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1.
Figure 7

Figure 7. From: An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

De-novo genomic sites of H3K27me3 methylation in Dnmt3a/b−/− ES cells. Genome-wide maps of H3K27me3 were generated for Dnmt3a/b−/− and wild-type ES (E14-TG2a) cell lines. For each locus, the profile of H3K27me3 (reads/10 million) is displayed for wild-type and mutant cells and compared with CpG density (CpGs/500 bp). (A) Positive control region (Hox A locus). (B) Negative control region (Hbb locus). (CJ) Examples of de-novo PRC2 recruitment at intragenic CpG islands that are methylated in wild-type cells and become demethylated in mutant cells. The locations of bisulphite amplicons displayed in Supplementary Figure S6 are indicated.

Magnus D Lynch, et al. EMBO J. 2012 January 18;31(2):317-329.
2.
Figure 4

Figure 4. From: An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

Chromatin bivalency at the HBA2 gene reflects competition between Polycomb recruitment and transcriptional activation. (A) Fragments tested: (1) HBA2 gene with promoter deletion (Fragment IV in Figure 3). (2) Intact HBA2 gene (Fragment I in Figure 3). (3) Intact HBA2 gene with an ∼200-bp fragment encoding the MC1 promoter inserted upstream of the full 4-kb HBA2 fragment with orientation towards the gene. (B) Expression of spliced HBA2 RNA was quantified with a species-specific qPCR probe. Expression is quantified relative to mouse Gapdh. For each fragment tested, data are shown for three separately derived and analysed cell lines ±s.d. (C) ChIP was performed with antibodies to H3K4me3, Ezh2 and H3K27me3. qPCR amplicons present in all of the tested fragments are indicated (green). Enrichment was quantified as percentage of input DNA. Data are shown for three separately derived and analysed cell lines ±s.d.

Magnus D Lynch, et al. EMBO J. 2012 January 18;31(2):317-329.
3.
Figure 6

Figure 6. From: An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

DNA methylation prevents the PcG-associated H3K27me3 histone modification in pluripotent cells. (A) Chromatin state (Ernst et al, 2011) and DNA methylation (Lister et al, 2009) at the human RHBDF1 gene in human ES cells and erythroid cell types. ChIP-seq data are normalized to mapped reads per 10 million. The same scales are employed for ES and erythroid cell types. (B, C) For human H1 ES cells, DNA methylation was quantified at all CpG islands in the human genome (Lister et al, 2009) and compared with density of either (B) H3K4me3 or (C) H3K27me3 histone modifications at the corresponding CpG islands (Ernst et al, 2011). (D, E) H3K27me3 read density is plotted against H3K4me3 read density for (D) all CpG islands and (E) CpG islands with >80% methylated CpG dinucleotides. Read density is displayed as reads per 10 million per 500 bp window.

Magnus D Lynch, et al. EMBO J. 2012 January 18;31(2):317-329.
4.
Figure 3

Figure 3. From: An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

Chromatin state is redundantly encoded in the 4-kb HBA2 fragment. (A) The indicated subfragments were separately integrated into the mouse α globin locus using the RMCE system. The positions of tested subfragments relative to the original 4-kb HBA2 fragment are shown. (B) Following Flp-mediated excision of the Hprt selective marker gene, ChIP was performed with antibodies to Ezh2, Cbx7, H3K4me3 and H3K27me3 and enrichment was quantified with species-specific qPCR probes. qPCR probes 1–3 are located within the endogenous mouse α globin locus as indicated in Figure 1. Other probe positions are identical to Figure 2. Enrichment was quantified as percentage of input DNA. Data are the shown for three separately derived and analysed cell lines ±s.d. (C) ChIP-seq was performed with an antibody to Ezh2 for a cell line with Fragment (IV) inserted into the mouse α globin locus (orange bar). Reads were mapped to the transgenic locus (middle track). For comparison, ChIP-seq data are shown for Ezh2 at the wild-type mouse α globin locus (upper track; data from Ku et al). An input track is also displayed (lower track). ChIP-seq enrichment is normalized to mapped reads per 10 million.

