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

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Abstract
The immunoglobulin κ light chain intronic enhancer (iEκ) 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 iEκ regulates κ rearrangement, we employed homologous recombination to mutate individual functional motifs within iEκ 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 iEκ, 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.