Advances in the joint profiling technologies of 5mC and 5hmC

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Overview of the joint profiling technologies of 5mC and 5hmC.

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Cite this: DOI: 10.1039/d4cb00034j
Advances in the joint profiling technologies
of 5mC and 5hmC
Bo He,
ab
Haojun Yao
c
and Chengqi Yi *
ade
DNA cytosine methylation, a crucial epigenetic modification, involves the dynamic interplay of 5-methyl-
cytosine (5mC) and its oxidized form, 5-hydroxymethylcytosine (5hmC), generated by ten-eleven
translocation (TET) DNA dioxygenases. This process is central to regulating gene expression, influencing
critical biological processes such as development, disease progression, and aging. Recognizing the
distinct functions of 5mC and 5hmC, researchers often employ restriction enzyme-based or chemical
treatment methods for their simultaneous measurement from the same genomic sample. This enables a
detailed understanding of the relationship between these modifications and their collective impact on
cellular function. This review focuses on summarizing the technologies for detecting 5mC and 5hmC
together but also discusses the limitations and potential future directions in this evolving field.
1. Introduction
5-Methylcytosine (5mC), often termed the fifth base, plays
crucial biological roles like regulating tissue-specific gene
expression and silencing retroviral elements. The formation
of 5mC occurs when a methyl group is added to the 5th carbon
of cytosine. This process is facilitated by DNA methyltrans-
ferases (DNMTs) and predominantly takes place within CpG
dinucleotides.
1,2
The ten-eleven translocation (TET) enzymes
are known for their ability to iteratively oxidize DNA, leading to
demethylation.
3–5
This process results in the production of
5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and
5-carboxylcytosine (5caC).
6
The formation of 5fC and 5caC
allows for their active removal by thymine DNA glycosylase
(TDG) through the base excision repair (BER) pathway, facili-
tating active DNA demethylation.
7
Recently, 5hmC has gained
significant attention, being recognized as the sixth base in the
mammalian genome.
8
As the second most abundant DNA
modification following 5mC, 5hmC is now acknowledged to
be a distinct epigenetic mark with functions that contrast with
those of 5mC.
9–11
Several base-conversion chemistries are used to differentiate
unmodified cytosine (C) from its epigenetic variants. These
methods include: (1) bisulfite-based methods like whole-
genome bisulfite sequencing (WGBS), where bisulfite treatment
converts C to U, but not 5mC or 5hmC;
12
and (2) bisulfite-free
techniques such as enzymatic methyl-sequencing (EM-seq),
using Tet2 to oxidize 5mC and 5hmC to 5caC, followed by
A3A treatment, which converts C to U, excluding the newly
generated 5caC.
13
This method can be optimized for single-cell
5mC detection.
14
In TET-assisted pyridine borane sequencing
(TAPS), Tet2 oxidizes 5mC and 5hmC to 5caC, and then borane
reduction converts 5caC to DHU.
15
However, traditional meth-
ods for measuring 5mC often fail to distinguish between 5mC
and 5hmC. Thus, several base-conversion techniques have been
developed to separate 5mC and 5hmC, such as oxidative
bisulfite sequencing (oxBS-seq) and Tet-assisted bisulfite
sequencing (TAB-seq) under bisulfite-based methods,
16,17
as
well as APOBEC-coupled epigenetic sequencing (ACE-seq),
18
chemical-assisted C-to-T conversion of 5hmC sequencing
(hmC-CATCH),
19
chemical-assisted pyridine borane sequen-
cing (CAPS) in bisulfite-free approaches,
20
and single-step
deamination sequencing (SSD-seq).
21
OxBS-seq employs potas-
sium perruthenate for the oxidation of 5hmC to 5fC, followed
by bisulfite treatment.
16
TAB-seq uses beta-glucosyltransferase
(b-GT) to safeguard 5hmC, while Tet2 oxidizes 5mC to 5caC.
17
ACE-seq also utilizes b-GT for 5hmC protection, then applies
A3A treatment to deaminate C and 5mC.
18
hmC-CATCH
involves potassium ruthenate oxidation of 5hmC to 5fC, fol-
lowed by indanedione labeling.
