Single Nucleotide Changes in the Human Iγ1 and Iγ4 Promoters Underlie Different Transcriptional Responses to CD40

Analysis of subclass-specific germline transcription (GLT) in activated peripheral B cells revealed a highly biased expression pattern of the four Iγ transcripts to signals through CD40 and IL-4. This difference was most pronounced when comparing the profile of Iγ1 and Iγ4 transcripts and not expected given the very high degree of sequence conservation between promoters. In this report, the influence of sequence differences on the regulation of the Iγ1 and Iγ4 promoters has been investigated given the highly muted transcriptional activity of the Iγ4 promoter. Two regions were analyzed where single nucleotide differences corresponded to major changes in transcriptional activity. These regions were the previously defined CD40RR containing three putative NF-κB binding sites and the downstream 36 bp region containing CREB/ATF and κB6 sites. Mutation of a single nucleotide at position 6 within the Iγ4 κB6 site increased promoter activity to approximately 50% the activity of the Iγ1 promoter. Furthermore, elevated promoter strength corresponded with increased binding of p50, p65, c-Rel, RelB and p300 proteins to a level comparable to that of Iγ1. Importantly, minor nucleotide changes to both the Iγ4 CD40RR and the 36 bp element resulted in a response undistinguishable from an Iγ1 response suggesting cooperation between the two regulatory regions for optimal transcriptional activity. Collectively, these mutational analyses suggest that minor sequence differences contribute to the composition and affinity of transcriptional protein complexes regulating subclass-specific GLT, which in part impacts the overall level of class switch recombination to targeted CH regions.