The epigenetic basis of cellular heterogeneity

Abstract

Single-cell sequencing-based methods for profiling gene transcript levels have revealed substantial heterogeneity in expression levels among morphologically indistinguishable cells. This variability has important functional implications for tissue biology and disease states such as cancer. Mapping of epigenomic information such as chromatin accessibility, nucleosome positioning, histone tail modifications and enhancer-promoter interactions in both bulk-cell and single-cell samples has shown that these characteristics of chromatin state contribute to expression or repression of associated genes. Advances in single-cell epigenomic profiling methods are enabling high-resolution mapping of chromatin states in individual cells. Recent studies using these techniques provide evidence that variations in different aspects of chromatin organization collectively define gene expression heterogeneity among otherwise highly similar cells.

Fig. 1 |. Epigenetic landscapes of transcribed and silent genes in eukaryotes.

Diagram depicting the epigenetic landscapes present at transcriptionally repressed and active chromatin. a | Transcriptionally repressed, enzymatically inaccessible cis-regulatory elements are characterized by regularly spaced nucleosome arrays enriched for DNA methylation and specific histone modifications, such as trimethylation of lysine 27 on histone 3 (H3K27me3). b | Transcriptionally active genes exhibit enzymatically accessible cis-regulatory elements with positioned, flanking nucleosomes enriched for specific histone modifications such as acetylation. CCCTC-binding factor (CTCF) and cohesin are depicted facilitating a chromatin contact between an active enhancer and an active promoter. 5mC, 5-methylcytosine; Pol II, Polymerase II; TSS, transcription start site.

Fig. 2 |. Single-cell techniques provide greater resolution than bulk-cell methods.

Three morphologically homogeneous cell populations exhibiting varying patterns of enrichment for a particular epigenetic characteristic, with darker shading indicating greater enrichment. Bulk-cell analysis generates a population-average value and fails to differentiate between the different scenarios. By contrast, single-cell approaches can readily distinguish variable levels of enrichment (middle) from rare subpopulations (right) and homogeneous enrichment (left).

Fig. 3 |. scATAC-seq and scDNase-seq measure the dynamics of accessible chromatin sites.

a | Assay for transposase-accessible chromatin sequencing (ATAC-seq) and DNase I hypersensitive site sequencing (DNase-seq) use enzymes (Tn5 transposase and DNase I, respectively) that cut DNA but can only do so at accessible chromatin sites. The activity of these enzymes produces fragments for sequencing that correspond to open chromatin. b | Model of interactions between transcription factors and accessible chromatin. Closed chromatin sites can be bound by sequence-specific pioneer transcription factors. Pioneer transcription factor binding facilitates the opening of chromatin and the creation of a new accessible site. Chromatin-modifying enzymes and other transcription factors that may not possess pioneering activity are able to bind to the accessible site. scATAC-seq, single-cell ATAC-seq; scDNase-seq, single-cell DNase-seq.

Fig. 4 |. scMNase-seq reveals nucleosome positioning dynamics.

a | Diagram depicting how nucleosome-bound DNA sequences are isolated for sequencing using micrococcal nuclease digestion deep sequencing (MNase-seq). Open chromatin and linker DNA is digested using micrococcal nuclease (MNase), and the resulting nucleosome-occluded DNA fragments are isolated and sequenced. b | Illustration of variable nucleosome positioning around accessible chromatin sites among a morphologically homogeneous cell population. The spacing mode of nucleosomes flanking the enhancer informs expression levels of the associated gene. Arrow thickness represents elevated levels of transcription. c | Illustration of enhancer priming in a morphologically homogeneous population. The yellow-shaded region represents nucleosome depletion at a lineage-specific transcription factor binding motif. Such epigenetic priming, which can produce cells with different developmental potentials, is not detectable using single-cell RNA sequencing because the gene is not expressed in either cell. scMNase-seq, single-cell MNase-seq.

Fig. 5 |. Single-cell Hi-C-based techniques measure the variability of enhancer–promoter contacts.

a | Simple depiction of how a chromatin contact is captured by Hi-C. Distal genomic regions in close spatial proximity are indicated using different colours. The chromatin is crosslinked to preserve the interactions, followed by a sequence-specific restriction digest to cut the chromatin into fragments. The cut ends are then ligated together and labelled with biotin. Finally, the crosslinking is reversed and the DNA fragments are purified using the biotin label and sequenced. b | Depiction of topologically associating domains (TADs), which are chromatin regions comprising many intra-regional spatial interactions. TAD exhibiting enhancer–promoter contacts facilitated by CCCTC-binding factor (CTCF) and cohesin. CTCF and cohesin bring distal genomic elements together into domains containing elevated local concentrations of enhancer (orange) and promoter (blue) elements (left). Absence of CTCF-mediated and cohesin-mediated interactions affects transcriptional heterogeneity without disrupting overall TAD structure (right). Hi-C, high-throughput chromosome conformation capture.