Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development
- University of Cambridge, United Kingdom
Figures
Figure 1
G9a-dependent programming occurs at implantation.
(A) Schematic of early mouse development and their in vitro equivalents. Genome-wide DNA demethylation after fertilisation leads to an epigenetic basal state with low 5meC in ICM of blastocysts. …
Figure 2 with 3 supplements
G9a represses germline and proliferation-related genes in the postimplantation epiblast.
(A,B) bright-field images of Ehmt2+/+and Ehmt2−/− embryos at E7.5 (A) (scale bar = 0.1 mm). At least nine embryos of each type were staged (B) (*Chi2 test p-value= <0.05). (C) Scatter plot showing …
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Figure 2—source data 1
List of differentially expressed genes in E6.25 Ehmt2−/− epiblast from RNA-seq analysis data is based on four individual Ehmt2−/− and control (Ehmt2+/+ or Ehmt2+/−) epiblasts.
Differentially expressed genes were identified using a minimum Log2(FC)>1.4 and maximum Fisher combined test of p-value<0.05. In the upregulated sample, expression was 1<log (RPKM). RNA-seq: RNA sequencing; RPKM: Reads Per Kilobase of transcript per Million mapped reads; FC: fold change.
- https://doi.org/10.7554/eLife.09571.005
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Figure 2—source data 2
List of enriched GO terms in genes upregulated in E6.25 Ehmt2−/− epiblast GO term enrichment for biological processes was calculated using DAVID software with minimum five genes in a category and EASE p-value<0.05.
EASE: Expression Analysis Systematic Explorer; GO: gene ontology.
- https://doi.org/10.7554/eLife.09571.006
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Figure 2—source data 3
List of differentially expressed genes in E6.25 Ezh2−/−epiblast from RNA-seq analysis data is based on 3 individual Ezh2−/− epiblasts and 3 individual control (Ezh2+/+ or Ezh2+/−) epiblasts.
Differentially expressed genes were identified using a minimum Log2(FC)>1.4 and maximum Fisher combined test of p-value<0.05. In the upregulated sample, expression was 1<log(RPKM). RNA-seq: RNA sequencing; RPKM: Reads Per Kilobase of transcript per Million mapped reads; FC: fold change.
- https://doi.org/10.7554/eLife.09571.007
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Figure 2—source data 4
List of enriched GO terms in genes upregulated in E6.25 Ezh2−/− epiblast GO term enrichment for biological processes was calculated using DAVID software with minimum five genes in a category and EASE p-value<0.05.
EASE: Expression Analysis Systematic Explorer; GO: gene ontology.
- https://doi.org/10.7554/eLife.09571.008
Figure 2—figure supplement 1
Epigenetic programming by G9a in postimplantation epiblast.
(A) Cryosection IF of E6.25 Ehmt2+/+ and Ehmt2−/− embryos stained with antibodies raised against G9a and H3K9me2. Scale bar = 20 μm. (B) Top 10 enriched GO terms in genes upregulated in Ehmt2−/− E6.2…
Figure 2—figure supplement 2
Loss of G9a does not affect the exit from pluripotency and germline specification.
(A,B) Whole-mount IF of E6.5 GGOF Ehmt2+/+ and Ehmt2−/− embryos stained with antibodies raised against Nanog (A), GFP and G9a (B). Shown is a sum of an equal number of z-stacks. Scale bar 20 = μm. (C…
Figure 2—figure supplement 3
EZH2 represses multiple developmental regulators in vivo.
(A) Scatter plot showing transcript expression levels in Ezh2−/− versus Ezh2cntr E6.25 epiblast. Red points are differentially expressed genes (Log2RPKM>1, p-value<0.05, Log2(FC)>1.4). Shown is …
Figure 3 with 3 supplements
In vivo lcChip-seq from E6.25 epiblast reveals distinct epigenetic state of primed pluripotent cells.
(A) Density contour plot showing the relationship between H3K9me2 and H3K27me3 enrichment. Shown are all promoters associated with genes repressed in the epiblast. (B) Density contour plots showing …
Figure 3—figure supplement 1
LcChIP-seq on E6.25 epiblast.
(A) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 106 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold …
Figure 3—figure supplement 2
H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states.
(A) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. (B) Density contour plots showing correlation between levels of H3K9me2 (blue) …
Figure 3—figure supplement 3
Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content.
(A) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 …
Figure 4 with 1 supplement
EpiSC show aberrant H3K27me3 distribution and DNA hypermethylation at germline genes.
(A,D) Distribution of H3K9me2 (A) and H3K27me3 (D) by metagene analysis in epiblast and EpiSCs. Genes were classified based on promoter CpG density. (Scale = base pairs.) (B,E) Scatter plots showing …
Figure 4—figure supplement 1
H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC.
(A) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with …
Figure 5 with 2 supplements
H3K9me2 and H3K27me3 are directly involved in repression of genes and transposable elements.
