1. Chromosomes and Gene Expression
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Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity

  1. Jeffrey M Alexander
  2. Juan Guan
  3. Bingkun Li
  4. Lenka Maliskova
  5. Michael Song
  6. Yin Shen
  7. Bo Huang
  8. Stavros Lomvardas
  9. Orion D Weiner  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Chan Zuckerberg Biohub, United States
  3. Columbia University, United States
Research Article
Cite this article as: eLife 2019;8:e41769 doi: 10.7554/eLife.41769
12 figures, 5 videos, 1 table, 10 data sets and 8 additional files

Figures

Figure 1 with 3 supplements
The Sox2 Locus As a Model for Visualization of Enhancer-Promoter Regulation in Mouse Embryonic Stem Cells.

(A) To visualize chromosome loci in living cells, we have used tetO/TetR and cuO/CymR genetic labels. Our pipeline for insertion of these labels into the mouse genome is shown. First, CRISPR-Cas9 is used to place an attP integrase landing site. Second, a targeting plasmid bearing the compatible attB sequence, the tetO or cuO array, and a selection cassette is introduced along the integrase (Int) to mediate site-specific integration. The selection cassette can then be subsequently removed by Cre/Flp recombinase. (B) The Sox2 locus in mouse ESCs. Genomic browser tracks of epigenomic and expression data demonstrate high levels of histone acetylation, RNA polymerase II, and transcription factor (OCT4, SOX2, NANOG, CTCF) and cohesin (RAD21) occupancy at Sox2 and the distal Sox2 Control Region enhancer (tan boxes). Data from 4C and HiC experiments demonstrate chromosomal contacts at the Sox2 locus. For 4C data, read density indicates contact frequency with a fixed position near the Sox2 promoter (red triangle). Y-axis for browser tracks is reads per million. For HiC, all pairwise contact frequencies are shown using a heatmap. The intensity of each pixel represents the normalized number of contacts detected between a pair of loci. The maximum intensity is indicated in red square. At bottom, locations of the cuO- and tetO-arrays for the three cell lines utilized for this study. Sox2-8CcuO/+; Sox2-117TtetO/+ (Sox2-SCR) ESCs were used to track Sox2/SCR location. Two control lines, Sox2-43TtetO/+; Sox2-164TcuO/+ (Control-Control) and Sox2-117TtetO/+; Sox2-242TcuO/+ (SCR-Control) were analyzed for comparison. H3K27ac, RNA polymerase II (RNAP), and RNAseq data from GSE47949 (Wamstad et al., 2012); DNase data from GSE51336 (Vierstra et al., 2014); SOX2, OCT4, NANOG, CTCF data from GSE11431 (Chen et al., 2008b), and RAD21 data from GSE90994 (Hansen et al., 2017); 4C data from GSE72539 (de Wit et al., 2015); and HiChIP data from GSE96107 (Bonev et al., 2017).

https://doi.org/10.7554/eLife.41769.002
Figure 1—figure supplement 1
Characterization of Modified Embryonic Stem Cell Lines.

(A) Schematic of modified cell lines used in this study. Primer sets used to amplify recombination arms for tetO- and cuO- integration are shown. (B,C) PCR genotyping of ESC lines shown in A.

https://doi.org/10.7554/eLife.41769.003
Figure 1—figure supplement 2
Sox2 Expression Characterization for Modified Embryonic Stem Cell Lines.

(A) Ratio of Sox2 expression from the 129 allele and the CastEiJ allele measured by qPCR for modified ESC lines. Sox2-SCR cell line has cuO array inserted 8 kb centromeric to Sox2 TSS and tetO array inserted 5 kb telomeric to SCR. Control-Control cell line has cuO and tetO located 43 kb and 164 kb telomeric to Sox2 TSS. SCR-Control has tetO inserted 5 kb telomeric to SCR and cuO located 242 kb telomeric to Sox2 TSS. Samples labeled with CymR/TetR coexpress CymR-GFP and TetR-tdTom. (B) Sox2 expression relative to control gene (Tbp) for various cell lines. E14 (129/129) mESCs are included to demonstrate specificity of allele-specific qPCR assay. SCR deletion cell line are in the context of cuO and tetO integrations in the Sox2-SCR configuration. Deletion of SCR region leads to loss of expression from the Sox2 allele in cis. Bars show mean of at least three biological replicates. Error bars show the standard error. N.D. is not detected.

https://doi.org/10.7554/eLife.41769.004
Figure 1—figure supplement 3
Sox2-SCR Contacts Are Maintained in Modified Embryonic Stem Cell Lines.

(A) Near-cis plots of 4C analysis using the Sox2 promoter region (red arrowhead) as bait shows elevated contacts with the SCR region (black arrowhead) in all cell lines investigated. Individual black points show fragment-based raw data, while blue points show a running median. The blue line and grey ribbon shows a loess-smoothed trendline for the data with the 20–80% quantile range. (B) Proportion of 4C reads that can be unambiguously assigned to a parental genome for each allele. These data demonstrate roughly half of Sox2-SCR contacts as measured by 4C come from the modified (129) allele and that our genome modifications do not significantly affect Sox2-SCR interactions.

https://doi.org/10.7554/eLife.41769.005
Figure 2 with 3 supplements
Visualization of the Sox2 Region in ESCs Reveals Minimal Evidence for Sox2/SCR Interactions.

