Transforming Growth Factor β-Mediated Transcriptional Repression of c-myc Is Dependent on Direct Binding of Smad3 to a Novel Repressive Smad Binding Element

Abstract

Smad proteins are the most well-characterized intracellular effectors of the transforming growth factor β (TGF-β) signal. The ability of the Smads to act as transcriptional activators via TGF-β-induced recruitment to Smad binding elements (SBE) within the promoters of TGF-β target genes has been firmly established. However, the elucidation of the molecular mechanisms involved in TGF-β-mediated transcriptional repression are only recently being uncovered. The proto-oncogene c-myc is repressed by TGF-β, and this repression is required for the manifestation of the TGF-β cytostatic program in specific cell types. We have shown that Smad3 is required for both TGF-β-induced repression of c-myc and subsequent growth arrest in keratinocytes. The transcriptional repression of c-myc is dependent on direct Smad3 binding to a novel Smad binding site, termed a repressive Smad binding element (RSBE), within the TGF-β inhibitory element (TIE) of the c-myc promoter. The c-myc TIE is a composite element, comprised of an overlapping RSBE and a consensus E2F site, that is capable of binding at least Smad3, Smad4, E2F-4, and p107. The RSBE is distinct from the previously defined SBE and may partially dictate, in conjunction with the promoter context of the overlapping E2F site, whether the Smad3-containing complex actively represses, as opposed to transactivates, the c-myc promoter.

This work was supported by National Institutes of Health grants CA75368 and CA83770 to X.-F. Wang. J.P.F. and D.S.W. were supported by predoctoral fellowships from the Department of Defense Breast Cancer Research Program, DAMD17-98-1-8067 and DAMD17-00-1-0229, respectively. N.T.L was supported by a National Science Foundation predoctoral fellowship.

We thank Bert Vogelstein, Joan Massagué, David J. Loskutoff, Mitsuyasu Kato, Harold L. Moses, and Ning Zheng for providing reagents. We are also grateful to Suzanne D. Neill and John E. Bisi of GlaxoSmithKline for generating the Smad-expressing adenovirus constructs and Allan Balmain and Sheelagh Frame for advice on primary keratinocyte isolation. Joseph R. Nevins, Paloma M. Giangrande, and Ester P. Black were a source of helpful information regarding E2F function.