The ECS-Iγ 3 sequence upstream of the human Ig Sγ 3 region is a functional promoter. Synergistic activation by CD40L and IL-4 via distinct cis regulatory elements

Ig isotype switching is a central event in the generation of a mature antibody response, and it is effected by switch DNA recombination. This is targeted by proceeding transcriptional activation of germline CH genes, possibly regulated by the transcription promoting activity of sequences lying 5′ of I_H regions. In the human, the identity and functionality of Cγ upstream sequences that putatively regulate the transcription of the γ loci have not been determined. We have transfected CL-01 cells, our human monoclonal model of germinal center B cell differentiation, with a luciferase reporter gene vector carrying the cloned human evolutionarily conserved (ECS) - Iγ 3 sequence to analyze the functionality and cis regulatory elements of this region. We found that the upstream ECS-Iγ 3 region is a potent transcriptional promoter which is synergistically induced by CD40L and IL-4. Deletional mutant analysis showed that the CD40 responsive element contains two tandem κB sites, while the IL-4 responsive element consists of the same two κB sites and a STAT-6 binding site. Thus, our findings show that the human upstream ECS-Iγ3 region is an inducible promoter which contains all cis regulatory elements required for independent transcriptional activation by IgG class switch inducing stimuli.