The Igκ Gene Enhancers, E3′ and Ed, Are Essential for Triggering Transcription

The mouse Ig kappa gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). Previous studies on B lymphocytes derived from mutant embryonic stem cells have shown that deletion of either Ei or E3' significantly reduces Ig kappa gene rearrangement, whereas the combined deletion of both Ei and E3' eliminates such recombination. Furthermore, deletion of either E3' or Ed significantly reduces rearranged Ig kappa gene transcription. To determine whether the combined presence of both E3' and Ed are essential for Ig kappa gene expression, we generated homozygous double knockout (DKO) mice with targeted deletions in both elements. Significantly, homozygous DKO mice were unable to generate kappa(+) B cells both in bone marrow and the periphery and exhibited surface expression almost exclusively of Ig lambda-chains, despite the fact that they possessed potentially functional rearranged Ig kappa genes. Compared with their single-enhancer-deleted counterparts, Ig kappa loci in homozygous DKO mice exhibited dramatically reduced germline and rearranged gene transcription, lower levels of gene rearrangement and histone H3 acetylation, and markedly increased DNA methylation. This contributed to a partial developmental block at the pre-B cell stage of development. We conclude that the two downstream enhancers are essential in Ig kappa gene expression and that Ei in homozygous DKO mice is incapable of triggering Ig kappa gene transcription. Furthermore, these results reveal unexpected compensatory roles for Ed in E3' knockout mice in triggering germline transcription and V kappa gene rearrangements to both J kappa and RS elements. The Journal of Immunology, 2010, 185: 7544-7552.