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. 2015 Nov 9;4:e09571. doi: 10.7554/eLife.09571

Figure 5. 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 overlaps are statistically significant (p-value< 0.01 using Chi2 test). (B) Genome browser tracks showing H3K9me2 and H3K27me3 enrichment at genes that are: derepressed in Ehmt2−/− (Asz1, Casp14, Cdkn1a), active epiblast markers (Otx2), and PRC2 targets (Hoxc10). Data is shown as a sliding window (1kb and 300bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM Input). (C) lcChIP-qPCR validation for H3K9me2 and H3K27me3 at the promoter of Asz1 and gene body of Cdkn1a during EpiLC and EpiSC induction. Signal was scaled relative to average enrichment on negative (Gapdh: H3K9me2 and H3K27me3) and positive control regions (Pcsk5: H3K9me2, Hoxc10: H3K27me3). Data are represented as mean (± SEM) from three independent biological replicates (*Student’s t-test p-value<0.05 relative to 2i/LIF ESC sample). (D) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ehmt2−/− E6.25 epiblast. Red points are subfamilies with significant H3K9me2 enrichment and increased proportion of upregulated loci. (E) Single cell RT-qPCR validation of repeat upregulation in individual E6.25 Ehmt2+/+ and Ehmt2−/−epiblast cells. Statistical significance was calculated using Wilcoxon rank sum test for IAP where the majority of WT and KO cells show detectable expression. For remaining repeats, a Chi2 test was used.(*p-value<0.05). (F) LcChIP-qPCR measuring H3K9me2 levels at selected repeat elements in Ehmt2F/− CreER+ve d2 EpiLCs treated with EtOH or TAM. Data are mean (± SD) from two independent biological replicates. (*Student’s t-test p-value<0.05 of EtOH compared with TAM treated sample). Also see Figure 5—figure supplement 1,2 and Figure 5—source data 1,2. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EZH2: Enhancer of zeste homolog 2; ChIP: chromatin immunoprecipitation; EpiLCs: epiblast-like cells; EpiSCs: epiblast stem cells; SEM: standard error of the mean; TAM: tamoxifen; EtOH: ethanol; IAP: intracisternal A particle.

DOI: http://dx.doi.org/10.7554/eLife.09571.018

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.
DOI: http://dx.doi.org/10.7554/eLife.09571.019
elife-09571-fig5-data1.xls (1.5MB, xls)
DOI: 10.7554/eLife.09571.019
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.
DOI: http://dx.doi.org/10.7554/eLife.09571.020
elife-09571-fig5-data2.xls (4.4MB, xls)
DOI: 10.7554/eLife.09571.020

Figure 5.

Figure 5—figure supplement 1. H3K9me2 accumulates at G9a-regulated promoters in the epiblast.

Figure 5—figure supplement 1.

(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) Heatmap showing H3K9me2 and H3K27me3 enrichment at promoters of genes upregulated in Ehmt2−/−. Data shown is from two biological replicates of lcChIP-qPCR from 2i/LIF ESCs and day 1–3 EpiLCs. Scale is in relative enrichment normaliseormalized to a positive control region of Pcsk5 (for H3K9me2) and Hoxc10 (for H3K27me3).
DOI: http://dx.doi.org/10.7554/eLife.09571.021
Figure 5—figure supplement 2. H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast.

Figure 5—figure supplement 2.

(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 showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2−/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. (C,D) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 (C) or H3K27me3 (D) and upregulated in Ehmt2−/− (C) or Ezh2−/−(D) E6.25 epiblast. (E) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. (F) Scatter plots FC in expression levels of unique loci in Ehmt2−/− versus Ehmt2+/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated.
DOI: http://dx.doi.org/10.7554/eLife.09571.022