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.
Chiaramello
A
A
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, USA.
Neuman
T
T
Peavy
D R
DR
Zuber
M X
MX
D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002874:Chromosome Mapping; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins; D015183:Restriction Mapping; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D019922:GAP-43 Protein; D051797:Inhibitor of Differentiation Protein 2; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; C497450:ID2 protein, human; C497451:Id2 protein, rat; C073366:TCF12 protein, human