Characterization of the human aldose reductase gene promoter

The promoter region of the human aldose reductase gene has been identified upstream of the translation start ATG codon. The promoter contains a TATA box, a CCAAT promoter element, and three Sp1 protein binding consensus sequences upstream of the capsite. A 640-base pair insert spanning +31 to -609 directs expression of the reporter gene chloramphenicol acetyltransferase in an orientation-specific manner in transfected Hep G2 cells. The promoter activity remained constant with deletions from base pairs -609 to -186. The TATA and the CCAAT consensus sequences show significant promoter activity, whereas the three Sp1 binding consensus sequences, individually, have no significant promoter activity. A GA-rich region (-186 to -146) contains two CGGAAA/G motifs, which show promoter activity and interaction with Hep G2 nuclear extract and GA-binding proteins (GABP alpha and GABP beta 1) as shown by mobility shift assays and DNase I footprinting. Similar cis-elements in herpes simplex virus type 1 interact with rat liver GABP and the viral VP16 protein to mediate the induction of immediate early viral genes. A GC-rich region (-87 to -31) is identified by mobility shift assay, and a consensus sequence of an androgen response element is present at -396 to -382. The human aldose reductase promoter, thus, has regulatory response elements that may be important during early development and puberty. These regulatory elements may play a significant role in the development of certain diabetic complications.     

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.     

Transactivation of the urokinase-type plasminogen activator receptor gene through a novel promoter motif bound with an activator protein-2alpha-related factor

The urokinase receptor overexpressed in invasive cancers promotes laminin degradation. The current study was undertaken to identify cis elements and trans-acting factors activating urokinase receptor expression through a footprinted (-148/-124) region of the promoter containing putative activator protein-2- and Sp1-binding motifs. Mobility shifting experiments using nuclear extract from a high urokinase receptor-expressing cell line (RKO) indicated that Sp1, Sp3, and a factor similar to, but distinct from, activator protein-2alpha bound to this region. Mutations preventing the binding of the activator protein 2alpha-related factor diminished urokinase receptor promoter activity. In RKO cells, the expression of a negative regulator of activator protein-2 function diminished urokinase receptor promoter activity, protein, and laminin degradation. Conversely, urokinase receptor promoter activity in low urokinase receptor-expressing GEO cells was increased by activator protein-2alphaA expression. Although using GEO nuclear extract, little activator protein-2alpha-related factor bound to the footprinted region, phorbol 12-myristate 13-acetate treatment, which induces urokinase receptor expression, increased complex formation. Mutations preventing the activator protein-2alpha-related factor and Sp1/Sp3 binding reduced urokinase receptor promoter stimulation by this agent. Thus, the constitutive and phorbol 12-myristate 13-acetate-inducible expression of the urokinase receptor is mediated partly through trans-activation of the promoter via a sequence (-152/-135) bound with an activator protein-2alpha-related factor.     

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.