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.
Highlights
There is accumulating evidence that gene transcription is discontinuous and occurs as irregular bursts in a pulsatile manner ([1,2,3,4,5,6] reviewed in [7,8,9])
Digestion with HindIII, which is not found on the integrated provirus, yields a single band of different size for both clones, confirming the two proviruses are integrated at different integration sites
In this study we used the MS2 system to demonstrate that discontinuous transcription occurs from highly expressed retroviral transgenes in embryonic stem (ES) cells
Summary
There is accumulating evidence that gene transcription is discontinuous and occurs as irregular bursts in a pulsatile manner ([1,2,3,4,5,6] reviewed in [7,8,9]). The MS2 system is an established tool to track transcription in live cells over time [10] In this system, the MS2 stem-loop repeat is integrated into a reporter gene or part of an endogenous gene. Discontinuous transcription is observed when the fluorescence intensity of the focal dot returns to background level Using this system, bursts of transcription were detected in several eukaryotic cell types in both reporter constructs [3,5] and in endogenous genes [2,6]. Of the handful of genes studied using the MS2 method to date, all reported transcriptional pulses are longer than a few minutes, and whether there are shorter transcriptional pulses has yet to be studied It is still unknown how the dynamics of pulsing at the individual cell level contributes to the overall expression at the population level
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