Important roles for E protein binding sites within the immunoglobulin kappa chain intronic enhancer in activating Vkappa Jkappa rearrangement.

The immunoglobulin kappa light chain intronic enhancer (iEkappa) activates kappa rearrangement and is required to maintain the earlier or more efficient rearrangement of kappa versus lambda (lambda). To understand the mechanism of how iEkappa regulates kappa rearrangement, we employed homologous recombination to mutate individual functional motifs within iE(kappa) in the endogenous kappa 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 kappa rearrangement. Surprisingly, mutation of the kappaB site had no apparent effect on kappa rearrangement. Comparable to the deletion of the entire iEkappa, simultaneous mutation of kappaE1 and kappaE2 reduces the efficiency of kappa 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

Global fitness profiling of fission yeast deletion strains by barcode sequencing

A genome-wide deletion library is a powerful tool for probing gene functions and one has recently become available for the fission yeast Schizosaccharomyces pombe. Here we use deep sequencing to accurately characterize the barcode sequences in the deletion library, thus enabling the quantitative measurement of the fitness of fission yeast deletion strains by barcode sequencing

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

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