Simultaneous Analysis of Higher-order Chromatin Structure and DNA Methylation Analysis in the Same Cell?
Simultaneous Analysis of Higher-order Chromatin Structure and DNA Methylation Analysis in the Same Cell?
Higher-order chromatin structure and DNA methylation represent critical epigenetic layers that regulate the gene expression profiles that establish specific cell type/subtype identities in the human body; however, how these modes of regulation differ across cell types/subtypes remains somewhat unclear. Single-cell analyses of DNA methylation and 3D chromatin structure had remained limited to specific tissues or cell types, which represented an obstacle to a more global epigenetic view. While a recent study profiled DNA methylation patterns across a broad spectrum of human cell types (Loyfer et al.), the requirement for cell sorting made the applied strategy unworkable for human tissue samples. Cell sorting and related techniques had also offered insights into cell type-specific patterns of 3D chromatin compartments, domains, and chromatin loops in cultured cells/tissues (Schmitt et al. and Rao et al.); however, this approach has not been comprehensively applied to distinguish human cell types from in vivo tissue samples. Furthermore, the lack of simultaneous analysis of higher-order chromatin structure and DNA methylation in the same single cell represents another obstacle to a deeper understanding of the impact of these layers of epigenetic regulation.
The Epigenome Technologies blog now brings you a three-part summary of a recent pre-print article from researchers from the laboratories of Jingtian Zhou, Jesse R. Dixon, and Joseph R. Ecker, who sought to solve these thorny problems by evaluating 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 fascinating new study applied single-nucleus methyl-3C sequencing (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. Overall, they hoped this resource would help to explain the inherent variability of these epigenetic features in human cell types and explore how they contribute to the establishment of human cell identity.
In Part 1 of this blog series, we introduce this fascinating study and report the steps and techniques employed to generate this first single-cell human body map of 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 the 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?
Single-cell Human Body Map of DNA Methylation and Chromatin Conformation
- The authors created the first single-cell map of DNA methylation or chromatin conformation across human tissues from over 87,000 cells with 195 billion methylation reads and 18 billion long-range chromatin contacts
- The cells profiled for each tissue came from at least two human donors
- 12 of the 16 tissue samples profiled came from the ENTEx collaborative project (Rozowsky et al.), with the remaining four tissue types chosen to broaden the diversity of cell types and lineages represented in the map (primary motor cortex, placenta, pancreatic islets, and peripheral blood)
- Analysis of data across donors, tissues, and modalities required the development of novel/improved computational methods
- Combining DNA methylation and chromatin conformation data classified cells into 35 major types
- Major cell types included cells exclusive to single tissues (e.g., excitatory neurons from primary motor cortex) and shared across tissues (e.g., memory T-cells)
- Integrating single-cell RNA-seq and single nucleus assay for transposase-accessible chromatin using sequencing data sub-classified major type cells into 206 cell subtypes
- An interactive cell browser allows the exploration of all data to study cell-type-resolved patterns of DNA methylation and chromatin conformation across diverse tissues and cell types
The Power of Simultaneous Epigenetic Analysis at the Single-cell Level
Epigenome Technologies reported on the use of single-nucleus methyl-3C sequencing to simultaneously profile multiple levels of epigenetic information in single cells and, as such, create the first single-cell human body map of DNA methylation and chromatin conformation. This accomplishment provides a highly useful reference for future explorations into their variability in human cells and how gene regulatory mechanisms establish cellular identity. Parts 2 and 3 of this blog series will discuss how this map improves our understanding of DNA methylation and chromatin conformation diversity.
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 requirements. 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 creation of the first-ever single-cell human body map of DNA methylation and chromatin conformation, see BioRxiv, March 2025.