Developmental regulation of the kappa locus involves both positive and negative sequence elements in the 3 ' enhancer that affect synergy with the intron enhancer

Expression of the mouse immunoglobulin kappa locus is regulated by the intr on and 3' enhancers. Previously, we have reported that these enhancers can synergize at mature B cell stages. Here we present our recent studies on th e identification and characterization of the 3' enhancer sequences that pla y important roles in this synergy. By performing mutational analyses with n ovel reporter constructs, we find that the 5' region of the cAMP response e lement (CRE), the PU.1/PIP, and the E2A motifs of the 3' enhancer are criti cal for the synergy. These motifs are known to contribute to the enhancer a ctivity. However, we also show that mutating other functionally important s equences has no significant effect on the synergy. Those sequences include the 3' region of the CRE motif, the BSAP motif, and the region 3' of the E2 A motif. We have further demonstrated that either the 5'-CRE, the PU.1/PIP, or the E2A motif alone is sufficient to synergize with the intron enhancer . Moreover, the PU.1 motif appears to act as a negative element at pre-B ce ll stages but as a positive element at mature B cell stages. We have also i dentified a novel negative regulatory sequence within the 3' enhancer that contributes to the regulation of synergy, as well as developmental stage an d tissue specificity of expression. While the levels of many of the 3' enha ncer binding factors change very little in cell lines representing differen t B cell stages, the intron enhancer binding factors significantly increase at more mature B cell stages.