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Abstract
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.
Highlights
Identify the molecular mechanisms by which the gp130 cytokines regulate gene transcription in the nervous system, we have examined ciliary neurotrophic factor (CNTF) induction of vasoactive intestinal peptide (VIP) gene expression
We have previously characterized the STAT and AP-1 sites within the VIP cytokine response element (CyRE) and shown both sites to contribute to the CNTF-mediated induction of CyRE-dependent transcription [38, 40]
One 4-bp motif (G9 site) is conserved between species, suggesting functional significance of this site. To investigate whether this 4-bp motif is important in mediating CNTF induction of CyRE transcription, we transfected NBFL neuroblastoma cells with a Cy1luc luciferase reporter plasmid mutated within these 4 bp (Cy1mG9luc) (Table I)
Summary
CCGGGTACCTAAAAAAGATTTCCTGG CCGGGTACCTAAAAAAGATGTACTGG CACCTGCAGCGTTCAAAGTCAGAC CCG GGT ACC TAA AAA AGA TGT ACT GGT ATT AAG CCA CAG GAA CTC TGG CAG CTG CAG CGT TCA AAG TCA GAC TGA TCT TAG AAA AGA ATA TCC TGC CAG CTG CAG GTA TCA AAG TCA GAC TGA TCC AGT AAA A CAG CTG CAG CGT AGT AAG TCA GAC TGA TCC AGT AAA A CAG CTG CAG CGT TCA TTC TCA GAC TGA TCC AGT AAA A CAG CTG CAG CGT TCA AAG ATT GAC TGA TCC AGT AAA A CAG CTG CAG CGT TCA AAG TCA CTA TGA TCC AGT AAA A CAG CTG CAG CGT TCA AAG TCA GAC ACT TCC AGT AAA A CAG CTG CAG CGT TCA AAG TCA GAC TGA ATG AGT AAA A CAG CTG CAG CGT TCA AAG TCA GAC TGA TCC TCA AAA A CAG CTG CAG CGT TCA AAG TCA GAC TGA TCC AGT TTC A CAG CTG CAG CGT TCA AAG TCA GAC TGA TCC AGT AAA TCG ATA T examined the proteins binding to the 3Ј-region and characterized the sequences to which they bind to understand the combinatorial mechanisms through which CNTF induces VIP gene expression
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