2 DISTINCT CLASS-A HELIX-LOOP-HELIX TRANSCRIPTION FACTORS, E2A AND BETA1, FORM SEPARATE DNA-BINDING COMPLEXES ON THE INSULIN GENE E BOX

Mutations in the RIPE3a element have shown it to be crucial for effici ent tissue-specific expression of the insulin gene. In order to isolat e factors binding to this element, we used a labeled RIPE3 probe to sc reen an expression library derived from a hamster insulinoma cell line . We isolated a clone encoding beta-cell E-box transcriptional activat or1 (BETA1). This clone is a member of the class A subfamily of the he lix-loop-helix superfamily of transcriptional activators, as determine d both by sequence analysis and by functional association with a class B member (myogenin). This clone is related to, but distinct from, oth er clones isolated from the same library which are also capable of bin ding RIPE3a. Analysis showed these additional clones to be the hamster homologs of E12 and E47 (German, M. S., Blaner, M. A., Nelson, C., Mo ss, L. G., and Rutter, W.J (1991) Mol. Endocrinol. 5, 292-299). Antibo dies were raised against BETA1 and against a common epitope of E12 and E47 to determine which proteins were contained in the native RIPE3a b inding complex. Using these antibodies, we were able to separate the c omplex into major and minor fractions which contained either E12/47 or BETA1, respectively. Thus, these two gene products are found in separ ate fractions of the tissue-specific binding activity and are therefor e both likely to be important in insulin gene regulation.