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Nucleolin binds specifically to an AP‐1 DNA sequence and represses AP1‐dependent transactivation of the matrix metalloproteinase‐13 gene
Author(s) -
Samuel Shaija,
Twizere JeanClaude,
Beifuss Katherine K.,
Bernstein Lori R.
Publication year - 2008
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/mc.20358
Subject(s) - nucleolin , biology , microbiology and biotechnology , transactivation , chromatin immunoprecipitation , immunoprecipitation , binding site , promoter , electrophoretic mobility shift assay , reporter gene , ap 1 transcription factor , dna , gene , transcription factor , gene expression , biochemistry , nucleolus , cytoplasm
Abstract Transcriptional 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.