Single-allele analysis of transcription kinetics in living mammalian cells

Abstract

We generated a system for in vivo visualization and analysis of mammalian mRNA transcriptional kinetics of single alleles in real time, using single-gene integrations. We obtained high-resolution transcription measurements of a single cyclin D1 allele under endogenous or viral promoter control, including quantification of temporal kinetics of transcriptional bursting, promoter firing, nascent mRNA numbers and transcription rates during the cell cycle, and in relation to DNA replication.

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Figure 1: Real-time transcription kinetics of single CCND1 alleles.
Figure 2: mRNA quantification at transcription sites.
Figure 3: Transcriptional activity during the cell cycle.

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Acknowledgements

We thank R. Pestell (Thomas Jefferson University) for the cyclin D1 promoter, C. Cardoso (Technische Universität Darmstadt) for RFP-PCNA and R. Drummer (Bar-Ilan University) for statistical analysis. This work was supported by grants to Y.S.-T. by the Israel Cancer Research Fund (Research Career Development Award), the Israel Ministry of Health, the Israel Cancer Association, the Alon Fellowship and the Jane Stern Lebell Family Fellowship of Bar-Ilan University; and to Y.S.-T. and Y.G. by the Israel Science Foundation Bikura grant. We thank the Israel Science Foundation for the fluorescence live-cell imaging microscopes.

Author information

S.Y. generated the cell system and performed the experiments; L.R. and Y.G. performed FRAP and diffusion experiments, and model analysis; and Y.S.-T. designed the project and wrote the paper.

Correspondence to Yaron Shav-Tal.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–2, Supplementary Discussion and Supplementary Note 1 (PDF 5186 kb)

Supplementary Video 1

Transcription sites release mRNPs. Release of mRNPs (left, CCND1pr-CCND1-MS2 cell) and nucleoplasmic mRNPs (right, CMVpr-CCND1-MS2 cell) detected in transcribing cells. (MOV 287 kb)

Supplementary Video 2

CMVpr-driven active transcription site. A CMVpr-CCND1-MS2 cell was imaged in three dimensions every 1 min (11 z-dimension steps, 0.4 Am). The movie is 59 min long. (MOV 173 kb)

Supplementary Video 3

CCND1pr-driven active transcription site; gene activation. A CCND1pr-CCND1-MS2 cell was imaged in three dimensions every 3 min (23 z-dimension steps, 0.45 Am). The movie is 108 min long. (MOV 163 kb)

Supplementary Video 4

CCND1pr-driven active transcription site gene inactivation and then reactivation. A CCND1pr-CCND1-MS2 cell was imaged in three dimensions every 1 min (31 z-dimension steps, 0.43 Am). The movie is 54 min long. (MOV 197 kb)

Supplementary Video 5

The CCND1pr-driven transcription site cycles between 'on' and 'off' states. A CCND1pr-CCND1-MS2 cell (bottom cell) was imaged in three dimensions every 10 min (37 z-dimension steps, 0.5 Am). The movie is 380 min long. (MOV 1236 kb)

Supplementary Video 6

Detection of mRNAs in cell volumes. A 3D representation of a total CMVpr-CCND1-MS2 cell volume from an RNA FISH experiment with a Cy3-MS2 probe (green) with raw images shown on the left and deconvolved images on the right. Bright dot is the transcription site. (MOV 289 kb)

Supplementary Video 7

Transcription sites during cell division. Two adjacent active transcription sites persisted in a cell up until mitosis. Thereafter, daughter cells had one active transcription site. The movie is 146 min long. First frame is repeated three times so that the two sites are easily identified when playing the movie. (MOV 207 kb)

Supplementary Video 8

Tracking the diffusion of the two active transcription sites. CMVpr-CCND1-MS2 cells are imaged in three dimensions every 30 s (26 z-dimension steps, 0.4 Am). The movie is 25 min long. Time is represented by a colored bar. (MOV 289 kb)

Supplementary Video 9

Simultaneous 'turning on' of a second transcription site in two different cells. CMVpr-CCND1-MS2 cells were imaged in three dimensions every 15 s (7 z-dimension steps). The movie is 65 min long. (MOV 2701 kb)

Supplementary Video 10

Activation of a second transcription site. Original movie (left) and deconvloved and enlarged data (presented as a 'fire' lookup table) of CMVpr-CCND1-MS2 imaged in three dimensions every 15 s (7 z-dimension steps). The movie is 44 min long. (MOV 2759 kb)

Supplementary Video 11

FRAP at two transcription sites. CMVpr-CCND1-MS2 cells imaged in three dimensions every 13 s (50 z-dimension steps, 0.2 Am). The movie is 32 min long. (MOV 524 kb)

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Yunger, S., Rosenfeld, L., Garini, Y. et al. Single-allele analysis of transcription kinetics in living mammalian cells. Nat Methods 7, 631–633 (2010). https://doi.org/10.1038/nmeth.1482

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