Clark et al. Genome Biology (2016) 17:72 DOI 10.1186/s13059-016-0944-x OPINION Open Access Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity Stephen J. Clark1, Heather J. Lee1,2*, Sébastien A. Smallwood1,3, Gavin Kelsey1,4 and Wolf Reik1,2,4,5 Abstract are very limited and where epigenetic heterogeneity may be most pronounced. Emerging single-cell epigenomic methods are being High-throughput sequencing has revolutionized the developed with the exciting potential to transform field of epigenetics with methods for genome-wide map- our knowledge of gene regulation. Here we review ping of DNA methylation, histone modifications, chro- available techniques and future possibilities, arguing matin accessibility, and chromosome conformation that the full potential of single-cell epigenetic studies (Table 1). Initially, the input requirements for these will be realized through parallel profiling of genomic, methods meant that samples containing hundreds of transcriptional, and epigenetic information. thousands or millions of cells were required; but in the last couple of years this has changed with numerous epi- genetic features now assayable at the single-cell level Introduction (Fig. 1). Combined single-cell methods are also emerging Epigenetics involves the study of regulatory systems that that allow analyses of epigenetic–transcriptional correla- enable heritable changes in gene expression within geno- tions thereby enabling detailed investigations of how epi- typically identical cells. This includes chemical modifica- genetic states are associated with phenotype. tions to DNA and the associated histone proteins, as In this article, we review current and emerging well as changes in DNA accessibility and chromatin con- methods for mapping epigenetic marks in single cells formation [1]. Until recently, our understanding of these and the challenges these methods present. We subse- epigenetic modifications has depended entirely upon quently discuss applications of these technologies to the correlations between bulk measurements in populations study of development and disease. of cells. These studies have classified epigenetic marks as being associated with active or repressed transcriptional states, but such generalizations often conceal a more Single-cell methodologies and future complex relationship between the epigenome and gene technological developments expression. Cytosine methylation and other DNA modifications Arguably, and as for many biological questions, DNA methylation of cytosine (5mC) residues can be investigation of epigenetic regulation in general is most mapped genome-wide using several methods such as usefully studied at the single-cell level, where intercellu- methylation-specific restriction enzymes [3], affinity lar differences can be observed leading to a more refined purification [4], or by using bisulfite conversion followed understanding compared with bulk analysis [2]. Add- by sequencing (BS-seq) [5]. The latter is considered the itionally, the development of single-cell technologies is gold-standard method as it allows single base resolution key to investigating the profound remodeling of the epi- and absolute quantification of DNA methylation levels. genome during the early stages of embryonic develop- While investigation of DNA methylation at the single- ment, including in human samples where cell numbers cell level was motivated by important biological ques- tions, until recently it was unfeasible due to the large * Correspondence: [email protected] amount of DNA degradation caused by the bisulfite 1Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK conversion, which was traditionally performed after pre- 2 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK paring adapter-tagged libraries. Full list of author information is available at the end of the article © 2016 Clark et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Clark et al. Genome Biology (2016) 17:72 Page 2 of 10 Table 1 Survey of current and emerging single-cell epigenetics techniques Technique Epigenomic feature Method Approach Single cell Cytosine modification 5mC BS-seq Bisulfite converts C but not 5mC Yes [6–8] (or 5hmC) to U so only methylated sites are sequenced as “C” 5mC MeDIP-seq Immunoprecipitation of 5mC DNA Not currently possible followed by sequencing 5mC Methyl-seq Restriction enzyme specific for 5mC Possible followed by sequencing 5hmC oxBS-seq 5hmC is oxidized to 5caC so that only Not possible for measuring 5mC survives bisulfite conversion. 