Telomere dysfunction cooperates with epigenetic alterations to impair murine embryonic stem cell fate commitment
- Institut de Recherche en Immunologie et Cancérologie (IRIC), Département de biologie moléculaire, Faculté de Médecine, Université de Montréal, Canada
- Princess Margaret Cancer Centre, University Health Network, Canada
- Princess Margaret Genomics Centre, Princess Margaret Cancer Centre, University Health Network, Canada
- Structural Genomics Consortium, Princess Margaret Cancer Centre, University of Toronto, Department of Medical Biophysics, Canada
- Department of Medical Biophysics, University of Toronto, Canada
Figures
Figure 1 with 1 supplement
Murine ESCs with short telomeres fail to complete differentiation commitment and to suppress pluripotency gene expression.
(A) Schematic representation of the experimental design. Wild-type (WT) and Tert-/- mESCs transduced with Pou5f1 -GFP were maintained in a pluripotent state in the presence of leukemia inhibitory …
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Figure 1—source data 1
Murine ESCs with short telomeres fail to complete differentiation commitment and to suppress pluripotency gene expression.
Source data of (i) the GFP percentage values represented in the histogram in Figure 1B and Figure 1—figure supplement 1F–G; (ii) Raw Ct values and information relative to the Qiagen qPCR array relative to Figure 1C,D,E and Figure 1—figure supplement 1C,D; (iii) Raw telomere signal intensities presented in Figure A-figure supplement 1A-B.
- https://cdn.elifesciences.org/articles/47333/elife-47333-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Telomere status, gene expression, and gamma-irradiation response in WT and Tert-/- mESCs.
(A) Relative telomere length measurement by Quantitative Fluorescence In-situ Hybridization (Q-FISH) analysis of WT and Tert-/- mESCs at passage 50. Representative photo of metaphase preparations. …
Figure 2 with 1 supplement
Murine ESCs with short telomeres exhibit altered H3K27me3 levels and incomplete differentiation that is exacerbated by PRC2 inhibition.
(A) Western blot analysis of H3K27me3 levels in WT and Tert-/- mESCs, in the presence of active PRC2 inhibitors (GSK343, UNC1999 and A395), their corresponding controls (UNC2400 and A395N), or the …
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Figure 2—source data 1
Murine ESCs with short telomeres exhibit altered H3K27me3 levels and incomplete differentiation that is exacerbated by PRC2 inhibition.
Source data of (i) the GFP percentage values represented in the histogram in Figure 2B and Figure 2—figure supplement 1E; (ii) Fold change values presented in Figure 2—figure supplement 1B; (iii) Raw data from the western blot quantification presented in Figure 2—figure supplement 1D,F; (iv) Raw Ct values and information relative to the Qiagen qPCR array relative to Figure 2D and Figure 2—figure supplement 1G,H.
- https://cdn.elifesciences.org/articles/47333/elife-47333-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
The impact of PRC2 inhibitors on H3K27me3, gene expression, and differentiation of WT and Tert-/- mESCs.
(A) Western blot of H3K27me3 in pluripotent (maintained in LIF) or ATRA treated (6 DA) WT and Tert-/- mESCs. H3 was probed as a loading control. Representative blot from n = 3. A western blot of the …
Figure 3 with 1 supplement
Inhibition of Kdm6a/b histone demethyltransferase activity partially rescues cell fate commitment.
(A, C) Western blot analysis of H3K27me3 or Nanog protein levels in WT and Tert-/- mESCs, in the presence of DMSO only, GSKJ4 or GSKJ5. For clarity, no image cropping was performed between lanes and …
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Figure 3—source data 1
Inhibition of Kdm6a/b demethylase activity partially rescues cell fate commitment.
Source data of (i) the GFP percentage values represented in the histogram in Figure 3C and Figure 3—figure supplement 1L; (ii) Raw Ct values and information relative to the Qiagen qPCR array relative to Figure 3E,F and Figure 3—figure supplement 1B,D,E; (iii) Raw data from the western blot quantification presented in Figure 3—figure supplement 1A,C,J,K; (iv) Fold change values presented in Figure 3—figure supplement 1A,B; (v) Indel frequency as showed in Figure 3—figure supplement 1I–L.
- https://cdn.elifesciences.org/articles/47333/elife-47333-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
The impact of PRC2 or Kdm6a/b inhibition on H3K27me3, gene expression, and differentiation in WT and Tert-/- mESCs.
(A, B) Assessment of relative fold change in Kdm6b and Kdm6a expression (relative to WT 2 DL) by RT-qPCR, normalized to two housekeeping genes: B2m and Gapdh, Data are represented as mean + SD (n = 3…
Figure 4 with 1 supplement
Telomere dysfunction affects the chromatin accessibility landscape during mESC differentiation.
(A) Spearman correlation analysis of called peaks following ATAC-seq analysis of Tert-/- and WT mESCs assessed throughout differentiation, with or without the addition of PRC2 or Kdm6a/b inhibitors, …
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Figure 4—source data 1
Supplemental information for high throughput sequencing metadata related to ATAC-seq.
- https://cdn.elifesciences.org/articles/47333/elife-47333-fig4-data1-v2.xls
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Figure 4—source data 2
Supplemental Table 1 related to ATAC-seq data.
- https://cdn.elifesciences.org/articles/47333/elife-47333-fig4-data2-v2.xlsx
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Figure 4—source data 3
Supplemental Table 1 related to ChIP-seq data.
- https://cdn.elifesciences.org/articles/47333/elife-47333-fig4-data3-v2.xlsx
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Figure 4—source data 4
Supplemental information for high-throughput sequencing metadata related to ChIP-seq.
- https://cdn.elifesciences.org/articles/47333/elife-47333-fig4-data4-v2.xls
Figure 4—figure supplement 1
Chromatin accessibility, including at the Pou5f1 promoter, in WT and Tert-/- mESCs during differentiation.
(A, B) IGV screenshot of the genomic region surrounding the Pou5f1 promoter, derived from either ATAC-seq data (A) or H3K27me3 ChIP-seq data (B) as shown in Figure 4. One representative replicate …
Figure 5
The unstable differentiation of mESC with short telomeres is exacerbated by epigenetic remodelling.
(A) Schematic of the cell state of WT and Tert-/- ESCs upon differentiation induction, based on our data. The slope is intended only to illustrate hypothetical relative differences between cell …
Additional files
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Supplementary file 1
Key resources table.
Supplemental information about sequence-based reagents, cells lines, antibodies, chemical compounds, software, algorithms and commercial kits used in this study.
- https://cdn.elifesciences.org/articles/47333/elife-47333-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/47333/elife-47333-transrepform-v2.docx