(A) Top, confocal Z slices of CymR-GFP and TetR-tdTom in Sox2-SCR ESCs, labeling the Sox2 promoter and SCR region with bright puncta, respectively. Middle, 3D surface rendering of the ESC nucleus shown above. A single fluorescence channel was rendered white and transparent to outline the nucleus, and GFP and tdTom surfaces were rendered with high threshold to highlight the cuO and tetO arrays, respectively. Bottom, tracking data is rendered for the nucleus shown above. Inset shows example of calculated 3D separation distance between the two labels. Scale bar is 1 µm. (B) Normalized histogram of 3D separation distance for Sox2-SCR ESCs demonstrates a single peak (Hartigan’s Dip Test for multimodality, p=1). Schematic for an hypothetical looping enhancer-promoter pair is shown as an inset, with two peaks. Tan box indicates regime where distance measurement error is expected to be greater than 50%. (C) Cumulative density of 3D separation distance for Sox2-SCR versus control comparisons. Mean distance for each sample shown on bottom right. (D) Mean 3D separation distance per cell for each label pair. Population means and standard deviations are shown for each sample. Mann-Whitney, *p<0.05, **p<0.01, ***p<0.001.

https://doi.org/10.7554/eLife.41769.007
Figure 2—figure supplement 1
Tracking Lengths for tetO and cuO Spots Across Cell Lines.

(A–B) Histograms of the cuO-array (A) or tetO-array (B) track lengths for cell lines used in the study as ESCs, NPCs, and MES. Tracking lengths were often shorter in NPCs or MES due to increased nuclear movement in these cell types compared to ESCs.

https://doi.org/10.7554/eLife.41769.008
Figure 2—figure supplement 2
Estimate of Localization Precision for cuO and tetO.

(A) Histograms of X, Y, and Z position uncertainty for fluorescent beads with signal-to-noise ratios comparable to cuO/CymR-GFP or tetO/TetR-tdTom. Data plotted are the standard deviation values measured using five frame sliding windows collected from 9 to 10 beads. (B) Histogram of X, Y, Z position uncertainty derived for tracking cuO/CymR-GFP and tetO/TetR-tdTom position in fixed cells. Data plotted are standard deviations using a five frame sliding window collected for 10 loci. (C) Histogram of X, Y, Z position uncertainty for fluorescent beads with signal-to-noise ratios comparable to cuO/CymR-Halox2 or tetO/TetR-GFPx2. In all cases, error bars show median and interquartile range of the computed position uncertainties, which are reported in the upper right of each panel.

https://doi.org/10.7554/eLife.41769.009
Figure 2—figure supplement 3
Impact of Localization Precision on 3D Distance Measurements.

(A) A plot of true distance versus predicted measured distance after localization error is included demonstrate significant overestimation of very small distances. These values were derived by sampling X, Y, and Z measurements for cuO and tetO from normal distributions centered on positions that are separated by the true distance and standard deviations consistent with estimated uncertainty (median values from Figure 2—figure supplement 2 panel A). (B) The interquartile range of stimulated distance measurements after localization error is included demonstrates that measured 3D distance uncertainty is distance dependent and plateaus at approximately 52 nm.

https://doi.org/10.7554/eLife.41769.010
Figure 3 with 2 supplements
Sox2 Locus Compacts upon ESC Differentiation.

(A) ESCs were differentiated into neural progenitor cells (NPCs), which maintain expression of Sox2 but inactivate the SCR, and cardiogenic mesodermal precursors (MES), which inactivate both Sox2 and the SCR. (B) Browser tracks of H3K27ac and RNA-seq data from ESCs, NPCs, and MES demonstrate the activation status of Sox2 and SCR in each cell type. Y-axis is 0–5 reads per million for H3K27ac data and 0–10 reads per million for RNA-seq data. (C) Cumulative density of 3D separation distance for Sox2-SCR and two control pairs for NPCs (left) and MES (right). ESC data are shown for comparison as solid lines on each graph and reproduced from Figure 2C. Tan box indicates regime where distance measurement error is expected to be greater than 50%. (D) Mean 3D separation distance per cell for each label pair, organized by cell type. Statistical analysis is for each matched pair-wise comparison between cell types. All p-values are below reported value. Mann-Whitney (**p<0.01, ***p<0.001). H3K27ac data from GSE47949 (Wamstad et al., 2012) and GSE24164 (Creyghton et al., 2010). RNAseq data from GSE47949 and GSE44067 (Zhang et al., 2013).

https://doi.org/10.7554/eLife.41769.011
Figure 3—figure supplement 1
Characterization of ESC-derived Neural Progenitor Cell Lines.

(A) Immunofluorescence of fixed neural progenitor cells (NPCs) for the NPC markers SOX2 and PAX6. (B-C) Immunofluorescence for the neuron marker β3-tubulin (B) or the astrocyte marker GFAP (C) on fixed cultures after 12 days of differentiation towards neurons or astrocytes, respectively. Scale bar is 100 µm.

https://doi.org/10.7554/eLife.41769.012
Figure 3—figure supplement 2
SCR Inactivation Does Not Drive Locus Compaction Upon Differentiation.

(A) Potential models for Sox2 locus compaction observed upon differentiation to NPCs or MES. At left, cellular differentiation may lead to global changes in chromatin structure that are not dependent of Sox2/SCR activation status. Alternatively, Sox2 and SCR inactivation could lead to changes to chromatin structure within the Sox2 locus, driving locus-specific compaction. (B) Strategy for CRISPR/Cas9-mediated SCR deletion. Two gRNAs were designed to flank the SCR region and generate a deletion of SCR. Below, the SCR deletion allele shows a novel junction near the locations of expected Cas9 cutting, indicating a loss of the intervening SCR sequence. (C) Scatterplot of mean and standard deviation of 3D distance measurements for each cell line visualizes similarity between Sox2 label pairs across cell types. (D) Dendrogram visualizing hierarchical clustering of Earth Mover’s distances between 3D separation distance histograms of distinct Sox2 label pairs across cell types. SCR-deleted ESCs show greatest similarity to other ESCs as opposed to differentiated cells with inactivation of the SCR element.

https://doi.org/10.7554/eLife.41769.013
Figure 4 with 1 supplement
Slow Sox2 Locus Conformation Dynamics Lead to Limited Exploration and Variable Encounters.

