Estrogen induces adenosine deaminase gene expression in MCF-7 human breast cancer cells: role of estrogen receptor-Sp1 interactions

Adenosine deaminase (ADA) gene expression is induced by 17beta-estradiol (E2) in MCF-7 human breast cancer cells, whereas the antiestrogens 4'-hydroxytamoxifen and ICI 182,780 exhibit partial estrogen receptor (ER) agonist/antagonist and antagonist activities, respectively. Previous studies have shown that the -211 to +11 region of the ADA gene promoter contains six GC-rich sites (I-VI) that bind Sp1 protein, and these elements are required for high basal expression. In transient transfection studies with pADA211, which contains the -211 to +11 ADA gene promoter linked to a bacterial chloramphenicol acetyl transferase (CAT) reporter gene, E2 and tamoxifen (but not ICI 182,780) induced CAT activity. Ligand-induced transactivation was observed only in cells cotransfected with expression plasmids for wild-type ER or HE11, which does not contain the DNA-binding domain of the ER. Cotransfection with HE15 and HE19, which contain the DNA-binding domain and activation function-1 (AF-1) and AF-2 of the ER, respectively, did not result in E2-induced activity. Subsequent deletion analysis of the ADA gene promoter showed that Sp1 binding site IV (-79 to -73) was primarily responsible for hormone responsiveness. ER activation of ADA gene expression is another example of an E2-induced gene that is dependent on ER/Sp1 interactions with a site-specific GC-rich motif.     

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.