Two 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 efficient tissue-specific expression of the insulin gene. In order to isolate factors binding to this element, we used a labeled RIPE3 probe to screen an expression library derived from a hamster insulinoma cell line. We isolated a clone encoding beta-cell E-box transcriptional activator1 (BETA 1). This clone is a member of the class A subfamily of the helix-loop-helix superfamily of transcriptional activators, as determined both by sequence analysis and by functional association with a class B member (myogenin). This clone is related to, but distinct from, other clones isolated from the same library which are also capable of binding RIPE3a. Analysis showed these additional clones to be the hamster homologs of E12 and E47 (German, M. S., Blaner, M. A., Nelson, C., Moss, L. G., and Rutter, W. J. (1991) Mol. Endocrinol. 5, 292-299). Antibodies were raised against BETA 1 and against a common epitope of E12 and E47 to determine which proteins were contained in the native RIPE3a binding complex. Using these antibodies, we were able to separate the complex into major and minor fractions which contained either E12/47 or BETA 1, respectively. Thus, these two gene products are found in separate fractions of the tissue-specific binding activity and are therefore both likely to be important in insulin gene regulation.