(A) Maximum-intensity projection images (top) centered on the Sox2 locus and associated 3D distance measurements (bottom) highlight distinct conformations and dynamics of the Sox2 locus across cells. Scale bar is 1 µm. (B) 3D separation distance measurements for individual cells for Sox2-SCR, Control-Control, and SCR-Control highlight the heterogeneity of Sox2 locus organization across the cell population. The three cells depicted in A are boxed. (C) Cartoon description of autocorrelation analysis. Distance measurement between two time points are correlated using population statistics, revealing the time scale over which local measurements diverge from the population mean. A cell with low autocorrelation will randomly fluctuate around the population mean, leading the autocorrelation function to quickly decay to zero. A cell with high autocorrelation will deviate substantially from the expected value, only slowly relaxing back to the population mean. In this case, the autocorrelation function will stay significantly above zero for large lag times. (D) Autocorrelation function for Sox2-SCR, Control-Control, and SCR-Control pairs demonstrates significant autocorrelation at large lag times, indicating significant memory in 3D conformation across a 20 min window. The plotted values are mean ± 95% CI. E) Percent of cells with an encounter between tetO and cuO labels shown as a function of the initial separation distance measured for the cell. Likelihood of an encounter depends on the initial conformation of the locus across all label pairs and encounter thresholds.

https://doi.org/10.7554/eLife.41769.014
Figure 4—figure supplement 1
Dynamics Statistics for Each Sox2 Locus Pair in ESCs.

(A–B) Normalized histograms of relative step size (A) and change in 3D separation distance (B) for adjacent frames. Mean value is highlighted by a red line.

https://doi.org/10.7554/eLife.41769.015
Figure 5 with 1 supplement
Visualizing Sox2 Expression in Single Living ESCs Reveals Intermittent Bursts of Transcription.

(A) Sox2 locus with cuO-labeled Sox2 promoter and tetO-labeled SCR was further modified to introduce an MS2 transcriptional reporter cassette into the Sox2 gene. Transcription of Sox2 leads to visible spot at the Sox2 gene due to binding and clustering of MS2 coat protein to the MS2 hairpin sequence. (B) Maximum-intensity projection images centered on the Sox2 promoter (cuO) show intermittent bursts of MS2 signal, which are quantified on the right. Scale bar is 1 µm. (C) Single cell trajectories of Sox2 transcriptional bursts as representatively shown in B. (D) Aligned Sox2 transcriptional bursts. Randomly selected Sox2 bursts are shown as color traces (n = 50). Black line is mean MS2 signal for all annotated bursts. (E) Percent time Sox2 transcriptional bursting for various experimental conditions. Bars are mean ± standard error of ≥3 independent experiments. Sox2MS2/+ indicates cell line harbors the Sox2-MS2 reporter allele. SCRdel/+ indicates presence of an SCR deletion in cis with the Sox2-MS2 reporter. DRB indicates treatment with the transcriptional inhibitor 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB).

https://doi.org/10.7554/eLife.41769.017
Figure 5—figure supplement 1
Generation and Characterization of Sox2-MS2 Transcriptional Reporter ESCs.

(A) Targeting strategy for Sox2 transcriptional reporter. A targeting plasmid was used with Sox2 homology arms and a P2A peptide puromycin resistance gene cassette (2Apuro) inserted in frame with Sox2. Downstream of 2A puro is a 24x MS2 stem loop array, which is inserted into the 3’ UTR. (B) PCR genotyping assay to identify a targeted Sox2 allele. A primer set was used that recognized the MS2 stem loop array and a genomic region downstream of the 3’ homology arm. (C) Western blotting for SOX2 protein in parental 129/CastEiJ ESCs or ESCs heterozygous for the Sox2-MS2 allele. Actin was used as a loading control. (D) Normalized histogram of the percentage of time each individual cell has a detectable Sox2 transcriptional burst.

https://doi.org/10.7554/eLife.41769.018
Figure 6 with 1 supplement
Sox2 Transcription Is Not Associated with SCR Proximity.

(A) Schematic illustrating the expected relation between Sox2/SCR distance and MS2 transcription for a looping enhancer model. (B) Maximum-intensity projection images centered on the Sox2 promoter (cuO) show transcriptional activity without correlation to Sox2/SCR distance changes. The measured distance and MS2 signal are shown at bottom. The mean separation distance across the cell population is shown as a dotted red line. Scale bar is 1 µm. (C) Percent time with Sox2 transcriptional burst as a function of Sox2/SCR distance. Weighted mean + SE for seven experiments are shown. Weights were determined based on the proportion of frames in each bin contributed by individual experiments. (D) Mean separation distance per cell, separated into bursting and non-bursting frames. (Mann-Whitney, p=0.68). (E) Mean separation distance across a 25 min window for all transcriptional bursts (black) or randomly select time points (red), aligned according the burst initiation frame. Values plotted are mean ± 95% CI. (F) Single cell trajectories of Sox2 transcriptional bursts ranked by number of bursting frames per cell. At right, matched mean separation distances for each cell shown at left. Spearman’s correlation coefficient for each is shown. (G) Mean separation distance per cell for transcribing and non-transcribing cells. (Mann-Whitney, p=0.15). (H) Potential models of SCR regulation of Sox2 that would uncouple Sox2/SCR proximity from transcriptional activity. Above, SCR leads to long-lived activation of the Sox2 promoter that can persist long after Sox2/SCR contact is disassembled. Below, SCR nucleates a large hub of activator proteins that can modify the Sox2 promoter environment despite large distances between Sox2 and SCR.

https://doi.org/10.7554/eLife.41769.021
Figure 6—figure supplement 1
Relative Displacement between Frames for Bursting and Non-Burst Time Points.

