YB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence.
Involvement of the AP-1 (activator protein-1) transcription factor has been demonstrated previously in the regulation of cell proliferation and cell-cycle progression, in the control of cell migration, invasion and metastasis, and in signal transduction, stress responsiveness, DNA replication and DNA repair. YB-1 (Y-box-binding protein-1) has also been implicated in many of these processes. However, the mechanism by which YB-1 mediates these processes is poorly understood. In the present study, we report that overexpression of a transfected gene encoding YB-1 in human HeLa cervical carcinoma cells significantly represses the transactivation of a minimal AP-1 reporter construct in response to the tumour promoter PMA. YB-1 also represses mRNA expression and PMA-induced promoter transactivation of the endogenous AP-1 target gene encoding matrix metalloproteinase-12 (metalloelastase). YB-1 transrepression of both the minimal and matrix metalloproteinase-12 promoter reporter constructs is dependent on the AP-1 sequence. To identify new nuclear proteins that bind specifically to the AP-1 DNA-binding site, we devised a DNA-affinity-chromatography-based assay termed NAPSTER (nucleotide-affinity preincubation specificity test of recognition) and discovered a 49 kDa protein from human cancer cells that binds in a sequence-specific manner to the AP-1 DNA sequence. By tandem MS fragmentation sequencing analyses we determined that p49 is a YB-1. Immunoblotting of the NAPSTER-purified p49 protein using anti-YB-1 antibodies confirmed YB-1 binding to the AP-1 DNA sequence, as did gel mobility-supershift assays using YB-1 antibodies. This is the first report of YB-1 transrepression and interaction at the AP-1 DNA-binding site.
Samuel
Shaija
S
Department of Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA.
Twizere
Jean-Claude
JC
Bernstein
Lori R
LR
D001483:Base Sequence; D002467:Cell Nucleus; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter / Q000235:genetics; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D053508:Matrix Metalloproteinase 12; D008666:Metalloendopeptidases / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins; D021122:Protein Subunits; D021381:Protein Transport; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D051840:Y-Box-Binding Protein 1
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D004247:DNA; D008666:Metalloendopeptidases; C507122:MMP12 protein, human; D053508:Matrix Metalloproteinase 12