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High-throughput RNA FISH analysis by imaging flow cytometry reveals that pioneer factor Foxa1 reduces transcriptional stochasticity.

ABSTRACT: 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 µm), 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.

SUBMITTER: Lalmansingh AS

PROVIDER: S-EPMC3779185 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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High-throughput RNA FISH analysis by imaging flow cytometry reveals that pioneer factor Foxa1 reduces transcriptional stochasticity.

Lalmansingh Avin S AS Arora Kamalpreet K Demarco Richard A RA Hager Gordon L GL Nagaich Akhilesh K AK

PloS one 20130920 9

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 ...[more]

PMID: 24073287

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Project description:Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to directly measure mitochondrial fusion in intact cells. Given how many cellular processes are being linked mitochondrial dynamics, this technique will provide a powerful new tool in the study of this important organelle. © 2016 International Society for Advancement of Cytometry.

Dataset Information

High-throughput RNA FISH analysis by imaging flow cytometry reveals that pioneer factor Foxa1 reduces transcriptional stochasticity.

Publications

High-throughput RNA FISH analysis by imaging flow cytometry reveals that pioneer factor Foxa1 reduces transcriptional stochasticity.

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