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. 2013;9(7):e1003161.
doi: 10.1371/journal.pcbi.1003161. Epub 2013 Jul 25.

Cell-cycle dependence of transcription dominates noise in gene expression

C J Zopf  1 , Katie Quinn, Joshua Zeidman, Narendra Maheshri
Affiliations

Cell-cycle dependence of transcription dominates noise in gene expression

C J Zopf et al. PLoS Comput Biol. 2013.

Abstract

The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ∼2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. An instantaneous transcription rate is calculated from single-cell fluorescence time series.
(A) In the 3-color diploid strain, tet-Trans-Activator (tTA) expressed from PMYO2 activates P7xtetO and is repressed by doxycycline. Homologous copies of P7xtetO drive expression of either CFP or YFP while PPGK1 drives RFP expression constitutively. (B) Monitoring growth of single cells in microfluidic culture. The segmented mother cell, daughter, and contiguous whole cell regions are outlined in blue, green, and red, respectively. For the mother and its daughters, (C) raw volume and (D) CFP concentration time series were smoothed to remove measurement noise; (E) integrated CFP fluorescence was calculated as their product (corresponding regions shown in B). The sum of bud and mother values until division constitutes a whole cell trace, which when extended past division, yields a running total trace that is fit to a differentiable smoothing spline. (F) Calculated relative mRNA levels and (G) instantaneous transcription rate.
Figure 2
Figure 2. Instantaneous transcription rate in single yeast cells correlates with growth across the cell cycle.
Single-cell data was binned by cell-cycle progression and averaged. (A) Because of the small (<10%) decrease in CFP concentration across the cell cycle, (B) total CFP rises rapidly with volume post-bud formation, similar to , but at slightly different rates. (C) Mean instantaneous growth and protein production rates are lower in G1 and peak in S/G2/M. Instantaneous transcription rate of P7xtetO and PPGK1 correlates with instantaneous growth rate for growth in (D) 2% glucose (N = 171 cell cycles/bin) and in (E) 2% raffinose (N = 246 cell cycles/bin). YFP and RFP transcription averages were normalized to that of CFP in glucose (D). Error bars represent the bin SEM from bootstrapping. Dotted lines indicate the bin s.d.
Figure 3
Figure 3. Transcriptional bursts from homologous loci are cell-cycle dependent and partially correlated.
The 3-color diploid strain was grown in microfluidics with 50 ng/mL dox, reducing expression from P7xtetO. (A) The probability that each P7xtetO's transcription rate is above background (Text S1), computed by averaging binarized individual cell responses (ON = 1, or OFF = 0) in each cell-cycle progression bin, increases after G1. (B) A 2D histogram of activation time for each promoter when both activate (t = 0 at budding). Most activation occurs near budding and is correlated. (C) Classifying all single-cell S/G2/M periods (from (A) plus those following an unobserved G1) by whether each P7xtetO activates reveals correlations in sporadic expression. Error bars represent SEM from bootstrapping.
Figure 4
Figure 4. Large differences in transcriptional activity between S/G2/M and G1 depend on promoter.
(A) YFP mRNA distributions in a haploid yeast with integrated P1xtetO-YFP and no tTA are shown in a column as a function of cell-cycle phase. Horizontal lines above each distribution are the experimental (gray) and predicted mean/standard deviation for different models, with colors shown in the legend at the bottom, calculated by assuming each bud phase represents 1/3 of S/G2/M. (B) As in (A) but with P7xtetO. (C&D) As in (A&B) but with tTA and 100 or 500 ng/mL dox added for P1xtetO and P7xtetO, respectively. (E) Integrated PDOA1-YFP with native DOA1 expressed from a plasmid. Mid log-phase cells were analyzed. (F) As in (E) but late log-phase cells.
Figure 5
Figure 5. Gene activation depends on cycle phase.
P1xtetO and P7xtetO were activated in the kinetic strain background (A) by a step change in phosphate concentration. (B) The time to localization of chimeric Pho4-tetR-YFP and (C) the subsequent kinetic transcription activation are identical for both promoters, when grown in 2% glucose and transcription rate traces are normalized by the average first peak height. Distributions of all single-cell (D) localization and (E) transcription activation delay times were calculated from the time of the phosphate switch to the time to cross an effective threshold (Fig. S11). (F) Each cell's delay to respond is calculated as the difference between the cell's transcription and localization times. (G) Separating the response delay time distributions by cell-cycle phase at the time of localization reveals faster activation post-budding. (H) Long response delays occur only when Pho4-tetR-YFP localizes in G1. Each cell is plotted as a stem where the distance along the abscissa corresponds to the cell-cycle progression at the time of TF localization; the height of the stem endpoint along the ordinate corresponds to the cell-cycle progression at the time of transcription activation; and the length of the stem from the green diagonal to the endpoint represents the response delay. Colors depict phases of localization and activation as in the inset legend. Data shown are for P7xtetO in raffinose.
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