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Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.

Shin I, Ray J, Gupta V, Ilgu M, Beasley J, Bendickson L, Mehanovic S, Kraus GA, Nilsen-Hamilton M - Nucleic Acids Res. (2014)

Bottom Line: Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression.Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals.The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

View Article: PubMed Central - PubMed

Affiliation: Ames Laboratory, US Department of Energy, Ames, IA 50011, USA Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, 1210 Molecular Biology Building, Iowa State University, Ames, IA 50011, USA.

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Time-dependent change in IMAGEtag RNA level after activation of the GAL1 promoter. Yeast cells transformed with a 2-µm plasmid for expression of control RNA (A) or 6xPDC IMAGEtags (B) both under the control of the GAL1 promoter were induced with galactose. FRET: yeast cells (n = 15–27) expressing control RNA (A) or 6xPDC IMAGEtags (B) were incubated for 90 min with 5-μM Cy3-PDC and 5-μM Cy5-PDC in medium containing 1% raffinose and no glucose. The cells were induced by the inclusion of 2% galactose for the last 10, 40 and 90 min of the incubation. The FRET values for individual cells are shown as circles and the average FRET is the uninterrupted line. The amount of IMAGEtag RNA in each population was determined by RT-qPCR and normalized to ACT1 mRNA. Fold change of RNA level indicated with a dashed line and black triangles. (C) Image showing FRET signals in cells with control RNA and 6xPDC IMAGEtags after 90-min induction with 2% galactose. (D) FRET efficiency determined by acceptor photobleaching: the average FRET efficiency from three independently performed experiments is shown with the standard deviation in error bars. ***, P < 0.0001. FRET efficiencies were calculated using the formula FRET efficiency = 1–FD/F'D, where FD and F'D are donor intensity before and after photobleaching the acceptor, respectively. (E) Quantification of expression of IMAGEtags from three yeast promoters. 6xPDC IMAGEtags were expressed under the control of the GAL1, ACT1 or ADH1 promoters and are imaged in the presence of 10-μM Cy3-PDC and 4-μM Cy5-PDC. Box plots are shown of compiled data from experiments in which 15–20 cells were quantified for each estimate. Quantification used the formula FRET = (B−A·b−c·C)/C described in the Materials and Methods section. P values are shown for the statistical significance of 6xPDC IMAGEtags induced versus uninduced (GAL1) or control RNA versus 6xPDC IMAGEtags (ACT1 and ADH1). •:median; ▪: minimum; ○: maximum; ♦: q1; Δ; q3.
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Figure 3: Time-dependent change in IMAGEtag RNA level after activation of the GAL1 promoter. Yeast cells transformed with a 2-µm plasmid for expression of control RNA (A) or 6xPDC IMAGEtags (B) both under the control of the GAL1 promoter were induced with galactose. FRET: yeast cells (n = 15–27) expressing control RNA (A) or 6xPDC IMAGEtags (B) were incubated for 90 min with 5-μM Cy3-PDC and 5-μM Cy5-PDC in medium containing 1% raffinose and no glucose. The cells were induced by the inclusion of 2% galactose for the last 10, 40 and 90 min of the incubation. The FRET values for individual cells are shown as circles and the average FRET is the uninterrupted line. The amount of IMAGEtag RNA in each population was determined by RT-qPCR and normalized to ACT1 mRNA. Fold change of RNA level indicated with a dashed line and black triangles. (C) Image showing FRET signals in cells with control RNA and 6xPDC IMAGEtags after 90-min induction with 2% galactose. (D) FRET efficiency determined by acceptor photobleaching: the average FRET efficiency from three independently performed experiments is shown with the standard deviation in error bars. ***, P < 0.0001. FRET efficiencies were calculated using the formula FRET efficiency = 1–FD/F'D, where FD and F'D are donor intensity before and after photobleaching the acceptor, respectively. (E) Quantification of expression of IMAGEtags from three yeast promoters. 6xPDC IMAGEtags were expressed under the control of the GAL1, ACT1 or ADH1 promoters and are imaged in the presence of 10-μM Cy3-PDC and 4-μM Cy5-PDC. Box plots are shown of compiled data from experiments in which 15–20 cells were quantified for each estimate. Quantification used the formula FRET = (B−A·b−c·C)/C described in the Materials and Methods section. P values are shown for the statistical significance of 6xPDC IMAGEtags induced versus uninduced (GAL1) or control RNA versus 6xPDC IMAGEtags (ACT1 and ADH1). •:median; ▪: minimum; ○: maximum; ♦: q1; Δ; q3.

