Single nucleotide changes in the human Igamma1 and Igamma4 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 Igamma transcripts to signals through CD40 and IL-4. This difference was most pronounced when comparing the profile of Igamma1 and Igamma4 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 Igamma1 and Igamma4 promoters has been investigated given the highly muted transcriptional activity of the Igamma4 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-kappaB-binding sites and the downstream 36-bp region containing CREB/activating transcription factor and kappaB6 sites. Mutation of a single nucleotide at position 6 within the Igamma4 kappaB6 site increased promoter activity to approximately 50% of the activity of the Igamma1 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 Igamma1. Minor nucleotide changes to both the Igamma4 CD40 response region and the 36-bp element resulted in a response undistinguishable from an Igamma1 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.

c-Rel plays a key role in deficient activation of B cells from a non-X-linked hyper-IgM patient.

Our previous results demonstrated that B cells from a patient (pt1) with non-X-linked hyper-IgM syndrome (HIGM) possess an atypical CD23(lo) phenotype that is unaffected by CD40-mediated activation. To investigate the molecular mechanism underlying defective CD23 expression in pt1 B cells, we used lymphoblastoid cell lines that express LMP1 under the control of a tetracycline-inducible promoter (LCL(tet)). Our analysis revealed that the CD23(lo) phenotype in the pt1-LCL(tet) cells is a direct consequence of diminished CD23 transcription. We demonstrate a marked decrease in c-Rel-containing complexes that bind to the proximal CD23a/b promoters in pt1-LCL(tet) extracts, resulting from an overall lower expression of c-Rel in pt1-LCL(tet) cells. Analysis of c-Rel mRNA revealed relatively equal amounts in pt1-LCL(tet) and control LCL(tet) cells, indicating that diminished c-Rel protein expression is unrelated to decreased transcription. Finally, a critical role for c-Rel in CD23 regulation was demonstrated by effectively altering c-Rel expression that resulted in the direct modulation of CD23 surface expression. Collectively, these findings demonstrate that low levels of c-Rel are the underlying cause of aberrant CD23 expression in pt1 B cells and are likely to play a critical role in the pathophysiology of this form of HIGM.

Use of chromatin immunoprecipitation (ChIP) to detect transcription factor binding to highly homologous promoters in chromatin isolated from unstimulated and activated primary human B cells.

The Chromatin Immunoprecipiation (ChIP) provides a powerful technique for identifying the in vivo association of transcription factors with regulatory elements. However, obtaining meaningful information for promoter interactions is extremely challenging when the promoter is a member of a class of highly homologous elements. Use of PCR primers with small numbers of mutations can limit cross-hybridization with non-targeted sequences and distinguish a pattern of binding for factors with the regulatory element of interest. In this report, we demonstrate the selective in vivo association of NF-kappaB, p300 and CREB with the human Igamma1 promoter located in the intronic region upstream of the Cgamma1 exons in the immunoglobulin heavy chain locus. These methods have the ability to extend ChIP analysis to promoters with a high degree of homology.

A novel NF-kappa B-regulated site within the human I gamma 1 promoter requires p300 for optimal transcriptional activity.

Transcriptional activation of germline (GL) promoters occurs through binding of NF-kappaB to three evolutionarily conserved sites within a CD40 response region in the human and mouse GL Igamma and Iepsilon promoters. Here we identify and characterize a novel NF-kappaB binding site (kappaB6) within the human GL Igamma1 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 Igamma1 but not the Igamma3 promoter, which are important for the amplification of transcription. Our data suggest a cohesive protein complex regulating Igamma1 promoter activity because disruption of any individual NF-kappaB 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-kappaB, proteins contributes directly to promoter activity. We found that CREB and p50 transactivators, as well as coactivator p300, interact in vivo with the Igamma1 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 Igamma1 construct-specific transcription. Together these data support a model whereby CREB and multiple NF-kappaB complexes bind to the Igamma1 promoter and recruit p300. CD40 signals induce p300-dependent changes that result in optimal Igamma1 promoter activity.

Maintenance of the CD40-related immunodeficient response in hyper-IgM B cells immortalized with a LMP1-regulated mini-EBV.

Our previous investigation of a patient (pt1) with non-X-linked hyper-immunoglobulin M syndrome revealed a CD40-mediated defect in B cell activation that resulted in low CD23 expression and absence of germ-line transcription and class-switch recombination. These deficiencies were complemented in vitro by a high threshold of sustained signaling through CD40. To further analyze the signaling defect in pt1 B cells, two types of Epstein-Barr virus lymphoblastoid cell lines (LCLs) were generated that either constitutively expressed the viral transforming protein latent membrane protein-1 (LMP1; pt1-LCL) or expressed it under the control of a tet-inducible promoter (pt1-LCL(tet)). Because LMP1 signals through the CD40 pathway, the pt1-LCL and pt1-LCL(tet) lines allow comparison of downstream functions in response to either constitutive LMP1 signals or regulated LMP1 and CD40 signals. Immortalized pt1-LCLs were initially CD23(lo)/CD38(hi) and reverted to a CD23(hi)/CD38(lo) phenotype upon extended growth in culture, suggesting that the CD40 defect was reversed by selection and/or constitutive expression of LMP1. In contrast, pt1-LCL(tet) cells retained the CD23(lo)/CD38(hi) phenotype after extended periods of culture and failed to up-regulate CD23 in response to CD40 signals. Analysis of pt1-LCL(tet) cells in response to the CD40 signals in the presence or absence of LMP1 revealed that mitogenic activation resulted only from LMP1 and not CD40, indicating a difference in the response of pt1 B cells to these two distinct signals. Together, these data demonstrate that the pt1-LCL(tet) cells maintain the CD40-related defect and provide a unique approach to study the independent effects of LMP1- and CD40-directed signals.