19
Similarly, CAPS uses potas-
sium ruthenate for 5hmC oxidation to 5fC and then employs
borane reduction to convert it to DHU.
20
SSD-seq utilized a
screened engineered A3A protein (eA3A-v10) to selectively
a
Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary
Studies, Peking University, Beijing, China. E-mail: chengqi.yi@pku.edu.cn
b
Peking University Chengdu Academy for Advanced Interdisciplinary
Biotechnologies, Chengdu, China
c
College of Chemistry and Chemical Engineering, Hunan University, Changsha,
China
d
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences,
Peking University, Beijing, China
e
Department of Chemical Biology and Synthetic and Functional Biomolecules
Center, College of Chemistry and Molecular Engineering, Peking University,
Beijing, China
Received 2nd February 2024,
Accepted 21st March 2024
DOI: 10.1039/d4cb00034j
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deaminate C and 5mC, but not 5hmC.
21
Each method provides
unique approaches for accurate differentiation of these epige-
netic marks. These approaches are crucial in detecting and
differentiating various DNA modifications, enhancing our
understanding of epigenetic regulation.
5mC and 5hmC are different types of DNA modifications
that play important roles in epigenetic regulation. 5mC is
concentrated in promoter regions and is associated with repres-
sive gene expression.
2,22
However, 5hmC, concentrated in spe-
cific genomic regions, plays a unique role in gene expression
and cellular functions.
23,24
An example of its importance is
observed with the methyl-CpG binding protein 2 (MECP2),
where mutations cause Rett syndrome.
25
MECP2 binds strongly
to 5mC but not to 5hmC in the CG context, impacting epige-
netic regulation through its differential affinity.
26,27
Therefore,
distinguishing between 5mC and 5hmC is crucial for accurately
measuring cell-type-specific epigenomic profiles in health and
disease contexts. Thus, this review aims to summarize methods
for simultaneously profiling 5mC and 5hmC based on next-
generation sequencing methods and to discuss the challenges,
limitations, and potential prospects.
2. Detection methods based on
restriction-enzymes
These methods utilize various types of restriction enzymes,
capitalizing on their different responses to specific DNA modi-
fications. This approach enables selective detection by lever-
aging the unique activities of these enzymes towards distinct
epigenetic marks.
DARESOME
The DARESOME method achieves simultaneous profiling of
5mC and 5hmC by using a sequence of enzymatic reactions and
DNA tagging.
28
This process involves three key steps: (1) initial
digestion and tagging: the DNA is first digested using the Hpa
II enzyme, which targets unmodified CCGG sites, and then
tagged with specific adapters (U-tags). (2) Secondary digestion
and tagging: the remaining CCGG sites, which may contain
5mC, or 5hmC, are digested with Msp I enzyme and tagged
with H-tags. (3) Glycosylation and final tagging: 5hmC bases
are glycosylated to form b-glucosyl-5-hydroxymethyl-cytosine
(5gmC), protecting them from further digestion. A second
Msp I digestion is then performed, followed by tagging with
M-tags for fragments containing 5mC. These steps result in
tagged DNA fragments, which can be sequenced to identify the
modification states of CCGG sites across the genome. The use
of different tags for unmodified cytosine, 5mC, and 5hmC
allows for their simultaneous detection and differentiation.
However, this method primarily captures CCGG sites, repre-
senting only about 10% of all CG sites, thus limiting its scope.
Furthermore, the reliance on restriction-enzyme cutting
restricts the analysis to only one site per DNA fragment, making
it challenging to analyze multiple neighboring 5mC and 5hmC
sites within short ranges. This highlights a significant
limitation in the method’s ability to provide comprehensive
epigenetic profiling.
Dyad-seq
Dyad-seq combines enzymatic detection of modified cytosines
with traditional nucleobase conversion techniques
29
(Fig. 1).
It is designed to quantify all combinations of 5mC and 5hmC at
individual CpG dyads. Dyad-seq utilizes the different methods
to detect the DNA modifications on different strand. Firstly, it
uses two restriction enzymes, MspJI and AbaSI, to digest DNA.
MspJI is a unique restriction endonuclease recognized 5mC on
the top strand.
30
It distinctively cleaves DNA at a fixed distance
from a 5mC site. AbaSI is a specialized modification-dependent
restriction endonuclease that targets DNA containing 5hmC
and 5gmC on the top strand.
31
Following the digestion of DNA
with MspJI or AbaSI, the bottom strand of the fragmented DNA
molecules is captured through ligation to a well-designed
adapter, which is specific liagted to the bottom strand.
This adapter contains a random overhang, a sample barcode,
a unique molecule identifier (UMI), and a sequence for PCR
amplification. Next, to identify methylated cytosines on the
opposing DNA strand, the samples are treated in one of two
ways. They are either enzymatically processed with Tet2, b-GT
and A3A or they undergo sodium bisulfite treatment, which
specifically converts unmodified cytosines into uracil, while
methylated cytosines remain read as C (M-M-dyad-seq and
H-M-dyad-seq). Moreover, a minor adjustment to the enzymatic
conversion reaction, involving b-GT and A3A, specifically
allows for the detection of 5hmC on the opposite DNA strand
(M-H-dyad-seq and H-H-dyad-seq). Each combination uniquely
targets and identifies specific cytosine modifications, facilitat-
ing comprehensive analysis. The M-M-dyad-seq method was
adopted for single-cell methylome detection and further inte-
grated with single-cell RNA-seq. This method, termed scDyad
&T-seq, enables the simultaneous analysis of both the tran-
scriptome and the methylome at the single-cell level. This
method offers multiple assays for simultaneously detecting
5mC or 5hmC, and it can simultaneously quantify genome-
wide methylation levels and mRNA from the same cells.
However, this method, like DARESOME, depends on restriction-
enzyme cutting, which limits its ability to detect only one site per
DNA fragment. This restriction prevents the analysis of multiple
neighboring 5mC and 5hmC sites in close proximity, representing
a significant limitation in understanding the complex patterns of
these epigenetic marks. Additionally, the presence of natural DNA
breaksinthesamplecanleadtofalsepositiveresults,presentinga
challenge for accurate interpretation of the data.
3. Detection methods based on
chemical processing
Innovative chemical reactions play a crucial role in developing
new epigenome sequencing tools. This section highlights
chemical methods for simultaneously detecting 5mC and
5hmC, emphasizing the importance and potential of these
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techniques in advancing our understanding of the epigenome
and its implications in various biological processes and
diseases.
Six-letter-seq
The six-letter seq workflow, designed to resolve the four genetic
bases and the epigenetic modifications 5mC and 5hmC,
involves a series of sophisticated steps
32
(Fig. 2). It starts with
the fragmentation of DNA, followed by ligation with synthetic
DNA hairpin adapters. The strands are separated, and a com-
plimentary copy strand, lacking epigenetic modifications, is
synthesized by a synthetic hairpin. A pivotal aspect of the six-
letter seq method is its ability to accurately distinguish between
5mC and 5hmC, while simultaneously ensuring precise genetic
base calling within the same DNA fragment. DNA methylation
modifications are added to the CpG position of newly synthe-
sized DNA strands that do not contain modification information,
utilizing DNA methyltransferase 5 (DNMT5) for its specificity
in copy methylation.
33
Concurrently, 5hmC is safeguarded from
this copying process through glycosylation, executed by beta-
glycosyltransferase.
34
This selective glycosylation effectively pre-
vents the replication of 5hmC modifications onto the copy strand,
enabling a distinct differentiation between 5mC and 5hmC in the
sequencing analysis. Additionally, the 5mC is enzymatically oxi-
dized by a TET2 mutant
35
and subsequently uses b-GT to protect
all 5hmCs. Furthermore, the cytosines are deaminated to uracil by
APOBEC3A(A3A).
36
Meanwhile, UvrD helicase is added to facilitate
the generation of a single-stranded DNA substrate, which is
necessary for the efficient action of A3A.