Mentions: To evaluate the cell to cell variability of Pol II promoter activity in living cells, we used IMAGEtags as reporters from three promoters: GAL1, ACT1 and ADH1. FRET signals in individual cells were quantified with time after induction of the GAL1 promoter and compared with the IMAGEtag RNA levels in the same cell populations (Figure 3A–C). Yeast cells expressing the control RNA containing no IMAGEtags (Figure 3A) or 6xPDC IMAGEtags (Figure 3B) were induced for different time periods and the FRET signal was measured in individual cells. There was a large variation in the range of individual cellular FRET signals at each time point under these conditions of preculture in glucose. However, the average increase in FRET, which represents the ensemble of induced cells, was proportional to the increase in IMAGEtag RNA level of the population measured by RT-qPCR (Figure 3A and B. This result indicates that the sampling of cells for FRET in these experiments was representative of the population and is consistent with the conclusion that the observed FRET is due to newly synthesized IMAGEtag RNA. Unlike for the IMAGEtags, the average FRET output of the control population did not increase in parallel with the mRNA content of the cell population. At each time point, the average FRET output from cells expressing the 6xPDC IMAGEtags was significantly higher than from cells expressing the control RNA (P < 0.001). The ability of IMAGEtags to detect the activity of two constitutive promoters, ACT1 and ADH1, was also tested (Figure 3E). The statistical significance of these results is reflected in the low P values that vary from 10−4 to 10−10. The larger variation of FRET signal from individual cells when the promoter was GAL1 (Figure 3A–C) was associated with an experimental design in which the cells were taken directly from a glucose containing medium to one with galactose replacing glucose. The GAL1 promoter is not activated until the intracellular glucose is depleted. The cell to cell variation in time to depletion of intracellular glucose may be the basis for larger variations in cell response in this experimental design compared with others. In experiments where the cells were first cultured in raffinose to allow glucose depletion, the variations in FRET signals were much smaller with an average coefficient of variation of 15% from the compiled results from seven conditions of groups of 11–21 cells (Supplementary Figure S14).


Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.

Shin I, Ray J, Gupta V, Ilgu M, Beasley J, Bendickson L, Mehanovic S, Kraus GA, Nilsen-Hamilton M - Nucleic Acids Res. (2014)

