Interphase Chromatin Undergoes a Local Sol-Gel Transition upon Cell Differentiation

Abstract

Cell differentiation, the process by which stem cells become specialized cells, is associated with chromatin reorganization inside the cell nucleus. Here, we measure the chromatin distribution and dynamics in embryonic stem cells in vivo before and after differentiation. We find that undifferentiated chromatin is less compact, more homogeneous, and more dynamic than differentiated chromatin. Furthermore, we present a noninvasive rheological analysis using intrinsic chromatin dynamics, which reveals that undifferentiated chromatin behaves like a Maxwell fluid, while differentiated chromatin shows a coexistence of fluidlike (sol) and solidlike (gel) phases. Our data suggest that chromatin undergoes a local sol-gel transition upon cell differentiation, corresponding to the formation of the more dense and transcriptionally inactive heterochromatin.

FIG. 1.

Chromatin distribution before and after cell differentiation. (a) & (c) Micrographs of (a) an undifferentiated (ESC) and (c) a differentiated nucleus with fluorescently labeled chromatin (H2B-GFP). (b) & (d) Color-coded normalized pixel intensities I_n (color bar, black to white) for (b) nucleus from (a), and (d) nucleus from (c). (e) Distribution of normalized pixel intensities I_n for 47 undifferentiated and 52 differentiated nuclei. (f) Distribution of σ/μ for population of undifferentiated (blue) and differentiated (magenta) nuclei. Solid and dashed lines correspond to the median and quartiles of the distribution, respectively.

FIG. 2.

Chromatin dynamics before and after cell differentiation. (a–b) DCS maps $\overrightarrow{d}(\overrightarrow{r},\Delta t)$ for nuclei from Fig. 1, vectors are color-coded by their magnitude, Δt = 0.25 s. (c–d) Heat maps of the average local speed v at Δt = 0.25 s averaged over all time frames. (e–f) Heat maps of local correlation length l of chromatin motion measured at Δt = 0.25 s. (g) The average local speed v as a function of the normalized pixel intensity I_n for 47 undifferentiated and 52 differentiated nuclei. (h) The local correlation length l as a function of I_n for population of undifferentiated (blue) and differentiated (magenta) nuclei. Error bars show standard error.

FIG. 3.

Analysis of chromatin dynamics before and after cell differentiation. (a) Average MSND for chromatin in 47 undifferentiated (blue squares) and 52 differentiated (magenta circles) nuclei and their fits (solid lines) to MSND(Δt) = A + BΔt^α. Black line presents the MSND of formalehyde-fixed cells establishing the noise floor. (b) Heat maps of normalized intensity I_n and local correlation length l in and around HC region outlined by grey contour. Inset shows the HC region trajectory. (c) Average MSND for heterochromatin (HC) regions (N = 93, green markers) and euchromatin (EC) regions (N = 72, blue markers). Yellow markers show the average MSD of the HC centroids and purple markers present the HC MSND corrected for the HC region motion and averaged over all HC regions. (d) Heat maps of I_n and l in and around EC region outlined by grey line. Error bars show standard error. Scale bar, (b) & (d) 500 nm, inset 50 nm.

FIG. 4.

Rheological models of undifferentiated and differentiated chromatin. (a) G′ and G″ measured for undifferentiated chromatin (blue markers) and fitted to the Maxwell fluid model (blue lines). Inset shows tan(δ) = G″/G′, dashed line represents ω⁻¹. (b) Diagram of the Maxwell fluid as a spring-dashpot system. (c) G′ and G″ measured for differentiated chromatin (magenta markers) and fitted to the Burgers model (magenta lines). Inset shows tan(δ) = G″/G′, dashed line indicates ω⁻¹. (d) Diagram of the Burgers model as a spring-dashpot system. (e) Cartoon illustrating the fluid-like (blue) state of undifferentiated chromatin and the emergence of local solid-like phase (magenta) upon differentiation. Error bars show standard error.