Human Single-cell Mapping of Chromatin and DNAm: Part 3 - Exploring Chromatin Conformation

July 7, 2025 By Stuart P. Atkinson

snmC-seq3 workflow schematic and myeloid contact/methylation tracks. — snmC-seq3 protocol and example genome-browser of Hema myeloid contact map with CG/CH methylation tracks. From Zhou and Wu et al.

Simultaneous Analysis of Higher-order Chromatin Structure and DNA Methylation Analysis in the Same Cell?

The Epigenome Technologies blog brings you the third of a three-part summary of a pre-print article from researchers led by Jingtian Zhou, Jesse R. Dixon, and Joseph R. Ecker, who sought to evaluate how DNA methylation and higher-order chromatin structure contribute to cell type-specific gene expression profiles in single cells from tissues across the human body (Zhou and Wu et al.). The authors of this study applied single-nucleus methyl-3C (sn-m3C-seq; Lee et al.) - which permits the simultaneous analysis of two distinct epigenetic regulatory layers - to cells resident in 16 human tissues to generate the first ever single-cell human body map of DNA methylation and chromatin conformation. They hoped this resource would help explain the inherent variability of these epigenetic features in human cell types and explore how they help to establish human cell identity. Part 3 of this blog series now explores chromatin conformation patterns in this data set and any relationships between DNA methylation and chromatin conformation.

parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing or " Paired-Tag " from Epigenome Technologies generates joint epigenetic and gene expression profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with comparable efficiency to single-nucleus RNA-seq/ChIP-seq assays. Paired-Tag also avoids any requirement for cell sorting. Applying Paired-Tag technology may enable researchers to take giant leaps forward in our understanding of gene regulatory mechanisms and significantly improve disease management. What additional insight could Paired-Tag provide to this sn-m3C-seq-based study?

Contact decay heatmap and contact-distance ratio boxplots per cell type. — Heatmap of chromatin contact frequency versus genomic distance - and contact ratios - for each cell type. From Zhou and Wu et al.

3D Chromatin Conformation Variety Across Cell Types

Genome-wide chromatin interactions involve two types: interactions at 100kb-1Mb (chromatin loops and topologically associating domains; TADs) and interactions over 10Mb (A/B chromatin compartments)

The relative enrichment of short- and long-range signals varies across cell types; some display domain dominance or compartment dominance, and others display a shared profile

Analysis of chromatin loops revealed considerable variability in number and size for each lineage

Comparing chromatin loops across lineages identified over 280,000 differential chromatin loops in major cell types, with many differential chromatin loops shared amongst related cell types
The presence of loops in a specific lineage corresponds to the enrichment of those lineages in the A compartment (more active regions of the genome)

The identification of loops present in specific lineages allowed a comparison with gene expression changes

Differentially expressed genes between cell subtypes associate with subtype-specific chromatin looping

These data suggest the existence of variable chromatin loops that differ according to major cell type and subtype lineages and strongly associate with differential gene expression

Loop-length boxplots and loop-strength heatmap across cell types. — Loop lengths (Mb) and loop (differential) strengths across cell types. From Zhou and Wu et al.

Associating Chromatin Conformation and DNA Methylation

Major cell types displayed a negative correlation between mCG and compartment score (agreeing with the presence of active chromatin in A compartment and mCG depletion)

Seven cell types displayed a positive correlation between mCG levels and compartment score, with all possessing PMDs (agreeing with the presence of PMDs in repressive chromatin with lower mCG levels)

Analysis of memory T cells revealed that the partially methylated compartment matched the B compartment, and the fully methylated compartment matched the A compartment

Expanding the comparison to all major cell types revealed that most genome regions displayed a stable status regarding compartments (although a subset of regions does switch in selected lineages)
Strong associations exist between B compartments and regions considered partially methylated DNA methylation compartments

Comparing DNA methylation patterns at chromatin domain boundaries explored their ability to separate A and B compartment regions

