Nucleolin binds specifically to an AP-1 DNA sequence and represses AP1-dependent transactivation of the matrix metalloproteinase-13 gene.
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.
Samuel
Shaija
S
Department of Molecular and Cellular Medicine, Texas A & M University System Health Science Center, College Station, Texas, USA.
Twizere
Jean-Claude
JC
Beifuss
Katherine K
KK
Bernstein
Lori R
LR
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D001483:Base Sequence; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D053509:Matrix Metalloproteinase 13 / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D000097796:Nucleolin
D004247:DNA; D053509:Matrix Metalloproteinase 13; D010446:Peptide Fragments; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D018808:Transcription Factor AP-1; D000097796:Nucleolin