TRANSCRIPTION OF THE HUMAN FOLYLPOLY-GAMMA-GLUTAMATE SYNTHETASE GENE

In mammals, folylpoly-gamma-glutamate synthetase (FPGS) activity is fo und in any cell undergoing sustained proliferative phases, but this en zyme also displays a tissue-specific pattern of expression in differen tiated tissues, It is now reported that the steady state levels of FPG S mRNA in normal and neoplastic cells reflect these patterns, supporti ng the concept that the control mechanisms underlying this distributio n are transcriptional. To initiate an understanding of these interacti ng levels of control, me have determined the position and properties o f the minimal FPGS promoter controlling transcription of the FPGS gene in human CEM leukemia cells, a line which expresses high levels of th is enzyme and its mRNA The TATA-less region immediately upstream of th e major transcriptional start site previously mapped in human tumor ce lls, which includes several GC- and Y-boxes, functioned as a remarkabl y efficient promoter when used to drive expression of a luciferase rep orter in transient expression studies in CEM cells, The minimal region of the FPGS promoter required for maximal transcriptional activation in CEM cells included the 80 base pairs over which the multiple transc riptional start sites were located, and the 43 base pairs immediately upstream. DNase I footprint analysis detected the binding of Sp1 at al l seven of the consensus sites within the probe used, two of which are contained within the minimal promoter region. The several Sp1 sites i mmediately upstream of the first major transcriptional start activated transcription in Drosophila cells when cotransfected with an Sp1 cons truct, including those in the region which functioned as a minimal pro moter in CEM cells, An additional region of the minimal promoter, situ ated between the two translational start codons of the FPGS gene, was bound by protein(s) from HeLa cell nuclear extracts. We conclude that transcription of the FPGS gene in CEM cells involves transactivation e vents over a limited upstream DNA sequence and that the FPGS promoter used in proliferating hu- man leukemic cells has strong similarity to other TATA-less promoters that utilize tandem, closely spaced Sp1 site s to initiate transcription.