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Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

Lo MY, Rival-Gervier S, Pasceri P, Ellis J - PLoS ONE (2012)

Bottom Line: These transcription foci colocalized with the transgene integration site detected by immunoFISH.We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression.These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

View Article: PubMed Central - PubMed

Affiliation: Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada.

ABSTRACT
Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

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Transcription dynamics of Clone 3A10.A. Representative detection of transcriptional pulses in one cell from Clone 3A10 at 2.5 minute intervals. Transcription foci indicated by arrows. Scale bar = 2.5 µM. B. Transcription foci of the cell depicted in Figure 4A was quantified for EGFP intensity over time. EGFP intensity plots were determined by subtracting background fluorescence at each time point (Blue line). Red line represents visual scoring of inferred transcriptional activity. C. Transcriptional activity of cells possessing EGFP foci at the start of live imaging. D. Summary of transcriptional dynamics displayed by all pulsing cells in Clone 3A10. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represents 2.5 minutes. The cell shown in Figure 4A is denoted with an asterisk. E. Representative image of transcriptional pulsing of a Clone 3A10 cell recorded at 30 sec intervals. Scale bar = 2.5 µM. F. Intensity time series data (blue line) and visual scoring of inferred transcriptional activity (red line) on the pulsing cell depicted in Figure 4E. G. Cumulative periods of transcriptional activity recorded by the two image intervals. 60 cells were examined for 2.5 minutes interval and 12 cells were examined for 30 seconds interval.
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pone-0037130-g004: Transcription dynamics of Clone 3A10.A. Representative detection of transcriptional pulses in one cell from Clone 3A10 at 2.5 minute intervals. Transcription foci indicated by arrows. Scale bar = 2.5 µM. B. Transcription foci of the cell depicted in Figure 4A was quantified for EGFP intensity over time. EGFP intensity plots were determined by subtracting background fluorescence at each time point (Blue line). Red line represents visual scoring of inferred transcriptional activity. C. Transcriptional activity of cells possessing EGFP foci at the start of live imaging. D. Summary of transcriptional dynamics displayed by all pulsing cells in Clone 3A10. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represents 2.5 minutes. The cell shown in Figure 4A is denoted with an asterisk. E. Representative image of transcriptional pulsing of a Clone 3A10 cell recorded at 30 sec intervals. Scale bar = 2.5 µM. F. Intensity time series data (blue line) and visual scoring of inferred transcriptional activity (red line) on the pulsing cell depicted in Figure 4E. G. Cumulative periods of transcriptional activity recorded by the two image intervals. 60 cells were examined for 2.5 minutes interval and 12 cells were examined for 30 seconds interval.

Mentions: To investigate whether the mRFP-MS2 retrovirus transcribes discontinuously, we performed real time imaging studies to detect transcriptional dynamics. Fields of EGFP-positive Clone 3A10 cells were imaged in 3D-stacks every 2.5 minutes for at least 20 minutes, as transcriptional pulsing could be observed previously with this acquisition setup [2] (Video S1). To increase the efficiency of analysis, we chose fields in which most cells possess transcription foci. While transcription foci were mostly visible throughout the course of imaging, foci were also found to fluctuate in fluorescence intensity, in which optimal levels of transcriptional activity appear as a green focal dot or become undetectable and appear as background, thus reflecting changing levels of transcriptional output (Figure 4A). We scored for the appearance and disappearance of transcription foci that were observed in at least 2 consecutive focal planes. To ensure accuracy in visual scoring, we also quantitated the intensity of fluorescence at the EGFP foci relative to the background fluorescence of a random area in the cell nucleus (Figure 4B). The absence of visible foci coincides with a drop in fluorescence at the focal dot.


Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

Lo MY, Rival-Gervier S, Pasceri P, Ellis J - PLoS ONE (2012)

Transcription dynamics of Clone 3A10.A. Representative detection of transcriptional pulses in one cell from Clone 3A10 at 2.5 minute intervals. Transcription foci indicated by arrows. Scale bar = 2.5 µM. B. Transcription foci of the cell depicted in Figure 4A was quantified for EGFP intensity over time. EGFP intensity plots were determined by subtracting background fluorescence at each time point (Blue line). Red line represents visual scoring of inferred transcriptional activity. C. Transcriptional activity of cells possessing EGFP foci at the start of live imaging. D. Summary of transcriptional dynamics displayed by all pulsing cells in Clone 3A10. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represents 2.5 minutes. The cell shown in Figure 4A is denoted with an asterisk. E. Representative image of transcriptional pulsing of a Clone 3A10 cell recorded at 30 sec intervals. Scale bar = 2.5 µM. F. Intensity time series data (blue line) and visual scoring of inferred transcriptional activity (red line) on the pulsing cell depicted in Figure 4E. G. Cumulative periods of transcriptional activity recorded by the two image intervals. 60 cells were examined for 2.5 minutes interval and 12 cells were examined for 30 seconds interval.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351450&req=5

pone-0037130-g004: Transcription dynamics of Clone 3A10.A. Representative detection of transcriptional pulses in one cell from Clone 3A10 at 2.5 minute intervals. Transcription foci indicated by arrows. Scale bar = 2.5 µM. B. Transcription foci of the cell depicted in Figure 4A was quantified for EGFP intensity over time. EGFP intensity plots were determined by subtracting background fluorescence at each time point (Blue line). Red line represents visual scoring of inferred transcriptional activity. C. Transcriptional activity of cells possessing EGFP foci at the start of live imaging. D. Summary of transcriptional dynamics displayed by all pulsing cells in Clone 3A10. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represents 2.5 minutes. The cell shown in Figure 4A is denoted with an asterisk. E. Representative image of transcriptional pulsing of a Clone 3A10 cell recorded at 30 sec intervals. Scale bar = 2.5 µM. F. Intensity time series data (blue line) and visual scoring of inferred transcriptional activity (red line) on the pulsing cell depicted in Figure 4E. G. Cumulative periods of transcriptional activity recorded by the two image intervals. 60 cells were examined for 2.5 minutes interval and 12 cells were examined for 30 seconds interval.
Mentions: To investigate whether the mRFP-MS2 retrovirus transcribes discontinuously, we performed real time imaging studies to detect transcriptional dynamics. Fields of EGFP-positive Clone 3A10 cells were imaged in 3D-stacks every 2.5 minutes for at least 20 minutes, as transcriptional pulsing could be observed previously with this acquisition setup [2] (Video S1). To increase the efficiency of analysis, we chose fields in which most cells possess transcription foci. While transcription foci were mostly visible throughout the course of imaging, foci were also found to fluctuate in fluorescence intensity, in which optimal levels of transcriptional activity appear as a green focal dot or become undetectable and appear as background, thus reflecting changing levels of transcriptional output (Figure 4A). We scored for the appearance and disappearance of transcription foci that were observed in at least 2 consecutive focal planes. To ensure accuracy in visual scoring, we also quantitated the intensity of fluorescence at the EGFP foci relative to the background fluorescence of a random area in the cell nucleus (Figure 4B). The absence of visible foci coincides with a drop in fluorescence at the focal dot.

Bottom Line: These transcription foci colocalized with the transgene integration site detected by immunoFISH.We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression.These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

View Article: PubMed Central - PubMed

Affiliation: Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada.

ABSTRACT
Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

Show MeSH
Related in: MedlinePlus