Platelet-Derived Growth Factor-Stimulated Expression of the MCP-1 Immediate-Early Gene Involves an Inhibitory Multiprotein Complex

Abstract

We have demonstrated previously that the seven-nucleotide (nt) motif TTTTGTA (the heptamer) that is present within the proximal 3′ untranslated sequences of numerous immediate-early genes is essential for platelet-derived growth factor (PDGF)-stimulated induction of the MCP-1 immediate-early gene. On this basis, the heptamer was suggested to be a conserved regulatory element involved in immediate-early gene expression, although its mechanism of action was unknown. Herein, we demonstrate that the heptamer functions to remove an inhibition of PDGF induction of MCP-1maintained by two independently acting inhibitory elements present in the MCP-1 5′ flanking sequences (designated I* elements). PDGF treatment relieves the I*-mediated inhibition of MCP-1expression only if the heptamer is also present. One inhibitory element is contained within a 59-nt portion of MCP-1 5′ flanking sequences and functions in an orientation-independent and heptamer-regulated manner. Significantly, proteins binding to two DNA sequences contribute to the formation of a single multiprotein complex on the 59-nt I* element. The I*-binding complex contains Sp3, an Sp1-like protein, and a novel DNA-binding protein. Moreover, the complex does not form on two 59-nt sequences containing mutations that reverse the inhibition of PDGF induction maintained by the wild-type I* element. We propose to call the multiprotein I*-binding complex a repressosome and suggest that it acts to repress PDGF-stimulated transcription of MCP-1 in the absence of the heptamer TTTTGTA.

ACKNOWLEDGMENTS

We thank John Alberta, Mike Carey, Rolf G. Freter, Charles Stiles, I. Bernard Weinstein, and Howard J. Worman for critical reading of the manuscript. We also thank Andrea Troxel for help with statistical analysis, Xiaoqin Qu for assistance with the Western analyses, and Christine Chen and Roger Lucas for technical assistance.

This research was supported in part by grants from the NIH (Shannon Award 1R55CA76045-01), the March of Dimes (Basil O’Connor Starter Scholar Award 5-FY97-0031), and the Council for Tobacco Research (to R.R.F.). R.R.F. is a Scholar of the V Foundation for Cancer Research and the James S. McDonnell Foundation. Additional support for this work was provided by grants from the Elsa U. Pardee Foundation for Cancer Research, The Milheim Foundation for Cancer Research, and the Ruth Estrin Goldberg Memorial for Cancer Research (to R.R.F.). H.A.S. is supported by a grant from the NIH (HL03806-01).