PROMOTION OF V(D)J RECOMBINATIONAL ACCESSIBILITY BY THE INTRONIC E-KAPPA ELEMENT - ROLE OF THE KAPPA-B MOTIF

The accessibility of a chromosomally integrated TCR beta minilocus rec ombination substrate in a V(D)J recombinase-inducible cell line (HDR37 ) depends on incorporation of transcriptional enhancer elements such a s the Ig kappa light chain intronic enhancer (E kappa). The E kappa el ement contains several functional motifs including the kappa B motif, which binds the NF-kappa B transcription factor. To assess molecular m echanisms by which E kappa promotes V(D)J recombinational accessibilit y, we compared the abilities of the wild-type E kappa, a corresponding E kappa sequence with a mutant kappa B motif (E kappa-kappa B-) and a kappa B motif dimer (kappa B2) to function in the context of the TCR beta minilocus/HDR37 system. The E kappa-containing minilocus underwen t demethylation, transcription and V(D)J recombination, independently of copy number or integration site. Transfectants containing low copy numbers (one or two) of the E kappa-kappa B--containing minilocus, lik e enhancerless or kappa B2-containing miniloci at any copy number, wer e inactive with respect to all three processes. In contrast, high-copy -number integrants of the E kappa-kappa B- substrates showed an integr ation-site dependent activation of all three processes. Together these data show that the kappa B motif plays a critical role in the ability of E kappa to confer V(D)J recombinational accessibility, but that it is not sufficient to mediate this process by itself.