BETA3, A NOVEL HELIX-LOOP-HELIX PROTEIN, CAN ACT AS A NEGATIVE REGULATOR OF BETA2 AND MYOD-RESPONSIVE GENES

Using degenerate PCR cloning we have identified a novel basic helix-lo op-helix (bHLH) transcription factor, BETA3, from a hamster insulin tu mor (HIT) cell cDNA library. Sequence analysis revealed that this fact or belongs to the class B bHLH family and has the highest degree of ho mology with another bHLH transcription factor recently isolated in our laboratory, BETA2 (neuroD) (J. E. Lou, S. M. Hollenberg, L. Snider, D . L. Turner, N. Lipnick, and H. Weintraub, Science 268:836-844, 1995; F. J. Naya, C. M. M. Stellrecht, and M.-J. Tsai, Genes Dev. 8:1009-101 9, 1995). BETA2 is a brain- and pancreatic-islet-specific bHLH transcr iption factor and is largely responsible for the tissue-specific expre ssion of th. insulin gene. BETA3 was found to be tissue restricted, wi th the highest levels of expression in HIT, lung, kidney, and brain ce lls. Surprisingly, despite the homology between BETA2 and BETA3 and it s intact basic region, BETAS is unable to bind the insulin E box in ba ndshift analysis as a homodimer or as a heterodimer with the class A b HLH factors E12, E47, or BETA1. Instead, BETA3 inhibited both the E47 homodimer and the E47/BETA2 heterodimer binding to the insulin E box. In addition, BETA3 greatly repressed the BETA2/E47 induction of the in sulin enhancer in HIT cells as well as the MyoD/E47 induction of a mus cle-specific E box in the myoblast cell line C2C12. In contrast, expre ssion of BETA3 had no significant effect on the GAL4-N P16 transcripti onal activity. Immunoprecipitation analysis demonstrates that the mech anism of repression in via direct protein-protein interaction, presuma bly by heterodimerization between BETA3 and class A bHLH factors.