Magnus D Lynch, et al. EMBO J. 2012 January 18;31(2):317-329.
5.
Figure 5

Figure 5. From: An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

CpG island erosion during mammalian evolution is associated with loss of chromatin bivalency. (A) In all, 2088 peaks of H3K27me3 enrichment associated with a CpG density of 6% or greater (500 bp window) were identified in human ES cells (Ernst et al, 2011). Data for each peak are plotted on a single line with flanking regions of 5 kb (X axis). The corresponding genomic intervals in the mouse genome were identified (UCSC liftOver tool). CpG density in a 500-bp window (percent) and H3K27me3 read density in a 500-bp window (percentage of maximal enrichment) for mouse ES cells are plotted adjacent to the corresponding human peaks (Ku et al, 2008). Peaks are displayed in order of maximum CpG density in the mouse genomic interval. CpG erosion in the mouse genome is associated with a diminution of H3K27me3 recruitment in mouse ES cells. The locations of selected genes are indicated. (B) Pileup analysis of the same data comparing mouse genomic regions associated with a maximum CpG density (500 bp window) of >3% (green) to those with <3% (blue). (C) A map of H3K27me3 was generated for rat ES cells and an analogous pileup analysis was performed. (D, E) Selected examples of CpG island erosion between human and mouse genomes at the (D) MYO1G, (E) CLEC4G and (F) MYF6 genes associated with loss of chromatin bivalency. ChIP-seq data are displayed as mapped reads per 10 million.

Magnus D Lynch, et al. EMBO J. 2012 January 18;31(2):317-329.
6.
Figure 2

Figure 2. From: An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

Establishment of a novel bivalent chromatin domain in the mouse α globin locus. (A) An RMCE cassette was targeted to the wild-type mouse α globin locus deleting the 3′ homology block containing the Hba2 and 3′ theta genes. The position of qPCR primers located within the mouse α globin locus upstream of the RMCE exchange site (1–2) and in exon 1 of the mouse α globin gene (3) are indicated. (BD) DNA fragments encoding the human (A) HBA2, (B) FERD3L and (C) HBB genes were separately integrated into this locus using the RMCE system. Following Flp-mediated excision of the Hprt selective marker gene, ChIP was performed with antibodies to Ezh2, H3K4me3 and H3K27me3 and enrichment quantified with species-specific qPCR probes. The positions of qPCR probes within the tested fragments are indicated (numbered 4–14). Other numbered probes are control points in the mouse genome as follows: (15) 5′ β globin; (16) β globin exon 1; (17) 3′ β globin; (18) β actin promoter; (19) HoxB7; (20) HoxC5 and (21) Gata6 promoter. Test probes are shown in green along with mouse genomic negative control points (red) and genomic positive control points (grey). Other points are shown as unfilled bars. Enrichment was quantified as percentage of input DNA. Data are shown for three separately derived and analysed cell lines ±s.d.

Magnus D Lynch, et al. EMBO J. 2012 January 18;31(2):317-329.
7.
Figure 1

Figure 1. From: An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

Differences in chromatin state at human and mouse α globin loci in humanized mouse ES cells. (A) The human α globin cluster is located close to the telomere (16p13.3), whereas the mouse cluster lies at an interstitial chromosomal position (11qA4). The positions of α globin genes (HBA1/Hba1 and HBA2/Hba2) and other globin genes within the loci are indicated. The α globin genes in both species are arranged in duplicated homology blocks. In the human locus, these blocks contain only the α globin genes whereas in the mouse, the θ globin genes are also present. Published ChIP-seq data illustrate differences in the epigenetic status at human versus mouse α globin genes in human and mouse ES cells. To facilitate analysis of duplicated homology blocks, reads were remapped with Bowtie permitting up to two copies of a sequence in the genome. Read count is normalized to reads mapped per 10 million. (B) For one copy of mouse chromosome 11, the illustrated region was replaced with the 120-kb orthologous region of human chromosome 16 including the α globin genes (Wallace et al, 2007) permitting comparison of human and mouse globin loci within the same nucleus of pluripotent cells. (CF) ChIP was performed with antibodies to (C) Ezh2, (D) Cbx7, (E) H3K4me3 and (F) H3K27me3 in these transgenic mouse ES cells bearing one wild-type allele and one humanized allele. Enrichment was quantified as percentage of input DNA and is plotted according to the position of species-specific qPCR amplicons relative to the human or mouse α globin gene. Positive and negative control points are shown on the right side of each panel (from left to right: 5′ mouse β globin gene, mouse β globin exon1, mouse β actin, mouse Gata6). Data are the result of at least two biological replicates ±s.d.

Magnus D Lynch, et al. EMBO J. 2012 January 18;31(2):317-329.

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