Benchmarks
Benchmarks
Enzymatic approaches and bisulfite
sequencing cannot distinguish
between 5-methylcytosine and
5-hydroxymethylcytosine in DNA
Colm Nestor1,2, Alexey Ruzov1, Richard R. Meehan1, and
Donncha S. Dunican1
1MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine,
Western General Hospital, Edinburgh, Scotland and 2Breakthrough Breast
Cancer Research Unit, University of Edinburgh, Western General Hospital,
Edinburgh, Scotland
BioTechniques 48:317-319 (April 2010) doi 10.2144/000113403
Keywords: cytosine hydroxymethylation; cytosine methylation; hmC; epigenetics; transcriptional repression
Supplementary material for this article is available at www.BioTechniques.com/article/113403.
DNA cytosine methylation (5mC) is highly abundant in mammalian cells and
is associated with transcriptional repression. Recently, hydroxymethylcytosine
(hmC) has been detected at high levels in certain human cell types; however,
its roles are unknown. Due to the structural similarity between 5mC and hmC,
it is unclear whether 5mC analyses can discriminate between these nucleotides.
Here we show that 5mC and hmC are experimentally indistinguishable using
established 5mC mapping methods, thereby implying that existing 5mC data
sets will require careful re-evaluation in the context of the possible presence of
hmC. Potential differential enrichment of 5mC and hmC DNA sequences
may be facilitated using a 5mC monoclonal antibody.
DNA cytosine methylation (5mC)
is a highly abundant heritable epigenetic mark frequently associated with
transcriptional repression, the genomic
location of which is central to the
processes it regulates, including development, X chromosome inactivation,
and human cancers (1,2). Genomic
5mC is commonly mapped using differential enzymatic digestion, bisulfite
sequencing, or a combination of both
methods (3). The recent discovery
of hydroxymethylcytosine (hmC) in
Purkinje neurons and embryonic stem
cells (4,5) via thin-layer chromatography has made it a priority to map this
mark on a genome-wide scale to understand its compartmentalization, tissue
specificity, and function. Considering
the similar structures of hmC and
5mC, it remains unclear how hmC
Vol. 48 | No. 4 | 2010
behaves in the classical assays used to
measure 5mC. Critically, the presence
of hmC in DNA can inhibit the binding
of the methyl-CpG binding protein
MeCP2 and the enzymatic function
of the maintenance methyltransferase DNMT1 (6,7). To test whether
restriction digestion can discriminate
between 5mC and hmC, we developed
an in vitro assay (Supplementary Figure
S1) based on PCR amplification, which
generates DNA templates that are
either unmethylated (dCTP), methylated (dmCTP), or hydroxymethylated
(dhmCTP) at cytosine.
We analyzed the human BRCA1
CpG island promoter since it contains
numerous methyl-sensitive restriction
sites and is known to be hypermethylated in cancer (8). BRCA1 PCR
products were digested with the
317
methylc y tosine-sensitive enzyme
HpaII or its methyl-insensitive isoschizomer MspI (Figure 1A and Supplementary Figure S2). As predicted,
digestion of the unmethylated BRCA1
amplicon is complete for both HpaII
and MspI, while methylated BRCA1 is
resistant to MspI digestion due to PCR
incorporation of modified cytosine at
the external nucleotide of its recognition site (hmCCGG) (9). Crucially,
HpaII digestion of hydroxymethylated
BRCA1 is completely inhibited to the
same degree as methylated BRCA1,
as evidenced by a 60-fold excess of
enzyme (Figure 1A). We extended
this analysis using the methyl-sensitive enzymes HpyCH4IV, HhaI,
and HaeIII and found that digestion
of unmethylated BRCA1 is complete
for all three enzymes (Figure 1B). In
contrast, hydroxymethylated BRCA1
is completely resistant to digestion,
indicating a generality in the refractory
nature of hydroxymethylated DNA to
digestion by methylcytosine-sensitive
restriction enzymes. A hydroxymethylated CDH1 (E-cadherin) substrate
is also resistant to HpyCH4IV, HhaI,
and HaeIII digestion, indicating that
hmC inhibition of these enzymes is not
sequence-specific (Figure S3). These
results suggest that existing vertebrate
DNA methylation data generated using
methylcytosine-sensitive enzymes may
have to take into account the potential
presence of hmC in DNA.
