High-Throughput RNA FISH Analysis by Imaging Flow Cytometry Reveals That Pioneer Factor Foxa1 Reduces Transcriptional Stochasticity

Journal Article · Fri Sep 20 00:00:00 EDT 2013 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0076043· OSTI ID:1904960

Lalmansingh, Avin S. ^[1]; Arora, Kamalpreet ^[1]; DeMarco, Richard A. ^[2]; Hager, Gordon L. ^[3]; Nagaich, Akhilesh K. ^[1]

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1. U.S. Food and Drug Administration (FDA), Bethesda, MD (United States). Center for Drug Evaluation and Research (CDER)
2. Amnis, EMD Millipore Corporation, Seattle, WA (United States)
3. National Institutes of Health (NIH), Bethesda, MD (United States). National Cancer Institute

Genes are regulated at the single-cell level. Here, we performed RNA FISH of thousands of cells by flow cytometry (flow-RNA FISH) to gain insight into transcriptional variability between individual cells. These experiments utilized the murine adenocarcinoma 3134 cell line with 200 copies of the MMTV-Ras reporter integrated at a single genomic locus. The MMTV array contains approximately 800–1200 binding sites for the glucocorticoid receptor (GR) and 600 binding sites for the pioneer factor Foxa1. Hormone activation of endogenous GR by dexamethasone treatment resulted in highly variable changes in the RNA FISH intensity (25–300 pixel intensity units) and size (1.25–15 mm), indicative of probabilistic or stochastic mechanisms governing GR and cofactor activation of the MMTV promoter. Exogenous expression of the pioneer factor Foxa1 increased the FISH signal intensity and size as expected for a chromatin remodeler that enhances transcriptional competence through increased chromatin accessibility. In addition, specific analysis of Foxa1-enriched cell sub-populations showed that low and high Foxa1 levels substantially lowered the cell-to-cell variability in the FISH intensity as determined by a noise calculation termed the % coefficient of variation. These results suggest that an additional function of the pioneer factor Foxa1 may be to decrease transcriptional noise.

• Research Organization: Oak Ridge Insitute. for Science and Education (ORISE), Oak Ridge, TN (United States)
• Sponsoring Organization: USDOE Office of Science (SC); U.S. Food and Drug Administration (FDA)
• Grant/Contract Number: SC0014664
• OSTI ID: 1904960
• Journal Information: PLoS ONE, Vol. 8, Issue 9; ISSN 1932-6203
• Publisher: Public Library of Science
• Country of Publication: United States
• Language: English

Related Subjects

54 ENVIRONMENTAL SCIENCES
Chromatin
fluorescent in situ hybridization
Histones
hormone receptor signaling
transcriptional control
DNA transcription
gene expression
RNA analysis