Promoter Specificity and Biological Activity of Tethered AP-1 Dimers

Abstract

Activator protein 1 (AP-1) is a group of dimeric transcription factors composed of Jun, Fos, and ATF family proteins. Both gain- and loss-of-function studies have revealed specific roles for individual AP-1 components in cell proliferation, differentiation, apoptosis, and other biological processes. However, little is known about the functions of specific AP-1 dimers. To test the importance of AP-1 composition in transcriptional activation, AP-1 monomers were joined via a flexible polypeptide tether to force specific pairing. The resultant single-chain AP-1 molecules showed DNA binding specificity and transcriptional activation potentials similar to those of untethered dimers, even in the presence of dominant-negative AP-1 monomers. c-Jun-containing dimers showed distinct promoter specificity in transient-transfection experiments, depending on the Fos, Fra, or ATF partner. When stably expressed in NIH 3T3 cells, c-Jun∼Fra2, but not c-Jun∼Fra1 and c-Jun∼cFos (the tilde indicates a tethered dimer), inhibited G₀ arrest at confluency and under low-serum conditions and specifically activated cyclin A expression. These data suggest that the choice of dimerization partner defines the role of c-Jun in gene activation and cell cycle regulation and that single-chain AP-1 molecules provide a powerful tool for assessing the role of specific AP-1 dimers.

We are grateful to J.-P. Abastado for advice in the design of the peptide linker and to D. Lallemand for the gift of antibodies. We thank D. Bohmann for the gift of the RSVcJunDB-4 mutant construct and R. Müller, H. van Dam, P. Herrlich, and J. Sobczak-Thepot for different reporter constructs. We thank J. Weitzman and A. Szremska for critical reading of the manuscript and C. Coffinier, B. Arcangioli, and M. Pontoglio for fruitful discussions.

This work was supported by grants from the EEC Biomed and Training and Mobility Programs. L.B. was the recipient of awards from the Pasteur Weitzmann Council, the Fondation des Treilles, and the EMBO Short Term Fellowship. M.W. was the recipient of an initial award from the French Foreign Ministry and was supported by the Association for International Cancer Research (AICR).