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Single Nucleotide Changes in the Human I1 and I4 Promoters Underlie Different Transcriptional Responses to CD40¹

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

Analysis of subclass-specific germline transcription in activated peripheral B cells revealed a highly biased expression pattern of the four I transcripts to signals through CD40 and IL-4. This difference was most pronounced when comparing the profile of I1 and I4 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 I1 and I4 promoters has been investigated given the highly muted transcriptional activity of the I4 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-B-binding sites and the downstream 36-bp region containing CREB/activating transcription factor and B6 sites. Mutation of a single nucleotide at position 6 within the I4 B6 site increased promoter activity to 50% of the activity of the I1 promoter. Furthermore, elevated promoter strength corresponded with increased binding of p50, p65, c-Rel, RelB, and p300 proteins to a level comparable with that of I1. Minor nucleotide changes to both the I4 CD40 response region and the 36-bp element resulted in a response undistinguishable from an I1 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 costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by National Institutes of Health Grant R01-AI37081 (to L.R.C.) and National Institutes of Health Training Fellowship T32AI07403 (to R.L.D.). This study was initiated as part of the Ph.D. dissertation work of R.L.D.

² F.L.S. and R.L.D. contributed equally to this article.

³ Address correspondence and reprint requests to Dr. Lori R. Covey, Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, B314 Nelson Hall, 604 Allison Road, Piscataway, NJ 08854. E-mail address: covey{at}biology.rutgers.edu

⁴ Abbreviations used in this paper: CSR, class switch recombination; GLT, germline transcription; CD40RR, CD40 response region; ATF, activating transcription factor; ChIP, chromatin immunoprecipitation; nChIP, native ChIP; wt, wild-type sequence; mut, mutated sequences.