The GAP-43 Gene Is a Direct Downstream Target of the Basic Helix-Loop-Helix Transcription Factors

The GAP-43 promoter region contains seven E-boxes (E1 to E7) that are organized in two clusters, a distal cluster (E3 to E7) and a proximal cluster (E1 and E2). Deletion analysis and site-directed mutagenesis of the GAP-43 promoter region showed that only the most proximal E1 E-box significantly modulates GAP-43 promoter activity. This E-box is conserved between the rat and human GAP-43 promoter sequences in terms of flanking sequence, core sequence (CAGTTG), and position. We found that endogenous E-box-binding proteins present in neuronal N18 cells recognize the E1 E-box and activate the GAP-43 promoter. The transcriptional activity of the GAP-43 promoter was repressed not only by the negative regulator Id2 protein, but also by two class A basic helix-loop-helix proteins, E12 and ME1a. In vitro analyses showed that both ME1a and E12 bind to the E1 E-box as homodimers. By Northern analyses, we established an inverse correlation between the level of E12 and ME1a mRNAs and GAP-43 mRNA in various neuronal cell lines as well as in ME1a-overexpressing PC12 cells. Therefore, we have identified a cis -acting element, the E1 E-box, located in the GAP-43 promoter region that modulates either positively or negatively the expression of the GAP-43 gene depending on which E-box-binding proteins occupy this site. Together, these data indicate that basic helix-loop-helix transcription factors regulate the expression of the GAP-43 gene and that the class A ME1a and E12 proteins act as down-regulators of GAP-43 expression.