Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter.

The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays, mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I) gene may depend on the relative activities of NF-I and Sp1.

A simplified method for the preparation of transcriptionally active liver nuclear extracts.

We have developed a simplified method for the preparation of liver nuclear extracts to study gene regulation and protein-DNA interactions. This protocol uses conventional laboratory equipment and standard reagents. The liver tissue is homogenized in a low-salt solution at physiological molarity with subsequent adjustment of the molarity and purification of nuclei by density sedimentation. The nuclear extracts are transcriptionally active in a validated cell-free transcription assay and contain functional DNA-binding proteins. This protocol results in the rapid preparation of highly reproducible and active liver nuclear extracts.

Analysis of the collagen alpha 1(I) promoter.

The collagen alpha 1(I) gene is regulated at a developmental and tissue specific level. We have previously demonstrated that only 220bp of the promoter region of the collagen alpha 1(I) gene are required for efficient expression in NIH 3T3 cells. DNAse I protection assays demonstrated 4 footprinted segments in the promoter region. Deletional analysis revealed that the 3 most proximal footprints were required for maximal expression. The most proximal footprint contains a CCAAT sequence and a 12bp segment that forms a direct repeat with the preceding footprint. Ligation of the proximal footprint sequence to a heterologous promoter enhanced transcription of the reporter gene. These studies, therefore, identify and characterize elements in the promoter region of the collagen alpha 1(I) gene that interact with DNA binding proteins and are required for efficient expression.

Stimulation of the collagen alpha 1 (I) endogenous gene and transgene in carbon tetrachloride-induced hepatic fibrosis.

Cirrhosis is characterized by a marked increase in the deposition of type I collagen and in the expression of the type I collagen genes alpha 1(I) and alpha 2(I). Although alpha 1(I) gene regulation has been extensively studied in cultured cells, these results may not be applicable to hepatic fibrogenesis in vivo. Therefore the regulation of the alpha 1(I) endogenous gene and an alpha 1(I) transgene was studied in a transgenic mouse model that has a single copy of a human alpha 1(I) gene segment containing the structural gene and 1.6 Kb of 5' DNA and 20 Kb of 3' DNA. To initiate hepatic fibrogenesis, we treated mice with the hepatotoxin carbon tetrachloride, either in a single dose or in biweekly doses for a period of 3 to 8 wk. Subsequently, hepatic alpha 1(I) messenger RNA levels were determined by a species-specific RNase protection assay. Carbon tetrachloride injections coordinately increased the messenger RNA levels of the alpha 1(I) endogenous gene and the transgene, both immediately and after 8 wk. These experiments demonstrate that this alpha 1(I) transgene fragment contains information sufficient for appropriate basal and carbon tetrachloride-stimulated hepatic expression. They further demonstrate that sufficient homology exists between the human and mouse regulatory elements for the recognition of human cis-acting elements by mouse trans-acting factors. Thus transgenic mice provide a unique model in which to characterize the collagen alpha 1(I) regulatory elements that are required in vivo for pathophysiological responses.

Activation of activating protein 1 during hepatic acute phase response.

During an acute phase response following inflammatory stimuli, specific changes occur in the synthesis and secretion of many hepatic proteins. Because the expression of differentiated function requires the coordinated regulation of many genes, we investigated the activity of general and tissue-specific transcription factors using a rat liver model of the acute phase response induced by Freund's adjuvant. Nuclear extracts and RNAs were prepared throughout a 48-h posttreatment period. Mobility shift assays revealed increased binding activity by nuclear factor-kappa B, interleukin-6 (IL-6) responsive element binding protein, and activating protein 1 (AP-1). Two AP-1 complexes were induced during the acute phase response, and correlation between their presence and transcription activity was demonstrated by transfection studies. Elevated binding activity of AP-1 also correlated with elevated levels of c-jun, junD, junB, and c-fos mRNAs. Western blots showed elevated hepatic levels of c-Jun but not c-Fos proteins during the acute phase response. In addition, IL-6, tumor necrosis factor-alpha, and IL-1 beta, cytokine regulators of the acute phase response, stimulated expression of an AP-1 responsive reporter gene introduced by DNA-mediated transfection into adult rat hepatocytes in primary culture. These findings demonstrate the complexity of AP-1 hepatic transcription factor responses to humoral regulators with direct hepatocellular effects.