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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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Visualization of transcription foci.A. Transcription foci were detected as focal dots (yellow arrows) in Clone B6 and 3A10 after transient transfection of MS2-EGFP. Uninfected J1 ES cells were used as a control. Scale bar = 2.5 µM. B. Sample images from multiple z-stacks of a cell from Clone 3A10 displaying a focal dot over 6 consecutive focal planes. Z-stacks were 0.3 µM apart. Scale bar = 5 µM. C. Quantification of the percentage of cells with transcription foci in EGFP-positive cells. (n = 2; at least 47 cells were examined for each n).
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pone-0037130-g002: Visualization of transcription foci.A. Transcription foci were detected as focal dots (yellow arrows) in Clone B6 and 3A10 after transient transfection of MS2-EGFP. Uninfected J1 ES cells were used as a control. Scale bar = 2.5 µM. B. Sample images from multiple z-stacks of a cell from Clone 3A10 displaying a focal dot over 6 consecutive focal planes. Z-stacks were 0.3 µM apart. Scale bar = 5 µM. C. Quantification of the percentage of cells with transcription foci in EGFP-positive cells. (n = 2; at least 47 cells were examined for each n).

Mentions: To determine whether we could detect transcription foci from the mRFP-MS2 retrovirus, we transiently transfected plasmid coding for the MS2-EGFP-NLS fusion protein. A green fluorescent focal dot can be detected in several consecutive focal planes in EGFP-positive cells of Clone B6 and Clone 3A10, but not in uninfected J1 ES cells (Figure 2A), indicating that transcription foci in the system are dependent on the presence of both the mRFP-MS2 virus and the MS2-EGFP-NLS fusion protein (Figure 2A). To further examine the cells, we fixed the two populations 16–18 hours after MS2-EGFP transfection. We collected images in multiple z-stacks to ensure all transcription foci were captured (Figure 2B). We quantified the number of EGFP-positive cells that possessed focal dots and showed that more of these were detected in Clone B6 (59%) than Clone 3A10 (32%) (Figure 2C). Thus, some EGFP-positive transfected cells contained no focal dots, suggesting that these cells were not transcribing the provirus at the time of fixation, which is consistent with the possibility of the provirus being between pulses of transcription. Overall, Clone B6 has a higher frequency of transcription foci correlating with a tighter peak of consistent expression detected by flow cytometry. In contrast, Clone 3A10 has a lower frequency of transcription foci and shows more variable expression by flow cytometry. Since both clones do not have any mRFP-negative cells in the population, these data indicate that the cells without active transcription foci have recently expressed high levels of the provirus in order to maintain such a high MFI of mRFP. We cannot exclude that the cells transcribe at very low levels in this situation, producing undetectable focal dots that are not distinguishable from the nuclear EGFP background.


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)

Visualization of transcription foci.A. Transcription foci were detected as focal dots (yellow arrows) in Clone B6 and 3A10 after transient transfection of MS2-EGFP. Uninfected J1 ES cells were used as a control. Scale bar = 2.5 µM. B. Sample images from multiple z-stacks of a cell from Clone 3A10 displaying a focal dot over 6 consecutive focal planes. Z-stacks were 0.3 µM apart. Scale bar = 5 µM. C. Quantification of the percentage of cells with transcription foci in EGFP-positive cells. (n = 2; at least 47 cells were examined for each n).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351450&req=5

pone-0037130-g002: Visualization of transcription foci.A. Transcription foci were detected as focal dots (yellow arrows) in Clone B6 and 3A10 after transient transfection of MS2-EGFP. Uninfected J1 ES cells were used as a control. Scale bar = 2.5 µM. B. Sample images from multiple z-stacks of a cell from Clone 3A10 displaying a focal dot over 6 consecutive focal planes. Z-stacks were 0.3 µM apart. Scale bar = 5 µM. C. Quantification of the percentage of cells with transcription foci in EGFP-positive cells. (n = 2; at least 47 cells were examined for each n).
Mentions: To determine whether we could detect transcription foci from the mRFP-MS2 retrovirus, we transiently transfected plasmid coding for the MS2-EGFP-NLS fusion protein. A green fluorescent focal dot can be detected in several consecutive focal planes in EGFP-positive cells of Clone B6 and Clone 3A10, but not in uninfected J1 ES cells (Figure 2A), indicating that transcription foci in the system are dependent on the presence of both the mRFP-MS2 virus and the MS2-EGFP-NLS fusion protein (Figure 2A). To further examine the cells, we fixed the two populations 16–18 hours after MS2-EGFP transfection. We collected images in multiple z-stacks to ensure all transcription foci were captured (Figure 2B). We quantified the number of EGFP-positive cells that possessed focal dots and showed that more of these were detected in Clone B6 (59%) than Clone 3A10 (32%) (Figure 2C). Thus, some EGFP-positive transfected cells contained no focal dots, suggesting that these cells were not transcribing the provirus at the time of fixation, which is consistent with the possibility of the provirus being between pulses of transcription. Overall, Clone B6 has a higher frequency of transcription foci correlating with a tighter peak of consistent expression detected by flow cytometry. In contrast, Clone 3A10 has a lower frequency of transcription foci and shows more variable expression by flow cytometry. Since both clones do not have any mRFP-negative cells in the population, these data indicate that the cells without active transcription foci have recently expressed high levels of the provirus in order to maintain such a high MFI of mRFP. We cannot exclude that the cells transcribe at very low levels in this situation, producing undetectable focal dots that are not distinguishable from the nuclear EGFP background.

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