Single cell analysis of transcriptional activation dynamics.
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.


RESULTS
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 alpha-amanitin pre-treatment, the VP16-activator, GCN5, and Brd2 are still recruited to the transcription site but the chromatin does not decondense.


CONCLUSIONS
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 alpha-amanitin pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription drives large-scale chromatin decondensation.
Rafalska-Metcalf
Ilona U
IU
Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania, United States of America.
Powers
Sara Lawrence
SL
Joo
Lucy M
LM
LeRoy
Gary
G
Janicki
Susan M
SM
D053959:Alpha-Amanitin / Q000494:pharmacology; D001665:Binding Sites; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D042002:Chromatin Assembly and Disassembly; D003584:Cytological Techniques; D005047:Etoposide / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D012319:RNA Polymerase II / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D050880:p300-CBP Transcription Factors / Q000378:metabolism; D000099224:p300-CBP-Associated Factor; D000092468:Bromodomain Containing Proteins
D053959:Alpha-Amanitin; D018797:Cell Cycle Proteins; D005047:Etoposide; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D012319:RNA Polymerase II; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C098415:BRD2 protein, human; D000092468:Bromodomain Containing Proteins; C441289:BRD4 protein, human