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. 2016 Feb 12;351(6274):720-4.
doi: 10.1126/science.aab2956.

Dynamics of epigenetic regulation at the single-cell level

Lacramioara Bintu  1 , John Yong  1 , Yaron E Antebi  1 , Kayla McCue  1 , Yasuhiro Kazuki  2 , Narumi Uno  2 , Mitsuo Oshimura  2 , Michael B Elowitz  3
Affiliations

Dynamics of epigenetic regulation at the single-cell level

Lacramioara Bintu et al. Science. .

Abstract

Chromatin regulators play a major role in establishing and maintaining gene expression states. Yet how they control gene expression in single cells, quantitatively and over time, remains unclear. We used time-lapse microscopy to analyze the dynamic effects of four silencers associated with diverse modifications: DNA methylation, histone deacetylation, and histone methylation. For all regulators, silencing and reactivation occurred in all-or-none events, enabling the regulators to modulate the fraction of cells silenced rather than the amount of gene expression. These dynamics could be described by a three-state model involving stochastic transitions between active, reversibly silent, and irreversibly silent states. Through their individual transition rates, these regulators operate over different time scales and generate distinct types of epigenetic memory. Our results provide a framework for understanding and engineering mammalian chromatin regulation and epigenetic memory.

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Figures

Fig. 1
Fig. 1. Time-lapse analysis reveals that silencing occurs through stochastic all-or-none events in individual cells
(A) Direct recruitment of a CR (black oval) to a reporter gene enables analysis of subsequent silencing dynamics (upper panel). After silencing, releasing the CR allows analysis of epigenetic memory and reactivation dynamics (lower panel). White and black dots represent changes in chromatin modifications. Wavy lines represent transcribed mRNA. (B) Construct for constitutive coexpression of H2B-mCherry and one of four CRs fused with the DNA binding protein rTetR (top). Engineered cells also contained a target H2B-citrine reporter gene driven by the mammalian elongation factor 1a promoter (pEF) (35) with five upstream rTetR binding sites (5x TetO). The reporter is flanked by insulators (ins) and is site-specifically integrated on a HAC in CHO-K1 cells (bottom). H2B domains localize fluorescent protein signals to the nucleus, improving quantitation. (C) (Top) Typical filmstrip of silencing dynamics (from movie S1). (Bottom) Total citrine fluorescence (a.u., arbitrary units) is plotted (dotted line) for the cell lineage circled in white, before (gray shading) and during (yellow shading) recruitment. A cumulative fluorescence trace (solid blue) was obtained by computationally restoring the fluorescence signal lost to the sister cell at each division. This procedure facilitates continuous quantification of the reporter production rate (slope of cumulative trace) and identification of silencing events (black circle) (see materials and methods). Numbers correspond to frames in the filmstrip above. (D) Representative single-cell traces showing silencing events (circles) induced by recruitment of the indicated CR. Only cells silenced during the corresponding movie (see movies S1 to S4) are shown. For clarity, traces are offset by arbitrary amounts on the y axis. The percentage of traces that resemble those shown here is indicated on each plot (see fig. S4 for other behaviors). (E) Distributions of silencing times, Toff (mean ± SD). n indicates the number of events for each histogram. (F) Single-cell cumulative fluorescence traces (gray lines) were aligned at the silencing event (0 on the x axis) and superimposed. The median of all traces is plotted as a colored line. (G) Median reporter production rates, obtained from all slopes of the individual traces in (F), showing the all-or-none nature of silencing events. (H) Fraction of cells silenced as a function of time (dots, mean ± SD). Curves represent exponential fits to a single silencing rate with a time delay for each CR (materials and methods).
Fig. 2
Fig. 2. Chromatin regulators produce distinct time scales of memory
(A) (Top) Filmstrip and (bottom) corresponding fluorescence trace before and after EED release (from movie S9). Traces, numbers, and shading are similar to those in Fig. 1C. (B) Representative single-cell traces showing reactivation events for EED, KRAB, and HDAC4 (circles; only reactivated cells are shown). No reactivation events were observed for DNMT3B, so only silent cells are plotted. Traces are vertically offset for clarity. (C) Flow cytometry enables classification of cells as silent (low-fluorescence peak) or active (high-fluorescence peak), using a threshold (gray line). (D) Fraction of silenced cells measured by flow cytometry at various time points after CR release. Each dot represents one flow cytometry measurement. Data from three independent experiments are shown. Spontaneous background silencing rates have been subtracted (fig. S7D and materials and methods). Solid lines are fits to the model in Fig. 3A.
Fig. 3
Fig. 3. A three-state model explains gene expression dynamics across different recruitment durations and strengths
(A) Proposed model based on stochastic transitions between actively expressing (A), reversibly silent (R), and irreversibly silent (I) states. Silencing (at rates kS and kI) depends on recruitment, whereas reactivation (at rate kA) is independent of recruitment. (B) Experimental strategy: The duration of recruitment was varied from 1 to 5 days (colored arrows). After removal of dox, the fraction of cells remaining silenced was measured for up to 30 days. (C to F) Flow cytometry measurements show the fraction of silent cells over time after CR release. Colors indicate recruitment duration, as in (B). Data from two or more independent experiments are shown. Each set of solid lines represents a single fit of all data for that factor to the model, with rate constants indicated above each panel (see materials and methods for details of fitting). (G to I) Silencing and reactivation dynamics are measured at different dox concentrations. For each concentration, these data are fit with the corresponding model for each CR to extract the kinetic rates indicated in the diagram (dots, see materials and methods). Error bars represent the 95% confidence interval of the fit. Curves [(G) and (I)] are fits to a Michaelis-Menten–like equation. Lines in (H) are fits to a constant value.
Fig. 4
Fig. 4. Chromatin regulators generate diverse modes of duration-dependent fractional silencing and memory
(A) The four CRs can be represented in a three-dimensional parameter space defined by the rate constants in the model (axis labels) over a range of recruitment strengths. The curve occupied by each CR in this space encapsulates its dynamic effects on gene expression and epigenetic memory. The colored dot at the end of each curve represents rate constants at full recruitment strength (saturating dox concentration). (B) For each regulator, the response to a pulse of recruitment is computed and plotted using the rate constants at full recruitment strength. The total fraction of silent cells in states R and I is indicated by the solid color line; the fractions of cells in each of these states are approximately indicated by the fraction of black and gray circles. Time is indicated on the x axis (log scale). The final fraction of cells in the I state is indicated by the red hatched bar. (C) Different durations of recruitment (upper and lower panels) can similarly produce full silencing on shorter time scales but can generate different amounts of permanent memory (red hatched bars).
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