STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE HUMAN ALPHA-3 NICOTINIC SUBUNIT GENE PROMOTER

We describe the structural and functional features of the human alpha 3 nicotinic receptor subunit promoter. A 0.35-kb region immediately up stream of the start codon was identified that when transfected in huma n neuroblastoma cells was able to drive the expression of the lucifera se reporter gene with a strength comparable to that of the well-charac terized simian virus 40 promoter/enhancer. This region displayed the f eatures of a multistart-site, GC-rich, TATA-less, and CAAT-less promot er, containing many overlapping Sp1 and AP-2 putative binding sites. F urther dissections of the 0.35-kb fragment revealed that its 3' region , specifying the 5' UT of the mRNA, plays a relevant positive effect i n determining the strength of the promoter. This region contains putat ive cis-acting elements for AP-2, nuclear factor-kappa B, and the rece ntly described multiple-start site element downstream-1. By mutation a nalysis, we showed that these sites are functional and when combined i ncrease the promoter activity by 4-fold. The 0.35-kb promoter was foun d to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha 3 Alu repeat works as a composite regio n, containing both positive and negative elements that control the act ivity of the downstream promoter. Finally, we investigated the tissue- specific activity of the human alpha 3 gene 5' regulatory sequences, s howing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.