Promotion of V(D)J recombinational accessibility by the intronic Ek element: Role of the κB motif

The accessibility of a chromosomally integrated TCRβ minilocus recombination substrate in a V(D)J recombinase-inducible cell line (HDR37) depends on incorporation of transcrlptlonal enhancer elements such as the Igk light chain intronic enhancer (Ek). The Ek element contains several functional motifs including the kB motif, which binds the NF-kB transcription factor. To assess molecular mechanisms by which Ek promotes V(D)J recombinational accessibility, we compared the abilities of the wild-type Ek, a corresponding Ek sequence with a mutant kB motif (Ek-kB^-) and a kB motif dlmer (kB2) to function in the context of the TCRβ minilocus/HDR37 system. The Ek-containing mlnilocus underwent demethylation, transcription and V(D)J recombination, independently of copy number or integration site. Transfectants containing low copy numbers (one or two) of the Ek-kB^--contalning minilocus, like enhancerless or kB2-containing miniloci at any copy number, were inactive with respect to all three processes. In contrast, high-copy-number integrants of the Ek-kB^- substrates showed an integration-site dependent activation of all three processes. Together these data show that the kB motif plays a critical role in the ability of Ek to confer V(D)J recombinational accessibility, but that it is not sufficient to mediate this process by itself.