Simultaneous profiling of 3D genome structure and DNA methylation in single human cells

Abstract

Dynamic three-dimensional chromatin conformation is a critical mechanism for gene regulation during development and disease. Despite this, profiling of three-dimensional genome structure from complex tissues with cell-type specific resolution remains challenging. Recent efforts have demonstrated that cell-type specific epigenomic features can be resolved in complex tissues using single-cell assays. However, it remains unclear whether single-cell chromatin conformation capture (3C) or Hi-C profiles can effectively identify cell types and reconstruct cell-type specific chromatin conformation maps. To address these challenges, we have developed single-nucleus methyl-3C sequencing to capture chromatin organization and DNA methylation information and robustly separate heterogeneous cell types. Applying this method to >4,200 single human brain prefrontal cortex cells, we reconstruct cell-type specific chromatin conformation maps from 14 cortical cell types. These datasets reveal the genome-wide association between cell-type specific chromatin conformation and differential DNA methylation, suggesting pervasive interactions between epigenetic processes regulating gene expression.

Figure 1.. Outline of the single-nucleus methyl-3C sequencing (sn-m3C-seq) method.

Samples are processed with a typical in situ 3C/Hi-C procedure following by single-cell DNA methylome library preparation using snmC-seq2.

Figure 2.. Data processing and analysis of m3C-seq sequencing reads.

Reads derived from non-bisulfite treated regular Hi-C sequencing are converted C to T (read1) and G to A (read2) in silico and aligned using BWA-meth, Bismark (bowtie1), and Bismark (bowtie1) followed by split-read alignment. Alignment of non-converted reads using conventional alignment pipeline is used as a standard (Conventional, Non-converted). For (a-d), the mapping algorithms were applied to a common test dataset (n=1) to make a fair comparison of their performance. (a) Percent of aligned reads as a pair. (b) Alignment accuracy of different alignment strategies compared with conventional Hi-C alignment using in silico converted reads. (c) Fraction of read pairs with cis short-range reads (cis < 1kb), cis long-range interactions (cis > 1kb), and trans interactions (trans) using different alignment strategies. (d) Similar to panel (c), but for 3C-seq (without conversion), bulk m3C-seq (with conversion, from the same sample as bulk 3C-seq), and combined 192 single-nucleus m3C-seq results. (e) Contact maps from chromosome 17 for conventional bulk Hi-C and bulk m3C data. (f) mC profiles near the Pou5f1 gene for conventional bulk MethylC-seq as well bulk m3C-seq. The experiment was repeated twice independently with similar results.

Figure 3.. Bulk and single-nucleus m3C-seq of mouse embryonic stem cells.

(a) Comparison of HiC and bulk m3C-seq chromatin contact profiles . Green bar plot shows CpG methylation level from m3C-seq. (b) Reconstructed mESC chromatin conformation map from sn-m3C-seq profiles compared to Hi-C or bulk m3C-seq. Red bar plot shows CpG methylation level from sn-m3C-seq. (c) Bulk and single-nucleus m3C-seq chromatin contact profiles of the Sox2 locus in mESC compared to published HiC data generated from mESC, cortical neurons (CN) and neural progenitor cells (NPC). . (d) Bulk and single-nucleus m3C-seq DNA methylation profiles at Dppa2/4 locus compared to published methylome data generated from mESC, mouse CN and frontal cortex. The experiment was repeated twice independently with similar results.

Figure 4.. Single-nucleus m3C-seq reconstructs cell-type specific chromatin conformation maps.

(a) tSNE of single-cell mC profiles of mouse ES cells and NMuMG cells . (b) Chromosome wide Pearson correlation matrix from pooled sc-m3C-seq maps for ES cells and NMuMG cells . (c-d) Principal component analysis (PCA) of whole genome contact matrices from sc-m3C-seq from ES from NMuMG cells (Percentage of variance are marked on the axis). PC1 and PC2 are shown in (c); PC1 and PC3 are shown in (d). (e) PCA of local interactions (<2Mb) from sc-m3C-seq data from NMuMG cells showing PC1 and PC2. (f) Correlation of PC1 and per cell contacts . For (a) and (c-f), n=2 independently prepared mouse ES cell cultures were analyzed. The two mESC replicates each contained 379 and 93 cells. One (n=1) replicate of NMuMG cells containing 96 cells was analyzed.

Figure 5.. Single-nucleus m3C-seq in human brain prefrontal cortex (PFC).

(a-c) Dimension reduction (t-Distributed Stochastic Neighbor Embedding, tSNE) visualization of single human PFC cells using mCH (a) and mCG (b) of non-overlapping 100kb genomic bins, or chromatin interaction at 1Mb resolution (c). L2/3, L4, L5 and L6: excitatory neuron subtypes located in different cortical layers. Ndnf and Vip: CGE derived inhibitory sub-types. Pvalb and Sst: MGE derived inhibitory sub-types. Astro: astrocyte. ODC: oligodendrocyte. OPC: oligodendrocyte progenitor cell. MG: microglia. NN1: non-neuronal cell type 1. Endo: endothelial cell. (d) looping between the SATB2 and LINC01923 locus in excitatory neuron (L2/3, L4, L5 and L6) (e) chromatin looping between PROX1 and RPS6KC1 region in CGE derived inhibitory cell types - Vip and Ndnf. (f-g) mCH (f) and mCG (g) levels at SATB2 locus in excitatory neuron clusters. (h-i) mCH (h) and mCG (i) levels at PROX1 locus in CGE derived inhibitory neuron clusters. All analyses were performed with 4,238 sn-m3C-seq profiles generated from n=2 independent human specimen.

Figure 6.. Differential mC signature associated with cell-type specific chromatin interactions .

(a The violin plot of the distribution of the overlap between permuted differential interacting region anchor sites and DMRs ; the labelled point indicates the observed overlap. Violin plot elements: maximum=14,234; minimum=13,822; mean=14038.7. (DMRs, p<0.0001, two-sided permutation test, n=10,000 permutations). (b-d) Heatmap visualization of cell-type specific chromatin interaction (b), CG methylation at anchor regions (c) and CG methylation at CTCF binding sites overlapping with the anchor regions (d).