H3.3K27M mutant proteins reprogram epigenome by sequestering the PRC2 complex to poised enhancers
- Irving Cancer Research Center, Columbia University, United States
- Mayo Clinic, United States
Figures
Figure 1 with 1 supplement
H3.3K27M mutant proteins are enriched at highly transcribed genes compared to lowly expressed genes in DIPG cells and mouse ES cells with H3.3K27M mutation.
(A–C) H3.3K27M mutant proteins are enriched at highly transcribed genes compared to lowly expressed genes in DIPG cells. The average read density of H3.3K27M ChIP-seq in two H3.3K27M mutant lines …
Figure 1—figure supplement 1
Site-specific mutation at the H3F3A gene, resulting in expression of the H3.3K27M proteins in mouse ES cells does not change cell identity.
(A) Antibodies against H3K27M are specific for ChIP analysis. ChIP assays were performed in three cell lines (astrocyte, astrocytes expressing wild type histone H3.3 (H3.3WT) and H3.3K27M mutant …
Figure 2 with 1 supplement
Ezh2 is sequestered at poised enhancers with H3.3K27M mutant proteins.
(A) A large fraction of Ezh2 peaks unique in H3.3K27M mutant mouse ES cells was identified. The percentage of peaks (Y-axis) in each category (WT unique peaks, H3.3K27M unique peaks, and overlap …
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Figure 2—source data 1
The source data to plot the bar chart in Figure 2A.
- https://doi.org/10.7554/eLife.36696.006
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Figure 2—source data 2
The source data to plot the bar chart in Figure 2F.
- https://doi.org/10.7554/eLife.36696.007
Figure 2—figure supplement 1
A large number of Ezh2 peaks uniques in H3.3K27M mutant mouse ES cells is identified.
(A) Venn diagram illustration of H3K27me3 peaks in the wild type (WT) and two H3.3K27M mutant mouse ES clones and their overlap. The number of peaks identified in each cell line was shown in the …
Figure 3 with 2 supplements
H3.3K27M levels on chromatin are low at gene promoters with H3K27me3.
(A) Representative Integrative Genomics Viewer shows the distribution of H3K27me3 in two DIPG cancer cell lines (SF7761 and SF8628) and one DIPG primary tissue. RefSeq genes are shown at the bottom. …
Figure 3—figure supplement 1
A small fraction of H3K27me3 peaks shows an equal or higher level of H3K27me3 in DIPG tumor cells than the corresponding reference cells.
(A) The level of H3K27me3 is reduced dramatically in H3.3K27M cell line PED8 and in two distinct regions of a DIPG primary tissue. Primary tissues from a patient autopsy sample were separated into …
Figure 3—figure supplement 2
In silico identification of H3K27me3 peaks at tumor-initiating cells of SF8628 and SF7761.
The diagram shows how gene promoters with H3K27me3 were identified in tumor-initiating cells of SF8628 (A) and SF7761 (B). Based on the average level of H3K27me3 at these gene promoters, gene …
Figure 4 with 1 supplement
Several TSGs including WT1 are silenced through H3K27me3-mediated mechanism in DIPG cells with H3.3K27M mutation.
(A) Analysis of expression levels of 12 different tumor suppressor genes (TSGs) in different DIPG lines and human NSC. Expressions of 12 different TSGs in tumor lines, with the mutation status of …
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Figure 4—source data 1
The source data to plot the bar chart in Figure 4B.
- https://doi.org/10.7554/eLife.36696.013
Figure 4—figure supplement 1
H3K27me3 and Ezh2 are enriched at the promoters of TSGs in DIPG cells.
(A) A diagram shows how the candidate TSGs were identified. Combining TSGs listed in three datasets, 1,245 TSGs were identified. Based on H3K27me3 ChIP-seq in two DIPG cell lines (SF7761, SF8628) …
Figure 5 with 1 supplement
An increase in WT1 expression inhibits the proliferation of H3.3K27M mutant cells.
