AbstractTranscriptional regulation via activator protein‐1 (AP‐1) protein binding to AP‐1 binding sites within gene promoter regions of AP‐1 target genes plays a key role in controlling cellular invasion, proliferation, and oncogenesis, and is important to pathogenesis of arthritis and cardiovascular disease. To identify new proteins that interact with the AP‐1 DNA binding site, we performed the DNA affinity chromatography‐based Nucleotide Affinity Preincubation Specificity TEst of Recognition (NAPSTER) assay, and discovered a 97 kDa protein that binds in vitro to a minimal AP‐1 DNA sequence element. Mass spectrometric fragmentation sequencing determined that p97 is nucleolin. Immunoblotting of DNA affinity‐purified material with anti‐nucleolin antibodies confirmed this identification. Nucleolin also binds the AP‐1 site in gel shift assays. Nucleolin interacts in NAPSTER with the AP‐1 site within the promoter sequence of the metalloproteinase‐13 gene (MMP‐13), and binds in vivo in chromatin immunoprecipitation assays in the vicinity of the AP‐1 site in the MMP‐13 promoter. Overexpression of nucleolin in human HeLa cervical carcinoma cells significantly represses AP‐1 dependent gene transactivation of a minimal AP‐1 reporter construct and of an MMP‐13 promoter reporter sequence. This is the first report of nucleolin binding and transregulation at the AP‐1 site. © 2007 Wiley‐Liss, Inc.