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