(A) Targeting p300 to the WT1 promoter results in an increase in H3K27ac and a reduction of H3K27me3. Top: a schematic strategy for targeting dCas9-HA-p300-HA fusion proteins to the promoter of WT1. …
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Figure 5—source data 1
The source data to plot the bar chart in Figure 5A.
- https://doi.org/10.7554/eLife.36696.016
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Figure 5—source data 2
The source data to plot the bar chart in Figure 5B.
- https://doi.org/10.7554/eLife.36696.017
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Figure 5—source data 3
The source data to plot the bar chart in Figure 5C.
- https://doi.org/10.7554/eLife.36696.018
Figure 5—figure supplement 1
Forced expression of WT1 inhibits the proliferation of DIPG cells with H3.3K27M mutation.
(A–B) Cell proliferations of two DIPG cell lines were inhibited by overexpression of different WT1 isoforms. The expression of different Flag-tagged WT1 isoforms in two DIPG lines (SF7761 and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H. sapiens) | SF7761 | PMID: 23603901 | RRID:CVCL_IT45 | Growth in neural stem cell media as neurosphere |
| Cell line (H. sapiens) | SF8628 | PMID: 25401693 | RRID:CVCL_IT46 | Growth in DMEM as monolayer |
| Cell line (H. sapiens) | KNS42 | PMID: 25401693 | RRID:CVCL_0378 | Growth in DMEM as monolayer |
| Cell line (H. sapiens) | SF9427 | PMID: 25401693 | Growth in DMEM as monolayer | |
| Cell line (H. sapiens) | PED8 | PMID: 26376656 | Growth in neural stem cell media as neurosphere | |
| Antibody | H3K27me3 | Cell Signaling | C36B11, RRID:AB_2616019 | 1.4 µg for ChIP, 1:1000 for Western blot |
| Antibody | Ezh2 | Cell Signaling | 5246, RRID:AB_10694683 | 5 µg for ChIP, 1:1000 for Western blot |
| Antibody | Suz12 | Cell Signaling | 3737S, RRID:AB_2196850 | 1:1000 for Western blot |
| Antibody | H3K27ac | Abcam | ab4729, RRID:AB_2118291 | 2 µg for ChIP, 1:1000 for Western blot |
| Antibody | H3K4me1 | Abcam | ab8895, RRID:AB_306847 | 2 µg for ChIP, 1:1000 for Western blot |
| Antibody | α-Tubulin | Sigma | T9026, RRID:AB_477593 | 1:1000 for Western blot |
| Antibody | FLAG | Sigma | F1804, RRID:AB_262044 | 2 µg for ChIP, 1:1000 for Western blot |
| Antibody | H3K27M | Millipore | ABE419, RRID:AB_2728728 | 2 µg for ChIP, 1:1000 for Western blot |
| Antibody | HA | This study | 12CA5 | 2 µg for ChIP, 1:1000 for Western blot |
| Antibody | H3 | PMID: 23603901 | 1:10000 for Western blot | |
| Commercial assay or kit | SuperScript III First-Strand kit | Invitrogen | 18080–051 | |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | 74104 | |
| Commercial assay or kit | QiaQuick PCR purification kit | Qiagen | 28104 | |
| Commercial assay or kit | ssoAdvanced Universal SYBR Green Supermix | Bio-Rad | 1725274 | |
| Commercial assay or kit | cell titer blue assay kit | Promega | G8081 | |
| Software, algorithm | Bowtie2 | PMID: 22388286 | RRID:SCR_005476 | |
| Software, algorithm | MACS2 | PMID: 18798982 | RRID:SCR_013291 | |
| Software, algorithm | Bedtools | PMID: 20110278 | RRID:SCR_006646 |
Additional files
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Supplementary file 1
Tables of cell lines, tissues and oligonucleotides used.
- https://doi.org/10.7554/eLife.36696.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.36696.020
