A novel NF-kappa B-regulated site within the human I gamma 1 promoter requires p300 for optimal transcriptional activity.

Transcriptional activation of germline (GL) promoters occurs through binding of NF-kappaB to three evolutionarily conserved sites within a CD40 response region in the human and mouse GL Igamma and Iepsilon promoters. Here we identify and characterize a novel NF-kappaB binding site (kappaB6) within the human GL Igamma1 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 Igamma1 but not the Igamma3 promoter, which are important for the amplification of transcription. Our data suggest a cohesive protein complex regulating Igamma1 promoter activity because disruption of any individual NF-kappaB 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-kappaB, proteins contributes directly to promoter activity. We found that CREB and p50 transactivators, as well as coactivator p300, interact in vivo with the Igamma1 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 Igamma1 construct-specific transcription. Together these data support a model whereby CREB and multiple NF-kappaB complexes bind to the Igamma1 promoter and recruit p300. CD40 signals induce p300-dependent changes that result in optimal Igamma1 promoter activity.