Characterization of a promoter for the human glial cell line-derived neurotrophic factor gene

To address the regulation of glial cell line-derived neurotrophic factor (G DNF) gene expression, we have isolated 5' extended cDNAs, cloned the human GDNF gene, and characterized the promoter. GDNF-encoding 5' extended cDNAs containing a novel exon were isolated via reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from human fetal kidney and adult skeletal muscle. The GDNF gene was isolated from a human genomic library in a P1 bac teriophage vector. Analysis of the 5' flanking sequence revealed a promoter that lacks a CCAAT-box motif and is GC rich. Consensus binding sites for a variety of transcription factors have been identified in the promoter. Pro moter/reporter plasmids have been constructed by fusion of the promoter and a portion of exon I to a luciferase gene. The promoter/reporter construct and a number of promoter deletions were transiently transfected into two hu man cell lines known to express GDNF. Multiple enhancer and silencer region s were revealed as well as a minimal promoter sufficient for basal transcri ption. Finally, a RT-PCR assay, specific for transcripts originating from t his GDNF promoter, was developed and used to show that this promoter is act ive in vivo. The results suggest GDNF is regulated in a complex manner. (C) 1999 Elsevier Science B.V. All rights reserved.