Regulation of the promoter for human immunoglobulin gamma 3 germ-line transcription and its interaction with the 3 'alpha enhancer

The mechanism underlying the differential regulation of switching to human IgG subclasses is still largely unknown. We demonstrate that the region ups tream of the initiation sites for gamma 3 germ-line (GL) transcripts contai ns a functional promoter which is synergistically induced by IL-4, antibody to CD40 and phorbol dibutyrate in transient transfection assays in the hum an DG75 cell line. Linker-scanning mutations identified multiple elements i n the 3' half of the evolutionarily conserved sequence that are required fo r inducibility. Electrophoretic mobility shift assays showed that Stat6 and NF-xB p50/p65 are induced after stimulation, and bind to specific sequence motifs within the promoter. Overexpression of Stat6, NF-xB p50/p65 and C/E BP gamma synergistically induced the GL gamma 3 promoter. Insertion of DNA segments from the human 3' IgH regions, which may function as a locus contr ol region for switch recombination, greatly activated the promoter in an or ientation-independent manner. Duplication of the enhancer fragments resulte d in a further increase of promoter activity. The greater enhancement of th e HS1,2 fragment from the 3' alpha 1 rather than the alpha 2 locus may sugg est a mechanistic explanation for the differential expression of various is otypes.