Development of a fluorescence reporter system to quantify transcriptional activity of endogenous p53 in living cells
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Abstract
The tumor suppressor p53 (also known as TP53) plays a central role in cellular stress responses by regulating transcription of multiple target genes. The temporal dynamics of p53 are thought to be important for its function; these encode input information and are decoded to induce distinct cellular phenotypes. However, it remains unclear to what extent the temporal dynamics of p53 reflect the activity of p53-induced gene expression. In this study, we report a multiplexed reporter system that allows us to visualize the transcriptional activity of p53 at the single-cell level. Our reporter system features simple and sensitive observation of the transcriptional activity of endogenous p53 to the response elements of various target genes. Using this system, we show that the transcriptional activation of p53 exhibits strong cell-to-cell heterogeneity. The transcriptional activation of p53 after etoposide treatment is highly dependent on the cell cycle but this is not seen after UV exposure. Finally, we show that our reporter system allows simultaneous visualization of the transcriptional activity of p53 and cell cycle. Our reporter system can thus be a useful tool for studying biological processes involving the p53 signaling pathway.
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We developed a novel reporter stable cell line using the Fucci reporter system for visualization of the cell cycle (Fig. 6A and Fig. S6A). Due to the limitation of available fluorescence channels, we used only the Cdt1 (30-120) fragment, which is stabilized in G1 phase and degraded in S/G2/M phase (Sakaue-Sawano et al., 2008).
One idea that would allow for greater resolving power of cell cycle kinetics while simultaneously monitoring p53 transcription would be to engineer the DNA Helicase B based (DHB) CDK1/2 biosensor (Spencer et al. 2013 doi: 10.1016/j.cell.2013.08.062) into the last position fused to mCerulean instead of the CDT1 component of the FUCCI system. The added benefit, after bookmarking the CDK sensor, would be a more precise read-out of cell cycle state using DHB as well as discriminating between cells that exit in G0 …
We developed a novel reporter stable cell line using the Fucci reporter system for visualization of the cell cycle (Fig. 6A and Fig. S6A). Due to the limitation of available fluorescence channels, we used only the Cdt1 (30-120) fragment, which is stabilized in G1 phase and degraded in S/G2/M phase (Sakaue-Sawano et al., 2008).
One idea that would allow for greater resolving power of cell cycle kinetics while simultaneously monitoring p53 transcription would be to engineer the DNA Helicase B based (DHB) CDK1/2 biosensor (Spencer et al. 2013 doi: 10.1016/j.cell.2013.08.062) into the last position fused to mCerulean instead of the CDT1 component of the FUCCI system. The added benefit, after bookmarking the CDK sensor, would be a more precise read-out of cell cycle state using DHB as well as discriminating between cells that exit in G0 (cdk-low) vs G1 (cdk-increasing). If you wanted an even more precise construct for delineating cell cycle state, you could consider swapping the NLS-mCherry for PCNA::mScarlet-I, as it seems like there is a lot of interesting biology associated with S-phase in your p53 sensor in this pre-print, and you could precisely define S-phase based on nuclear puncta formation in PCNA (I might avoid mCherry in this case due to mCherry aggregates) while still maintaining the nuclear mask, as PCNA is expressed and nuclear localized throughout the cell cycle.
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We developed a novel reporter stable cell line using the Fucci reporter system for visualization of the cell cycle (Fig. 6A and Fig. S6A). Due to the limitation of available fluorescence channels, we used only the Cdt1 (30-120) fragment, which is stabilized in G1 phase and degraded in S/G2/M phase (Sakaue-Sawano et al., 2008).
One idea that would allow for greater resolving power of cell cycle kinetics while simultaneously monitoring p53 transcription would be to engineer the DNA Helicase B based (DHB) CDK1/2 biosensor (Spencer et al. 2013 doi: 10.1016/j.cell.2013.08.062) into the last position fused to mCerulean instead of the CDT1 component of the FUCCI system. The added benefit, after bookmarking the CDK sensor, would be a more precise read-out of cell cycle state using DHB as well as discriminating between cells that exit in G0 …
We developed a novel reporter stable cell line using the Fucci reporter system for visualization of the cell cycle (Fig. 6A and Fig. S6A). Due to the limitation of available fluorescence channels, we used only the Cdt1 (30-120) fragment, which is stabilized in G1 phase and degraded in S/G2/M phase (Sakaue-Sawano et al., 2008).
One idea that would allow for greater resolving power of cell cycle kinetics while simultaneously monitoring p53 transcription would be to engineer the DNA Helicase B based (DHB) CDK1/2 biosensor (Spencer et al. 2013 doi: 10.1016/j.cell.2013.08.062) into the last position fused to mCerulean instead of the CDT1 component of the FUCCI system. The added benefit, after bookmarking the CDK sensor, would be a more precise read-out of cell cycle state using DHB as well as discriminating between cells that exit in G0 (cdk-low) vs G1 (cdk-increasing). If you wanted an even more precise construct for delineating cell cycle state, you could consider swapping the NLS-mCherry for PCNA::mScarlet-I, as it seems like there is a lot of interesting biology associated with S-phase in your p53 sensor in this pre-print, and you could precisely define S-phase based on nuclear puncta formation in PCNA (I might avoid mCherry in this case due to mCherry aggregates) while still maintaining the nuclear mask, as PCNA is expressed and nuclear localized throughout the cell cycle.
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