An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene.
An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular and viral genes. This element, 5'-TTTCGCGCCAAA-3', is comprised of two overlapping, oppositely oriented sites which match the consensus E2F site (5'-TTT(C/G)(C/G)CGC-3'). Recent work has shown that E2F binding activity is composed of at least six related cellular polypeptides which are capable of forming DNA-binding homo- and heterodimers. We have investigated the binding of cellular E2F activity and of homo- and heterodimers of cloned E2F proteins to the inverted repeat E2F element. We have demonstrated that mutations in this element that abolish its inverted repeat nature, while preserving a single consensus E2F site, significantly decrease the binding stability of all of the forms of E2F tested. The rate of association of E2F-1/DP-1 heterodimers with the inverted repeat wild type site was not significantly different from those with the two single site mutated probes. Furthermore, the mutations decrease in vitro transcription and transient reporter gene expression 2-5-fold, an effect equivalent to that of abolishing E2F binding altogether. These data suggest a functional role that may explain the conservation of inverted repeat E2F elements among the DHFR promoters and several other cellular and viral promoters.
Wade
M
M
Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Blake
M C
MC
Jambou
R C
RC
Helin
K
K
Harlow
E
E
Azizkhan
J C
JC
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D056465:Retinoblastoma-Binding Protein 1; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase