Functional characterization of the human thiopurine S-methyltransferase (TPMT) gene promoter.
Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S-methylation of aromatic and heterocyclic sulfhydryl compounds, including anticancer and immunosuppressive thiopurines. We recently isolated the human TPMT promoter, which does not contain TATA box or CCAAT element consensus sequences, but is GC rich with multiple GC boxes and other putative cis-regulatory elements. Here, we report the functional characterization of the TPMT promoter, revealing several positive regulatory elements and identifying stimulating protein 1 (Sp1) as an important trans-activator essential for constitutive activity in cell culture. One major and two closely located minor transcription start points were identified in HepG2 cells. Deletion analysis revealed positive cis-regulatory elements located in the regions -85 to -75, -68 to -58, -58 to -51 and +34 to +60 relative to the transcription start site. DNaseI footprinting analysis and cotransfection in Drosophila Schneider SL2 cells documented that Sp1 binds to the TPMT promoter and is important for constitutive activity. We conclude that constitutive transcription of the TPMT gene involves a limited upstream GC-rich DNA sequence, containing multiple GC boxes, and that transcription factor Sp1 [or related protein(s)] is an important trans-activator of this TATA-less promoter.
Fessing
M Y
MY
Department of Pharmaceutical Sciences, St Jude Children's Research Hospital, University of Tennessee, Memphis 38105, USA.
Krynetski
E Y
EY
Zambetti
G P
GP
Evans
W E
WE
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004330:Drosophila; D005809:Genes, Regulator; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation
D016329:Sp1 Transcription Factor; D008780:Methyltransferases; C022745:thiopurine methyltransferase