Identification of a transcriptional initiator element in the cytochrome c oxidase subunit Vb promoter which binds to transcription factors NF-E1 (YY-1, delta) and Sp1.
We have mapped the basal promoter activity of the mouse cytochrome c oxidase (COX) subunit Vb gene to the -17 to +20 region which contains two putative ets binding sites flanking an NF-E1 site fused to an Sp1 site. A 17-nucleotide sequence flanking the major transcription start site (-8 to +9), referred to as 17Inr (initiator sequence) was able to drive CAT activity in 3T3 cells to a level comparable to the construct containing sequences -17 to +20. This suggests that the 17Inr sequence contains the initiator activity. The 17Inr contains a pyrimidine-rich sequence, commencing with a CA that corresponds to the major transcription start site. Primer extension of RNA from transfected cells demonstrated that transcription initiation with the 17Inr template occurs at a site identical to the endogenous gene. DNA-protein binding by gel mobility shift and methylation interference analyses indicated that the pyrimidine-rich sequence immediately flanking the transcription start site consists of an NF-E1 factor binding motif with an overlapping upstream Sp1 binding site. A 13-nucleotide sequence, 13Inr (-4 to +9), which retains the NF-E1 binding activity but does not bind Sp1, was able to promote chloramphenicol acetyltransferase gene expression at levels similar to the 17Inr sequence, suggesting that NF-E1 factor binding is critical for initiator function. Finally, using an in vitro transcription system from Drosophila embryos we demonstrate that NF-E1 is necessary for transcription activation of both the 17Inr and the 13Inr initiator templates. Thus NF-E1 binding appears to be important for basal promoter function of the mouse COXVb gene.
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6047.
A
A
Basu
K
K
Park
M L
ML
Atchison
R S
RS
Carter
N G
NG
Avadhani
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA; D003576:Electron Transport Complex IV