In vitro sodium metabisulfite
(Na 2 S 2 O 5) treatment of both free
and native hmC nucleotides generate
the intermediate 5′-methylenesulfonate, which is resistant to deamination (similar kinetics to 5mC
deamination) (10). This is in contrast
to cytosine, which is readily deaminated. One prediction from this study
was that both hydroxymethylated and
methylated genomic sequences may
be protected from deamination in
bisulfite DNA sequencing reactions,
suggesting that this technique cannot
distinguish these marks. To test this
possibility, we bisulfite-treated PCR
templates based on the mouse Oct4
(Pou5F1) promoter (prepared with
dCTP, dmCTP, or dhmCTP) followed
by sequence analysis of cloned products
(Figure 2). As anticipated, all cytosine
bases in unmethylated Oct4 are successfully converted to uracil bases, which
are interpreted as thymine by Taq DNA
polymerase. In contrast, cytosine bases
in the 5mC-methylated Oct4 template
remain unconverted subsequent to
www.BioTechniques.com
Benchmarks
B
A
Figure 1. Hydroxymethylcytosine-rich BRCA1 DNA sequences are resistant to digestion by methylcytosine-sensitive restriction enzymes. (A) Shown is a representative restriction digest of differentially cytidine-labeled human BRCA1 CpG island amplicons using HpaII and its methyl-insensitive isoschizomer
MspI. Unmethylated BRCA1 is efficiently cleaved by HpaII and MspI compared with the mock digested amplicons (compare dCTP lanes). Methylated
BRCA1 is resistant to HpaII (dmCTP lane, left panel). Methylated BRCA1 is resistant to MspI due to PCR incorporation of methylcytosine at the external
C in the CCGG HpaII/MspI recognition site (compare dmCTP lanes). Hydroxymethylated BRCA1 is resistant to HpaII cleavage at up to a 60-fold excess of
enzyme (compare dhmCTP and dmCTP lanes). (B) Representative restriction digests of BRCA1 amplicons using the methyl-sensitive enzymes HpyCh4IV,
HhaI, and HaeIII are shown. Similar to the HpaII result in panel A, these methyl-sensitive enzymes are inhibited by the presence of hydroxymethylcytosine in BRCA1 DNA (compare hmCTP and CTP lanes). -, no enzyme; +, 1 U enzyme; +++, 10 U enzyme. Arrow indicates BRCA1 amplicon; * indicate
digested BRCA1 fragments. L, DNA ladder.
bisulfite treatment (Figure 2A). Significantly, hydroxymethylated Oct4 DNA
is also completely resistant to chemical
modification and is indistinguishable
from methylated Oct4 after bisulfite
conversion (Figure 2A).
Due to PCR incorporation, all
cytosine bases in the Oct4 amplicons
will be unmethylated, methylated, or
hydroxymethylated, including those
in non-CpG contexts. To selectively
incorporate cytosine in CpG contexts,
we used a plasmid clone containing
a bisulfite-treated mouse Tex19.1
promoter sequence that contains
cytosine bases in the context of CpG
dinucleotides only. This approach
allowed us to determine if local
non-CpG modification of cytosines in
Oct4 PCR products impairs bisulfite
conversion. Notably, as observed for
the Oct4 template, the hydroxymethylated and methylated Tex19.1-derived
sequences are completely protected from
conversion to uracil/thymine subsequent to bisulfite incubation (Figure
2B). Therefore, non–CpG-modified
cytosines do not compromise bisulfite
conversion reactions. Together these
results indicate that methyl-sensitive
enzymes (used in Southern blotting,
methyl-sensitive PCR , COBR A
meu
analysis, etc.) and the gold-standard
bisulfite sequencing technique (locusspecific or whole-genome bisulfite
analysis) are unable to account for the
presence of cytosine hydroxymethylation in the genome.
Non-CpG cytosine methylation in
human embryonic stem cells has been
reported previously using bisulfite
sequencing (11,12); from our data we
infer that non-CpG hydroxymethylation
may exist in these cells and perhaps other
tissue types. Moreover, 98% of non-CpG
cytosine methylated sites in human ES
cell DNA are methylated on one strand
only (10). Experiments using the methylcytosine-sensitive enzyme PstI (cuts
CTGCAG but not MTGCAG; M =
methylcytosine) with CDH1 indicate
that hydroxymethylation and hemi-hydroxymethylation in non-CpG contexts
inhibit this enzyme activity (Figure
S4). Our enzymatic analyses show that
many methylcytosine-sensitive enzymes
cannot discriminate between methylated, hydroxymethylated, or—in one
case—hemi-hydroxymethylated DNA.
