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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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Related in: MedlinePlus

Focal doublets on sister chromatids associate with transcription factories.A. Transcription doublets can be seen in a cell that has undergone replication at the integration site. The pair of transcription foci (green) overlaps the replicated DNA FISH signals (red) on one pair of alleles. The nucleus is counterstained with DAPI (cyan). B. ImmunoFISH of Clone B6 focal doublets (green) show they associate with distinct RNA Pol II factories (violet). A single focal plane with focal doublets is shown. The nucleus is counterstained with DAPI (cyan). Scale bar = 2.5 µM. C. Detection of transcriptional doublets in Clone 3A10 persisting through 10 minutes of image acquisition. Scale bar = 2.5 µM. D. Dynamics of transcription doublets in Clone B6. Scale bar = 2.5 µM.
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pone-0037130-g007: Focal doublets on sister chromatids associate with transcription factories.A. Transcription doublets can be seen in a cell that has undergone replication at the integration site. The pair of transcription foci (green) overlaps the replicated DNA FISH signals (red) on one pair of alleles. The nucleus is counterstained with DAPI (cyan). B. ImmunoFISH of Clone B6 focal doublets (green) show they associate with distinct RNA Pol II factories (violet). A single focal plane with focal doublets is shown. The nucleus is counterstained with DAPI (cyan). Scale bar = 2.5 µM. C. Detection of transcriptional doublets in Clone 3A10 persisting through 10 minutes of image acquisition. Scale bar = 2.5 µM. D. Dynamics of transcription doublets in Clone B6. Scale bar = 2.5 µM.

Mentions: Pairs of adjacent active transcription sites or “doublets" were previously reported and were interpreted to represent simultaneous transcription sites on sister chromatids [3]. We also observed such focal doublets in our cultures of asynchronously dividing cells. Doublets were found in 32% of Clone B6 cells and 25% of Clone 3A10 cells indicative of transgene transcription from sister alleles in S phase. ImmunoFISH signals of the integration site revealed two pairs of alleles as expected for cells in S phase (Figure 7A). EGFP focal doublets colocalize with one pair of alleles that bear the provirus, confirming that sister chromatids can be co-expressed during S phase. Doublets can also be found to occupy distinct transcription factories, as uncovered by RNA Pol II and EGFP co-staining (Figure 7B). Live imaging revealed that some focal doublets can be observed to persist for at least 30 minutes of imaging (Figure 7C). Remarkably, some doublets also undergo rapid changes, as doublets often rapidly reappear as a single focal dot or disappear synchronously (Video S3). Upon inspection, doublets were also observed in the z-axis, although they may appear as a single focal dot in the xy-plane (Figure S4). The disappearance of the focal doublet is not due to shifting in and out of the sections, as this would be captured by our 3D time-lapse images (Figure 7D, Video S4). These results are consistent with transcriptional pulsing occurring soon after replication, and that both sister chromatids can be transcribed or silenced at the same time. Overall, the dynamics of focal doublets on sister chromatids further corroborates our finding that transcriptional pulses from retroviral transgenes in ES cells can be very rapid.


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)

Focal doublets on sister chromatids associate with transcription factories.A. Transcription doublets can be seen in a cell that has undergone replication at the integration site. The pair of transcription foci (green) overlaps the replicated DNA FISH signals (red) on one pair of alleles. The nucleus is counterstained with DAPI (cyan). B. ImmunoFISH of Clone B6 focal doublets (green) show they associate with distinct RNA Pol II factories (violet). A single focal plane with focal doublets is shown. The nucleus is counterstained with DAPI (cyan). Scale bar = 2.5 µM. C. Detection of transcriptional doublets in Clone 3A10 persisting through 10 minutes of image acquisition. Scale bar = 2.5 µM. D. Dynamics of transcription doublets in Clone B6. Scale bar = 2.5 µM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351450&req=5

pone-0037130-g007: Focal doublets on sister chromatids associate with transcription factories.A. Transcription doublets can be seen in a cell that has undergone replication at the integration site. The pair of transcription foci (green) overlaps the replicated DNA FISH signals (red) on one pair of alleles. The nucleus is counterstained with DAPI (cyan). B. ImmunoFISH of Clone B6 focal doublets (green) show they associate with distinct RNA Pol II factories (violet). A single focal plane with focal doublets is shown. The nucleus is counterstained with DAPI (cyan). Scale bar = 2.5 µM. C. Detection of transcriptional doublets in Clone 3A10 persisting through 10 minutes of image acquisition. Scale bar = 2.5 µM. D. Dynamics of transcription doublets in Clone B6. Scale bar = 2.5 µM.
Mentions: Pairs of adjacent active transcription sites or “doublets" were previously reported and were interpreted to represent simultaneous transcription sites on sister chromatids [3]. We also observed such focal doublets in our cultures of asynchronously dividing cells. Doublets were found in 32% of Clone B6 cells and 25% of Clone 3A10 cells indicative of transgene transcription from sister alleles in S phase. ImmunoFISH signals of the integration site revealed two pairs of alleles as expected for cells in S phase (Figure 7A). EGFP focal doublets colocalize with one pair of alleles that bear the provirus, confirming that sister chromatids can be co-expressed during S phase. Doublets can also be found to occupy distinct transcription factories, as uncovered by RNA Pol II and EGFP co-staining (Figure 7B). Live imaging revealed that some focal doublets can be observed to persist for at least 30 minutes of imaging (Figure 7C). Remarkably, some doublets also undergo rapid changes, as doublets often rapidly reappear as a single focal dot or disappear synchronously (Video S3). Upon inspection, doublets were also observed in the z-axis, although they may appear as a single focal dot in the xy-plane (Figure S4). The disappearance of the focal doublet is not due to shifting in and out of the sections, as this would be captured by our 3D time-lapse images (Figure 7D, Video S4). These results are consistent with transcriptional pulsing occurring soon after replication, and that both sister chromatids can be transcribed or silenced at the same time. Overall, the dynamics of focal doublets on sister chromatids further corroborates our finding that transcriptional pulses from retroviral transgenes in ES cells can be very rapid.

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