DNA methylation signatures follow preformed chromatin compartments in cardiac myocytes

Abstract

Storage of chromatin in restricted nuclear space requires dense packing while ensuring DNA accessibility. Thus, different layers of chromatin organization and epigenetic control mechanisms exist. Genome-wide chromatin interaction maps revealed large interaction domains (TADs) and higher order A and B compartments, reflecting active and inactive chromatin, respectively. The mutual dependencies between chromatin organization and patterns of epigenetic marks, including DNA methylation, remain poorly understood. Here, we demonstrate that establishment of A/B compartments precedes and defines DNA methylation signatures during differentiation and maturation of cardiac myocytes. Remarkably, dynamic CpG and non-CpG methylation in cardiac myocytes is confined to A compartments. Furthermore, genetic ablation or reduction of DNA methylation in embryonic stem cells or cardiac myocytes, respectively, does not alter genome-wide chromatin organization. Thus, DNA methylation appears to be established in preformed chromatin compartments and may be dispensable for the formation of higher order chromatin organization.

Fig. 1

Distinct DNA methylation patterns in A and B compartments in adult CM. Hi-C contact maps uncover topologically associated domains (TADs) and multi-TAD A/B compartments in adult CM. Principal component analysis characterizes the A/B status of compartments (A, PC1 > 0; B PC1 < 0). Histone modifications, CTCF, and the cohesion subunit (SMC1) (RPKM) predict the chromatin state. Presence of RNA expression (FPKM) and marking with H3K36me3, H3K27ac, H3K4me1, H3K4me3 classifies A compartments as active. The inactive chromatin status of B compartments is indicated by H3K9me3 enrichment. CpG methylation data (%) shows low-methylated regions (LMRs), characteristic for cis-regulatory elements, and partially methylated domains (PMDs) overlapping with A and B compartments, respectively. Non-CpG methylation (mCHH, %), and 5-hydroxymethylcytosine (5hmC, RPKM) mark A compartments. Data shown are from n = 3 Hi-C, n = 3 RNA-seq, n = 3 WGBS; n = 2 5hmC-seq and n = 1–2 ChIP-seq experiments. RPKM reads per kilobase per million, FPKM fragments per kilobase per million mapped reads

Fig. 2

DNA methylation and gene expression succeeds formation of A/B compartments. a Dynamic A/B status precedes with the formation of LMRs in A compartments and PMDs in B compartments during differentiation and maturation of CM. Depicted is a representative locus harboring the CM-specific genes laminin subunit alpha 2 (Lama2) and triadin (Trdn). b, c Analysis of selected compartments with A/B switch manifestation at the progenitor stage (upper panels). B compartments present at the progenitor stage gain PMDs and concordantly loose gene expression after exit from the multipotent stage b. Establishment of LMRs and induction of gene expression succeeds formation of A compartments c. d Genome-wide principle component analysis (PCA) of A/B compartment values results in a tight cluster of differentiated cardiac myocytes and distant pluripotent ES and multipotent progenitor cells (left graph). Performing PCA analysis of base-pair resolution CpG methylation data results in a trajectory of CM differentiation and maturation with the smallest distance between postnatal stages (right graph). e Averaging CpG methylation data for A and B compartment present at all assessed stages (Common-A, Common-B) reveals hypermethylation of B compartments as compared to A compartments in undifferentiated ES and progenitor cells, while differentiated CM show a hypomethylation in B vs. A compartments. Data shown are from n = 2–3 Hi-C, n = 1–3 RNA-seq and n = 2–3 WGBS experiments. Shown are mean ± SEM. FPKM fragments per kilobase per million

Fig. 3

DNMT3A/B establishes non-CpG methylation in A compartments of post mitotic CM. a Originial traces of PC1 values corresponding to A/B compartments (A, PC1 > 0; B PC1 < 0), regions with differential CpG methylation (Δ > 40%) and CHH methylation (%). Differential CpG methylation during CM maturation and upon fetal ablation of DNMT3 were found in A compartments. CHH methylation is present in A compartments of adult cardiac myocytes in a DNMT3-dependant manner. b Gene expression occurs mainly in A compartments of postnatal CM. Ablation of DNMT3A/B had no significant effect on gene expression in CM. c, d Statistical analysis of CHH-methylation relative to the genome shows significant enrichment in A compartments and in fully methylated regions (mCpG > 85%). No differential CHH methylation is detectable in CM-DKO. Data shown are from n = 2–3 Hi-C, n = 3 RNA-seq, n = 2–3 WGBS experiments. Shown are boxplots with 10–90 percentile whiskers of FPKM values. *P < 0.05, ***P < 0.001 (ANOVA). FPKM fragments per kilobase per million mapped reads

Fig. 4

Loss of DNA methylation does not alter the chromatin organization in mouse ES cells. a Chromatin interaction maps of mouse embryonic stem cells (ES cell line1, upper panel) and ES cells with a complete loss of DNA methylation (ES-TKO cell line 1, Dnmt1 ^−/− /Dnmt3a ^−/− /Dnmt3b ^−/−) are indistinguishable. b, c Genome-wide correlation show that ablation of DNMT-isoenzymes has no effect of A/B pattern (b scatter plot and pearson correlation of PC1 values) and insulation of topologically associated domains (c TADscore). d CTCF peak signals (RPKM) in ES and ES-TKO cells correlates very well suggesting CpG methylation independent CTCF binding. Data shown are merged from n = 2 Hi-C experiments