Trends in Biotechnology
ReviewSingle-Cell DNA Methylation Profiling: Technologies and Biological Applications
Section snippets
DNA Methylation as an Epigenetic Modification
Epigenetic modifications are changes in the genetic material that cause a heritable phenotype without changing the DNA sequence itself. DNA methylation is an important epigenetic mark that comprises the coupling of a methyl group (CH3) to deoxyribonucleosides. Although methylation can take place at multiple positions on any of the bases, 5-methylcytosine (5meC; see Glossary) is the most abundant methylated DNA base in vertebrates [1]. Methylation of cytosines generally occurs in the context of
The Traditional DNA Methylation Profiling Toolbox
A large variety of traditional tools exist to study DNA methylation, based on a multitude of techniques such as chromatography, mass spectrometry, ELISA, restriction digestion, immunoprecipitation, and bisulfite conversion 10, 11. Continuous technical progress also means continuous development of new techniques, such as those based on single-molecule imaging [12] or nanopore technology [13]. Although every method has its own advantages and disadvantages, the toolbox as a whole is well suited to
Bisulfite-Based Single-Cell Methods
Even though bisulfite sequencing quickly became established as the method of choice for bulk DNA methylation analysis, single-cell adaptations faced the major hurdle of bisulfite-induced DNA degradation, which initially prohibited the development of low-input methods. In 2013, the first single-cell RRBS (scRRBS; Table 1) protocol was established through the integration of all experimental steps up to and including the bisulfite conversion into a single-tube reaction, followed by two rounds of
Bisulfite-Free Single-Cell Methods
Even so, bisulfite treatment remains relentlessly harsh and conversion rates can vary, causing inconsistency across samples and fuelling the search for bisulfite-free single-cell methods. An early study utilised restriction digestion by methylation-sensitive restriction enzymes (MSREs) coupled to PCR amplification in a single reaction mixture on a microreaction slide for high-throughput DNA methylation analysis of single cells [37]. Although relatively affordable and easy to implement, this
Biological Applications
While many single-cell DNA methylation papers focussed primarily on technical improvements, new biological insights have also been obtained. Most studies up to now have investigated either early embryonic development or tumour tissues. Here, we categorise these observations based on their biological context and briefly discuss them.
DNA Demethylation and Multiomics Approaches
All in all, numerous approaches, both bisulfite-based and bisulfite-free, exist for the analysis of DNA methylation on the single-cell level, accommodating a wide variety of research questions. Yet DNA methylation is a dynamic modification, and additional methods are needed to monitor these dynamics and integrate them with other data sets to obtain an accurate overview of the DNA methylation network and its biological consequences. One main disadvantage shared by the aforementioned methods is
Concluding Remarks and Future Perspectives
These are exciting times for the field of DNA methylation: recent technological advances have generated unprecedented possibilities for studying this epigenetic modification at the single-cell level. Despite the great progress that has been made, there always remains room for improvement (see Outstanding Questions).
Single-cell DNA methylation technologies can be categorised in several ways (Figure 3, Key Figure). Rather than there being one supreme method, the protocols are complementary and
Acknowledgements
Work in the Vermeulen lab is supported by a European Research Council Starting Grant (no. 309384) and the Netherlands Organisation for Scientific Research (NWO Gravitation Programme Cancer Genomics Netherlands). The Vermeulen lab is part of the Oncode Institute, which is partly funded by the Dutch Cancer Society (KWF).
Glossary
- 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC)
- oxidised derivatives of 5meC. During active DNA demethylation, ten-eleven translocation (TET) methylcytosine dioxygenases catalyse the oxidation of 5meC into first 5hmC, then 5fC and 5caC, which can be converted into unmethylated cytosine.
- 5-methylcytosine (5meC)
- cytosine base with a methyl group (CH3) covalently coupled to the 5′ position of its pyrimidine ring. The most abundant methylated DNA base in
References (62)
Methylated bases in DNA of animal origin
Arch. Biochem. Biophys.
(1970)Highly integrated single-base resolution maps of the epigenome in Arabidopsis
Cell
(2008)Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics
Cell Rep.
(2015)Tracing dynamic changes of DNA methylation at single-cell resolution
Cell
(2015)Methylation dynamics of imprinted genes in mouse germ cells
Genomics
(2002)FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency
Cell Stem Cell
(2013)Genome-wide, single-cell DNA methylomics reveals increased non-CpG methylation during human oocyte maturation
Stem Cell Rep.
(2017)Simultaneous profiling of transcriptome and DNA methylome from a single cell
Genome Biol.
(2016)CpG-rich islands and the function of DNA methylation
Nature
(1986)- et al.
CpG islands and the regulation of transcription
Genes Dev.
(2011)