XB-ART-46753Elife. February 26, 2013; 2 e00348.
Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates.
Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation on chromatin. In cold-blooded vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution. DOI:http://dx.doi.org/10.7554/eLife.00348.001.
PubMed ID: 23467541
PMC ID: PMC3583005
Article link: Elife.
Grant support: A15603 Cancer Research UK, MC_U137981013 Medical Research Council , 15603 Cancer Research UK, 202218 European Research Council, MC_UU_12009/8 Medical Research Council , MC_U137761446 Medical Research Council , WT0834922 Wellcome Trust , G1000902 Medical Research Council , 090018/Z/09/Z Wellcome Trust , CRUK_15603 Cancer Research UK, MRC_MC_U137981013 Medical Research Council , CRUK_A15603 Cancer Research UK, MRC_MC_U137761446 Medical Research Council , MRC_MC_UU_12009/8 Medical Research Council , ERC_202218 European Research Council
Genes referenced: act3 aptx dnaja1 ezh2 gnao1 gsx1 nkx2-2 nmi rnf2 smu1 sp9 suz12
Article Images: [+] show captions
|Figure 1. CpG island predictions do not accurately identify non-methylated islands of DNA in vertebrate genomes.(A) Non-methylated DNA profiles in testes at a representative syntenic region for seven vertebrate species. Genes are shown in black (improved annotation of gene TSSs using RNA-seq data is shown in red), CpG island predictions in green (CGI), and non-methylated DNA profiles are shown in blue. A phylogenetic tree (left) highlights the evolutionary relationship among the seven species. Dashed grey lines highlight the relationship between the gene TSSs across the species. A gap in the zebrafish profile indicates that aptx is found at a separate locus from dnaja1 and smu1. (B) The genome-wide overlap between CpG islands (green) and non-methylated islands (blue) is depicted as a Venn diagram for each of the species. (C) Nucleotide properties of non-methylated islands and control regions are depicted as density plots. CpG observed/expected (left) and GC content (right) are shown for NMI and control regions of the genome. Median values are shown as dark vertical lines. Thresholds for CpG island prediction are indicated (black dashed line).DOI: http://dx.doi.org/10.7554/eLife.00348.003Figure 1—figure supplement 1. NMIs are a conserved feature of vertebrate promoters as illustrated by two syntenic loci.(A) and (B) Profiles of non-methylated DNA are shown in testes at two representative syntenic regions for seven vertebrate species. Genes are shown in black (improved annotation of gene TSSs using RNA-seq data is shown in red), CpG island predications in green, and non-methylated DNA profiles are shown in blue. A phylogenetic tree (left) highlights the evolutionary relationship among the seven species and dashed grey lines highlight the relationship between the gene TSSs across the species.DOI: http://dx.doi.org/10.7554/eLife.00348.004|
|Figure 2. Non-methylated islands are associated with gene promoters in vertebrate genomes.(A) A histogram depicting the proportion of protein-coding transcription start sites (TSSs) which are overlapped by an NMI for all seven species. Blue bars indicate overlap with annotated TSSs and red bars indicate overlap with additional TSSs identified using RNA-seq data (platypus, chicken and lizard) or Xtev gene sets (frog). (B) Profiles of non-methylated DNA were plotted over a 6-kb window centred on all TSSs with an NMI (dark blue), without an NMI (blue), and for all transcription termination sites (TTS, black). The non-methylated DNA signal peaks at the TSS of gene promoters in all vertebrates.DOI: http://dx.doi.org/10.7554/eLife.00348.005|
|Figure 4. Intergenic NMIs are associated with distal regulatory elements, non-coding RNAs, and unannotated transcripts.(A) Most NMIs are associated with known protein-coding genes (left) but a substantial proportion are located within intergenic regions of the genome (right). (B) NMIs (green) are found at 45% and 64% of all known long non-coding RNA (lncRNA) TSSs (black) in mouse and zebrafish respectively. (C) A pie chart depicting the proportion of intergenic NMIs (>5 kb from a protein-coding gene) associated with different genomic features in mouse embryonic stem (ES) cells and zebrafish 24 hpf embryos. The association was performed hierarchically in the following order: lncRNA TSSs, other non-coding RNA TSSs (miRNAs, rRNAs, snRNAs, or snoRNAs), other TSSs (pseudogenes and processed transcripts), putative enhancer mark H3K4me1 and novel RNA-seq TSSs. This analysis indicates that intergenic NMIs mark novel transcriptional units or regulatory elements.DOI: http://dx.doi.org/10.7554/eLife.00348.007|
