Identification of a novel gp130-responsive site in the vasoactive intestinal peptide cytokine response element.
The neuropoietic cytokine ciliary neurotrophic factor (CNTF) potently induces transcription of the vasoactive intestinal peptide (VIP) gene through a 180-base pair (bp) cytokine response element (CyRE) in the VIP promoter. We have previously shown that CNTF induction of STAT and AP-1 protein binding within the CyRE is necessary to mediate CNTF induction of VIP gene transcription. We now show that a third, previously uncharacterized site at the 3'-end of the CyRE is also critical to CNTF induction of CyRE transcription. A 4-bp mutation in this 3'-region reduced CNTF-mediated induction of transcription approximately 80%. Whereas mutations in both the STAT and AP-1 sites substantially reduced CNTF induction of transcription, mutations in these sites together with the novel 3'-site completely abolished the ability of CNTF to induce CyRE-mediated transcription. Gel shift analysis indicated that a complex in neuroblastoma cells bound specifically to this 3'-site. This complex was not altered by CNTF treatment. Mutations in an 8-bp sequence (TTACTGGA) eliminated binding of this protein complex and markedly reduced transcriptional activation of the CyRE by CNTF. Thus, we have identified a protein complex binding to a novel DNA sequence that is necessary for full CNTF induction of VIP gene transcription.
Jones
E A
EA
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Conover
J
J
Symes
A J
AJ
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D020934:Ciliary Neurotrophic Factor / Q000494:pharmacology*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology; D017930:Genes, Reporter; D006801:Humans; D046911:Macromolecular Substances; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics*; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000235:genetics*
D020934:Ciliary Neurotrophic Factor; D015342:DNA Probes; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014660:Vasoactive Intestinal Peptide