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J Biol Chem, Vol. 274, Issue 6, 3285-3293, February 5, 1999

Developmental Regulation of the  Locus Involves Both Positive and Negative Sequence Elements in the 3' Enhancer That Affect Synergy with the Intron Enhancer

From the Department of Biochemistry, Institute of Human Genetics and the Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455

Expression of the mouse immunoglobulin  locus is regulated by the intron and 3' enhancers. Previously, we have reported that these enhancers can synergize at mature B cell stages. Here we present our recent studies on the identification and characterization of the 3' enhancer sequences that play important roles in this synergy. By performing mutational analyses with novel reporter constructs, we find that the 5' region of the cAMP response element (CRE), the PU.1/PIP, and the E2A motifs of the 3' enhancer are critical for the synergy. These motifs are known to contribute to the enhancer activity. However, we also show that mutating other functionally important sequences 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 E2A 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 cell stages but as a positive element at mature B cell stages. We have also identified a novel negative regulatory sequence within the 3' enhancer that contributes to the regulation of synergy, as well as developmental stage and tissue specificity of expression. While the levels of many of the 3' enhancer binding factors change very little in cell lines representing different B cell stages, the intron enhancer binding factors significantly increase at more mature B cell stages.

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