Single Nucleotide Changes in the Human Iγ1 and Ιγ4 Promoters Underlie Different Transcriptional Responses to CD40

Abstract

Analysis of subclass-specific germline transcription 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 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 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 CD40 response region containing three putative NF-κB-binding sites and the downstream 36-bp region containing CREB/activating transcription factor and κB6 sites. Mutation of a single nucleotide at position 6 within the Iγ4 κB6 site increased promoter activity to ∼50% of 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 with that of Iγ1. Minor nucleotide changes to both the Iγ4 CD40 response region 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 germline transcription, which in part impacts the overall level of class switch recombination to targeted CH regions.

The N-terminus of hTERT contains a DNA-binding domain and is required for telomerase activity and cellular immortalization

“…retardation of mobility) upon binding to the DNA:protein complex. Although not as common, antibody recognition can perturb the intact nucleoprotein complex leading to the disappearance of complexes, or appearance of faster mobility complexes, owing to an alteration of relative DNA:protein stoichiometry ( 69–71 ). These data show that hTEN was present in the telomeric DNA complex, and argue for a specific interaction between the N-terminus of hTERT and telomeric DNA.…”

Heat-KilledSaccharomyces cerevisiae, A Dectin-1 Agonist, Selectively Induces IgG4 Production by Human B Cells

“…The nucleotide sequences of the 2 promoters showed an identity of 77.8%. The highly conserved sequences contain 3 previously identified NF-κB binding sites (293064) (underlined in Supplementary Fig. 1) and 2 putative C-Ets-1 binding sites.…”

“…The human GLTγ4 promoter DNA fragment (−1076 to +100) was amplified from human tonsil genomic DNA using PCR. PCR primers (Supplementary Table 1) were derived from previously reported human GLTγ4 promoter nucleotide sequences (2930). The GLTγ4 promoter fragment was subcloned into the pGL3-basic vector (Promega, Madison, WI, USA), and the reporter plasmid was named pGL3-hγ4[−1076/+100].…”

“…The putative C-Ets-1 binding sites were identified using the MATCH™ public version 1.0 (BIOBASE GmbH, Wolfenbüttel, Germany). The underlined sequences indicate previously reported NF-κB binding sites (293064).…”

B cells defined by immunoglobulin isotypes

“…The I H promoters have cytokine-responsive elements that differ between subclasses, hence the efficiency of CSR directly correlates with germline transcription [ 62 ]. For example, single nucleotide differences in the Iγ1 and Iγ4 promoters confer divergent responses to CD40 signaling, resulting in much lower germline transcription from Iγ4 [ 63 ]. In addition, while all switch regions have transcription factor binding sites responsive to IL-4 and CD40 signaling, these vary in length and in the number of tandem repeats between individual subclasses.…”