Nascent DNA methylome mapping reveals inheritance of hemimethylation at CTCF/cohesin sites

Abstract

The faithful inheritance of the epigenome is critical for cells to maintain gene expression programs and cellular identity across cell divisions. We mapped strand-specific DNA methylation after replication forks and show maintenance of the vast majority of the DNA methylome within 20 minutes of replication and inheritance of some hemimethylated CpG dinucleotides (hemiCpGs). Mapping the nascent DNA methylome targeted by each of the three DNA methyltransferases (DNMTs) reveals interactions between DNMTs and substrate daughter cytosines en route to maintenance methylation or hemimethylation. Finally, we show the inheritance of hemiCpGs at short regions flanking CCCTC-binding factor (CTCF)/cohesin binding sites in pluripotent cells. Elimination of hemimethylation causes reduced frequency of chromatin interactions emanating from these sites, suggesting a role for hemimethylation as a stable epigenetic mark regulating CTCF-mediated chromatin interactions.

Fig. 1.. The vast majority of the DNA methylome is maintained 20 min after replication.

(A) Correlation of methylation frequency between pCs and dCs within the same pdCpGs in pulse. (B) Count of pdCpGs in pulse with differential ΔmC values. (C) Different types of hemiCpGs with all 4 Cs mapped at least 4 times. (D) For concordant hemiCpGs in pulse, the distribution of methylation frequency of4Csin chase is shown (left), and vice versa (right). (E) All concordant hemiCpGs were intersected with WGBS datasets from other human cells. The distribution of ΔmC values is shown for each dataset.

Fig. 2.. HemiCpG is a significant component of the DNA methylome.

(A) Schematic representation of the principles underlying the iSA method. (B) The fraction of all 4 types of intraCpGs in pulse and chase. (C) The frequency of 3 types of intraCpGs (with 2 types of intraCpGs^hemi combined) at different mouse embryonic stages. ICM: inner cell mass. MEF: mouse embryonic fibroblast. mPGC/fPGC: male/female primordial germ cell. (D) The frequency of 3 types of intraCpGs at genic regions at different mouse embryonic stages.

Fig. 3.. Transient interactions between DNMTs and substrate dCs in both maintenance and de novo methylation.

(A) The fraction of all 4 types ofDNMT- targeted intraCpGs in pulse and chase. (B) Counts of all DNMT-targeted intraCpGs^hemi-pC in pulse allocated to the appropriate cells according to their methylation frequency in pulse nasBS-seq. (C) Reduction of methylation under DNMT1 KO (24 hr) or DNMT3A/3B double KO (late) is shown for all CpGs, DNMT-targeted intraCpGs^hemi-pC and intraCpGs^me in pulse and chase, and unmapped CpGs. ***, p<0.001. NS, not significant. (D) Distribution of methylation frequency of the 4 Cs in concordant hemiCpGs viewed through nasBS-seq and DNMT3A/3B nasChIP-BS-seq. The asterisks mark the 2 Cs inspected in each panel.

Fig. 4.. Inherited hemiCpGs flanking CTCF/cohesin sites may regulate chromatin interactions.

(A) Frequency of motif or opposite strand-methylated (same me or oppo me) intraCpGs^hemi around oriented CTCF motifs co-occupied by CTCF/SMC1A from the two nascent DNA duplexes. Frequency of hairpinCpG^hemi from CTCF ChIP-hairpinBS-seq is also shown. (B) All CTCF motifs co-occupied by CTCF/SMC1A in pulse were ranked by their Hemi Index. ΔmC of CpGs from the two nascent DNA duplexes, and reads per million (RPM) for CTCF and SMC1A nasChIP-seq within al kb window surrounding the motifs are shown. Black in the ΔmC heatmaps represents missing data points. (C) All hemiCpGs (ΔmC≥67% or ≤−67%) from two flanking regions in (B) were retrieved. Methylation frequency of the 2 Cs in the other dyad in pulse (left) or the same dyad in chase (right) are shown. ***,_p<0.001. NS, not significant. (D) The Hemi Index of CTCF motifs showing HI>50 in the pooled data were compared between two dyads, from pulse to chase, and across 5 passages. (E) Occupancy of WT and R133C mutant MeCP2 in WT mESC, and MeCP2 in DNMT1/3A/3B triple KO (TKO) mESCs was profiled around CTCF motifs showing upstream- or downstream-only hemi-methylation in mESCs. (F) The ratio between interaction contacts from Hi-ChIP in WT and DNMT3B-KO HUES64 hESCs emanating from occupied CTCF motifs and extending up to ±l Mb window is shown.