Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle
- Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland
Figures
Figure 1 with 3 supplements
Interplay between OCT4 and SOX2 in regulating ES cell chromatin accessibility.
(A) Experimental strategy to compare the effect of OCT4 and SOX2 depletion on chromatin accessibility. (B) Number of regions significantly changed in accessibility upon OCT4 (left) and SOX2 (right) …
Figure 1—figure supplement 1
Immunofluorescence analysis of OCT4 OFF and SOX2 OFF cell lines and comparison of ATAC-seq changes between culture conditions and treatment times.
(A) Immunofluorescence of 2TS22C cells stained for DNA (DAPI), OCT4, and SOX2 without dox treatment (left), and after 26 hr (middle), and 40 hr (right) of dox treatment. (B) Violin plot of …
Figure 1—figure supplement 2
Heatmaps of ATAC-seq and ChIP-seq profiles in OCT4 OFF and SOX2 OFF cell lines at affected loci.
Heatmaps of RPKM-normalized ATAC-seq and ChIP-seq binding profiles upon OCT4 (A) and SOX2 (B) depletion 5 kb around OCT4-regulated (A) and SOX2-regulated (B) loci. Each row represents one individual …
Figure 1—figure supplement 3
Classification of OCT4/SOX2 binding sites.
(A) Classification of all OCT4 and SOX2 binding sites into OD, CD, and SD loci as well as loci that were discarded due to differences in untreated cells between conditions or cell lines (Discarded), …
Figure 2 with 2 supplements
Characterization of OCT4/SOX2-dependent loci.
(A) Heatmap of RPKM-normalized OCT4 and SOX2 ChIP-seq binding profiles in untreated ZHBTc4 cells 5 kb around OD, CD, and SD loci. Each row represents one individual locus. (B–C) Average …
Figure 2—figure supplement 1
Additional characterization of OD, CD, and SD loci.
(A) Average ATAC-seq signal 2 kb around OD, CD, and SD loci in BRG1fl cells that were treated with tamoxifen (TAM) or left untreated. (B) Frequency of overlap (bar) and enrichment p-values (white …
Figure 2—figure supplement 2
Additional analyses of accessibility and binding changes upon SOX2 and OCT4 depletion.
(A) Correlation between log2 fold-change values of accessibility (x-axis) and OCT4 binding (y-axis) upon SOX2 depletion in 2TS22C cells with dox treatment for 26 hr. Coefficient (R) and p-value are …
Figure 3 with 4 supplements
Mitotic degradation of OCT4 results in different patterns of accessibility loss.
(A) Experimental strategy used to assess the impact of OCT4 depletion at the M-G1 transition. (B–C) Genome browser tracks of RPKM-normalized accessibility profiles across the cell cycle for one …
Figure 3—figure supplement 1
Characterization of MD-OCT4 and MD*-OCT4 cell lines.
(A) Gate used to sort SNAP-MD-OCT4 (left) and SNAP-MD*-OCT4 (right) cells for the same average SNAP-Cell 647-SiR signal. Y-axis: Signal amplitude at 405 nm excitation and 526/52 nm emission …
Figure 3—figure supplement 2
Analyses of distance to closest gene and histone modifications in clusters.
(A) Violin plot of distance to closest TSS in the clusters from Figure 3D. Dots: mean; Vertical lines: standard deviation; p-values: Mann-Whitney U. (B) Heatmap of ChIP-seq signal of H3K4me3, …
Figure 3—figure supplement 3
Additional analyses of clusters.
(A) Percentage of the closest gene in the clusters from Figure 3D and at non-OCT4 bound accessible regions whose nascent RNA levels are downregulated or upregulated upon 24 hr of OCT4 depletion. p-va…
Figure 3—figure supplement 4
Correlation between OCT4 binding and chromatin accessiblity, and analysis of results from random forest model.
(A) Correlation between the log of normalized OCT4 ChIP-seq reads per bp (x-axis) and the log2 fold-change values of accessibility loss upon OCT4 depletion (y-axis) at all OCT4 binding sites in …
Figure 4 with 2 supplements
Auxin-inducible degradation reveals pioneer activity of OCT4 at different cell cycle phases.
