Single cell analysis of transcriptional activation dynamics

Abstract

Background: Gene activation is thought to occur through a series of temporally defined regulatory steps. However, this process has not been completely evaluated in single living mammalian cells. Methodology/Principal Findings: To investigate the timing and coordination of gene activation events, we tracked the recruitment of GCN5 (histone acetyltransferase), RNA polymerase II, Brd2 and Brd4 (acetyl-lysine binding proteins), in relation to a VP16-transcriptional activator, to a transcription site that can be visualized in single living cells. All accumulated rapidly with the VP16 activator as did the transcribed RNA. RNA was also detected at significantly more transcription sites in cells expressing the VP16-activator compared to a p53-activator. After a-amanitin pre-treatment, the VP16-activator, GCN5, and Brd2 are still recruited to the transcription site but the chromatin does not decondense. Conclusions/Significance: This study demonstrates that a strong activator can rapidly overcome the condensed chromatin structure of an inactive transcription site and supercede the expected requirement for regulatory events to proceed in a temporally defined order. Additionally, activator strength determines the number of cells in which transcription is induced as well as the extent of chromatin decondensation. As chromatin decondensation is significantly reduced after a-amanitin pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription drives large-scale chromatin decondensation. © 2010 Rafalska-Metcalf et al.