A novel NF-κB-regulated site within the human Iγ1 promoter requires p300 for optimal transcriptional activity

Transcriptional activation of germline (GL) promoters occurs through binding of NF-κB to three evolutionarily conserved sites within a CD40 response region in the human and mouse GL Iγ and Iε promoters. Here we identify and characterize a novel NF-κB binding site (κB6) within the human GL Iγ1 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 Iγ1 but not the Iγ3 promoter, which are important for the amplification of transcription. Our data suggest a cohesive protein complex regulating Iγ1 promoter activity because disruption of any individual NF-κB 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-κB, proteins contributes directly to promoter activity. We found that CREB and p50 transactivators, as well as-coactivator p300, interact in vivo with the Iγ1 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 Iγ1 construct-specific transcription. Together these data support a model whereby CREB and multiple NF-κB complexes bind to the Iγ1 promoter and recruit p300. CD40 signals induce p300-dependent changes that result in optimal Iγ1 promoter activity.