(A) Experimental strategy used to assess the impact of OCT4 depletion and recovery at different cell cycle phases. (B) Red fluorescence (mCherry) signal in mCherry-OCT4-AID cells treated with IAA at …
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Figure 4—source data 1
Time-lapse microscopy source data of mCherry-OCT4-AID signal after IAA treatment (Figure 4B) and washout (Figure 4C).
Time is in hours; Signal is the background-subtracted mean intensity; Cell is the cell unique identifier of each tracked cell; Treatment indicates IAA or Washout just prior to imaging.
- https://cdn.elifesciences.org/articles/50087/elife-50087-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Characterization of mCherry-OCT4-AID cell line.
(A) Ratio of the number of colonies with and without dox treatment, for wt ZHBTc4 cells (Control) and ZHBTc4 cells expressing mCherry-OCT4-AID after one week in culture. n = 3 biological replicates; …
Figure 4—figure supplement 2
Additional data on accessibility changes upon rapid IAA-mediated OCT4 depletion.
(A) Genome browser tracks of accessibility profiles of a cluster 1 locus at chr10:95455826–95456819 after IAA treatment. (B) Heatmap of RPKM-normalized ATAC-seq signal 2 kb around regions in the …
Figure 5 with 1 supplement
Time course analysis of chromatin accessibility changes during OCT4 degradation reveals its highly dynamic pioneer activity.
(A) log2 fold-change values of accessibility compared to untreated cells in the four clusters from Figure 3D at different time points of IAA treatment. (B) Genome browser tracks of accessibility …
Figure 5—figure supplement 1
ATAC-seq data from different read sizes and RT-qPCR analysis upon rapid OCT4 depletion.
(A) Violin plot of normalized ATAC-seq signal of sequencing reads 0–100 bp and 180–250 bp in length in untreated cells and 2 hr IAA-treated cells in cluster 1 (A), cluster 2 (B), cluster 3 (C), and …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | ZHBTc4 ES cells | (Niwa et al., 2000) | RRID: CVCL_C715 | OCT4 OFF cell line |
| Cell line (Mus musculus) | 2TS22C ES cells | (Masui et al., 2007) | RRID: CVCL_E266 | SOX2 OFF cell line |
| Antibody | Rabbit anti-BRG1 | Abcam #ab110641 | RRID: AB_10861578 | ChIP (5 μg per 10 million cells) |
| Antibody | Rabbit anti-OCT4 monoclonal | Cell Signaling Technology | RRID: AB_10547892 | ChIP (20 μl per 10 million cells) |
| Antibody | Rabbit anti-H3K27ac polyclonal | Abcam #ab4729 | RRID: AB_2118291 | ChIP 2 μg/25 μg chromatin) |
| Antibody | Mouse anti-OCT4 monoclonal | Santa Cruz #sc-5279 | RRID: AB_628051 | IF (1:500) |
| Antibody | Rabbit anti-SOX2 polyclonal | Thermo Fisher #48–1400 | RRID: AB_2533841 | IF (1:200) |
| Recombinant DNA reagent | psPAX2 | Addgene | RRID: Addgene_12260 | For lentiviral production |
| Recombinant DNA reagent | pMD2.G | Addgene | RRID: Addgene_12259 | For lentiviral production |
| Recombinant DNA reagent | pLV-PGK-YPet-MD | (Deluz et al., 2016) | ||
| Recombinant DNA reagent | pLV-PGK-SNAP-MD-OCT4 | This paper | Used to generate lentiviral particles for the MD-OCT4 cell line (see Figure 3A). Available upon request | |
| Recombinant DNA reagent | pLV-PGK-SNAP-MD*-OCT4 | This paper | Used to generate lentiviral particles for the MD*-OCT4 cell line (see Figure 3A). Available upon request | |
| Recombinant DNA reagent | pLEX-mCherry-OCT4-AID | This paper | Used to generate lentiviral particles for the OCT4-AID cell line (see Figure 4A). Available upon request | |
| Software, algorithm | FiJi | (Schindelin et al., 2012) | RRID:SCR_002285 | Version 2.0.0-rc-69/1.52 p |
