In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation

doi:10.1073/pnas.0404192101

In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation

^*Division of Human Biology, Fred Hutchinson Cancer Research Center, and ^‡Departments of Pathology and Neurology, University of Washington School of Medicine, 1100 Fairview Avenue North C3-168, P.O. Box 19024, Seattle, WA 98109-1024

Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved June 29, 2004 (received for review June 11, 2004)

Abstract

The transcriptional coactivators p300 and pCAF are necessary for the myogenic factor MyoD to initiate the expression of skeletal muscle genes. In addition to mediating histone acetylation, both of these factors can acetylate MyoD; however, the complexity of cellular systems used to study MyoD has impeded delineation of the specific roles of these two acetyltransferases. Therefore, we established a MyoD-dependent in vitro transcription system that permits us to determine the roles of p300 and pCAF during MyoD-dependent transcriptional activation. Consistent with results from cellular systems, we demonstrate that maximal levels of transactivation in vitro require both p300 and pCAF, as well as the cofactor acetyl CoA. Dissection of the steps leading to transcription initiation revealed that the activities of p300 and pCAF are not redundant. During the initial stages of transactivation, p300 acetylates histone H3 and H4 within the promoter region and then recruits pCAF to MyoD. Once tethered to the promoter, pCAF acetylates MyoD to facilitate the transactivation process. Thus, we have established that pCAF and p300 carry out sequential and functionally distinct events on a promoter leading to transcriptional activation. Further dissection of this in vitro transcription system should be highly useful toward elucidating the mechanism by which coactivators facilitate differential gene expression by MyoD.

Footnotes

↵ † To whom correspondence may be sent at the * address. E-mail: stapscot{at}fhcrc.org or jdilworth{at}ohri.ca.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: MCK, muscle creatine kinase; AT, acetyltransferase; ChIP, chromatin immunoprecipitation.