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A Novel NF-B-Regulated Site within the Human I1 Promoter Requires p300 for Optimal Transcriptional Activity¹

Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854

Transcriptional activation of germline (GL) promoters occurs through binding of NF-B to three evolutionarily conserved sites within a CD40 response region in the human and mouse GL I and I promoters. Here we identify and characterize a novel NF-B binding site (B6) within the human GL I1 promoter that plays an essential role in basal- and CD40-induced transcription. This site is adjacent to identified CREB/activating transcription factor (ATF) sites, present in the I1 but not the I3 promoter, which are important for the amplification of transcription. Our data suggest a cohesive protein complex regulating I1 promoter activity because disruption of any individual NF-B or CREB/ATF site markedly lowers the overall inducible activity of the promoter. In addition, alteration of helical phasing within the promoter indicates spatial orientation of CREB/ATF and NF-B, proteins contributes directly to promoter activity. We found that CREB and p50 transactivators, as well as coactivator p300, interact in vivo with the I1 promoter in the presence and absence of CD40 signaling in Ramos and primary B cells. However, the level of CREB and p300 binding differs as a consequence of activation in primary B cells. Furthermore, overexpression of p300, and not a mutant lacking acetyltransferase activity, significantly increases I1 construct-specific transcription. Together these data support a model whereby CREB and multiple NF-B complexes bind to the I1 promoter and recruit p300. CD40 signals induce p300-dependent changes that result in optimal I1 promoter activity.

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