Furthermore, it is not possible to use
bisulfite sequencing to determine if
a particular unconverted cytosine
base (CpG and non-CpG contexts) in
post–bisulfite-treated DNA represents
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hydroxymethylated cytosine, methylated
cytosine, or even incomplete chemical
conversion.
In the future, 5mC and hmC
discrimination may involve selective
immunoprecipitation (with α-5mC)
of 5mC-enriched sequences followed
by bisulfite analysis of unbound
fractions that may be enriched for
hmC loci. The reciprocal experiment
may be possible when α-hmC becomes
available to enrich for bound hmC-enriched sequences. Such strategies
will be reliant on α-5mC and α-hmC
antibody specificities. To test α-5mC
specificity, we probed DNA dot blots
containing methylated, hydroxymethylated, and unmethylated BRCA1
and CDH1 DNA, demonstrating that
α-5mC (0.1 μg/mL) strongly detects
5mC compared with no affinity for
hmC and unmodified cytosine (Figure
2C). It is noted that at higher dilutions
(1 μg/mL), α-5mC can partially crossreact with hmC (Supplementary Figure
S5). To put this in context, the widely
used methylated DNA immunoprecipitation (MeDIP) protocol suggests using
the identical antibody at 20 μg/mL (13).
The difference in 5mC signal between
the BRCA1 and CDH1 templates may
reflect the relative cytosine content of
the amplicons. An additional caveat to
the immunoprecipitation approaches
is that 5mC and hmC may overlap at
common loci, which could hamper
selective enrichments.
In summary, current technologies
used to assay cytosine DNA methylation
do not account for hmC and now necessitate careful re-evaluation of existing
methylation datasets. The discovery of
hmC will require the development of
novel approaches to detect this novel
epigenetic mark.
Benchmarks
B
A
C
Figure 2. Hydroxymethylcytosine and methylcytosine are indistinguishable subsequent to bisulfite
conversion. (A) The organization of the Oct4 sequence analyzed is depicted (blue circles denote
CpG cytosines, red circles denote non-CpG cytosines, black circles denote methylated cytosine,
black circles with italicized H denote hydroxymethylated cytosine, and white circles denote unmodified cytosine). Genomic coordinates relative to the transcription start site are indicated. Multiple
(n = 10) representative independent bisulfite-converted clones derived from dmCTP, dhmCTP, or
dCTP Oct4 input DNA are shown. Efficient bisulfite conversion is confirmed in Oct4 dCTP bisulfiteconverted input DNA, which retains no cytosine bases (lower panel). In contrast, comparison of
bisulfite sequences derived from dmCTP and dhmCTP Oct4 input DNA indicates that hydroxymethylated and methylated cytosine is resistant to bisulfite conversion (top and middle panels). (B)
The organization of the Tex19.1 sequence analyzed is depicted (note that the analyzed sequence
contains cytosine bases in CpG dinucleotide contexts only). Similar to Oct4, hydroxymethylated and
methylated cytosines in Tex19.1 are completely protected from bisulfite conversion (top and middle
panels). In contrast, unmethylated Tex19.1 cytosines are completely deaminated by bisulfite conversion (bottom panel). (C) A representative DNA dot blot probed with α-5mC and α-ssDNA is shown.
Note that α-5mC has high specific affinity for 5mC BRCA1 and CDH1, with no affinity for hmC or
unmethylated BRCA1 and CDH1.
Acknowledgments
The authors wish to thank Hazel
Cruickshanks and Jamie Hackett for
critical assessment of the manuscript.
We thank James Reddington for the
Tex19.1 plasmid clone. We acknowledge
the funding provided by the Medical
Research Council and Breakthrough
Breast Cancer.
While this manuscript was in review, a
complementary yet non-overlapping study
was reported: Huang, Y., W.A. Pastor, Y.
Shen, M. Tahiliani, D.R. Liu, and A. Rao.
2010. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS
One 5:e8888.
Competing interests
The authors declare no competing
interests.
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Received 21 January 2010; accepted 26 February
2010.
Address correspondence to Donncha S. Dunican
or Richard R. Meehan, MRC Human Genetics
Unit, Western General Hospital, Crewe Road,
EH4 2XU, Edinburgh, Scotland. e-mail:
ddunican@hgu.mrc.ac.uk or richard.meehan@
hgu.mrc.ac.uk
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