Regulation of human glutathione S-transferase π gene transcription: influence of 5′-flanking sequences and trans-mactivating factors which recognize AP-1-binding sites

Charles S. Morrow, Merrill E. Goldsmith, Kenneth H. Cowan

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

To investigate the transcription regulation of human glutathione S-transferase π (GSTπ) gene expression, we fused the GSTπ promoter, including 2203 bp of the 5′-flanking region, exon 1, and most of intron 1, to the chloramphenicol acetyltransferase (CAT)-encoding reporter gene (cat). When transfected into human cell lines, this GST-cat construct (-2203 GST-cat) supported high level cat gene expression. RNase-protection and primer-extension experiments showed that the normal GSTπ transcriptional start point (tsp) is utilized, and furthermore, that intron 1 is faithfully removed by splicing from the majority of primary GST-cat transcripts. A series of constructs containing deletions in the GSTπ sequences of the -2203 GST-cat vector were prepared to define potential regulatory regions. Transfection of these deletion plasmids revealed that a region between GSTπ sequences -80 and -8 is absolutely required for cat expression. Furthermore, transfection of the -2203 GST-cat and deletion vectors into two human cell lines - one line which does not produce endogenous GSTπ(HeLa cells) and one which produces high levels of endogenous GSTπ (HS 578T cells) - failed to identify sequences that differentially influence the level of transcription in either cell line. A putative TRE (TPA responsive element or AP-1 recognition sequence) strategically situated upstream from the GSTπ tsp (-69 to -63) was examined by TPA treatment of HeLa cells transfected with GST-cat DNA. Additionally, the potential interaction of fos and jun proteins with the GSTπ promoter was examined by co-transfection of GST-cat constructs with jun and fos expression vectors in F9 cells. Both of these treatments, which are known to enhance transcription of several genes containing 5′-flanking TREs, failed to induce GST-cat expression. These data suggest that the putative TRE sequence in GSTπ is unresponsive both to phorbol esters and to these particular transcriptional activating factors of the fos and jun family.

Original languageEnglish (US)
Pages (from-to)215-225
Number of pages11
JournalGene
Volume88
Issue number2
DOIs
StatePublished - Apr 16 1990

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Keywords

  • Recombinant DNA
  • deletion constructs
  • oncogenes fos, jun
  • phorbol esters

ASJC Scopus subject areas

  • Genetics

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