April 1, 2016;
Active DNA demethylation at enhancers during the vertebrate phylotypic period.
The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2
in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.
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Figure 1: DNA methylome dynamics during vertebrate embryogenesis and the phylotypic stage. (a) Global DNA methylation (5mC) levels and fraction of total CpGs displaying high (≥0.8), intermediate (>0.2 and <0.8), low (>0 and ≤0.2) and no (0) 5mC at four stages of zebrafish, Xenopus and mouse embryogenesis. (b) Number, directionality and developmental stage of DMRs (FDR = 0.05, minimum ΔmCG = 0.2) identified in zebrafish, Xenopus and mouse embryos. The regions of developmental decrease in methylation or increase in methylation are denoted as phylo(−)DMRs and phylo(+)DMRs, respectively. (c) Conserved GO (Biological Process) terms associated with phylo(−)DMRs in zebrafish, Xenopus and mouse (FDR q ≤ 0.01). (d) Demethylation of phylo(−)DMRs in embryonic tissues and adult brains. The boxes show the interquartile range (IQR) around the median, and the whiskers extend from the minimum value to the maximum value unless the distance to the first or third quartile was more than 1.5 times the IQR. (e) 5mC levels of phylo(−)DMRs in adult mouse tissues.
Figure 2 : Phylo(−)DMRs are developmentally activated enhancers associated with vertebrate body plan formation. (a) Sorted heat maps of 5mC levels and normalized ChIP-seq read density for H3K4me1, H3K4me3, H3K27ac and p300 binding at phylo(−)DMRs in zebrafish, Xenopus and mouse embryos. (b) Mean CpG density (CpGs/100 bp) in zebrafish, Xenopus and mouse phylo(−)DMRs. (c) Comparisons of mean CpG density (CpGs/100 bp) for phylo(−)DMRs and CpG islands (CGIs). (d) Genomic overlaps and expression patterns (indicated by arrows) of validated VISTA enhancers associated with mouse phylo(−)DMRs. (e) Hierarchical clustering of transcript abundance (scaled TPMs) of orthologous genes (n = 138) linked to phylo(−)DMRs. Mm, mouse; Dr, zebrafish; Xt, Xenopus. (f) Evolutionary conservation (aggregate phastCons scores) of DMRs in zebrafish and mouse embryos (Kruskal-Wallis test, Dunn's post test, *P < 0.05, ***P < 0.001). The boxes show the IQR around the median, and the whiskers extend from the minimum value to the maximum value unless the distance to the first or third quartile was more than 1.5 times the IQR.
Figure 4 : Phylo(−)DMRs are characterized by 5hmC enrichment in vertebrate embryos. (a) Steady-state abundance (TPM) of Tet1, Tet2 and Tet3 transcripts during zebrafish, Xenopus and mouse embryogenesis. (b) Genome browser displays demonstrating the co-occurrence of phylo(−)DMRs, 5hmC (TAB-seq) and active enhancer marks (H3K27ac, p300). (c) 5hmC levels in developmentally hypomethylated DMRs. The boxes show the IQR around the median, and the whiskers extend from the minimum value to the maximum value unless the distance to the first or third quartile was more than 1.5 times the IQR. (d) Phylo(−)DMR-centered heat maps of 5hmC and 5mC enrichment. (e) Negative correlation of the fraction of 5hmC ((hmCG/CG)/(mCG/CG)) at the phylotypic stage and the fraction of 5mC (mCG/CG) at the tailbud stage for individual dinucleotides within phylo(−)DMRs.
5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development.