Important Roles for E Protein Binding Sites within the Immunoglobulin κ Chain Intronic Enhancer in Activating V_κ J_κ Rearrangement

The immunoglobulin κ light chain intronic enhancer (iE_κ) activates κ rearrangement and is required to maintain the earlier or more efficient rearrangement of κ versus lambda (λ). To understand the mechanism of how iE_κ regulates κ rearrangement, we employed homologous recombination to mutate individual functional motifs within iE_κ in the endogenous κ locus, including the NF-κB binding site (κB), as well as κE1, κE2, and κE3 E boxes. Analysis of the impacts of these mutations revealed that κE2 and to a lesser extent κE1, but not κE3, were important for activating κ rearrangement. Surprisingly, mutation of the κB site had no apparent effect on κ rearrangement. Comparable to the deletion of the entire iE_κ, simultaneous mutation of κE1 and κE2 reduces the efficiency of κ rearrangement much more dramatically than either κE1 or κE2 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 κ rearrangement.