPbeta in mediating the activation of the alpha1(I) collagen promoter by acetaldehyde. Nuclear extracts isolated from cultured activated rat hepatic stellate cells formed four protein-DNA complexes on electrophoretic mobility shift assay with an oligonucleotide including the C/EBP binding site between -365 and -335 in the alpha1(I) collagen promoter. The four complexes were identified to represent C/EBPbeta binding to the oligonucleotide by supershift with C/EBPbeta antibody. The principal C/EBP isoform found in the nuclear extracts from stellate cells was C/EBPbeta, with very low amounts of C/EBPalpha detected. Acetaldehyde (200 microM) increased C/EBPbeta protein in stellate nuclear extracts, increased its binding to the promoter, and activated the alpha1(I) collagen promoter in transfected stellate cells. Mutation of the C/EBPbeta binding site markedly decreased nuclear protein binding. A transfected promoter, mutated at the C/EBP binding site, had decreased basal activity, was not activated by acetaldehyde, and was not activated when cotransfected with a C/EBPbeta expression vector. This study shows that C/EBPbeta is the predominant C/EBP isoform found in activated stellate cells and that increased C/EBPbeta protein and C/EBPbeta binding to a proximal C/EBP binding site in the promoter mediates the activating effect of acetaldehyde.     

CRP2 (C/EBP beta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity.

Two members of the C/EBP family of basic region-leucine zipper proteins enriched in the liver, C/EBP (C/EBP alpha) and CRP2 (C/EBP beta), were previously shown to transactivate the albumin promoter in a cell type-dependent manner. These proteins function efficiently in HepG2 hepatoma cells, but inefficiently in HeLa (epithelial) and L (fibroblastic) cells. Here we have investigated the mechanism for cell-specific control of CRP2 activity. We show that CRP2 contains a negative regulatory region composed of two elements, RD1 and RD2. Deletions of RD2 relieve the inhibition of CRP2 activity in L cells, but do not affect CRP2 function in HepG2 cells. These deletions also increase the DNA binding activity of CRP2 approximately 3-fold, suggesting that RD2-mediated repression of DNA binding activity is responsible for CRP2 inhibition in L cells. The adjacent RD1 element functions independently of RD2 and modulates the CRP2 activation domain, which we show to be composed of three subdomains that are conserved within the C/EBP protein family. RD1 does not affect cell type specificity, but inhibits the transactivation potential of GAL4-CRP2 hybrid proteins by 50-fold. These findings suggest that CRP2 assumes a tightly folded conformation in which the DNA binding and activation domains are masked by interactions with the regulatory domain and that to function efficiently in HepG2 cells the protein must undergo an activation step. We propose that relief of inhibition conferred by the regulatory domains also accounts for CRP2 activation in response to extracellular signals.     

Characterization of the promoter region of the human peroxisomal multifunctional enzyme type 2 gene

Peroxisomal multifunctional enzyme type 2 (perMFE-2) catalyzes conversion of (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24-keto)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoyl-CoA, which are physiological intermediates in cholic acid synthesis. In contrast to long chain fatty acid oxidizing enzymes clofibrate does not induce peroxisomal enzymes metabolizing bile acid intermediates. We proposed the existence of PPAR-independent regulation of cholesterol side chain oxidation in the process of bile acid synthesis. In the present study, we characterized the promoter region of the human perMFE-2 gene. The promoter contains the Sp1/AP2 binding site (-151/-142) within 197 base pairs upstream of the translation start site. Mutation of the Sp1/AP2 binding site decreases the promoter activity. Analysis by the luciferase assay revealed that the activity of the promoter region is strong in HepG2 and HeLa cell lines, although the activity in HepG2 cells was five- to sixfold higher than that in HeLa cells. Transient transfection assays have confirmed that AP2alpha and AP2gamma were able to transactivate the perMFE-2 promoter/luciferase chimeric gene. Cotransfections with Sp1 expression plasmid decreased the promoter activity. We suggest that perMFE-2 promoter activity is the result of both the abundance of AP2 and Sp1 family members and their relative ratios.