Domain boundaries demarcate regions with mCG enrichment and depletion across nearly all lineages
Most lineages display a relative focal depletion of mCG at or immediately adjacent to the domain boundary
The association of mCG with A/B compartments varies between lineages when considering domain boundaries that separate A and B compartment regions
Lineages with PMDs possess lower mCG levels in the B compartment side adjacent to domain boundaries, while other lineages suffer from mCG depletion within the A compartment side
mCH depletion occurs at domain boundaries separating similar compartments, which agrees with the general depletion of mCH at candidate cis-regulatory regions and suggests the universality of mCH absence at regulatory elements

Higher-res analysis of chromatin conformation and DNA methylation revealed the enrichment of differentially methylated regions (DMRs) at loop summit anchors in all major types

Motif enrichment analysis at DMRs revealed that CTCF and lineage-specific transcription factors regulate chromatin loop formation and maintenance

DMRs displayed enrichment at chromatin loops with higher variability across subtypes in most major types, with variable relationships existing between mCG at DMRs and chromatin loops

Specific lineages displayed anticorrelations between DNA methylation and chromatin loops at loop anchors (agreeing with DNA methylation depletion at active regulatory elements)
Other lineages possessed no clear relationships or bimodal distributions

Bar plots of compartment-score vs mCG and mCH correlations by cell type. — Pearson correlations between compartment scores and (A) mCG or (B) mCH levels, across diverse cell types. From Zhou and Wu et al.

Chromatin Conformation and DNA Methylation: Discrepancies?

While frequent correlations existed between patterns of DNA methylation and chromatin conformation, distinct differences related to the clustering of major cell types into cell subtypes also existed

The association of DNA methylation and chromatin conformation with cell states may vary across lineages

Analysis of discrepancies in greater detail focused on skeletal muscle, with DNA methylation and chromatin conformation data demonstrating a significant mismatch between the subtype assignment
Other lineages displayed almost no correlation between DNA methylation and chromatin conformation

Placental epithelial cells form 2 clusters from chromatin conformation and 6 clusters for DNA methylation (driven by donor and global methylation level differences and not segregated in chromatin embedding)
3D genome embedding reflected the cell type differences more precisely in the trophoblast

Overall, global patterns of DNA methylation may lack information regarding cell type, but local methylation patterns over specific genes may be informative

DNA methylation may play a key role in the regulation of these genes and lineages; however, the variation in DNA methylation globally across single cells remains higher and is not primarily defined by cell type

UMAPs, track plots, heatmaps, and motif-enrichment dots for fast/slow subtypes. — UMAPs of DNA methylation and chromatin contacts for “fast” vs. “slow” muscle/endo subtypes; with local contact and methylation tracks. From Zhou and Wu et al.

Single-cell Human Body Map Reveals Chromatin Conformation Variations and Links to DNA Methylation

While Part 1 of this blog series from Epigenome Technologies reported on the creation of the first single-cell body map of DNA methylation and chromatin conformation in human cells and Part 2 reported on the DNA methylation patterns discovered, Part 3 explored chromatin conformation diversity and reported in the links discovered between DNA methylation and chromatin conformation. The study characterized chromatin conformation across an unprecedented number of cell types, identifying chromatin compartments, domains, and loops in major cell types and subtypes. The results suggested that chromatin loops play a significant role in establishing gene regulatory programs during type specification and that distinct lineages can display compartment-dominant or domain-dominant phenotypes or a mixture of the two. The comparison of DNA methylation and chromatin conformation also identified how these features correlate throughout the genome, including at the chromatin compartment and domain levels; however, interestingly, specific cell types display divergence between the two features.

A deeper understanding of DNA methylation and chromatin conformation at the single-cell level in humans offers a means to push groundbreaking research forward; can Epigenome Technologies help in this endeavor? The profiling of multiple histone modifications combined with simultaneous RNA sequencing at the single-cell level may provide an understanding of the complementary role of another level of epigenetic regulation. Paired-Tag from Epigenome Technologies generates joint epigenetic and gene expression profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Furthermore, Epigenome Technologies offers other single-cell products and services suitable for your research needs. As such, applying Paired-Tag technology may enable giant leaps forward in understanding gene regulation and complement the findings of this exciting study.

For more on the assessment of chromatin conformation and associations with DNA methylation in the first-ever single-cell human body map of DNA methylation and chromatin conformation, see BioRxiv, March 2025.