IL-4 induces both IgE and IgG4 in human B cells. The coregulation of these isotypes with functionally distinct properties is likely to be mediated at the level of germline transcription. To define the mechanisms of ∈/γ4 coregulation, it is necessary to analyze and compare the structure and function of the ∈ and γ4 germline promoters. We therefore cloned γ4 GLT by RT-PCR using a forward primer upstream of the splice donor site used by γ1 and γ3 GLT, and a reverse primer in the γ4 hinge region. The 411 bp fragment thus amplified from cDNA of PBMC stimulated with IL-4+anti-CD40 mAb contained the 3′ end of the Iγ4 exon correctly spliced to the 5′ portion of Cγ4. Sequence analysis located the human Iγ4 exon about 1.5 kb upstream of the switch γ4 region. RT-PCR analysis using additional 5′ primers indicated that γ4 GLT initiate at least 420 bp from the Iγ4 splice donor site and are colinear with the genomic sequence. Primer extension analysis on polyA+ RNA purified from BL-2 cells treated with IL-4+anti-CD40 mAb led to the identification of several transcription start sites located 550-590 bp upstream of the Iγ4 splice donor site. Reporter assays showed that a 2,064 bp BamHI/NaeI fragment that contains Iγ4 at the 3′ end has promoter activity and drives an 8-fold increase in transcription following stimulation of BL-2 cells with IL-4+anti-CD40 mAb. The BamHI/NaeI fragment contains one putative Stat6 and at least three putative NF-κB binding sites upstream of the transcription start sites. We are investigating the role of these sites in the regulation of γ4 germline transcription.
|Publication status||Published - Mar 20 1998|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology