The Retinoblastoma Susceptibility Gene Product Represses Transcription When Directly Bound to the Promoter

doi:10.1074/jbc.270.15.8837

The Retinoblastoma Susceptibility Gene Product Represses Transcription When Directly Bound to the Promoter (*)

From the (1) Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

^¶ To whom correspondence should be addressed. Tel.: 412-648-9268; Fax: 412-624-1401.

Abstract

Rb represses E2F-mediated transcription in part by blocking the trans-activation domain of E2F. In addition, Rb can convert an E2F binding site from a positive to a negative element. To examine the effect of a Rb-DNA-bound complex on transcription, full-length Rb was fused to the DNA binding domain of GAL4. Here, we report that GAL4-Rb can repress transcription mediated by either Sp1, AP-1, or p53, dependent upon the presence of both the GAL4 DNA binding domain and GAL4 binding sites. Moreover, GAL4-Rb inhibited the activity of the herpes simplex virus tk promoter from GAL4 binding sites located at a distance from the promoter. In contrast, GAL4-Rb was unable to repress basal transcription. Cotransfection of specific cyclins and cyclin-dependent kinases or SV40 T-antigen abolished the repressive activity of GAL4-Rb. The domains of Rb involved in mediating the repression of transcription were mapped to regions that are overlapping, but not identical, to those required for the interaction with E2F. We propose that Rb can function as a general repressor of transcription when bound to the promoter region.

Footnotes

↵^§ Supported by a postdoctoral fellowship from the Association pour la Recherche contre le Cancer.
↵* This work was supported by Public Health Service Award CA55227 from NCI, National Institutes of Health (to P. D. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CAT

chloramphenicol acetyltransferase

TAF

TATA binding protein-associated factor

HSV

herpes simplex virus

bp

pase pair(s)

cdk

cyclin-dependent kinase.