Abstract
In mammals, folylpoly-gamma-glutamate synthetase (FPGS) activity is found in any cell undergoing sustained proliferative phases, but this enzyme also displays a tissue-specific pattern of expression in differentiated tissues. It is now reported that the steady state levels of FPGS mRNA in normal and neoplastic cells reflect these patterns, supporting the concept that the control mechanisms underlying this distribution are transcriptional. To initiate an understanding of these interacting levels of control, we have determined the position and properties of the minimal FPGS promoter controlling transcription of the FPGS gene in human CEM leukemia cells, a line which expresses high levels of this enzyme and its mRNA. The TATA-less region immediately upstream of the major transcriptional start site previously mapped in human tumor cells, which includes several GC- and Y-boxes, functioned as a remarkably efficient promoter when used to drive expression of a luciferase reporter 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 transcriptional start sites were located, and the 43 base pairs immediately upstream. DNase I footprint analysis detected the binding of Sp1 at all seven of the consensus sites within the probe used, two of which are contained within the minimal promoter region. The several Sp1 sites immediately upstream of the first major transcriptional start activated transcription in Drosophila cells when cotransfected with an Sp1 construct, including those in the region which functioned as a minimal promoter in CEM cells. An additional region of the minimal promoter, situated 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 events over a limited upstream DNA sequence and that the FPGS promoter used in proliferating human leukemic cells has strong similarity to other TATA-less promoters that utilize tandem, closely spaced Sp1 sites to initiate transcription.
Highlights
Folylpoly-␥-glutamate synthetase (FPGS)1 catalyzes the ATPdependent formation of an amide bond between the ␥-carboxyl group of the naturally occurring folates and the amino group of glutamic acid
FPGS mRNA Levels in Normal and Neoplastic Tissues—The size and abundance of transcripts from the FPGS gene were determined by Northern blot analysis in normal tissues and neoplastic cell lines
When RNA from eight human tumor cell lines was hybridized with the human FPGS probe, there was a high level of FPGS mRNA detected in all of the samples (Fig. 1c)
Summary
Folylpoly-␥-glutamate synthetase (FPGS) catalyzes the ATPdependent formation of an amide bond between the ␥-carboxyl group of the naturally occurring folates and the amino group of glutamic acid. A previous study in this laboratory [6] concluded that FPGS levels are controlled by at least two mechanisms, one of which is linked to proliferation and the other results in a tissuespecific pattern of expression in differentiated tissues. Despite this complexity, all available evidence [13] appears to rule out the existence of multiple genes for FPGS function, a fact that has focused attention on control of the known FPGS locus. The limited information available implicates the level of transcription of this gene as the major determinant of cellular FPGS activity
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