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Volume 272, Number 48, Issue of November 28, 1997 pp. 30483-30490
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Constitutive Protection of E2F Recognition Sequences in the Human Thymidine Kinase Promoter during Cell Cycle Progression

(Received for publication, June 26, 1997, and in revised form, August 28, 1997)

From the Department of Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, California 91010

The sequences responsible for S phase-specific induction of the human thymidine kinase (TK) gene have been mapped to a small region that contains putative E2F binding sites. We have analyzed protein-DNA interactions at the TK promoter during cell cycle progression in human fibroblasts using an in vivo footprinting approach. We found 14 protein binding sites that were occupied in vivo. All of the sites (among them two inverted CCAAT boxes and several Sp1 sites) bound transcription factors constitutively throughout the cell cycle, i.e. none of the factor binding was cell cycle-dependent. An E2F-like site located between nucleotides 97 and 89 relative to the major transcription start site was protected in G₀, G₁, S, and G₂ phases. This cell cycle-independent protection of E2F sequences in the TK promoter differs from the G₀/G₁-restricted binding of E2F complexes observed for genes in which the E2F sites function as repressor elements (Tommasi, S., and Pfeifer, G. P. (1995) Mol. Cell. Biol. 15, 6901-6913; Zwicker, J., Liu, N., Engeland, K., Lucibello, F. C., and Müller, R. (1996) Science 271, 1595-1597). A comparison of several genes containing E2F motifs indicates that E2F sites located in proximity to the transcription initiation site (50 to +20) in TATA-less promoters predominantly function as repressor elements, while in other genes constitutively bound E2F complexes located further upstream mediate activation presumably in conjunction with a functional TATA box.

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