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.     

T3 receptor suppression of Sp1-dependent transcription from the epidermal growth factor receptor promoter via overlapping DNA-binding sites

Expression of the human epidermal growth factor receptor (EGFR) gene is inhibited by ligand-activated thyroid hormone receptor (T3R). Binding sites for Sp1 and for the T3R.retinoid X receptor (RXR) complex overlap in a functional core of the EGFR promoter. Sp1 inhibited binding of the T3R complex to this 36-base pair (bp) EGFR element in vitro but did not affect binding of the T3R complex to a positive thyroid hormone response element (TRE). In Drosophila SL2 cells, which lack Sp1 and T3R, function of the EGFR promoter was strongly dependent on Sp1. Sp1-dependent promoter function was inhibited by ligand-activated T3R but not by mutant T3R defective in DNA or T3 binding. RXR increased the extent of inhibition. Sp1 enhanced activity of the 36-bp element placed 5' to a minimal TATA promoter and this enhancement was also repressed by T3R. Mutations in the 36-bp element were unable to separate Sp1 and T3R functions. However, addition of a second half-site 5' to the existing site in an inverted repeat configuration created a positive TRE. In the absence of ligand, T3R inhibited Sp1 stimulation from this altered element; addition of T3 reversed the inhibition. When a dimeric TRE is separated from Sp1-binding sites strong synergism was observed. The nature and location of the TRE thus strongly influence biological responses. A TRE site in the EGFR promoter that overlaps an Sp1-binding site inhibits Sp1 function but is unable to direct positive function.     

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.     

Control elements of Dnmt1 gene are regulated in cell-cycle dependent manner

The Dnmt1 gene is constitutively expressed and is required for the maintenance of global methylation after DNA replication. We investigated here the effects of histone deacetylase (HDAC) inhibitor and DNA demetylation agent on promoter activity of mouse Dnmt1 gene in somatic cells. The promoter activity of Dnmt1 gene was increased approximately 2-fold in the treatment of cells by Tricostatin A (TSA) at 1 x 10(-8) M, as compared with that without treatment of TSA. By contrast, treatment with 5-azacytidne (5aza-C) did not affect the promoter activity of the Dnmt1 gene. This result indicates the Dnmt1 gene is possibly regulated by histone acetylation. We also examined the expression levels of Dnmt1 gene and of its control elements like Sp1, Sp3 and p300 by the chromatin immunoprecipitation and Western blot analysis. The expression of Dnmt1 gene is observed at early S phase. Sp1 is recruited mainly at the G1 phase and Sp3 is recruited at the early S phase. p300 is also obviously recruited at the second S phase. These data indicated that the regulators of Dnmt1 gene were controlled in cell-cycle dependent manner.