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

Analysis of subclass-specific germline transcription in activated peripheral B cells revealed a highly biased expression pattern of the four I gamma transcripts to signals through CD40 and IL-4. This difference was most pronounced when comparing the profile of I gamma 1 and I gamma 4 transcripts and was 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 I gamma 1 and I gamma 4 promoters has been investigated given the highly muted transcriptional activity of the I gamma 4 promoter. Two regions were analyzed where single nucleotide differences corresponded to major changes in transcriptional activity. These regions were the previously defined CD40 response region containing three putative NF-kappa B-binding sites and the downstream 36-bp region containing CREB/activating transcription factor and kappa B6 sites. Mutation of a single nucleotide at position 6 within the I gamma 4 kappa B6 site increased promoter activity to similar to 50% of the activity of the I gamma 1 promoter. Furthermore, elevated promoter strength corresponded with increased binding of p50, p65, c-Rel, ROB, and p300 proteins to a level comparable with that of I gamma 1. Minor nucleotide changes to both the I gamma 4 CD40 response region and the 36-bp element resulted in a response undistinguishable from an I gamma 1 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 germline transcription, which in part impacts the overall level of class switch recombination to targeted C-H regions. The Journal of Immunology, 2009, 182: 2185-2193.