(A) Displacement of the SCR element (tetO/TetR) relative to the Sox2 promoter (cuO/CymR) between successive frames shows no difference between bursting and non-bursting time points (Mann-Whitney, p=0.4172).

https://doi.org/10.7554/eLife.41769.022
Author response image 1
Author response image 2
Author response image 3
Author response image 4
Author response image 5
Author response image 6

Videos

Video 1
Visualization of Sister Chromatids at Sox2 Locus.

Maximum-intensity Z projection of 3D confocal Z-stacks of cuO/CymR-GFP (left) and tetO/TetR-tdTom (middle) labeling the Sox2 promoter region and SCR, respectively demonstrate two clear spots for the SCR label, suggesting cells in S/G2. These cells were excluded from analysis. Scale bar is 1 µm.

https://doi.org/10.7554/eLife.41769.006
Video 2
Variability in Sox2 Locus Organization Across Cells.

Maximum-intensity Z projection of 3D confocal Z-stacks of cuO/CymR (green) and tetO/TetR (magenta) labeling the Sox2 promoter region and SCR, respectively for three individual cells highlighted in Figure 3. The distance range explored by Cell1 and Cell2 is limited, while Cell3 shows large, abrupt changes in distance. Scale bar is 1 µm.

https://doi.org/10.7554/eLife.41769.016
Video 3
Identification of Sox2 Transcriptional Bursts in mESCs.

Maximum-intensity Z projection of 3D confocal Z-stacks of a tandem dimer of MS2 coat protein fused with two copies of tagRFP-T. The dashed yellow box highlights the ROI used for burst detection in our automated analysis pipeline, centered on the location of the Sox2 promoter (cuO/CymR location, not shown). Detected bursts are highlighted by red circles centered on the burst location, with color intensity indicating burst intensity. Scale bar is 1 µm.

https://doi.org/10.7554/eLife.41769.019
Video 4
High Transcriptional Output from Sox2 Locus.

Maximum-intensity Z projection of 3D confocal Z-stacks of a tandem dimer of MS2 coat protein fused with two copies of tagRFP-T demonstrate a period of high transcriptional activity for the highlighted Sox2 gene. The dashed yellow box highlights the ROI used for burst detection in our automated analysis pipeline, centered on the location of the Sox2 promoter (cuO/CymR location, not shown). Detected bursts are highlighted by red circles centered on the burst location, with color intensity indicating burst intensity. Scale bar is 1 µm.

https://doi.org/10.7554/eLife.41769.020
Video 5
Sox2 Transcriptional Bursts in the Absence of SCR Proximity.

Maximum-intensity Z projection of 3D confocal Z-stacks of cuO/CymR (green) and tetO/TetR (magenta) labeling the Sox2 promoter region and SCR, respectively (left), and MS2 coat protein highlighting Sox2 transcriptional activity (right). We detect clear Sox2 transcriptional bursts despite no colocalization of the Sox2/SCR labels. Scale bar is 1 µm.

