Class switching in B cells lacking 3′ immunoglobulin heavy chain enhancers

The 40-kb region downstream of the most 3' immunoglobulin (Ig) heavy chain constant region gene (C alpha) contains a series of transcriptional enhancers speculated to play a role in Ig heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR regulatory region, we generated mice with germline mutations in which one or the other of the two most 5' enhancers in this cluster (respectively referred to as HS3a and HS1,2) were replaced either with a pgk-neo' cassette (referred to as HS3aN and HS1,2N mutations) or with a loxP sequence (referred to as HS3a Delta and HS1,2 Delta, respectively). B cells homozygous for the HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic similarity of the two insertion mutations, both of which were cis-acting, suggested that inhibition might result from pgk-neo' cassette gene insertion rather than enhancer deletion. Accordingly, CSR returned to normal in B cells homozygous for the HS3a Delta or HS1,2 Delta mutations. In addition, induced expression of the specifically targeted pgk-neo' genes was regulated similarly to that of germline C-H genes. Our findings implicate a 3' CSR regulatory locus that appears remarkably similar in organization and function to the beta-globin gene 5' LCR and which we propose may regulate differential CSR via a promoter competition mechanism.