Gene activation requires the tight coordination of regulatory factors at the site of transcription. We propose to study the recruitment dynamics of transcriptional regulatory factors to a chromatinized transcription site in single living cells in order to produce a high-resolution temporal and spatial portrait of transcriptional activation. We will use shRNA depletion to evaluate the requirement of known regulatory factors on chromatin decondensation, pre-initiation complex assembly and RNA synthesis. We will also determine the kinetic and spatial dynamics of post-translational modifications (PTM) and PTM binding proteins during transcription activation and mitosis. In order to expand the use of the live cell imaging methodology, we also propose to use p53 as a model transcriptional activator and to interrogate the timing of its activation kinetics. These studies are significant because they cannot be done using other existing techniques that average effects in cell populations. Additionally, they will provide a portrait of unperturbed gene regulation in single cells. A comprehensive understanding of transcriptional regulation is important for the development of new strategies to cure human diseases.

Public Health Relevance

Numerous diseases, including cancer, are caused by aberrations in transcriptional regulation. Gene activation requires the tight coordination of regulatory factors at the site of transcription. We propose to study the recruitment dynamics of these factors at a transcription site in single living cells in order to produce a high- resolution map of their recruitment order. A comprehensive understanding of transcriptional regulation is important for the development of new strategies to cure human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM093000-05
Application #
8696869
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Carter, Anthony D
Project Start
2010-08-01
Project End
2015-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
5
Fiscal Year
2014
Total Cost
$347,985
Indirect Cost
$159,885
Name
Wistar Institute
Department
Type
DUNS #
075524595
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Newhart, Alyshia; Janicki, Susan M (2014) Seeing is believing: visualizing transcriptional dynamics in single cells. J Cell Physiol 229:259-65
Newhart, Alyshia; Rafalska-Metcalf, Ilona U; Yang, Tian et al. (2013) Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site. J Biol Chem 288:19882-99
Newhart, Alyshia; Negorev, Dmitri G; Rafalska-Metcalf, Ilona U et al. (2013) Sp100A promotes chromatin decondensation at a cytomegalovirus-promoter-regulated transcription site. Mol Biol Cell 24:1454-68
Rafalska-Metcalf, Ilona U; Janicki, Susan M (2013) Preparation of cell lines for single-cell analysis of transcriptional activation dynamics. Methods Mol Biol 977:249-58