Regulation of the neural-specific gene VGF in PC12 cells. Identification of transcription factors interacting with NGF-responsive elements.

Nerve growth factor (NGF) is an important regulator of differentiation and survival in both the peripheral and central nervous systems. We have begun to analyze the mechanism by which NGF induces the expression of a neural specific gene, VGF, in PC12 cells. Using DNase I footprinting and transient transfection analysis, we identified two VGF promoter regions, V1 and V2, that are required for basal promoter expression as well as gene induction by NGF, epidermal growth factor (EGF), and cAMP. The V1 element is essential for VGF promoter function, but it is not sufficient to confer NGF responsiveness to a heterologous promoter. In contrast, the V2 element can independently stimulate the expression of a linked herpes simplex virus (HSV) thymidine kinase promoter in response to NGF. We showed that the V2 region also contains a sequence that acts as a promoter-specific negative regulator of basal VGF gene expression. As determined by gel mobility shift and Southwestern analysis, the V1 sequence is recognized by a novel PC12 nuclear protein of about 110-kDa molecular mass. Using oligonucleotide competition and antibody supershift assays, we demonstrated that the cAMP-response element (CRE) motif within the V2 element interacted specifically with proteins related to cAMP-response element binding (CREB), JunB, and JunD transcription factors. The JunB-related binding activities were transiently induced by NGF, suggesting that part of the mechanism utilized by NGF to activate VGF transcription includes increased synthesis of a V2 binding protein. Taken together, our analysis suggests that the VGF promoter is regulated by a complex mechanism, and its activation requires combinatorial action of several transcription factors interacting with multiple promoter elements.