The evolutionarily conserved sequence upstream of the human Ig heavy chain Sγ3 region is an inducible promoter: Synergistic activation by CD40 ligand and IL-4 via cooperative NF-κB and STAT-6 binding sites

Germline Cγ gene transcription is a crucial event in the process that leads to switch DNA recombination to IgG, but its regulation in the human is poorly understood. We took advantage of our monoclonal model of germinal center B cell differentiation, IgM⁺ IgD⁺ CL-01 cells, to define the role of the Iγ3 evolutionarily conserved sequence (ECS) in the germline transcriptional activation of the human Cγ3 gene. The Iγ3 ECS lies upstream of the major Iγ3 transcription initiation site and displays more than 90% identity with the corresponding human Iγ1, Iγ2, and Iγ4 regions. Reporter luciferase gene vectors containing the human γ3 ECS were used to transfect CL-01 cells, which have been shown to undergo Sμ→Sγ3 DNA recombination, upon engagement of CD40 by CD40 ligand (CD40L) and exposure to IL-4. In these transfected CL-01 cells, CD40:CD40L engagement and exposure to IL-4 synergistically induced γ3 ECS-dependent luciferase reporter gene activation. Targeted mutational analysis demonstrated that a tandem NF- κB/Rel binding motif is critical for the γ3 ECS responsiveness to both CD40L and IL-4, while a STAT-6-binding site is additionally required for IL- 4 inducibility. Electrophoretic mobility shift assays showed that p50/p65/c- Rel and STAT-6 are effectively induced by CD40L and IL-4, respectively, and bind to specific DNA motifs within the ECS. These partially overlapping CD40L and IL-4 responsive elements are functionally cooperative as the disruption of one of them prevents synergistic promoter activation. Thus, the γ3 ECS is an inducible promoter containing cis elements that critically mediate CD40L and IL-4-triggered transcriptional activation of the human Cγ3 gene.