| Software, algorithm | CellProfiler | (Carpenter et al., 2006) | RRID:SCR_007358 | Version 3.1.8 |
| Software, algorithm | STAR | (Dobin et al., 2013) | RRID:SCR_015899 | Version 2.6.1 c |
| Software, algorithm | Picard | Broad Institute | RRID:SCR_006525 | Version 2.8.3 |
| Software, algorithm | MACS | (Zhang et al., 2008) | RRID:SCR_013291 | Version 2.1.1.20160309 |
| Software, algorithm | BEDTools | (Quinlan and Hall, 2010) | RRID:SCR_006646 | Version 2.26.0 |
| Software, algorithm | edgeR | (Robinson et al., 2010) | RRID:SCR_012802 | Version 3.18.1 |
| Software, algorithm | limma | (Ritchie et al., 2015) | RRID:SCR_010943 | Version 3.32.10 |
| Software, algorithm | HOMER | (Heinz et al., 2010) | RRID:SCR_010881 | Version 4.10.4 |
| Software, algorithm | biomaRt | (Durinck et al., 2005) | RRID:SCR_002987 | Version 2.32.1 |
| Software, algorithm | deepTools | (Ramírez et al., 2016) | RRID:SCR_016366 | Version 3.2.0 |
| Software, algorithm | SAMTools | (Li et al., 2009) | RRID:SCR_002105 | Version 1.8 |
| Software, algorithm | GenomicRanges | (Lawrence et al., 2013) | RRID:SCR_000025 | Version 1.28.6 |
| Software, algorithm | RStudio | RRID:SCR_000432 | Version 1.0.153 | |
| Software, algorithm | ggplot2 | (Wickham, 2009) | RRID:SCR_014601 | Version 3.2.0 |
Additional files
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Supplementary file 1
Additional statistics.
This file contains p-values and fold change values for all relevant comparisons in Figure 1D–G, Figure 2B,C,J,K, Figure 2—figure supplement 1A,C,D,F,H, Figure 2—figure supplement 2B–G, Figure 3F, Figure 3—figure supplement 2C, Figure 3—figure supplement 3D, Figure 3—figure supplement 4D–E, Figure 4D–E, and Figure 5—figure supplement 1F-G.
- https://cdn.elifesciences.org/articles/50087/elife-50087-supp1-v1.xlsx
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Supplementary file 2
Motif analysis.
This file contains enrichment values (logP) and frequencies of known motifs from HOMER in the following groups of loci: OD, CD, SD, OCT4 OFF upregulated (OCT4up), SOX2 OFF upregulated (SOX2up), and clusters 1–4. Only motifs with -logP < 50 in at least one group are shown.
- https://cdn.elifesciences.org/articles/50087/elife-50087-supp2-v1.xlsx
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Supplementary file 3
Random forest model results.
This file contains the top 500 features of the random forest model used to predict the cluster of regions based on overlap with ChIP-seq peaks from cistromeDB annotated as belonging to mouse ES cells.
Importance values are derived from the model. Sample name, Factor, Cell line, and GSMID refer to sample data in GEO and ID refers to the sample ID in cistromeDB. Cluster 1–4 columns indicate fraction of regions overlapping the sample peaks.
- https://cdn.elifesciences.org/articles/50087/elife-50087-supp3-v1.xlsx
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Supplementary file 4
Primers used for RT-qPCR.
This file contains the oligonucleotide sequences used to perform RT-qPCR experiments.
- https://cdn.elifesciences.org/articles/50087/elife-50087-supp4-v1.xlsx
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Supplementary file 5
Published datasets used.
This file contains descriptions of publicly available raw data that were aligned and processed according to the Materials and methods section as well as publicly available pre-processed data used in the study.
- https://cdn.elifesciences.org/articles/50087/elife-50087-supp5-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50087/elife-50087-transrepform-v1.docx
