C-Myc Mediates Activation of the cad Promoter via a Post-RNA Polymerase II Recruitment Mechanism

Scott R Eberhardy,Peggy J Farnham

doi:10.1074/jbc.m109014200

Scott R Eberhardy, Peggy J Farnham

Open Access

https://doi.org/10.1074/jbc.m109014200

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Abstract

The c-Myc protein is a site-specific DNA-binding transcription factor that is up-regulated in a number of different cancers. We have previously shown that binding of Myc correlates with increased transcription of the cad promoter. We have now further investigated the mechanism by which Myc mediates transcriptional activation of the cad gene. Using a chromatin immunoprecipitation assay, we found high levels of RNA polymerase II bound to the cad promoter in quiescent NIH 3T3 cells and in differentiated U937 cells, even though the promoter is inactive. However, chromatin immunoprecipitation with an antibody that recognizes the hyperphosphorylated form of the RNA polymerase II carboxyl-terminal domain (CTD) revealed that phosphorylation of the CTD does correlate with c-Myc binding and cad transcription. We have also found that the c-Myc transactivation domain interacts with cdk9 and cyclin T1, components of the CTD kinase P-TEFb. Furthermore, activator bypass experiments have shown that direct recruitment of cyclin T1 to the cad promoter can substitute for c-Myc to activate the promoter. In summary, our results suggest that c-Myc activates transcription of cad by stimulating promoter clearance and elongation, perhaps via recruitment of P-TEFb.

Highlights

C-Myc is a site-specific DNA-binding transcription factor that is conserved throughout evolution
When levels of RNA polymerase II (RNAP II) were examined on the mutated cad promoter, we found that RNAP II was bound to the mutated cad promoter despite the absence of c-Myc and USF, at similar levels found on the endogenous cad promoter
We have previously shown that transcriptional activation of the cad promoter correlates with binding of c-Myc to an E box located 65 base pairs downstream of the transcription start site

Summary

Introduction

C-Myc is a site-specific DNA-binding transcription factor that is conserved throughout evolution. Recent advances in microarray technology has allowed genome-wide studies of mRNA transcripts responsive to transcription factors, and a number of such experiments have been done to examine which genes are responsive to c-Myc [12,13,14]. A difficulty in studying c-Myc target genes is that overexpression of c-Myc in cells results in only modest increases in promoter activity, typically around 2–5-fold. This may be due to several factors, including the presence of highly abundant proteins, such as USF, which bind to target gene E boxes and may interfere with c-Myc binding, or a requirement for a coactivator which is at limiting concentrations in the cell.

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