If a better understanding of gene regulation is music to your ears, then tune in for the latest developments in single-cell epigenomics. It is well established that both DNA methylation and higher-order chromatin structure play crucial roles in development, differentiation and disease, and there are readily available technologies for both (whole-genome bisulfite sequencing and chromatin conformation capture, respectively). While these techniques are valuable on their own, together they strike a chord: two independent teams of researchers have developed methods for simultaneously characterizing genome-wide methylation and chromatin structure at the single-cell level, providing insight into the interplay between these two epigenetic performers.
Both research teams took advantage of the fact that traditional 3C techniques rely on fixation, restriction digest, and ligation to generate libraries — a process that does not affect DNA methylation. Thus, DNA methylation can be assessed in the same sample, allowing for simultaneous methylome mapping and generation of a cell-type-specific chromatin conformation map.
sc-Methyl-HiC for Studying DNA Methylation Coordination
Previous research has shown that adjacent CpG sites on linear DNA exhibit coordinated methylation; i.e. they are more likely to have the same methylation status as neighboring CpG sites when compared to distant CpG sites. When DNA is condensed into chromatin, DNA sequences that are linearly far apart may be brought into close proximity in the 3-dimensional chromatin structure, which prompted the lab of Bing Ren (University of California San Diego, USA) to question if these 3D-proximal CpG sites might also have coordinated methylation. Unfortunately, standard whole-genome bisulfite sequencing (WGBS) generates short sequence fragments, which makes it difficult to detect long-range coordinated DNA methylation. Not to be deterred, the talented ensemble developed a new technique combining in situ Hi-C and WGBS, which they termed “Methyl-HiC”, to allow for concurrent mapping of both the methylome and chromatin architecture.
After validating the Methyl-HiC approach against stand-alone Hi-C and WGBS, the team moved on to single-cell simultaneous profiling (sc-Methyl-HiC) of mESCs. The workflow begins with standard Hi-C followed by FACS sorting to obtain individual nuclei, which are then subjected to WGBS. They treated mESCs with either serum (serum mESCs) or a combination of GSK3 and MEK inhibitors (2i mESCs) and obtained sc-Methyl-HiC data for 103 serum mESCs and 47 2i mESCs.
Here’s what they found:
- Methylation is coordinated within chromatin loops, and they observed slightly higher CpG methylation within TADs when compared to between TADs
- Single-cell methylation data can be used to cluster cells into groups with similar methylomes. They identified three clear cell populations: the 2i mESCs, and two subgroups of serum mESCs
- The two serum-treated sub-clusters have unique methylomes and chromosome conformations; comparing the two clusters revealed differentially methylated regions (DMRs) and differential chromosome compartment organization
- One of the serum-treated subgroups is potentially related to embryonic limb development, based on methylome overlap with previously characterized cells
- Methyl-HiC data identified DMRs and active gene compartmentalization that is enriched for genes related to embryonic limb development
Aside from developing a valuable new technique for studying chromatin conformation and methylation, this study also revealed methylation concordance of DNA sequences that are separated by many base pairs but close in 3D space. Excitingly, they were also able to identify cell-type-specific chromatin interactions; data which will hopefully be generated for many other cell types in the future. The authors believe that new DNA methylation detection strategies need to be developed and integrated into this system for an even more robust and accurate method.
Cell Type Specifics Using sn-m3C-seq
The second team, led by Joseph Ecker and Jesse Dixon (The Salk Institute, La Jolla, USA) was driven by the desire to study the chromatin architecture of different cell types within a heterogeneous, complex tissue sample. While methylation maps can reliably determine cell type, it was unclear if a similar chromatin architecture map could do the same. Thus, they developed a new method called “sn-m3C-seq” to generate chromatin structure maps and assign them to the identified cell types.
The sn-m3C-seq method combines standard in situ chromatin conformation capture (3C) and WGBS to investigate both chromatin architecture and the methylome at the single-cell level. This method was validated against single-cell methylation and chromatin mapping techniques (scNMT-seq and Hi-C, respectively), then put to the test in distinguishing unique cell types.
Here are the highlights:
- While sn-m3C-seq accurately distinguishes mESCs and mouse epithelial cells, the methylation maps are far more effective than Hi-C contact maps for partitioning
- They applied sn-m3C-seq to human post-mortem prefrontal cortex tissue and identified 14 major cell types
- CpG methylation signatures separate neuronal and non-neuronal cell types, and CpH methylation provides further subdivision of neuronal cell types
- From the 14 cell types identified via methylome mapping, they generated cell-type-specific chromatin interaction maps and identified differences in domain boundaries, chromatin loops, and methylation patterns
- Methylation and chromatin interactions are coordinated, specifically at CTCF binding sites and differential domain boundaries
- Cell-type-specific enrichment of chromatin interactions correlates with hypomethylation at these sites
The emergence of chromatin conformation capture (3C) technologies has unveiled a variety of 3-dimensional chromatin structures, but until now it has been difficult to determine differences across cell types. The sn-m3C-seq method opens up the possibility of identifying cell-type-specific unique chromatin interactions, and the relationship to differentially methylated regions.
Single-cell Methylome and Chromatin Mapping: Moving Forward
While these two epigenetic methods were previously soloists, we think they make a great duet. Both studies developed single-cell methods for cell-type identification via methylome mapping plus detection of 3D chromatin structures, revealing crosstalk between these two epigenetic players and furthering our understanding of differential gene expression across various cell types. These methods also allow for the generation of cell-type-specific chromatin architecture maps, which can hopefully be integrated with other single-cell atlases to develop a more global view of unique cell-type properties.