Identification of two activating elements in the proximal promoter region of the human glutathione transferase-A1 and -A2 genes.

Promoter regions derived from the human glutathione S-transferase (GST) alpha gene cluster located on chromosome 6p12 were studied: the identical proximal promoters of the GST A1 and GST A2 genes and a proximal promoter of a pseudogene of this class. The sequence of the pseudogene promoter differs in four single nucleotides at positions -86, -66, -41, and -13, and a noncritical TTT insertion at positions -71 to -69 from the GST A1/A2 promoter. Here, it was shown that the GST A1/A2 proximal promoters differed by a factor of 3.4 in their activity from the proximal pseudogene promoter. Therefore, the functional significance of single base exchanges was examined by introducing individual point mutations at the four positions within the proximal GST A1/A2 promoter. In functional tests in transiently transfected human hepatoblastoma HepG2 cells the base exchange at position -13 showed no effect, whereas mutations at position -41 or -86 diminished the promoter activity to a level comparable to the pseudogene promoter. Promoter fragments of both genes spanning over these four sites were analyzed in a heterologous promoter context for their functionality in HepG2 cells. Moreover, gel shift experiments showed specific binding of nuclear proteins to these promoter fragments. The results show that in the proximal GST A1/A2 promoter the sites at position -41 or -86 are essential for the binding of activating transcription factor complexes.

A 47-amino-acid fragment of SV40 T antigen represses transcription from human GSTalpha promoters.

SV40 T antigen downregulates the expression of an important detoxification enzyme, glutathione S-transferase alpha (GSTalpha). We show here that the target of this repression is a 14-bp element common to the human GSTA1 and GSTA2 promoters. This element, which we have named TAGR, is also critical for high-level, constitutive expression from these promoters. The TAGR element does not appear to contain a binding site for any transcription factor known to be present in fibroblasts, although the TAGR element does resemble the binding site for the Ikaros transcription factor found in hematopoietic cells. We also have identified a 47-amino-acid fragment of T antigen that includes amino acids 83-100 and 119-147, which is sufficient to repress transcription from the GSTalpha promoter in transient transcription assays. Thus, GSTalpha repression does not require binding of T antigen to pRb, p300, or p53, since the domains of T antigen required for binding these cellular proteins are missing from this T antigen fragment. We show, however, that this fragment does bind to three cellular proteins with approximate molecular weights of 54, 59, and 94 kDa.

Positive and negative regulatory regions in promoters of human glutathione transferase alpha genes.

The human glutathione transferase (GST, EC 2.5.1.18) alpha class locus comprises several genes and pseudogenes. Genomic DNA encoding several human alpha-class-related genes and pseudogenes was cloned and characterized. Three distinct but highly similar 5'-flanking regions of GST alpha genes as well as a series of 5'-deletions were investigated for promoter activity by fusion to the luciferase reporter gene. Transient transfection of these luciferase constructs into human hepatoblastoma, kidney carcinoma, nephroblastoma or bladder carcinoma cells revealed that the promoters are active and contain both positive and negative regulatory regions that behave in a cell-type specific fashion. The 150 bp proximal promoter regions of the three sequences retained the same relative activities as the full length promoters. Two of them were equally active, whereas the third one showed only 20% of the activity of the two stronger promoters. Site-directed mutagenesis indicated that a conspicuous insertion of three nucleotides (TTT) in the weak promoter is not responsible for the different activities.