The CCAAT-binding proteins CP1 and NF-I cooperate with ATF-2 in the transcription of the fibronectin gene.
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 cross-linking 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.
Alonso
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Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina), Vuelta de Obligado 2490, Buenos Aires, Argentina.
Pesce
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Kornblihtt
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D051698:Activating Transcription Factor 2; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D022762:CCAAT-Enhancer-Binding Proteins; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014981:Xenopus; D051840:Y-Box-Binding Protein 1
D051698:Activating Transcription Factor 2; C497106:Atf2 protein, mouse; C497107:Atf2 protein, rat; D022762:CCAAT-Enhancer-Binding Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D005353:Fibronectins; D051839:NFI Transcription Factors; C497648:Nfic protein, mouse; C497649:Nfic protein, rat; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human