https://doi.org/10.7554/eLife.41769.023

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Cell line
(M. musculus)
129/Cast F1 ESCsPMID: 9298902
Cell line
(M. musculus)
E14 ESCsPMID: 3821905RRID:CVCL_C320
Cell line
(M. musculus)
Sox2-SCR ESCsthis paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2
TSS on the 129 allele
Cell line
(M. musculus)
Sox2-SCR ESCs;
CymR-GFP;
TetR-tdTom
this paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2
TSS on the 129 allele. Cells
stably express ePiggyBac
vectors epB-UbC-CymRV5
-nls-GFP-DEx2 and epB-
CAG-TetRFlag-nls-tdTom-DEx4
Cell line
(M. musculus)
Control-Control ESCsthis paper129/Cast F1 ESCs with
tetO array inserted 43 kb
telomeric to Sox2 TSS and
cuO array inserted 164 kb
telomeric to Sox2 TSS on
the 129 allele
Cell line
(M. musculus)
Control-Control ESCsthis paper129/Cast F1 ESCs with
tetO array inserted 43 kb
telomeric to Sox2 TSS and
cuO array inserted 164 kb
telomeric to Sox2 TSS on
the 129 allele. Cells stably
express ePiggyBac vectors
epB-UbC-CymRV5-nls-GFP-DEx2
and epB-CAG-TetRFlag-nls-
tdTom-DEx4
Cell line
(M. musculus)
SCR-Control ESCsthis paper129/Cast F1 ESCs with
tetO array inserted 117 kb
telomeric to Sox2 TSS and
cuO array inserted 242 kb
telomeric to Sox2 TSS on
the 129 allele
Cell line
(M. musculus)
SCR-Control ESCsthis paper129/Cast F1 ESCs with
tetO array inserted 117 kb
telomeric to Sox2 TSS and
cuO array inserted 242 kb
telomeric to Sox2 TSS on
the 129 allele. Cells stably
express ePiggyBac vectors
epB-UbC-CymRV5-nls-GFP-DEx2
and epB-CAG-TetRFlag-nls-td
Tom-DEx4
Cell line
(M. musculus)
SCR deletion ESCsthis paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2
TSS on the 129 allele.
SCR deletion (104 kb-112kb
from Sox2 TSS) is present
on 129 allele
Cell line
(M. musculus)
SCR deletion ESCsthis paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2
TSS on the 129 allele. SCR
deletion (104 kb-112kb
from Sox2 TSS) is present
on 129 allele. Cells stably
express ePiggyBac vectors
epB-UbC-CymRV5-nls-GFP-DEx2
and epB-CAG-TetRFlag-nls
-tdTom-DEx4
Cell line
(M. musculus)
Sox2-MS2 ESCsthis paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2
TSS on the 129 allele.
129 Sox2 allele has been
replaced with Sox2-P2A-
puro-24xMS2.
Cell line
(M. musculus)
Sox2-MS2 ESCsthis paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted 117 kb
telomeric to Sox2 TSS on the
129 allele. 129 Sox2 allele
has been replaced with
Sox2-P2A-puro-24xMS2. Cells
stably express ePiggyBac
vectors epB-UbC-CymRV5-
nls-Halox2-DEx4, epB-CAG-
TetRFlag-nls-GFPx2, and
epB-UbC-tdMS2cp-tagRFP-Tx2
Cell line
(M. musculus)
Sox2-MS2; SCR
deletion ESCs
this paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2 TSS
on the 129 allele. 129 Sox2
allele has been replaced
with Sox2-P2A-puro-24xMS2.
SCR deletion (104 kb-112kb
from Sox2 TSS) is present
on 129 allele
Cell line
(M. musculus)
Sox2-MS2; SCR
deletion ESCs
this paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2
TSS on the 129 allele.
129 Sox2 allele has been
replaced with Sox2-P2A-
puro-24xMS2. SCR deletion
(104 kb-112kb from Sox2 TSS)
is present on 129 allele.
Cells stably express
ePiggyBac vectors epB-UbC
-CymRV5-nls-Halox2-DEx4,
epB-CAG-TetRFlag-nls-GFPx2,
and epB-UbC-tdMS2cp-
tagRFP-Tx2
Cell line
(M. musculus)
Sox2-del-SCR ESCsthis paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2 TSS
on the 129 allele. Large
deletion (1 kb-112kb from
Sox2 TSS) is present on 129
allele. All genetic distances
based on reference genome.
Cell line
(M. musculus)
Sox2-del-SCR ESCsthis paper129/Cast F1 ESCs with
cuO array inserted 8 kb
centromeric to Sox2 TSS
and tetO array inserted
117 kb telomeric to Sox2
TSS on the 129 allele.
Large deletion (1 kb-112kb
from Sox2 TSS) is present
on 129 allele. All genetic
distances based on
reference genome. Cells
stably express ePiggyBac
vectors epB-UbC-CymRV5
-nls-GFP-DEx2 and epB-CAG
-TetRFlag-nls-tdTom-DEx4.
Cell line
(M. musculus)
Sox2-SCR NPCsthis paperNeural progenitor cells
derived from Sox2-SCR
ESCs. Cells stably express
ePiggyBac vectors epB-UbC
-CymRV5-nls-GFP-DEx2
and epB-CAG-TetRFlag-nls
-tdTom-DEx4.
Cell line
(M. musculus)
Sox2-SCR NPCsthis paperNeural progenitor cells
derived from Sox2-SCR
ESCs
Cell line
(M. musculus)
Control-Control NPCsthis paperNeural progenitor cells
derived from
Control-Control ESCs.
Cells stably express
ePiggyBac vectors
epB-UbC-CymRV5-nls-GFP-DEx2
and epB-CAG-TetRFlag-
nls-tdTom-DEx4.
Cell line
(M. musculus)
SCR-Control NPCsthis paperNeural progenitor cells
derived from SCR-Control
ESCs
Cell line
(M. musculus)
SCR-Control NPCsthis paperNeural progenitor cells
derived from SCR-Control
ESCs. epB-UbC-CymRV5
-nls-GFP-DEx2 and
epB-CAG-TetRFlag-nls-td
Tom-DEx4.
Antibodyrat monoclonal
PE-conjugated
anti-PDGFRα
Thermo Fisher12-1401-81;
RRID:AB_657615
Flow 1:400
Antibodymouse monoclonal
anti-SOX2
Santa Cruzsc-365823;
RRID:AB_10842165
WB 1:1000, IF 1:100
Antibodyrabbit polyclonal
anti-PAX6
Biolegend901301;
RRID:AB_2565003
IF 1:100
Antibodymouse monoclonal
anti-TUBB3
Biolegend801201;
RRID:AB_2313773
IF 1:100
Antibodymouse monoclonal
anti-GFAP
SigmaG3893;
RRID:AB_477010
IF 1:400
Antibodyrabbit polyclonal
anti-βactin
Abcamab8227;
RRID:AB_2305186
WB 1:2000
Antibodyanti-Flk1 biotinPMID: 17084363Hybridoma clone
D218 Flow 1:100
Recombinant
