Transcription of the mouse RFC-1 gene encoding a folate transporter. Multiplicity and properties of promoters with minimum requirements for their basal activity.
The mouse RFC-1 gene incorporates alternates of exon 1 (exon 1 and 1a) which encode different 5' ends. This finding, and the elucidation of a promoter-like sequence immediately upstream of these alternates of exon 1, suggest that two separate promoters drive transcription of this gene. The regions upstream of either exon 1 or exon 1a inserted in pGL3 will separately promote transcription in NIH3T3 cells of the luciferase reporter gene, with the region upstream of exon 1 having the strongest promoter activity. Tissue-specific expression in the form of RFC-1 mRNA splice variants reflects the separate action of each promoter. In the most upstream portion of the region proximal to exon 1a, elements were revealed that enhance transcription along with a more downstream element that suppresses transcription in NIH3T3 cells. Three Sp1 sites closely proximal to exon 1a within a region spanning 123 nucleotides were shown to be transcriptionally active by site-directed mutagenesis, with the middle SP1 site found to be the most important of the three in maintaining basal promoter activity. A poly (GT) 21 di-nucleotide repetitive element upstream of these Sp1 sites was found in a region which, when deleted, increased transcription. In the region upstream of exon 1, two elements were elucidated which enhanced transcription. Site-directed mutagenesis showed that two adjacent SP1 sites proximal to exon 1 were equally important in sustaining basal promoter activity. The role of each Sp1 site in maintaining basal activity of each promoter was confirmed by DNase I footprinting analysis. In addition, a binding site of unknown significance was identified by this analysis within the upstream promoter sequence between the two Sp1 sites proximal to exon 1a. These data show that both promoters regulating expression of the RFC-1 gene utilize closely spaced Sp1 sites in tandem to sustain basal transcription, at least in NIH3T3 cells, in a manner characteristic of TATA-less promoters.
Tolner
B
B
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Singh
A
A
Esaki
T
T
Roy
K
K
Sirotnak
F M
FM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D004247:DNA; D018983:DNA Footprinting; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D017384:Sequence Deletion; D014158:Transcription, Genetic
D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C120043:SLC19A2 protein, human; C492292:Slc19a2 protein, mouse; D004247:DNA; D008156:Luciferases