37
The DNA constructs
are sequenced using a paired-end format. This involves read 1,
primed by P7, representing the original DNA strand, and read 2,
primed by P5, representing the synthesized copy strand. These
reads are then aligned pairwise, ensuring that read 1 is matched
with its complementary read 2. This alignment is critical for
accurately reconstructing the sequence and identifying both
genetic and epigenetic information from the DNA sample. Com-
putational alignment generates a resolved read, which is then
aligned to the reference genome for genetic variant and methyla-
tion analysis. This method ensures accurate differentiation
between genetic and epigenetic information in DNA analysis. This
method can distinguish between four bases and their epigenetic
information, using the complementary DNA strand for correction.
However, it is not suitable for single-cell detection due to its
complex procedural steps. This limitation highlights a trade-off
between the method’s comprehensive detection capabilities
and its practical applicability in more streamlined or single-cell
contexts.
Joint-snhmC-seq
The methods used in Joint-snhmC-seq involve a combination of
technologies and processes to simultaneously profile 5mC and
5hmC in single cells
38
(Fig. 3). First, lysed individual cells/
Fig. 1 Overview of dyad-seq. This figure illustrates the schematic and sequencing features of the dyad-seq for combined detection of 5mC and 5hmC.
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nuclei were treated with bisulfite, which can potentially achieve
chemical protection of 5hmC through the formation of
cytosine-5-methylenesulfonate (CMS) and can chemically modify
cytosine to uracil.
39
The converted single-stranded DNA (ssDNA) is
divided into two portions. One part is used directly to detect
signals of 5mC and 5hmC(snmC-seq2). The other part undergoes
an optimized enzymatic deamination process, typically using A3A.
This enzymatic treatment is efficient in converting 5mC to
thymine (T), facilitating the differentiation and analysis of these
epigenetic marks. After enzymatic deamination using A3A, the
treated single-stranded DNA (ssDNA) can be effectively captured
through random priming or post-deamination adapter
tagging(snhmC-seq2). This step is crucial for enabling low-
input bulk or single-cell 5-hydroxymethylome sequencing ana-
lysis, enhancing the method’s efficiency and applicability
for detailed epigenetic studies. The analysis of APOBEC3A-
deaminated ssDNA is concurrently conducted by two separate
methods: snhmC-seq2 for mapping 5hmC and snmC-seq2 for
identifying true 5mC, achieved by subtracting 5hmC signals
from the combined 5hmC and 5mC signals. This dual approach,
Fig. 2 Overview of six-letter-seq. This figure illustrates the joint profiling of genetics and epigenetics information.
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by physically linking two distinct epigenetic modalities—5hmC
and true 5mC—from the same cell, effectively bypasses the
challenge of cross-modal computational integration. This is a
significant advancement for reconstructing single-cell 5hmC
and 5mC profiles. However, this method’s indirect approach in
detecting 5mC and 5hmC can lead to false positives if enzyme
efficiency is not optimal. Additionally, the conversion of all
cytosines to uracil reduces sequence complexity, which may
result in a lower mapping ratio. Moreover, this technique
necessitates dividing genomic materials into two separate parts
for analysis, adding complexity to the process. These factors
underscore the need for careful consideration of methodo-
logical limitations in epigenetic studies.
SIMPLE-seq
SIMPLE-seq is a high-throughput method for the simultaneous
detection of 5mC and 5hmC in single cells.
40
In this process,
ruthenate (VI) oxidizes 5hmC to 5fC, which is then labeled by
malononitrile
19,41
(Fig. 4). This generates a specific ‘‘20C-to-T’’
signal at 5hmC sites after PCR amplification, while leaving C
and 5mC sites unchanged. Primer extension is used to record
this ‘‘5hmC-to-T’’ transition on the complementary strand.
The subsequent step involves TET-mediated oxidation, which
transforms 5mC in the original DNA template into 5caC. This
5caC is then subjected to borane reduction, resulting in the
formation of DHU.
15
This process is crucial as it generates a
second ‘‘C-to-T’’ signal specifically at the 5mC sites within the
same DNA molecule, facilitating the differentiation and
identification of these sites. Unmodified cytosines and other
bases remain unaffected throughout this process. To differentiate
between 5mC and 5hmC, both of which produce ‘‘C-to-T’’
mutations, a specific primer pre-deposited with a 5caC base
is designed to record the 5mC signals in the extension pro-
ducts. During the subsequent reaction targeting 5mC, this 5caC
base is converted to a ‘‘T’’ signal. This transformation is key in
distinguishing between the amplification products derived
from 5mC and 5hmC, allowing for accurate identification and
analysis of these epigenetic modifications in the DNA template.
SIMPLE-seq is scalable and offers base-resolution analysis of
both modifications. This allows the identification of the types
and locations of modifications from the same DNA molecule
in single cells. However, in scenarios where endogenous 5fC is
labeled and undergoes a C-to-T transition, it is incorrectly
identified as 5hmC, leading to potential false positive results.
4. Discussion
Various studies suggest that increased DNA methylation in
promoter regions typically correlates negatively with gene
expression.
42,43
However, single-cell multi-omics sequencing
indicates that this negative association between promoter
methylation levels and gene expression is only evident in a
small percentage of cases.
44–46
A potential reason for this dis-
crepancy could be the inability of previous detection methods
to differentiate between 5mC and 5hmC. This limitation might
lead to mixed signals, thereby obscuring the true relationship
between 5mC levels and gene expression. Thus, distinguishing
between 5hmC and true 5mC enhances our understanding of
gene regulation. Furthermore, the dynamic changes of 5mC
and 5hmC during cell fate transition can well explain how these
two modifications vary in the process of cell fate change. The
dual-modality epigenetic sequencing approach provides clarity
Fig. 3 Overview of Joint-snhmC-seq. The figure demonstrates the concurrent profiling of 5mC and 5hmC at the single-cell level.
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in distinguishing between 5mC and 5hmC, addressing the
challenges posed by traditional DNA methylome sequencing
methods that often conflate these two modifications. This
enhanced resolution in identifying true 5mC and 5hmC
profiles significantly improves the accuracy of integrating mul-
timodal data, thereby offering a more precise understanding of
epigenetic modifications and their implications. This advance-
ment marks a significant step forward in the field of epigenetics.
Recently, the emergence of spatial omics has significantly
enhanced our understanding of disease mechanisms through
the spatial mapping of cells within tissues.
47
Nonetheless, the
scope of current spatial technologies predominantly focuses on
gene expression levels, leaving other aspects like the epigenome
less explored. This highlights the necessity for further advance-
ments in spatial omics, aiming to incorporate a broader range
of omics data such as epigenomic information, for a more
comprehensive and accurate profiling of tissue structures.
Moreover, these methods focusing on 5mC and 5hmC could
be integrated with single-molecule multiplexed detection
methods for different modifications. With further modification,
it has the potential for extensive single-cell multimodal integra-
tion, including transcriptome, 3D genome structure, chromatin
states, and protein abundances.
48–59
This approach opens up
possibilities for comprehensive analysis and understanding of
cellular processes at the single-cell level by monitoring various
molecular layers simultaneously. Progress in the field of multi-
omics will significantly contribute to the development of sophis-
ticated therapeutic strategies. Additionally, it will enable the
creation of comprehensive atlases that encompass various omics
layers and temporal scales, enhancing our understanding of
health and disease.
Furthermore, in recent years, an increasing number of
studies have indicated that 5mC and 5hmC can serve as
biomarkers for early cancer screening.
60–67
5mC exhibits signi-
ficant up-regulation, particularly at certain key oncogenes, while
5hmC experiences a notable decrease in tumors.
66
Accurately
distinguishing the signal changes of 5mC and 5hmC can signifi-
cantly improve the precision of tumor diagnosis. These methods
provide a precise and effective technique for future disease
detection.
Conflicts of interest
The authors declare that they have no conflicts of interest in
this work.
Acknowledgements
We thank Dongsheng Bai and Jinmin Yang for discussion. This
study is supported by Beijing Natural Science Founda-
tion (no. Z220013), the National Natural Science Founda-
tion of China (91953201, 22207003, 22107006 and 32200467),
and Ministry of Science and Technology of China (2023
YFC3402200). The authors apologize for not being able to cite
all the publications related to this topic owing to space con-
straints of the journal.
Fig. 4 Overview of SIMPLE-seq. The figure depicts the concurrent profiling of 5mC and 5hmC within a single molecule.
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