|Figure 5. Differential methylation of a subset of NMIs.(A) All vertebrate genomes have a subset of NMIs that are subject to differential methylation as illustrated by a heat map of non-methylated DNA signal from testes and liver in human, mouse and zebrafish. In each case NMIs are ranked according to length and clustered as shared (upper) or unique (lower) between the two tissues. A 5-kb window centred at the NMI is shown and read density is indicated by colour intensity. (B) The overlap of NMIs identified in liver and testes is depicted by Venn diagrams for NMIs associated with protein-coding TSSs (upper) and for NMIs away from TSSs (lower). NMIs at TSSs are generally non-methylated in both tissues whereas differentially methylated NMIs tend to be found away from TSSs. (C) NMI length distribution plots for shared (Shared NMIs, solid line) or unique (Unique NMIs, dashed line) NMIs from testes (blue) or liver (red). Shared NMIs tend to be longer than tissue-specific unique NMIs. (D) CpG density distribution plots for shared (solid line) or unique (dashed line) NMIs from testes (blue) or liver (red). Shared NMIs tend to have higher CpG density than unique NMIs.DOI: http://dx.doi.org/10.7554/eLife.00348.008Figure 5—figure supplement 1. Validation of differentially methylated NMIs between liver and testes in mouse and zebrafish by bisulfite sequencing.(A, i–iv) Mouse NMIs unique to liver or testes were analysed by bisulfite sequencing to verify that the regions were indeed differentially methylated. Traces of non-methylated DNA are depicted for differentially methylated regions in mouse liver (red) and testes (blue) with NMIs depicted as bars under the traces. The y-axis depicts read density. Methylation status of the unique NMIs was confirmed using the indicated bisulfite PCR amplicon (BA, black rectangle). Empty and filled circles represent non-methylated and methylated CpG dinucleotides, respectively. (B, (i–iii) Zebrafish NMIs unique to liver or testes were validated by bisulfite sequencing as in (A).DOI: http://dx.doi.org/10.7554/eLife.00348.009|
|Figure 5—figure supplement 2. Differential methylation of NMIs in platypus, chicken, lizard and frog and length distributions of NMIs from all seven vertebrates.(A) A heat map of non-methylated DNA signal from testes and liver in platypus, chicken, lizard and frog. In each case NMIs are ranked according to length and clustered as shared (upper) or unique (lower) between the two tissues. A 5-kb window centred at the NMI is shown and read density is indicated by colour intensity. (B) Venn diagrams demonstrate that shared NMIs are found predominantly at protein-coding gene TSSs (upper) and unique NMIs tend to be found away from TSSs (lower). (C) NMI length distribution plots for shared (Shared NMIs, solid line) or unique (Unique NMIs, dashed line) NMIs from testes (blue) or liver (red). Shared NMIs tend to be longer than tissue-specific unique NMIs. (D) CpG density distribution plots for shared (solid line) or unique (dashed line) NMIs from testes (blue) or liver (red). Shared NMIs tend to have higher CpG density than unique NMIs.DOI: http://dx.doi.org/10.7554/eLife.00348.010|
|Figure 6. Chromatin modification at NMIs depends on their underlying DNA methylation state.(A) H3K4me3 read density from testes (blue) and liver (red) is profiled over testes unique (left) and liver unique (right) NMIs for human (upper) and mouse (lower) and displayed as an average profile. At differentially methylated loci, the histone H3K4me3 modification is found preferentially in the tissue with the non-methylated NMI. (B) The H3K4me3 signal (profiled in frog stage 11–12 embryos and zebrafish 24 hpf) is present specifically at unique NMIs from frog stage 11–12 and zebrafish 24 hpf (green) and not at unique NMIs from the liver (red).DOI: http://dx.doi.org/10.7554/eLife.00348.012|