Organization of the transcription factor binding sites in the kappa Ig intron enhancer. Effects of position, orientation, and spacing.

The kappa Ig intron enhancer is comprised of multiple sequence motifs known to bind trans-acting factors that activate gene expression. A species comparison reveals a high level of conservation of the organization of the transcription factor binding sites within the enhancer. The importance of the conserved organization of the kappa intron enhancer was examined by using topologic mutations that disrupt the position, orientation, and spacing of individual binding sites within the enhancer. The effects of these changes were monitored by their effects on reporter gene activity at two distinct stages of B cell development. Previously, mutational analysis indicated the kappa B and kappa E2 sequence motifs to be the most crucial sites for intron enhancer function. We have demonstrated that intron enhancer activity is dependent on the position of the kappa B and kappa E2 sequence motifs within the enhancer. Intron enhancer function is, however, independent of kappa B and kappa E2 binding site orientation and is flexible in spacing requirements among binding sites.