3D imaging of Sox2 enhancer clusters in embryonic stem cells
- Howard Hughes Medical Institute, Janelia Research Campus, United States
- University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
Localization of Sox2 stable binding sites in 3D by lattice light-sheet, single-molecule imaging.
(A) Whole-nucleus single molecule imaging was performed by lattice light-sheet microscopy with 300 nm z steps and 50 ms per frame. HaloTag-Sox2 molecules were labeled by membrane permeable JF549 …
Figure 1—figure supplement 1
Optics layout, PSF, and localization uncertainty estimation.
(A) Schematic of the light path used for the optical lattice microscope. A collimated circular laser beam is passed through two pairs of cylindrical lenses to illuminate a thin stripe across the …
Figure 2 with 2 supplements
Clustering of Sox2 bound enhancers in the nucleus.
(A) 3D density map of H2B distribution (n = 7000) in single ES cell nucleus. The imaging condition and analysis parameter set-ups were the same as HaloTag-Sox2 in Figure 1. The color map reflects …
Figure 2—figure supplement 1
Quantification of clustering by pair correlation function.
(A) 3D density map of simulated uniform sites (n = 7000) in single ES cell nucleus. The color map reflects the number of local neighbors that was calculated by using a canopy radius of 400 nm. The …
Figure 2—figure supplement 2
Temporal profiles of individual clusters and the number of localization detections per frame.
(A–C) Time counting of the arrival events of Sox2 stable binding sites within individual clusters. Cumulative Density Function is plotted as the function of the frame number. The time interval …
Figure 3 with 2 supplements
Sox2 enhancer clusters and heterochromatin regions are not co-localized.
(A) Two color imaging to probe the spatial relationship between enhancer clusters and heterochromatin regions. Sox2 stable binding sites were mapped by low-excitation 2D single molecule imaging …
Figure 3—figure supplement 1
Heterochromatin and Sox2 EnC spatial relationship.
(A) Wide-field GFP-HP1 image was first processed by Matlab function to subtract background signals. Then, the normalized intensity map of heterochromatin was calculated (See details in ‘Materials …
Figure 3—figure supplement 2
Probing Sox2 levels in heterochromatin regions.
(A) Wide-field fluorescent images of GFP-HP1, over-labeled JF549-HaloTag-Sox2, and merged from single live cells. Upper: intensity profiles from the two separate channels along the indicated path …
Figure 4 with 1 supplement
Sox2 targets a subset of Pol II-enriched regions in the nucleus.
(A) Upper left: a live-cell 2D PALM super-resolution image of Dendra 2 Pol II. Upper Right: Sox2 enhancer clusters mapped by time-resolved, 2D single-molecule imaging/tracking. Stable binding events …
Figure 4—figure supplement 1
Spatial correlation between Sox2 EnCs and Pol II enriched regions.
(A) Left: the representative pixel-to-pixel intensity plot calculated from Pol II and EnC intensity maps shown in (B). Right: Pearson correlation coefficients calculated from the pixel-to-pixel …
Figure 5 with 1 supplement
Two-color imaging reveals differential Sox2 behavior within enhancer clusters vs heterochromatin.
(A) Two color single-molecule imaging to probe Sox2 binding and diffusion dynamics in enhancer clusters. EnC regions were first mapped by the low-excitation, long-acquisition time condition. Then, …
Figure 5—figure supplement 1
Regional specific diffusion and binding dynamics.
(A) Enhancer cluster regions were first mapped by low excitation, long acquisition (2 Hz) imaging of JF646-HaloTag-Sox2. Single-molecule stable binding localization events were used to generate the …
Figure 6 with 2 supplements
Enhancer clustering modulates global search efficiency and uncouples target search to a long-range and a local component.
(A) Monte Carlo simulation of TF target search in the nucleus to test the effects of target site distribution on the first passage 3D time (τ3D). Fold of Delay is defined as the ratio of the average …
Figure 6—figure supplement 1
TF 3D Brownian motion simulation.
