An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.
The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.
Jones
R M
RM
Massachusetts General Hospital Cancer Center, Charlestown, 02129, USA.
Branda
J
J
Johnston
K A
KA
Polymenis
M
M
Gadd
M
M
Rustgi
A
A
Callanan
L
L
Schmidt
E V
EV
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins; D039561:Eukaryotic Initiation Factor-4E; D005347:Fibroblasts; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014176:Protein Biosynthesis / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000502:physiology*; D012315:RNA Caps / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D016271:Proto-Oncogene Proteins c-myc; D012315:RNA Caps; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat