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Advances in the profiling of DNA modifications: cytosine methylation and beyond

Key Points

  • Cytosine methylation has an important role in the regulation of mammalian gene expression. Additional forms of cytosine modification, including 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), are intermediates of 5mC that occur during DNA demethylation, and are hypothesized to have additional functional roles in cellular development.

  • Recent efforts in method development for quantifying cytosine modifications and mapping their genomic location, preferentially at single-base resolution, have focused on improving accuracy, increasing throughput, lowering sample input and reducing costs.

  • One major class of DNA methylation assays relies heavily on bisulphite treatment and sequencing approaches that provide single-base resolution. Whole-genome bisulphite sequencing (WGBS) and reduced representation bisulphite sequencing (RRBS) are widely used to generate genome-wide maps of DNA methylation. Application of low input methods such as tagmentation-based WGBS (T-WGBS) and post-bisulphite adaptor tagging (PBAT) allows for the detection of 5mC using input DNA from hundreds to thousands of cells.

  • Various targeted methylation-sequencing approaches, such as liquid hybridization and parallel amplification, have been developed to characterize DNA methylation at selected regions. These approaches are more cost effective for analysing large numbers of samples than non-targeted methods.

  • Improvements in several techniques, including restriction enzyme-based single-cell methylation assay (RSMA), limiting dilution bisulphite (pyro) sequencing and single-cell RRBS (scRRBS) and single-cell BS-seq (scBS-seq), have enabled 5mC detection in single cells.

  • Array-based methods are widely used in many studies of large cohorts. A major improvement is the dramatic increase in features per array and, hence, genome coverage.

  • The combination of chromatin immunoprecipitation (ChIP) assay and bisulphite sequencing (BS-seq) in sequential order allows for simultaneous detection of DNA methylation and other epigenetic marks. Profiling of fluorescence-labelled DNA fragments using nanofluidic devices has helped to correlate multiple chromatin marks with DNA methylation. In addition, the incidence of DNA methylation and nucleosome occupancy can be mapped with nucleosome occupancy and methylome sequencing (NOMe-seq).

  • A major challenge in the quantification of 5mC oxidation derivatives is their rarity in mammalian genomes. The use of specific antibodies and chemical or enzymatic modifications has enabled the enrichment of 5mC oxidation derivatives and the determination of their relative abundance in the genome, albeit with limited resolution. Chemical modifications coupled with BS-seq can be used to identify 5mC oxidation derivatives at single-base resolution, as in the oxidative bisulphite sequencing (oxBS-seq) and Tet-assisted bisulphite sequencing (TAB-seq) approaches for 5hmC quantification, the 5fC chemical modification-assisted bisulphite sequencing (fCAB-seq) and redBS-seq approaches for 5fC quantification, and the chemical modification-assisted bisulphite sequencing (CAB-seq) approach for 5caC quantification.

  • Third-generation DNA sequencing technologies, including single-molecule, real-time (SMRT) sequencing and nanopore sequencing, are very appealing for direct reading of 5mC and other DNA modifications on the same DNA molecule, with the potential advantages of speed, read length and the lack of chemical treatment. Nevertheless, the throughput and accuracy of these technologies are still not sufficient for routine use.

Abstract

Chemical modifications of DNA have been recognized as key epigenetic mechanisms for maintenance of the cellular state and memory. Such DNA modifications include canonical 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Recent advances in detection and quantification of DNA modifications have enabled epigenetic variation to be connected to phenotypic consequences on an unprecedented scale. These methods may use chemical or enzymatic DNA treatment, may be targeted or non-targeted and may utilize array-based hybridization or sequencing. Key considerations in the choice of assay are cost, minimum sample input requirements, accuracy and throughput. This Review discusses the principles behind recently developed techniques, compares their respective strengths and limitations and provides general guidelines for selecting appropriate methods for specific experimental contexts.

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Figure 1: Methods for quantifying 5-methylcytosine (5mC) and its oxidized derivatives.
Figure 2: Simultaneous detection of 5mC and other epigenetic modifications.
Figure 3: Assays for mapping 5-methylcytosine (5mC) oxidation derivatives at single-base resolution.

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Acknowledgements

The authors gratefully acknowledge members of the Zhang laboratory for their proofreading of the manuscript. We apologize to those authors whose works were not covered here due to space constraints. This work is funded by the US National Institutes of Health grants R01GM097253 and R01AG042187. D.H.D. is supported by a UCSD-CIRM pre-doctoral fellowship.

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Correspondence to Kun Zhang.

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Glossary

Partially methylated domains

(PMDs). Large contiguous regions of the genome (mean length ~ 153 kb) that display an intermediate level of DNA methylation (average <70%).

Ten-eleven translocation

(TET). DNA-binding enzymes that have been found to be methylcytosine dioxygenases in mammals and include TET1, TET2 and TET3. They were named for the genetic variant found in the TET1 gene sequence in acute myeloid and lymphocytic leukaemia.

Short interspersed nuclear elements

(SINEs). A subtype of transposable elements reverse-transcribed from RNA molecules. They do not encode a functional reverse transcriptase protein and cover a substantial portion of primate genomes, including all Alu sequences.

Long terminal repeats

(LTRs). Stretches of DNA sequences that are identical and repeat in hundreds to thousands of copies. LTRs were founds at the end of retrotransposons and act as insertion sites for viruses to insert their genetic material into the host genome.

Type I errors

The errors that result when there are false positives or when falsely rejecting the null hypothesis.

Type II errors

The errors that result when there are false negatives or incorrect failure to reject the null hypothesis

Laser-capture microdissection (LCM)

A method for isolating specific cells or specific areas from cell, tissue or organism samples using laser cutting under microscopic visualization.

Ligation capture

A method for capturing restriction enzyme-digested DNA molecules via the annealing of an oligonucleotide containing complementary sequences to adaptor oligonucleotides to the DNA molecules and to the adaptor oligonucleotides. The adaptors and DNA molecules are then ligated together, allowing for PCR amplification of only the ligated products.

Bisulphite padlock probe (BSPP) capture

A method for capturing the target CpG sites of bisulphite treated genomic DNA with bisulphite padlock probes (BSPP). The two capturing arms of the BSPPs are designed to flank the region of interest. After annealing padlock probes to target regions, polymerization is preformed to fill the gap and two ends of the padlock probe are joined together to form circularized DNA. The captured regions are amplified with barcoded adaptor primers and sequenced.

Liquid hybridization

A method for capturing fragmented DNA molecules via the annealing of biotinylated oligonucleotides to the DNA molecules. The binding of biotin to streptavidin beads allows for washing and removal of uncaptured DNA molecules, and subsequent elution of the captured DNA molecules.

Microdroplet PCR

Massively parallel PCR amplification of target sequences in microdroplets. The process involves the preparation of template and PCR mix in picoliter volume and primer droplets, combination of individual template and primer pair droplet, pooling the fused droplets for thermal cycling, and releasing of PCR amplicons for purification and sequencing.

Barcoded primers

Unique DNA sequences that are incorporated into adaptor sequences for tagging of different samples before sample pooling and shotgun sequencing.

Pyrosequencing

A sequencing-by-synthesis method based on the detection of phyrophosphate released upon nucleotide incorporation.

Shotgun library construction

The generation of a sequencing library involving random fragmentation of DNA and addition of adaptor sequences to both ends of DNA fragments before sequencing.

Transposase-based library construction

A procedure to generate a sequencing library using the transposase Tn5 to insert common transposon sequences to DNA. DNA segments are then amplified by annealing of primers to the transposon sequences.

Tn5 transposase

A member of the RNase superfamily of proteins that harbours retroviral integrases to catalyse random insertion of transposon DNA into target DNA.

Binning

A computational technique frequently used to reduce noise by grouping sequencing reads mapped to contiguous genomic segments.

Nucleosome

A basic unit of DNA packaging in eukaryotes that consists of section of DNA (~ 166 bp) wrapping around a histone core. Nucleosome structure helps to condense DNA into smaller volume. Nucleosomes are subunits of chromatin.

GpC methyltransferase (M.CviPI)

An enzyme from Chlorella virus that methylates all cytosines within the double-stranded dinucleotide recognition sequence 5′... GC...3′.

CCCTC-binding factor

(CTCF). A chromatin binding factor with highly conserved zinc finger domains that control binding to consensus DNA target sequences. CTCF regulates transcription by binding to chromatin insulators and preventing interaction between the promoter and enhancers or silencers.

Third-generation sequencing

A new progression of sequencing technology that aims to improve throughput and reduce sequencing cost and time. The main goals of third-generation sequencing are to eliminate the DNA amplification step before sequencing and to enable real-time signal monitoring.

β-glucosyltransferase

(βGT). An enzyme that transfers the glucose residue of uridine diphosphosphate glucose (UDP-Glc) specifically to the hydroxyl group of 5-hydroxymethylcytosine to generate β-glucosyl-5hmC (5gmC).

Click chemistry

A nonspecific chemical reaction that combines small modular units and is used to generate or label a substance. Azide alkyne Huisgen cycloaddition, in which an azide and alkyne interact to form a 1,2,3-triazole (with 5-membered ring) is the most popular click chemistry reaction. Click chemistry has been used for selectively labelling biomolecules.

Glucosylation

The process of transferring a glucose residue from a nucleotide sugar derivative, such as from uridine diphosphate glucose (UDP-Glc) to a target molecule.

J-binding proteins

Proteins that specifically bind to the base J (β-D-glucopyranosyloxymethyluracil), a modified form of uracil found in the DNA of a number of organisms, such as human pathogen Trypanosoma and the kinetoplastids. Base J is formed by hydroxylation of thymidine and subsequent glycosylation by glycosyltransferase enzyme.

Isoschizomer

Restriction enzymes that have the same recognition sequences and cleave at the same positions.

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Plongthongkum, N., Diep, D. & Zhang, K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet 15, 647–661 (2014). https://doi.org/10.1038/nrg3772

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