Single-allele analysis of transcription kinetics in living mammalian cells

Journal name:
Nature Methods
Volume:
7,
Pages:
631–633
Year published:
DOI:
doi:10.1038/nmeth.1482
Received
Accepted
Published online

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.

At a glance

Figures

  1. Real-time transcription kinetics of single CCND1 alleles.
    Figure 1: Real-time transcription kinetics of single CCND1 alleles.

    (a) Scheme of the two constructs. HA sequence encodes the hemagglutinin tag, and MS2 encodes an array of MS2 protein binding sites. Green spheres represent MS2-GFP fusion protein binding to the MS2 stem-loops. (b) Live-cell fluorescence-image movie frames collected before and at indicated times after photobleaching of the transcription site (top; scale bar, 5 μm) and FRAP recovery curves. Goodness of fit evaluated by checking for a random distribution of residuals around 0 is plotted at the bottom. (c,d) MS2-GFP signal intensity profiles at transcription sites of three CMVpr-CCND1-MS2 cells (c; imaging frequency: 300 ms (left), 1 min (middle) and 20 min (right)) and three CCND1pr-CCND1-MS2 cells (d; imaging frequency: 3 min (left); 1 min (middle); 10 min (right)). Transcription site movie frames from random times during interphase show periods of transcription activity and inactivity (top). Scale bar, 1 μm.

  2. mRNA quantification at transcription sites.
    Figure 2: mRNA quantification at transcription sites.

    (a) Images show deconvolved 3D stacks of images from RNA FISH experiments (with MS2-Cy3 probe, pseudocolored green) in CMVpr-CCND1-MS2 (left) and CCND1pr-CCND1-MS2 (right) cells. Transcription sites (arrows) and cytoplasmic mRNAs (green) were identified. Hoechst staining of DNA is pseudocolored red. Scale bars, 5 μm. (b) Distribution of cellular CCND1-MS2 mRNAs (n = 25 cells). (c) Distribution of CCND1-MS2 mRNAs at the transcription sites. ANOVA (P = 0.000; F = 76.262, d.f. = 3, 90; n = 25).

  3. Transcriptional activity during the cell cycle.
    Figure 3: Transcriptional activity during the cell cycle.

    (a) Fluorescence images revealing two adjacent transcription sites (RNA FISH with a Cy3-MS2 probe) in a CMVpr-CCND1-MS2 (left) and CCND1pr-CCND1-MS2 (right) cell. (b) Stained nuclei from a CMVpr-CCND1-MS2 cell with one transcription site (green) in early to mid-S phase (left), and two sites in late S phase (right). RFP-PCNA fusion marks replication foci. (c) Fluorescence image showing MS2 FISH signal (red; arrows) and CENP-F labeling (green) of CMVpr-CCND1-MS2 cells in G2 phase (left). Same field (right) shows Hoechst staining (blue) and RNA FISH (red). (d) Frames from a time-lapse movie showing transcription site duplication. Magnification of the transcription sites is shown in the lower images (gray intensity levels were pseudocolored using the ImageJ 'fire' look-up table). (e) FRAP frames (13-s interval after bleach), recovery curves and fits of replicated transcription site doublets, site 1 (left) and site 2 (right). (f) Quantification of number of CCND1-MS2 transcripts on replicated sites (n = 20). ANOVA post-hoc Bonferroni test showed a significant difference between the intensities of the single and duplicated sites (P = 0.000 (F = 76.262, d.f. = 3,90), n = 20). Scale bars, 5 μm (insets in ac, e and magnifications in d, 1 μm).

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Author information

Affiliations

  1. The Mina and Everard Goodman Faculty of Life Sciences and Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel.

    • Sharon Yunger &
    • Yaron Shav-Tal
  2. The Department of Physics and Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel.

    • Liat Rosenfeld &
    • Yuval Garini

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

PDF files

  1. Supplementary Text and Figures (5M)

    Supplementary Figures 1–11, Supplementary Tables 1–2, Supplementary Discussion and Supplementary Note 1

Movies

  1. Supplementary Video 1 (288K)

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

  2. Supplementary Video 2 (176K)

    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.

  3. Supplementary Video 3 (164K)

    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.

  4. Supplementary Video 4 (200K)

    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.

  5. Supplementary Video 5 (1M)

    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.

  6. Supplementary Video 6 (2M)

    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.

  7. Supplementary Video 7 (292K)

    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.

  8. Supplementary Video 8 (208K)

    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.

  9. Supplementary Video 9 (3M)

    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.

  10. Supplementary Video 10 (3M)

    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.

  11. Supplementary Video 11 (528K)

    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.

Additional data