Time-dependent change in IMAGEtag RNA level after activation of the GAL1 promoter. Yeast cells transformed with a 2-µm plasmid for expression of control RNA (A) or 6xPDC IMAGEtags (B) both under the control of the GAL1 promoter were induced with galactose. FRET: yeast cells (n = 15–27) expressing control RNA (A) or 6xPDC IMAGEtags (B) were incubated for 90 min with 5-μM Cy3-PDC and 5-μM Cy5-PDC in medium containing 1% raffinose and no glucose. The cells were induced by the inclusion of 2% galactose for the last 10, 40 and 90 min of the incubation. The FRET values for individual cells are shown as circles and the average FRET is the uninterrupted line. The amount of IMAGEtag RNA in each population was determined by RT-qPCR and normalized to ACT1 mRNA. Fold change of RNA level indicated with a dashed line and black triangles. (C) Image showing FRET signals in cells with control RNA and 6xPDC IMAGEtags after 90-min induction with 2% galactose. (D) FRET efficiency determined by acceptor photobleaching: the average FRET efficiency from three independently performed experiments is shown with the standard deviation in error bars. ***, P < 0.0001. FRET efficiencies were calculated using the formula FRET efficiency = 1–FD/F'D, where FD and F'D are donor intensity before and after photobleaching the acceptor, respectively. (E) Quantification of expression of IMAGEtags from three yeast promoters. 6xPDC IMAGEtags were expressed under the control of the GAL1, ACT1 or ADH1 promoters and are imaged in the presence of 10-μM Cy3-PDC and 4-μM Cy5-PDC. Box plots are shown of compiled data from experiments in which 15–20 cells were quantified for each estimate. Quantification used the formula FRET = (B−A·b−c·C)/C described in the Materials and Methods section. P values are shown for the statistical significance of 6xPDC IMAGEtags induced versus uninduced (GAL1) or control RNA versus 6xPDC IMAGEtags (ACT1 and ADH1). •:median; ▪: minimum; ○: maximum; ♦: q1; Δ; q3.
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Figure 3: Time-dependent change in IMAGEtag RNA level after activation of the GAL1 promoter. Yeast cells transformed with a 2-µm plasmid for expression of control RNA (A) or 6xPDC IMAGEtags (B) both under the control of the GAL1 promoter were induced with galactose. FRET: yeast cells (n = 15–27) expressing control RNA (A) or 6xPDC IMAGEtags (B) were incubated for 90 min with 5-μM Cy3-PDC and 5-μM Cy5-PDC in medium containing 1% raffinose and no glucose. The cells were induced by the inclusion of 2% galactose for the last 10, 40 and 90 min of the incubation. The FRET values for individual cells are shown as circles and the average FRET is the uninterrupted line. The amount of IMAGEtag RNA in each population was determined by RT-qPCR and normalized to ACT1 mRNA. Fold change of RNA level indicated with a dashed line and black triangles. (C) Image showing FRET signals in cells with control RNA and 6xPDC IMAGEtags after 90-min induction with 2% galactose. (D) FRET efficiency determined by acceptor photobleaching: the average FRET efficiency from three independently performed experiments is shown with the standard deviation in error bars. ***, P < 0.0001. FRET efficiencies were calculated using the formula FRET efficiency = 1–FD/F'D, where FD and F'D are donor intensity before and after photobleaching the acceptor, respectively. (E) Quantification of expression of IMAGEtags from three yeast promoters. 6xPDC IMAGEtags were expressed under the control of the GAL1, ACT1 or ADH1 promoters and are imaged in the presence of 10-μM Cy3-PDC and 4-μM Cy5-PDC. Box plots are shown of compiled data from experiments in which 15–20 cells were quantified for each estimate. Quantification used the formula FRET = (B−A·b−c·C)/C described in the Materials and Methods section. P values are shown for the statistical significance of 6xPDC IMAGEtags induced versus uninduced (GAL1) or control RNA versus 6xPDC IMAGEtags (ACT1 and ADH1). •:median; ▪: minimum; ○: maximum; ♦: q1; Δ; q3.
Mentions: To evaluate the cell to cell variability of Pol II promoter activity in living cells, we used IMAGEtags as reporters from three promoters: GAL1, ACT1 and ADH1. FRET signals in individual cells were quantified with time after induction of the GAL1 promoter and compared with the IMAGEtag RNA levels in the same cell populations (Figure 3A–C). Yeast cells expressing the control RNA containing no IMAGEtags (Figure 3A) or 6xPDC IMAGEtags (Figure 3B) were induced for different time periods and the FRET signal was measured in individual cells. There was a large variation in the range of individual cellular FRET signals at each time point under these conditions of preculture in glucose. However, the average increase in FRET, which represents the ensemble of induced cells, was proportional to the increase in IMAGEtag RNA level of the population measured by RT-qPCR (Figure 3A and B. This result indicates that the sampling of cells for FRET in these experiments was representative of the population and is consistent with the conclusion that the observed FRET is due to newly synthesized IMAGEtag RNA. Unlike for the IMAGEtags, the average FRET output of the control population did not increase in parallel with the mRNA content of the cell population. At each time point, the average FRET output from cells expressing the 6xPDC IMAGEtags was significantly higher than from cells expressing the control RNA (P < 0.001). The ability of IMAGEtags to detect the activity of two constitutive promoters, ACT1 and ADH1, was also tested (Figure 3E). The statistical significance of these results is reflected in the low P values that vary from 10−4 to 10−10. The larger variation of FRET signal from individual cells when the promoter was GAL1 (Figure 3A–C) was associated with an experimental design in which the cells were taken directly from a glucose containing medium to one with galactose replacing glucose. The GAL1 promoter is not activated until the intracellular glucose is depleted. The cell to cell variation in time to depletion of intracellular glucose may be the basis for larger variations in cell response in this experimental design compared with others. In experiments where the cells were first cultured in raffinose to allow glucose depletion, the variations in FRET signals were much smaller with an average coefficient of variation of 15% from the compiled results from seven conditions of groups of 11–21 cells (Supplementary Figure S14).

Bottom Line: Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression.Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals.The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

View Article: PubMed Central - PubMed

Affiliation: Ames Laboratory, US Department of Energy, Ames, IA 50011, USA Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, 1210 Molecular Biology Building, Iowa State University, Ames, IA 50011, USA.

Show MeSH
Related in: MedlinePlus