The CCAAT-binding proteins CP1 and NF-I cooperate with ATF-2 in the transcription of the fibronectin gene

Alonso, Claudio R, Pesce, C Gustavo and Kornblihtt, Alberto R (1996) The CCAAT-binding proteins CP1 and NF-I cooperate with ATF-2 in the transcription of the fibronectin gene. Journal of Biological Chemistry, 271 (36). pp. 22271-22279. ISSN 0021-9258

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We have previously proposed a molecular interaction between the liver factors that bind to the cyclic AMP response element (CRE) and CCAAT sites of the fibronectin (FN) gene based on the following evidence: (i) the close spacing of 20 base pairs between CRE and CCAAT elements is conserved in the FN genes from rats, mice, and humans; (ii) footprinting competitions showed that CRE oligonucleotides are able to detach both liver factors; (iii) CCAAT binding and transcriptional activity of liver extracts are reduced when the distance between the CRE and CCAAT elements is increased; and (iv) CCAAT- binding is stimulated by the addition of a liver extract fraction containing the CRE-binding factor ATF-2. This report provides binding and immunochemical evidence that nuclear factor I (CTF/NF-I) and CP1 (NF-Y or CBF) are the only liver factors that bind to the -150 CCAAT element of the FN gene, forming distinct complexes. We show that these factors bind less efficiently to the CCAAT site of a FN promoter in which the -170 CRE has been disrupted by site- directed mutagenesis and that each element contributes positively to the liver transcriptional activity assessed in vitro with a G-less cassette construct and in vivo by transfection of hepatoma cells with CAT constructs. Furthermore, using a method that combines UV crosslinking and immunoprecipitation, we show that antibodies specific to ATF-2 are able to specifically precipitate protein-protein-DNA complexes containing NF-I and CP1. This simple method preserves weak macromolecular interactions, avoiding the disruptive electrophoresis conditions of gel mobility shifts assays.

Item Type: Article
Schools and Departments: School of Life Sciences > Evolution, Behaviour and Environment
Depositing User: Claudio Alonso
Date Deposited: 06 Feb 2012 20:55
Last Modified: 22 Mar 2012 12:35
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