FUNCTIONAL-CHARACTERIZATION OF THE HUMAN THIOPURINE S-METHYLTRANSFERASE (TPMT) GENE PROMOTER

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catal yzes S-methylation of aromatic and heterocyclic sulfhydryl compounds, including anticancer and immunosuppressive thiopurines. We recently is olated the human TPMT promoter, which does not contain TATA box or CCA AT element consensus sequences, but is GC rich with multiple GC boxes and other putative cis-regulatory elements, Here, we report the functi onal characterization of the TPMT promoter, revealing several positive regulatory elements and identifying stimulating protein 1 (Spl) as an important traits-activator essential for constitutive activity in cel l culture. One major and two closely located minor transcription start points were identified in HepG2 cells. Deletion analysis revealed pos itive cis-regulatory elements located in the regions -85 to -75, -68 t o -58, -58 to -51 and +34 to +60 relative to the transcription start s ite. DNasel footprinting analysis and cotransfection in Drosophila Sch neider SL2 cells documented that Spl binds to the TPMT promoter and is important for constitutive activity. We conclude that constitutive tr anscription of the TPMT gene involves a limited upstream GC-rich DNA s equence, containing multiple GC boxes, and that transcription factor S pl [or related protein(s)] is an important traits-activator of this TA TA-less promoter.