The CCAAT-binding Proteins CP1 and NF-I Cooperate with ATF-2 in the Transcription of the Fibronectin Gene

Abstract

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 CREbinding 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.Advances in our knowledge of the physiology of gene regulation will ultimately require the precise definition of the multimolecular processes that are built up on a given gene's DNA by both the well known and still unknown factors. Ideally, the model gene should be widely expressed and regulated at the levels of transcription and splicing. Besides fitting these requirements (1), the fibronectin (FN) 1 gene offers a suitable model because it has an essential role in development (2), and changes in its expression pattern are involved in pathology, particularly of the liver (3). The importance of studying FN regulation in liver is highlighted by the fact that although many tissues express FN, liver is the major source of the plasma form of FN (4), made of splicing variants different from those of the extracellular matrix forms. Moreover, liver fibrosis, a common pathological condition preceding cirrhosis, is accompanied by dramatic changes in FN expression (3), whose mechanisms are still poorly understood.The Ϫ170 cyclic AMP response element (CRE; 5Ј-TGACGTCA-3Ј) of the FN gene appears to be involved in different regulations of the promoter. Besides mediating cAMP stimulation (5-7), this element was unexpectedly shown to be essential for responsiveness to serum (8) in an additive way and therefore independent from cAMP. Viral induction by adenovirus E1a oncoprotein has also been shown to be mediated throug...

Induction of CHOP Expression by Amino Acid Limitation Requires Both ATF4 Expression and ATF2 Phosphorylation

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Downregulation of fibronectin transcription in highly metastatic adenocarcinoma cells

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