DNA reagent
pCAGGS-Bxb1o-nlsFlagthis paperAddgene: 119901Expresses Bxb1
integrase in mammalian
cells
Recombinant
DNA reagent
pDEST-tetOx224
_PhiC31attB_loxP-
PGKpuro-loxP
this paperAddgene: 119902PhiC31 integration
plasmid for tetO
array with Neo
selection cassette
Recombinant
DNA reagent
pDEST-cuOx144
_Bxb1attB_loxP-
PGKpuro-loxP
this paperAddgene: 119903Bxb1 integration plasmid
for cuO array with Puro
selection cassette
Recombinant
DNA reagent
pDEST-tetOx224
_PhiC31attB_FRT-
EF1a-GFP-FRT
this paperAddgene: 119904PhiC31 integration
plasmid for tetO array
with GFP expression
cassette
Recombinant
DNA reagent
pDEST-cuOx144_
Bxb1attB_loxP-
EF1a-tagRFP-T-loxP
this paperAddgene: 119905Bxb1 integration plasmid
for cuO array with
RFP expression cassette
Recombinant
DNA reagent
epB-UbC_CymRV5
-nls-GFP-DEx2
this paperAddgene: 119906ePiggyBac mammalian
expression plasmid for
CymR-GFP fusion
Recombinant
DNA reagent
epB-UbC_CymRV5
-nls-Halox2_DEx4
this paperAddgene: 119907ePiggyBac mammalian
expression plasmid
for CymR-Halo fusion
Recombinant
DNA reagent
epB-UbC_tdMS2cp
-tagRFP-Tx2
this paperAddgene: 119908ePiggyBac mammalian
expression plasmid for
tandem dimer
MS2cp-tagRFP-T fusion
Recombinant
DNA reagent
epB_CAG_TetRFlag-
nls-tdTom-DEx4
this paperAddgene: 119909ePiggyBac mammalian
expression plasmid for
TetR-tdTom fusion
Recombinant
DNA reagent
epB_CAG_TetRFlag-nls_GFPx2_DEx2this paperAddgene: 119910ePiggyBac mammalian
expression plasmid for
TetR-GFP fusion
Recombinant
DNA reagent
ePiggyBac-Transposasethis paperAddgene: 119911Mammalian expression
plasmid for the ePiggy
Bac transposes
Recombinant
DNA reagent
pKS_Sox2-P2A-puro-
24xMS_targeting_
vector_NoPAM
this paperTargeting vector for
generating Sox2-MS2
allele
Recombinant
DNA reagent
pX330-Sox2_3'
UTR_gRNA
this paperCas9/sgRNA expression
vector with gRNA that
targets the Sox2 3' UTR
Recombinant
DNA reagent
pX330-Sox2-
8C_gRNA
this paperCas9/sgRNA expression
vector with gRNA that
targets 8 kb centromeric
to Sox2 TSS
Recombinant
DNA reagent
pX330-Sox2-
43T_gRNA
this paperCas9/sgRNA expression
vector with gRNA that
targets 43 kb telomeric
to Sox2 TSS
Recombinant
DNA reagent
pX330-Sox2-
117T_gRNA
this paperCas9/sgRNA expression
vector with gRNA that
targets 117 kb telomeric
to Sox2 TSS
Recombinant
DNA reagent
pX330-Sox2-
164T_gRNA
this paperCas9/sgRNA expression
vector with gRNA that
targets 164 kb telomeric
to Sox2 TSS
Recombinant
DNA reagent
pX330-Sox2-
104T_gRNA
this paperCas9/sgRNA expression
vector with gRNA that
targets 104 kb telomeric
to Sox2 TSS
Recombinant
DNA reagent
pX330-Sox2-
112T_gRNA
this paperCas9/sgRNA expression
vector with gRNA that
targets 112 kb telomeric
to Sox2 TSS
Recombinant
DNA reagent
pX330-Sox2-
242T_gRNA
this paperCas9/sgRNA expression
vector with gRNA that
targets 242 kb telomeric
to Sox2 TSS
Sequence-based
reagent
Sox2 qPCR
Forward Primer
this paper5'-CTACGCGCACATGAACGG-3'
Sequence-based
reagent
Sox2 qPCR
Reverse Primer
this paper5'-CGAGCTGGTCATGGAGTTGT-3'
Sequence-based
reagent
Sox2 qPCR 129
allele Probe
this paper5'-/56-FAM/CAACCGATG
/ZEN/CACCGCTACGA/
3IABkFQ/−3'
Sequence-based
reagent
Sox2 qPCR
Cast allele Probe
this paper5'-/56-FAM/CAGCCGATG
/ZEN/CACCGATACGA/
3IABkFQ/−3'
Sequence-based
reagent
Tbp qPCR
Forward Primer
this paper5'-ACACTCAGTTACAGGTGGCA-3'
Sequence-based
reagent
Tbp qPCR
Reverse Primer
this paper5'-AGTAGTGCTGCAGGGTGATT-3'
Sequence-based
reagent
Tbp qPCR Pan
allele Probe
this paper5'-/56-FAM/ACACTGTGT/
ZEN/GTCCTACTGCA/3IABkFQ/−3'
Sequence-based
reagent
Genotyping
PCR Primers
this papersee Supplementary file 1
Sequence-based
reagent
CRISPR guide
sequences
this papersee Supplementary file 2
Peptide, recombinant
protein
Leukemia inhibitory
factor (Lif)
Peprotech250–02
Peptide, recombinant
protein
APC-StreptavidinBD-Biosciences554067;
RRID:AB_10050396
Flow 1:200
Peptide, recombinant
protein
InsulinSigmaI6634
Peptide, recombinant
protein
Epidermal growth
factor (EGF)
Peprotech315–09
Peptide, recombinant
protein
Fibroblast growth
factor basic (Fgfb)
R and D Systems233-FB
Peptide, recombinant
protein
Natural mouse lamininThermo Fisher23017015
Peptide, recombinant
protein
Bone morphogenetic
protein 4 (BMP4)
R and D Systems314 BP
Peptide, recombinant
protein
Vascular endothelial
growth factor (VEGF)
R and D Systems293-VE
Peptide, recombinant
protein
Activin AR and D Systems338-AC
peptide, recombinant
protein
Fibroblast growth
factor 10 (Fgf10)
R and D Systems345-FG
Peptide, recombinant
protein
Laminin-511iWaichemN-892011
Chemical
compound,
drug
Prolong Live
Antifade Reagent
Thermo FisherP36975
Chemical
compound,
drug
ascorbic acidSigmaA45-44
Chemical
compound,
drug
1-thioglycerolSigmaM6145
Chemical
compound,
drug
PD03259010SelleckchemS1036
Chemical
compound,
drug
CHIR99021SelleckchemS2924
Chemical
compound,
drug
5,6-Dichlorobenzimidazole
1-β-D-ribofuranoside
SigmaD1916
Chemical
compound,
drug
JF646PMID: 28869757
Software,
algorithm
MS2Reporter
AnalysisPipeline_knn
Model.py
this paperPython scripts can
be accessed on
github (Alexander, 2018; copy archived at https://github.com/elifesciences-publications/2018_eLife_Alexander_et_al)
OtherTetraspeck
fluorescent beads
Thermo FisherT7279
Commerical
assay, kit
KAPA Library
Quantification Kit
RocheKK4854
Commerical
assay, kit
SPRIselectBeckman CoulterB23319

