Interaction of the -170 cyclic AMP response element with the adjacent CCAAT box in the human fibronectin gene promoter

In the fibronectin gene promoter the cAMP response element (CRE) and the CCAAT box are separated by only 20 base pairs (bp), i.e. two turns of double helix. Binding of nuclear proteins to these elements, assessed by DNase I footprinting, differs in the different cell types. While in a variety of cells tested (HeLa, granulosa, brain, and adenocarcinoma) only CRE binding activity is observed, liver extracts show both CRE and CCAAT binding activities. Competitions with CRE oligonucleotides were able to prevent the binding of both liver factors, while competitions with CCAAT oligonucleotides only abolished the binding to the CCAAT box. Consistently, the occupation of the CCAAT box was reduced when the distance between the CRE and CCAAT elements was increased in a series of spacing mutants in which DNA fragments of 20, 28, or 44 bp were inserted, and in a construct where the CRE sequence was deleted. Furthermore, the mutants are less efficient than the wild type as templates for in vitro transcription elicited by liver nuclear extracts. Transcriptional activity decreases with the 20- and 28-bp insertions but is partially recovered with the 44-bp insertion. Partial purification of liver CRE- and CCAAT-binding proteins by high performance liquid chromatography on a Mono Q column and recombination of column fractions showed that a novel 73-kDa CRE-binding protein facilitates the association of the CCAAT-binding protein to the CCAAT site of the fibronectin gene.