Synergy among Nuclear Receptor Coactivators: Selective Requirement for Protein Methyltransferase and Acetyltransferase Activities

Abstract

Hormone-activated nuclear receptors (NR) bind to specific regulatory DNA elements associated with their target genes and recruit coactivator proteins to remodel chromatin structure, recruit RNA polymerase, and activate transcription. The p160 coactivators (e.g., SRC-1, GRIP1, and ACTR) bind directly to activated NR and can recruit a variety of secondary coactivators. We have established a transient-transfection assay system under which the activity of various NR is highly or completely dependent on synergistic cooperation among three classes of coactivators: a p160 coactivator, the protein methyltransferase CARM1, and any of the three protein acetyltransferases, p300, CBP, or p/CAF. The three-coactivator functional synergy was only observed when low levels of NR were expressed and was highly or completely dependent on the methyltransferase activity of CARM1 and the acetyltransferase activity of p/CAF, but not the acetyltransferase activity of p300. Other members of the protein arginine methyltransferase family, which methylate different protein substrates than CARM1, could not substitute for CARM1 to act synergistically with p300 or p/CAF. A ternary complex of GRIP1, CARM1, and p300 or CBP was demonstrated in cultured mammalian cells, supporting a physiological role for the observed synergy. The transfection assay described here is a valuable new tool for investigating the mechanism of coactivator function and demonstrates the importance of multiple coactivators, including CARM1 and its specific protein methyltransferase activity, in transcriptional activation.

We thank the following investigators for providing plasmids: Vittorio Sartorelli (NIH) for vectors encoding p/CAF, p300, and their mutants; Tso-Pang Yao (Duke University) for CBP expression vector; Adam Frankel, Steve Clarke, and Harvey Herschman (UCLA) for vectors encoding PRMT1, PRMT2, PRMT3, and RMT1; and Richard Goodman (University of Oregon Health Sciences Center) for the vector encoding the Gal4-CBP fusion protein. We thank Baruch Frenkel (University of Southern California) for critical comments on the manuscript.

This work was supported by U.S. Public Health Service Grant DK55274 (to M.R.S.) and GM61355 (to X.Z. and X.C.) from the National Institutes of Health. S.S.K. was supported by a predoctoral training fellowship from Grant AG00093 from the National Institutes of Health.