5hmC due to the need for Readout is pure 5mC and subtraction subtraction from BS-seq determines 5hmC 5hmC TAB-seq Maps 5hmC by enzymatic oxidation Possible prior to bisulfite treatment: only 5hmC survives conversion 5hmC hMeDIP-seq Immunoprecipitation of 5hmC DNA Not currently possible followed by sequencing 5hmC Aba-seq Restriction enzyme specific for 5hmC Possible Protein–DNA interaction Histone modification ChIP-seq Immunoprecipitation of DNA bound Yes [17] to a specific histone variant or transcription factor Transcription factor DamID Cells are transfected with a fusion of Yes for nuclear lamina binding a transcription factor gene and Dam interactions [18] protein which methylates adenine residues in proximity to the binding site. 6 mA specific restriction digest is used to map Chromatin structure Nucleosome positioning MNase-seq Microcococal nuclease digestion of Possible chromatin and sequencing of the product which are regions protected by nucleosomes Nucleosome positioning NOME-seq GpC methylation of DNA not protected Possible by nucleosomes followed by BS-seq DNA accessibility DNase-seq DNaseI digestion of open chromatin Yes [23] into small fragments suitable for library preparation and sequencing DNA accessibility FAIRE-seq Chromatin is crosslinked, sonicated, Not currently possible and then purified by phenol–chloroform extraction. The aqueous layer contains only DNA not associated with protein DNA accessibility ATAC-seq Tn5 transposase enzyme fragments Yes [21, 22] and attaches adapters to open chromatin Three-dimensional Chromosome HiC DNA is crosslinked, then restriction Yes [29] organization conformation digested to fragment before ligation and reversal of the crosslinks. Resulting fragments are hybrids from separate genomic locations that were in close proximity in three-dimensional space. Paired-end sequencing is used to link the two regions C cytosine, 5caC 5-carboxylcytosine, 5hmC hydroxymethylcytosine, 5mC methylcytosine, U uracil The first single-cell method for measuring genome- because it allows assessment of a large fraction of pro- wide 5mC used a reduced representation bisulfite se- moters with relatively low sequencing costs, but its limi- quencing (scRBBS) approach based on enrichment of tation is poor coverage of many important regulatory CpG dense regions (such as CpG islands) via restriction regions such as enhancers. digestion, and it allows the measurement of approxi- To develop true whole-genome single-cell approaches mately 10 % of CpG sites [6]. scRRBS is powerful [7, 8] technological developments have been based on a Clark et al. Genome Biology (2016) 17:72 Page 3 of 10 Manual Droplet manipulation encapsulation or FACS Parallel Physical separation Chromatin RNA DNA Immuno- Enzymatic precipitation treatment Chemical treatment scBS-seq scRNA-seq scDNA-seq scRRBS scChIP-seq scATAC-seq scDNase-seq scDamID scHiC Fig. 1 Epigenomics and the spectrum of single-cell sequencing technologies. The diagram outlines the single-cell sequencing technologies currently available. A single cell is first isolated by means of droplet encapsulation, manual manipulation, fluorescence-activated cell sorting (FACS) or microfluidic processing. The first examples of single-cell multi-omic technologies have used parallel amplification or physical separation to measure gene expression (scRNA-seq) and DNA sequence (scDNA-seq) from the same cell. Note that single-cell bisulfite conversion followed by sequencing (scBS-seq) is not compatible with parallel amplification of RNA and DNA, as DNA methylation is not conserved during in vitro amplification. Single-cell epigenomics approaches utilize chemical treatment of DNA (bisulfite conversion), immunoprecipitation or enzymatic digest (e.g., by DNaseI) to study DNA modifications (scBS-seq and scRRBS), histone modifications (scChIP-seq), DNA accessibility (scATAC-seq, scDNase-seq), chromatin conformation (scDamID, scHiC) post-bisulfite adapter-tagging (PBAT) approach in which detection of high variability between single cells in distal bisulfite conversion is performed before library prepar- enhancer methylation (not usually captured by scRRBS) ation so that DNA degradation does not destroy in mouse embryonic stem cells (ESCs) [7]. adaptor-tagged fragments [9]. As a result, methylation in Building on this method has allowed BS-seq and up to 50 % of the CpG sites in a single cell can now be RNA-seq in parallel from the