An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene.

TY - JOUR

T1 - An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene.

AU - Wade, M

AU - Blake, M C

AU - Jambou, R C

AU - Helin, K

AU - Harlow, E

AU - Azizkhan, J C

N1 - Keywords: 3T3 Cells; Animals; Base Sequence; CHO Cells; Carrier Proteins; Cell Cycle Proteins; Cloning, Molecular; Cricetinae; DNA; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Hela Cells; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Protein Binding; Repetitive Sequences, Nucleic Acid; Tetrahydrofolate Dehydrogenase; Transcription Factor DP1; Transcription Factors; Transcription, Genetic

PY - 1995

Y1 - 1995

N2 - 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.

AB - 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.

M3 - Journal article

C2 - 7730357

SN - 0021-9258

VL - 270

SP - 9783

EP - 9791

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 17

ER -