ISOLATION OF 2 E-BOX BINDING-FACTORS THAT INTERACT WITH THE RAT TYROSINE-HYDROXYLASE ENHANCER

The enhancer of the rat tyrosine hydroxylase gene (TH) in PC8b cells i s composed of the AP1 motif (TCAT-TCA, -205 to -199) and an overlappin g 20-base pair dyad symmetry element (TCAGAGGCAGGTGCCTGTGA, -201 to -1 82) whose core is an E-box. We have isolated two partial cDNA clones t hat encode factors which bind the TH dyad. One is rITF2 with a basic h elix-loop-helix motif and the other is CDP2 with a homeodomain. rITF2 is a rat homolog of human ITF2 (or E2-2), and CDP2 is a member of a ne w family of homeoproteins defined by histidine as the 9th residue of t he recognition helix and by unique 64 amino acid repeats related to th ose of the Drosophila cut gene. The binding affinity of CDP2 alone is relatively weak, but it enhances the binding of rITF2 to the TH-dyad. In transfected F9 cells, activation of a TH-driven reporter requires b oth rITF2 and CDP2, suggesting that the proteins may functionally inte ract. However, rITF2 and CDP2 are not restricted to TH-expressing tiss ues; hence they may not be involved in the tissue-specific expression of TH. In addition, CDP2 is phosphorylated in vitro and in vivo.