(A) An exemplary track of TF 3D Brownian motion simulated by using Equations 14–15 (See ‘Materials and methods’ for details of parameter set-ups). (B) Mean square displacement plot fitted with a …
Figure 6—figure supplement 2
Effects of number of clusters and distance-between-targets on TF target search.
(A) To exclude the possibility that increased search times that we observed as target sites become more clustered is due to direct contacts between targets, we performed simulation experiments as …
Figure 7
Epigenetic perturbation of enhancer clustering and genome-wide binding.
(A) The fluctuation range (x) and amplitude (y) were obtained by fitting the pair-correlation function of the indicated dataset with the fluctuation model. Figure 2 and Figure 2—figure supplement 1, …
Figure 8
Spatially modulated target search and gene regulation in ES cells.
(A) Sox2 stable binding sites form enhancer clusters that are segregated from heterochromatin regions. Sox2 searches for targets via a 3D diffusion dominant mode traveling between clusters and …
Videos
Video 1
Single-molecule light-sheet imaging of Sox2 in GFP-HP1 ES cells.
HaloTag-Sox2 is gradually labeled with JF549 ligand by diffusion. Light-sheet imaging was performed with a z step of 200 nm.
Video 2
Single-molecule, light-sheet imaging of HaloTag-Sox2 in single live ES cells.
The z step size is 300 nm.
Video 3
Reconstructed Sox2 stable binding sites in the live ES cell nucleus.
HaloTag-Sox2 stable binding sites (7000, >3 s) were localized, tracked, and reconstructed with a color map same as Figure 1C. The unit is nm. 2 cells were shown here.
Video 4
Reconstructed H2B distribution in the live ES cell nucleus.
HaloTag-H2B sites (7000) were localized, tracked, and reconstructed with a color map same as that of Figure 2A. The unit is nm.
Video 5
Uniformly distributed, simulated positions in a nucleus.
Uniformly distributed positions (7000) were presented with a color map same as that of Figure 1C. The unit is nm.
Video 6
Transient Sox2 binding sites in the live ES cell nucleus.
HaloTag-Sox2 transient binding sites (7000, <3 s) were displayed with a color map same as Figure 1C. The unit is nm.
Video 7
Map stable Sox2 binding sites in GFP-HP1 labeled cells.
Low excitation and long acquisition time (500 ms) wide-field imaging was used to map Sox2 stable binding sites in the GFP-HP1 labeled cells.
Video 8
Two color light-sheet imaging of Sox2 over-labeled GFP-HP1 ES cells.
HaloTag-Sox2 is over labeled with JF549 ligand. Light-sheet imaging was performed with a z step of 200 nm.
Video 9
3D spatial relationship between heterochromatin and Sox2 enhancer clusters.
(A) 3D reconstruction of over-labeled JF549 HaloTag-Sox2 and GFP-HP1 in single cell nucleus. (B) 3D reconstruction of JF549 HaloTag-Sox2 stable binding events (7000) (residence time >6 s) and …
Video 10
Tracking Sox2 binding/diffusion dynamics within enhancer clusters.
Two color single molecule imaging was performed with JF646 channel (Left) for mapping the enhancer cluster regions and JF549 (right) for tracking fast Sox2 diffusion/binding dynamics.
Video 11
Tracking Sox2 binding/diffusion dynamics in heterochromatin regions.
Two color imaging was performed with the GFP channel (upper) for mapping the heterochromatin regions and JF549 (lower) for tracking fast Sox2 diffusion/binding dynamics.
Video 12
TF target search simulation.
An example of TF target search simulation in a single nucleus.
Video 13
Reconstructed Sox2 stable binding sites in the TSA treated live cell nucleus.
HaloTag-Sox2 stable binding sites in the TSA treated live cell nucleus (7000, >3 s) were localized, tracked, and reconstructed with a color map same as that of Figure 1C. The unit is nm.
Additional files
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Supplementary file 1
The fluctuation model fitting results, localization parameters, and ChIP-Exo primers.
- https://doi.org/10.7554/eLife.04236.033