Data availability

All microscopy localization data utilized in this study are included as supplementary files. Example raw confocal stacks and denoised confocal stacks from Batch65 imaging available on Zenodo. Tracking data for cuO and tetO from these images can be found in Supplementary file 4. Details of microscopy acquisition in Materials and Methods. Sequencing data have been deposited in GEO under accession code GSE127901 and SRA under accession code PRJNA523665.Python scripts can be accessed on GitHub at https://github.com/JMAlexander/2018_eLife_Alexander_et_al (copy archived at https://github.com/elifesciences-publications/2018_eLife_Alexander_et_al).

The following data sets were generated
  1. 1
    NCBI Gene Expression Omnibus
    1. JM Alexander
    2. J Guan
    3. B Li
    4. L Maliskova
    5. M Song
    6. Y Shen
    7. B Huang
    8. S Lomvardas
    9. OD Weiner
    (2019)
    ID GSE127901. 4C on Sox2 Locus with tetO/cuO Modifications.
  2. 2
    Zenodo
    1. JM Alexander
    2. J Guan
    3. B Li
    4. L Maliskova
    5. M Song
    6. Y Shen
    7. B Huang
    8. S Lomvardas
    9. OD Weiner
    (2019)
    Live-Cell Imaging Reveals Enhancer-dependent Sox2 Transcription in the Absence of Enhancer Proximity.
    https://doi.org/10.5281/zenodo.2658814
The following previously published data sets were used
  1. 1
    NCBI Gene Expression Omnibus
    1. JA Wamstad
    2. JM Alexander
    3. RM Truty
    4. A Shrikumar
    5. F Li
    6. KE Ellertson
    7. H Ding
    8. JN Wylie
    9. AR Pico
    10. JA Capra
    11. G Erwin
    12. SJ Kattman
    13. GM Keller
    14. D Srivastava
    15. SS Levine
    16. KS Pollard
    17. AK Holloway
    18. LA Boyer
    19. BG Bruneau
    (2013)
    ID GSE47949. ChIP-seq analysis of histone modifications and RNA polymerase II at 4 stages of directed cardiac differentiation of mouse embryonic stem cells.
  2. 2
  3. 3
  4. 4
  5. 5
    NCBI Gene Expression Omnibus
    1. B Bonev
    2. Cohen N Mendelson
    3. Q Szabo
    4. L Fritsch
    5. G Papadopoulos
    6. Y Lubling
    7. X Xu
    8. X Lv
    9. J Hugnot
    10. A Tanay
    11. G Cavalli
    (2017)
    ID GSE96107. Multi-scale 3D genome rewiring during mouse neural development.
  6. 6
    NCBI Gene Expression Omnibus
    1. MP Creyghton
    2. AW Cheng
    3. GG Welstead
    4. T Kooistra
    5. BW Carey
    6. EJ Steine
    7. J Hanna
    8. MA Lodato
    9. GM Frampton
    10. PA Sharp
    11. LA Boyer
    12. RA Young
    13. R Jaenisch
    (2010)
    ID GSE24164. ChIP-Seq of chromatin marks at distal enhancers in Mouse Embryonic Stem Cells and adult tissues.
  7. 7
    NCBI Gene Expression Omnibus
    1. Y Zhang
    2. CH Wong
    3. RY Bimbaum
    4. G Li
    5. R Favaro
    6. CY Ngan
    7. J Lim
    8. E Tai
    9. HM Poh
    10. E Wong
    11. FH Mulawadi
    12. WK Sung
    13. S Nicolis
    14. N Ahituv
    15. Y Ruan
    16. CL Wei
    (2013)
    ID GSE44067. Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations.
  8. 8
    NCBI Gene Expression Omnibus
    1. AS Hansen
    2. I Pustova
    3. C Cattolico
    4. R Tjian
    5. X Darzacq
    (2017)
    ID GSE90994. CTCF and cohesion regulate chromatin loop stability with distinct dynamics.

Additional files

Supplementary file 1

Protocol for insert of cuO-/tetO-arrays into mouse ESCs.

Protocols for targeting the cuO and/or tetO array(s) into genomic regions of interest in mouse ESCs.

https://doi.org/10.7554/eLife.41769.024
Supplementary file 2

Primer sequences used in cell line characterization.

List of PCR primer sequences and expected amplicon size used in the study. Brief description of the purpose of each primer pair is included.

https://doi.org/10.7554/eLife.41769.025
Supplementary file 3

20 bp guide RNA sequences used in CRISPR/Cas9 genome engineering.

List of 20 bp sequences homologous to the mouse 129 genome designed into CRISPR/Cas9 sgRNAs. Targeted genomic location (mm9 coordinates), genome strand, and brief description of purpose for sgRNA is included.

https://doi.org/10.7554/eLife.41769.026
Supplementary file 4

Data table from 3D tracking of cuO/CymR and tetO/TetR labels.

