Research ArticleGENETICS

Single-molecule imaging of transcription at damaged chromatin

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Science Advances  09 Jan 2019:
Vol. 5, no. 1, eaau1249
DOI: 10.1126/sciadv.aau1249
  • Fig. 1 New reporter genes allow visualization of transcription upon DNA damage with single-molecule resolution.

    (A) Illustration of a HEK293 cell containing a single FRT genomic locus for single copy integration of the reporter gene. (B) Schematic of the two reporter genes with the I–SceI site and MS2/PP7 sequences highlighted. (C and D) Representative images of PROP (C) and EX2 (D) reporter cells. Images were acquired at the indicated time points after doxycycline treatment. Examples of pre-mRNAs diffusing throughout the nucleoplasm are numbered 1 to 4. The transcription site (TS) of the reporter gene is indicated in each cell. Corresponding images depicting the diffraction limited objects in the highest intensity plane are shown below. (E and F) Time-lapse series of imaged PROP (E) and EX2 (F) reporter cells upon doxycycline treatment. The number (n) of transcripts was plotted over time in line graphs. Enlarged line plots depict a complete cycle of fluorescence gain and loss for each of the time-lapse series.

  • Fig. 2 PROP DSBs silence transcription.

    (A) PROP reporter cells were transfected with the I–SceI–GR-RFP fusion protein and imaged for 8 min after TA treatment. Images acquired at the indicated time points illustrating the translocation of the enzyme into the nucleus are shown. (B) Dynamics of transcription revealed by MS2-RFP fluorescence intensity at the transcription site and of MDC1-GFP recruitment to the transcription site in PROP cells transfected with I–SceI–GR fusion proteins and live-imaged after TA treatment. Representative images acquired at the indicated time points are shown. The dual-line plot on the right-hand side represents the average labeling intensity of MDC1-GFP and MS2-RFP quantified for a region of interest (ROI) defined around the transcription site over time. One representative experiment from more than 30 individual cells recorded is shown. a.u., arbitrary units. (C and D) Time-lapse, multiplane, spinning disk confocal microscopy was used to monitor live cell transcription dynamics of PROP reporter cells expressing MS2-GFP in the absence of (C) or upon (D) TA treatment. Representative line plots of number (n) of transcripts over time are shown. Bar graphs on the right-hand side represent the frequency of cells that exhibit a pattern of continuous (Contin.) or suppressed/terminated (Term.) transcription revealed by the lack of MS2-GFP fluorescence. The absolute number (n) of imaged cells is shown above each bar.

  • Fig. 3 Break-induced transcription initiation.

    Time-lapse, multiplane, spinning disk confocal microscopy recordings of EX2 reporter transcription dynamics in live cells revealed by the dual labeling of nascent transcripts with MS2-RFP and PP7-GFP in the absence [(A) no TA] or upon [(B and C) +TA] DNA damage. The dual-line plots represent the number (n) of transcripts detected at the transcription site over time. The bar graph in (A) shows the frequency of cells that exhibit a pattern of continuous (Contin.) and suppressed/terminated (Term.) transcription. The bar graph in (B) shows the frequency of cells, where preexisting transcription was suppressed in response to TA treatment and either resumes (Resume) or remains silenced (Term.) during the entire imaging period. A representative pattern of transcription resuming is depicted on the images and dual-line plot in (B). (C) Representative images and dual-line plot of a recorded cell where only the PP7-GFP fluorescence is observed after the damage-induced termination and before dual-labeled transcripts are detected. The bar graph depicts the frequency of cells that exhibit a similar uncoupling of the MS2-RFP and PP7-GFP fluorescent signal (Uncoupl.) and those where transcription resumed as illustrated in (B) (Coupl.). (D) Western blot analysis of DNA ligase IV in control (ctrl) or DNA ligase IV small interfering RNA (siRNA)–depleted cells. α-Tubulin and total histone H3 serve as loading controls. The bar graph depicts the frequency of cells that either resume or irreversibly terminate transcription after induction of the DSB in DNA ligase IV–depleted cells. kd, knockdown; RNAi, RNA interference.

  • Fig. 4 Break-induced chromatin modifications.

    (A) Nucleosome occupancy measured by total histone H3 ChIP at two regions (marked A and B in the schematics of the reporter gene) flanking the I–SceI site of the EX2 reporter. (B) Levels of the indicated histone modifications at the A and B regions of the EX2 reporter gene measured by ChIP before and after (30 min and 1 hour) the addition of TA to induce a DSB. (C) Nucleosome occupancy and (D) levels of histone modifications at a region (marked C) immediately downstream the I–SceI site of the PROP reporter gene. All data were normalized against the nucleosome occupancy on non–TA-treated cells and represent mean and SEM from at least three independent experiments. *P < 0.05 and **P < 0.01, obtained using two-tailed Student’s t test.

  • Fig. 5 Intragenic DNA damage drives bidirectional break-induced transcription initiation.

    (A) Schematics of the EX2-AS reporter constructed to directly inspect break-induced antisense transcription initiation. (B) Representative images of a total of eight imaged cells and dual-line plot of a recorded cell where break-induced antisense transcription initiation was detected. The bar graph depicts the frequency of cells where antisense transcription was either not observed (No AS transcr., 50%) or observed (AS transcr., 50%).

  • Fig. 6 Pervasive antisense transcription at endogenous DSBs.

    (A) Metaprofiles and heatmaps of DSBs, DNase accessible regions, and antisense transcription centered on the position of intragenic DSBs. (B) Fold enrichment of antisense transcription upstream the DSB calculated as the number of transcribed DSB regions divided by the number of randomly shuffled DSBs overlapping transcribed regions. Error bars depict SEM of fold enrichment over random. Permutation analysis, ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau1249/DC1

    Fig. S1. Schematics of the reporter genes.

    Fig. S2. Detection of single RNAs and measurement of transcription rates.

    Fig. S3. I–Sce I cleaves the reporter genes driving MDC1 recruitment to damaged loci.

    Fig. S4. Single-molecule calibration measurements.

    Fig. S5. Single transcript calibration measurements.

    Fig. S6. Break-induced nucleosome depletion.

    Table S1. Sequence of primers used in this study.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Schematics of the reporter genes.
    • Fig. S2. Detection of single RNAs and measurement of transcription rates.
    • Fig. S3. I–Sce I cleaves the reporter genes driving MDC1 recruitment to damaged loci.
    • Fig. S4. Single-molecule calibration measurements.
    • Fig. S5. Single transcript calibration measurements.
    • Fig. S6. Break-induced nucleosome depletion.
    • Table S1. Sequence of primers used in this study.

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