Important roles for E protein binding sites within the immunoglobulin κ chain intronic enhancer in activating VκJκ rearrangement

The immunoglobulin Knight chain intronic enhancer (iE(kappa)) activates K rearrangement and is required to maintain the earlier or more efficient rearrangement of K versus lambda (X). To understand the mechanism of how iE(kappa) regulates K rearrangement, we employed homologous recombination to mutate individual functional motifs within iE(kappa) in the endogenous K locus, including the NF-kappaB binding site (kappaB), as well as kappaE1, kappaE2, and kappaE3 E boxes. Analysis of the impacts of these mutations revealed that kappaE2 and to a lesser extent kappaE1, but not kappaE3, were important for activating K rearrangement. Surprisingly, mutation of the kappaB site had no apparent effect on K rearrangement. Comparable to the deletion of the entire iE(kappa), simultaneous mutation of kappaE1 and kappaE2 reduces the efficiency of K rearrangement much more dramatically than either kappaE1 or kappaE2 mutation alone. Because E2A family proteins are the only known factors that bind to these E boxes, these findings provide unambiguous evidence that E2A is a key regulator of kappa rearrangement.