PROMOTER ANALYSIS OF THE NEURONAL NICOTINIC ACETYLCHOLINE-RECEPTOR ALPHA-4 GENE - METHYLATION AND EXPRESSION OF THE TRANSGENE

Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes compos e a family of genes. The major isoform of nAChR in the brain is made u p of the alpha 4 and beta 2 subunits and possesses a high affinity for nicotine. To investigate the mechanisms of the regulation of the nACh R alpha 4 gene expression in mouse, its genomic DNA was cloned and cha racterized. The transcription initiation site was mapped by primer ext ension and RNase protection experiments and localized at about 254 bp upstream of the translation initiation site. The 5' flanking region of this gene did not have typical TATA box but GC-rich sequences were fo und around the initiation site. Methylation analysis of this region re vealed that: genomic DNAs from liver and muscle are partially methylat ed, whereas little methylation was observed in genomic DNA from brain. To characterize the cis-acting elements driving cell-specific express ion of the alpha 4 subunit gene, we produced lines of transgenic mice which carry a series of fragments of the alpha 4 gene fused with bacte rial lacZ as a reporter gene. An 11.5-kb DNA fragment containing 9 kb of the region upstream of the transcription initiation site and the fi rst intron was found to confer an expression pattern which coincides r ather well with the endogenous gene expression pattern at early embryo nic stages, suggesting that the elements necessary For the onset of al pha 4 gene expression are located in this region. A DNA fragment conta ining the 1.8-kb upstream sequence and the first intron drove expressi on of lacZ in a limited subset of alpha 4 expressing cells, whereas th e 1.8-kb upstream sequence alone did not elicit any significant expres sion. These results show that both upstream and intronic sequences are important for cell-specific expression of the nAChR alpha 4 gene.