Direct observation of frequency modulated transcription in single cells using light activation
- Center for Cancer Research, National Cancer Institute, National Institutes of Health, United States
- Johannes Gutenberg University of Mainz, Germany
- University of North Carolina, United States
- Albert Einstein College of Medicine, United States
Figures
Figure 1
Reporter gene design enables observation of multiple steps in gene expression.
(A) The reporter gene consists of a SR response element multimerized 85 ×, upstream of the transcription start site. The gene is a bi-cistronic construct introduced via lentivirus infection. The …
Figure 2 with 3 supplements
Dynamic observation of integrated reporter genes shows transcriptional bursting.
(A–D) Time-lapse images of active transcription in two separate nuclei. TS are visualized as punctate fluorescent spots where MS2-YFP has coalesced onto multiple MS2-binding sites in nascent …
Figure 2—figure supplement 1
Time-dependent induction of reporter gene.
Cells are induced at t = 0 with 50 μM Ponasterone A at 37°C. z-stacks are acquired every hour, and the number of cells with a transcription site is recorded.
Figure 2—figure supplement 2
Dynamic observation of integrated reporter genes shows transcriptional bursting.
(A and B) Quantification of TS intensity for a cell treated with 12.5 μM and 3.125 μM PA, respectively. The left axis is the integrated intensity of the TS; the right axis is the normalized …
Figure 2—figure supplement 3
Dose dependence of on- and off- times for an alternative single cell clone.
Average on (black) and off (gray) duration for three different [PA] obtained from fitting experimental data to an exponential distribution. Error bars are the SEM.
Figure 3
Dynamic observation of transiently transfected reporter genes shows fast induction and no bursting.
Time-lapse images of transiently transfected cells induced with 50 μM PA at t = 0 min. Each cell contains multiple copies of the reporter plasmid, visible as diffraction limited spots (Video 6). …
Figure 4
Steady state distribution of mRNA from the steroid-activated reporter gene indicates frequency modulation of transcription.
(A) Dose response of the reporter gene. Total cellular reporter mRNA is plotted as a function of [PA] for three different clones isolated from a population of lentiviral-infected cells (clone 1, red;…
Figure 5 with 2 supplements
Caged PA is an ecdysone receptor antagonist.
(A) Molecular structures of the two forms of caged PA: DMNB-PonA and CNB-PonA. (B) Dose dependent competition between caged PA and unmodified PA. The competition experiment consists of an overnight …
Figure 5—figure supplement 1
Caged-PA can be switched from inactive to active with UV photolysis.
(A) Dose-response of CFK-SKL production as measured by western blot for PA (black circles and line) and the DMNB-PA (red-circles and line). The solid lines are fit to a first-order Hill response …
Figure 5—figure supplement 2
Caged-PA competes with unmodified PA.
Cells are incubated with 5 μM PA overnight, resulting in the appearance of transcription sites. At t = 0 hr, time-resolved measurements of transcription site intensity begin. At t = 2 hr, caged PA …
Figure 6
Photoactivation of single genes in vivo relates agonist kinetics to transcription dynamics.
(A) Schematic uncaging experiment in tissue culture. The target cell is photolyzed with a laser that has the physical dimensions of a single nucleus (dotted circle). The transcription site in that …
Videos
Video 1
Time-lapse sequence of reporter gene response to 50 μM PA shows a single pulse of transcription.
The nascent transcription site is visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA. Each frame is the maximum projection of 18 z-steps acquired at 0.5 μm …
Video 2
Time-lapse sequence of reporter gene response to 50 μM PA shows a gene which is on for almost the entire duration of the video.
The nascent transcription site is visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA. Each frame is the maximum projection of 18 z-steps acquired at 0.5 μm …
Video 3
Time-lapse sequence of reporter gene response to 50 μM PA for multiple cells showing uncorrelated transcription dynamics.
The nascent transcription site is visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA. Each frame is the maximum projection of 18 z-steps acquired at 0.5 μm …
Video 4
Time-lapse sequence of reporter gene response to 50 μM PA for cells in Figure 2.
The nascent transcription site is visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA. Each frame is the maximum projection of 18 z-steps acquired at 0.5 μm …
Video 5
Post-processing time-lapse sequence of reporter gene response to 50 μM PA for cells in Figure 2.
The nascent transcription site is visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA. Each cell is individually segmented and re-cropped to a separate image file, …
Video 6
Time-lapse sequence of a transfected reporter.
Multiple nascent transcription sites are visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA emerging from the plasmids. Each frame is a single z-plane. Exposure time …
Video 7
Time-lapse sequence of a transfected reporter.
Multiple nascent transcription sites are visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA emerging from the plasmids. Each frame is a single z-plane. Exposure time …
Video 8
Time-lapse sequence of reporter gene uncaging.
The nascent transcription site is visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA. Cells were incubated in 100 μM DMNB-PA overnight. The media were then removed, …
Video 9
Time-lapse sequence of reporter gene uncaging.
The nascent transcription site is visualized as the coalescence of MS2-YFP coat protein on newly synthesized pre-mRNA. Cells were incubated in 100 μM DMNB-PA overnight. The media were then removed, …