(A) Venn diagrams showing overlap between H3K27me3 enrichment at promoters (left panel) or H3K9me2 at gene bodies (right panel); genes upregulated in Ezh2−/− and Ehmt2−/− epiblasts are shown. The …
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Figure 5—source data 1
List of promoters enriched for H3K27me3 in lcChIP-seq from E6.25 epiblast data is based on two biological replicates of lcChIP-seq.
Enrichment was called by k-means clustering.
- https://doi.org/10.7554/eLife.09571.019
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Figure 5—source data 2
List of gene bodies enriched for H3K9me2 in lcChIP-seq from E6.25 epiblast Data is based on two biological replicates of lcChIP-seq.
Enrichment was called by k-means clustering.
- https://doi.org/10.7554/eLife.09571.020
Figure 5—figure supplement 1
H3K9me2 accumulates at G9a-regulated promoters in the epiblast.
(A) Venn diagrams showing overlap between H3K9me2 enrichment at promoters and genes upregulated in Ehmt2−/− epiblasts. The overlaps are statistically significant (p-value<0.01 using Chi2 test). (B) …
Figure 5—figure supplement 2
H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast.
(A) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. (B) Scatter plot …
Figure 6 with 2 supplements
H3K9me2 marks enhancers undergoing inactivation during exit from naïve pluripotency.
(A) Genome browser tracks showing p300, H3K27ac, and H3K4me1 at inactive Esrrb and Prdm1 putative enhancers (black boxes) in day 2 EpiLCs (GSE56138) (Buecker et al., 2014). Bottom two tracks show …
Figure 6—figure supplement 1
Genome-wide accumulation of H3K9me2 extends to multiple enhancer elements.
(A) Bar plot showing H3K9me2 levels in ESC, d2 EpiLC and EpiSC measured by quantifying total DNA in anti-H3K9me2 ChIP relative to the input sample. Shown is mean (± SD) from at least two independent …
Figure 6—figure supplement 2
H3K9me2 marks a distinct set of enhancers.
(A) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or …
Figure 7 with 1 supplement
H3K9me2 spreading to enhancers results in transient coenrichment with H3K27ac.
(A) Density contour plots showing correlation between H3K27ac enrichments at enhancers in 2i/LIF ESCs versus EpiLC (left panels) or 2i/LIF ESCs versus EpiSC (right panels). Green panels show all …
Figure 7—figure supplement 1
H3K9me2-marked enhancers retain some H3K27ac in EpiLCs.
Box plots showing H3K27ac levels at enhancers in 2i/LIF ESC, EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; …
Figure 8 with 1 supplement
G9a promotes transcriptional inactivation of enhancers.
(A,B) Box plots showing transcript levels of genes in epiblast (A) and EpiLCs (B), which lie in proximity of H3K9me2- or H3K27me3-marked enhancers. All comparisons are statistically significant (p<0.…
Figure 8—figure supplement 1
H3K9me2 enriched enhancers are preferentially linked to the p53 pathway.
(A) Selected enriched GO terms in H3K9me2-marked enhancers. (B,C) Bar plots showing top 20 enriched motifs in H3K9me2- (B) or H3K27me3- (C) marked enhancers. Source data file legends
Figure 9
Proposed model for G9a-mediated enhancer inactivation.
Distal regulatory elements active in ICM are typically associated with H3K4me1 and H3K27ac enrichment (green) as well as eRNA expression. Such elements activate promoters (purple) to increase gene …
Author response image 1
Western Blot of d2 G9aF/- CreER+ve EpiLCs treated with ethanol (EtOH) or tamoxifen (TAM).
Membranes were probed with anti-G9a, anti-phopho-p53 and anti-aTubuline antibodies.
Author response image 2
Bar plot showing changes in expression levels of genes nearest to analysed enhancers in G9acntrand G9a-/- E6.25 epiblast.
Data was extracted from RNAseq experiment. Shown are average Log2(Fold change) (+/- SEM). * pvalue<0.05 using Wilcoxon rank-sum test
Author response image 3
Plot showing background (expected) and observed number of transcripts reaching specific fold change (FC) deregulation thresholds.
Expected values were estimated based on variability of gene expression between G9acntr or Ezh2cntr RNAseq samples. Observed dataset are genes reaching FC threshold in G9a-/- or Ezh2-/- RNAseq …
Author response image 4
Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast.
https://doi.org/10.7554/eLife.09571.035
Author response image 5
Bar plot showing DNAseI hypersensitivity of selected enhancers in d2 G9aF/- CreER+ve EpiLCs treated with ethanol and tamoxifen.
Shown is average from two independent biological repeats (+/- StDev).
Procedure was performed as previously described (John et al., 2013)
Additional files
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Supplementary file 1
List of primers used in this study.
- https://doi.org/10.7554/eLife.09571.031
