Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter.
We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.
Fornasari
D
D
CNR Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Italy. diegof@farma9.csfic.mi.cnr.it
Battaglioli
E
E
Flora
A
A
Terzano
S
S
Clementi
F
F
D001483:Base Sequence; D005796:Genes / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000235:genetics*
D011978:Receptors, Nicotinic