All data used in the study for cuO/CymR and tetO/TetR localization. C1 refers to Channel 1 (cuO/CymR). C2 refers to Channel2 (tetO/TetR). For examples of raw and denoised data files that were used for this analysis, see doi: 10.5281/zenodo.2658814https://zenodo.org/record/2658814#.XNDLAhNKjyw. Columns are as follows:

Cell_Line– label used to identify cell line

Batch– unique microscopy session identifier

C1_T_Step-sec– step size between frames

Locus_ID– unique identifier for each Sox2 locus

C1_TrackID– track identifier from TrackMate

C1_Track_Length– track length from TrackMate

C1_SpotID– spot identifier from TrackMate

C1_X_Value_pixel – X position in pixels for C1 spot

C1_Y_Value_pixel – Y position in pixels for C1 spot

C1_Z_Value_slice – Z position in slices for C1 spot

C1_T_Value_frame – frame of measurement

C1_X_Value_um – X position in microns for C1 spot

C1_Y_Value_um – Y position in microns for C1 spot

C1_Z_Value_um – Z position in microns for C1 spot

C1_T_Value_sec – time point in seconds for measurement

C2_TrackID– track identifier from TrackMate

C2_Track_Length– track length from TrackMate

C2_SpotID– spot identifier from TrackMate

C2_X_Value_pixel – X position in pixels for C2 spot

C2_Y_Value_pixel – Y position in pixels for C2 spot

C2_Z_Value_slice – Z position in slices for C2 spot

C2_T_Value_frame – imaging frame

C2_X_Value_um – X position in microns for C2 spot

C2_Y_Value_um – Y position in microns for C2 spot

C2_Z_Value_um – Z position in microns for C2 spot

C2_T_Value_sec – time point in seconds

X_Distance_um– X distance between C1 and C2 labels

Y_Distance_um– Y distance between C1 and C2 labels

Z_Distance_um– Z distance between C1 and C2 labels

XY_Distance_um– XY distance between C1 and C2 labels

XYZ_Distance_um–XYZ distance between C1 and C2 labels,

C1_Corrected_X_Value_um – X position in microns for C1 spot after correcting for chromatic aberration,

C1_Corrected Y_Value_um–Y positfion in microns for C1 spot after correcting for chromatic aberration

C1_Corrected Z_Value_um–Z position in microns for C1 spot after correcting for chromatic aberration

Corrected_X_Distance_um–X distance after correcting for chromatic aberration

Corrected_Y_Distance_um – Y distance after correcting for chromatic aberration

Corrected_Z_Distance_um – Z distance after correcting for chromatic aberration

Corrected_XY_Distance_um – XY distance after correcting for chromatic aberration

Corrected_XYZ_Distance_um – XYZ distance after correcting for chromatic aberration

Relative_C1_Corrected_X_Value_um–X position of C1 label relative to the position of C2 in microns

Relative_C1_Corrected_Y_Value_um–Y position of C1 label relative to the position of C2 in microns

Relative_C1_Corrected_Z_Value_um–Z position of C1 label relative to the position of C2 in microns

Relative_XY_Displacement_um–Relative XY distance traveled by the C1 label between adjacent frames

Relative_XYZ_Displacement_um–Relative XYZ distance traveled by the C1 label between adjacent frames

Relative_XY_Angle_radians–Relative angle between two successive displacements for the C1 label in the XY plane

https://doi.org/10.7554/eLife.41769.027
Supplementary file 5

Data table for MS2 transcription analysis for all loci.

All data used in transcriptional analysis of Sox2 locus. Columns are as follows:

Cell_Line– label used to identify cell line

Locus_ID– unique identifier for each Sox2 locus

Gauss_Filter– whether the MS2 Gaussian fit passed the knn filter step

Noise_Filter–whether the MS2 Gaussian fit passed a high frequency noise filter step

Pass_Filter–whether the MS2 signal for the given frame was classified as transcriptional signal. Required both Gauss_Filter = TRUE and Noise_Filter = TRUE

Gaussian_Height_Threshold–minimum relative height above background allowed for Gaussian fit

Gaussian_Width_Threshold–maximum Gaussian variance allowed for Gaussian fit

Background–Offset calculated from Gaussian fit. If no Gaussian fit was found, set to median pixel intensity of ROI

Gaussian Height–Amplitude calculated from Gaussian fit. If no Gaussian fit was found, set to 0

Gaussian_Volume–Volume under fitted Gaussian. If no Gaussian fit was found, set to 0

Local_Median–Median pixel intensity of ROI

Norm_MS2_Signal–Relative height of MS2 gaussian normalized to background. For frames that did not pass filter, local median value was used in pace of gaussian height. See MATERIALS and METHODS for more details.

R_Squared–Coefficient of determination between 2D gaussian fit and experimental data

T_Value_frame– imaging frame

X_Value_pixel– X position in pixels for C2 spot (cuO/CymR)

X_Location– X position of peak of fit Gaussian

X_Sigma– X dimension variance of fit Gaussian

Y_Value_pixel– Y position in pixels for C2 spot (cuO/CymR)

Y_Location– Y position of peak of fit Gaussian

Y_Sigma– Y dimension variance of fit Gaussian

Z_Value_slice– Z position in slices for C2 spot (cuO/CymR)

Batch– unique microscopy session identifier.

https://doi.org/10.7554/eLife.41769.028
Supplementary file 6

Data table for MS2 transcription analysis and 3D localization for Sox2-SCR Singlets.

Data used to compare transcriptional activity of Sox2 locus to 3D distances between Sox2 and SCR. C1 refers to Channel 1 (tetO/TetR). C2 refers to Channel2 (cuO/CymR). Columns are as in Supplementary files 3 and 4 with one additional column: Active_Transcribing– Whether the locus demonstrated any MS2 signal that passed filter during imaging session.

https://doi.org/10.7554/eLife.41769.029
Supplementary file 7

Data table of atatistical comparison of distances centered on transcriptional bursts.

Summary statistics and associated Mann-Whitney p-values for pairwise comparisons between burst centered and random centered distances.

https://doi.org/10.7554/eLife.41769.030
Transparent reporting form
https://doi.org/10.7554/eLife.41769.031

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