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Pubmed ID :10721719
Publication Date : //

Two promoters regulate transcription of the mouse folylpolyglutamate synthetase gene three tightly clustered Sp1 sites within the first intron markedly enhance activity of promoter B.


The process of polyglutamylation mediated by folylpolyglutamate synthetase (FPGS) in mammalian cells has nutritional and pharmacological importance. In murine cells, FPGS expression is controlled by two promoters that, as we show here, vary substantially in their efficiency, at least in the context of a reporter gene assay. Characteristics of the most efficient promoter (promoter B) were examined in the present studies. Insertion in pGL3 of a 1635 bp segment of upstream sequence including the most upstream exon (B1c), intron B1c and only 26 bp of the more downstream exon Bla resulted in a 15-20-fold increase in transcription in NIH3T3 and Hep1-6 cells compared with the promoterless vector. Deletion analysis of DNA sequence upstream of exon B1c showed that transcription was regulated by putative cis active elements only within two distally located upstream segments which when deleted cumulatively increased transcription three- to four-fold. However, deletion of the 56 bp intron B1c immediately downstream of the most upstream exon (Blc) resulted in 1/10 the rate of transcription. Primer extension analysis with NIH3T3 cells revealed start sites for transcription appreciably upstream of and within exon B1c as well as downstream in exon B1a. This result is consistent with the frequent occurrence in murine cells of an FPGS variant (variant III) incorporating exon B1c [Roy et al., J. Biol. Chem. 271 (1996) 23820; 272 (1997) 5587]. Site-directed mutagenesis and DNAse I footprinting revealed that three canonical GC boxes, either overlapping or tightly clustered within intron B1c, bound Sp1 and markedly enhanced transcription, accounting for the maximal promoter B activity. Moreover, in a cellular background devoid of Sp1 activity, we demonstrate that Spl can induce high levels of promoter B activity in pGL3 transfectants, but only when intron B1c is included within the reporter gene construct used. These results suggest that the unusually tight cluster of active Sp1 sites within intron B1c are essential and sufficient for maximal activity of this promoter. These tightly clustered sites appear to act as an enhancer element in promoting transcription and efficiently stabilize transcription initiation complexes at both distal and proximal start sites.

Authors : Chen J , Hayes P , Roy K , Sirotnak F M ,

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