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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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Dynamics of transcriptional pulsing in Clone B6.A. Transcriptional activity of cells possessing an EGFP foci at the start of live imaging. B. Summary of transcriptional dynamics displayed by all pulsing cells in Clone B6. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represent 25–32 seconds depending on the cell examined and red line denotes 20 minutes of imaging. An asterisk marks the cell shown in 5A. C. Number of pulses detected in 20 minutes in all cells in Clone B6. One pulse represents a cell that is expressed continuously for 20 minutes. D. Distribution of duration of gene activity of Clone B6. E. Distribution of time gaps between transcriptional pulses of Clone B6.
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pone-0037130-g006: Dynamics of transcriptional pulsing in Clone B6.A. Transcriptional activity of cells possessing an EGFP foci at the start of live imaging. B. Summary of transcriptional dynamics displayed by all pulsing cells in Clone B6. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represent 25–32 seconds depending on the cell examined and red line denotes 20 minutes of imaging. An asterisk marks the cell shown in 5A. C. Number of pulses detected in 20 minutes in all cells in Clone B6. One pulse represents a cell that is expressed continuously for 20 minutes. D. Distribution of duration of gene activity of Clone B6. E. Distribution of time gaps between transcriptional pulses of Clone B6.

Mentions: For ease of comparison with Clone 3A10, we limited our analysis of the transcriptional pulses of Clone B6 to the first 20 minutes of image acquisition. Of the 43 Clone B6 cells which possessed a focal dot at the start of the imaging period, the majority (58%) displayed discontinuous transcription, while only 16% remained on for the whole 20 minutes (Figure 6A). A summary of the transcriptional activity of all pulsing B6 cells recorded are shown in Figure 6B. Up to 7 pulses could be detected within the 20 minutes, showing frequent fluctuation between the two transcriptional states (Figures 6C). Most transcriptional events and the gap time between such events were also rapid (<32 seconds), with an average pulse length of 144 seconds (2.4 minutes). The period of time between pulses was also rapid, with an average duration of 88 seconds or approximately 1.5 minutes (Figures 6D,E). The distribution of the lengths of gaps follows that of the Random Telegraph Model [9], in which the gene transitions stochastically between the on and off state. We conclude that rapid pulses of transgene transcription can be detected in both cell clones. The provirus in B6 cells has 24 MS2 stem cell loops and therefore would be expected to have the greatest sensitivity, but most pulses detected were rapid. In contrast, Clone 3A10 has 16 MS2 stem loops but primarily demonstrated longer pulses and gaps. These findings indicate that rapid pulses are not artifacts of reduced sensitivity.


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)

Dynamics of transcriptional pulsing in Clone B6.A. Transcriptional activity of cells possessing an EGFP foci at the start of live imaging. B. Summary of transcriptional dynamics displayed by all pulsing cells in Clone B6. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represent 25–32 seconds depending on the cell examined and red line denotes 20 minutes of imaging. An asterisk marks the cell shown in 5A. C. Number of pulses detected in 20 minutes in all cells in Clone B6. One pulse represents a cell that is expressed continuously for 20 minutes. D. Distribution of duration of gene activity of Clone B6. E. Distribution of time gaps between transcriptional pulses of Clone B6.
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Related In: Results  -  Collection

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pone-0037130-g006: Dynamics of transcriptional pulsing in Clone B6.A. Transcriptional activity of cells possessing an EGFP foci at the start of live imaging. B. Summary of transcriptional dynamics displayed by all pulsing cells in Clone B6. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represent 25–32 seconds depending on the cell examined and red line denotes 20 minutes of imaging. An asterisk marks the cell shown in 5A. C. Number of pulses detected in 20 minutes in all cells in Clone B6. One pulse represents a cell that is expressed continuously for 20 minutes. D. Distribution of duration of gene activity of Clone B6. E. Distribution of time gaps between transcriptional pulses of Clone B6.
Mentions: For ease of comparison with Clone 3A10, we limited our analysis of the transcriptional pulses of Clone B6 to the first 20 minutes of image acquisition. Of the 43 Clone B6 cells which possessed a focal dot at the start of the imaging period, the majority (58%) displayed discontinuous transcription, while only 16% remained on for the whole 20 minutes (Figure 6A). A summary of the transcriptional activity of all pulsing B6 cells recorded are shown in Figure 6B. Up to 7 pulses could be detected within the 20 minutes, showing frequent fluctuation between the two transcriptional states (Figures 6C). Most transcriptional events and the gap time between such events were also rapid (<32 seconds), with an average pulse length of 144 seconds (2.4 minutes). The period of time between pulses was also rapid, with an average duration of 88 seconds or approximately 1.5 minutes (Figures 6D,E). The distribution of the lengths of gaps follows that of the Random Telegraph Model [9], in which the gene transitions stochastically between the on and off state. We conclude that rapid pulses of transgene transcription can be detected in both cell clones. The provirus in B6 cells has 24 MS2 stem cell loops and therefore would be expected to have the greatest sensitivity, but most pulses detected were rapid. In contrast, Clone 3A10 has 16 MS2 stem loops but primarily demonstrated longer pulses and gaps. These findings indicate that rapid pulses are not artifacts of reduced sensitivity.

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