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 recombination substrate in a V(D)J recombinase-inducible cell line (HDR37) depends on incorporation of transcriptional enhancer elements such as the Ig kappa light chain intronic enhancer (E kappa). The E kappa element contains several functional motifs including the kappa B motif, which binds the NF-kappa B transcription factor. To assess molecular mechanisms by which E kappa promotes V(D)J recombinational accessibility, 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 underwent demethylation, transcription and V(D)J recombination, independently of copy number of integration site. Transfectants containing low copy numbers (one or two) of the E kappa-kappa B(-)-containing minilocus, like enhancerless or kappa B2-containing miniloci at any copy number, were inactive with respect to all three processes. In contrast, high-copy-number integrants of the E kappa-kappa B- substrates showed an integration-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.
Demengeot
J
J
Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Oltz
E M
EM
Alt
F W
FW
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004742:Enhancer Elements, Genetic; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D007438:Introns; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection; D045567:VDJ Recombinases
D017931:DNA Primers; D007145:Immunoglobulin kappa-Chains; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases