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www.sciencemag.org/cgi/content/full/science.1210944/DC1
Supporting Online Material for
Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA
Yu-Fei He, Bin-Zhong Li, Zheng Li, Peng Liu, Yang Wang, Qingyu Tang, Jianping Ding, Yingying Jia, Zhangcheng Chen, Lin Li, Yan Sun, Xiuxue Li, Qing Dai, Chun-
Xiao Song, Kangling Zhang, Chuan He, Guo-Liang Xu*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 4 August 2011 on Science Express
DOI: 10.1126/science.1210944
This PDF file includes:
Materials and Methods Figs. S1 to S14 References
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Materials and Methods
5hmC and 5caC oligonucleotides.
5hmC oligonucleotides were obtained from Invitrogen, Shanghai. 5caC oligonucleotides were
synthesized using a published procedure (27).
Recombinant protein expression and purification.
For expression of Flag-tagged recombinant proteins, cDNA was cloned into a modified pcDNA4
(Invitrogen) vector. The expression construct was transfected into HEK293T cells using
polyethylenimine (PEI, Sigma). After culturing for 40 hrs, the cells were harvested and lysed in
lysis buffer containing 50 mM Tris, pH 7.4, 500 mM NaCl, 1% NP40, 1X protease inhibitors
without EDTA (Roche) and 1 mM PMSF. The recombinant protein was purified by using the
FLAG M2 affinity gel (Sigma) according to the manufacturer’s instructions. The eluted
recombinant protein was dialyzed with storage buffer (20 mM HEPES, pH 7.4, 50 mM NaCl and
50% glycerol) for 4 hrs at 4°C and then stored at -20°C. The homogeneity of the eluted protein
was determined using SDS-PAGE followed by Coomassie blue staining and immunoblotting
using an anti-Flag antibody (Sigma). The expression and purification of the catalytic domain of
mouse Tet1 (Tet1CD) from insect cells were performed following the methods described
previously (8).
Generation of Tdg knockdown mouse ES cell lines.
Lentiviral constructs carrying a Tdg shRNA expression cassette driven by the U6 promoter were
constructed by following a published protocol (28). Two target siRNA sequences, corresponding
to nucleotides 573-593 and 1180-1200 of the mouse Tdg gene (NCBI accession no.:
NM_172552) respectively, were inserted between the AgeI and EcoRI sites of the modified
pLKO.1 vector in which the puromycin resistance gene had been replaced with EGFP. MPI-II
ES cells (129Sv/Pas derived) were infected with lentivirus at an MOI of 60. Multiple clones
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expressing EGFP were picked and expanded. Clones deficient in TDG were selected by Western
blotting assay.
Generation of iPS cell lines deficient in TDG.
A Tdg targeting vector was prepared as described (29) and electroporated into C57BL/6 ES cells
for selection of homologous recombination. In positive ES clones, a genomic region
encompassing exons 3-7 coding for amino acids 67-275 was flanked by LoxP sites. For
establishing TDG-null iPS cell lines, embryonic fibroblasts were isolated from E13.5 embryos
with double floxed Tdg on a mixed C57BL/6-ICR genetic background and were induced to
reprogramming with standard factors Oct4, Sox2 and Klf4. Tdg was then inactivated by
Cre-mediated deletion. Detailed procedure for Tdg targeting will be described elsewhere.
DNA oxidative reaction.
An in vitro enzymatic assay was performed using a procedure described previously (8) with a
slight modification. Briefly, 5mC or 5hmC-containing PCR fragments were incubated with
nuclear extracts or purified Tet proteins in 50 mM HEPES, pH 8.0, 50 mM NaCl, 2 mM ascorbic
acid, 1mM 2-oxoglutarate, 100 μM Fe(NH4)2(SO4)2, 1 mM ATP, and 1 mM DTT for 40 min at
37°C. After treated with proteinase K, the DNA substrate was purified by phenol-chloroform
extraction and precipitated with DNAmate (TaKaRa) according to the manufacturer's protocols.
Analysis of 5mC derivatives using thin-layer chromatography (TLC).
After incubation with a Tet enzyme, the biotin-labeled DNA substrates were digested with
EcoNI for 1 hour at 37°C, purified by phenol-chloroform extraction and precipitated with
DNAmate (TaKaRa). Digested DNA substrates were then treated with calf intestinal alkaline
phosphatase (CIAP) (TaKaRa) for 1 hour and purified again. The DNA oligonucleotides were
then end-labeled with T4 Polynucleotide Kinase (TaKaRa) in the presence of 10 μCi of [γ-32P]
4
ATP for 1 hour at 37°C. Labeled fragments were incubated with streptavidin Sepharose High
Performance beads (GE Healthcare) for 1 min at RT. After washed with TE buffer, the bound
DNA fragments were digested with nuclease S1 (TaKaRa) for 15 min at 23°C. One microliter of
the digestion product was spotted on cellulose TLC plates (Sigma) and developed in isobutyric
acid: water: ammonium hydroxide (66: 20: 2). Plates were analyzed by phosphorimager scanning
using FujiFilm Fluorescent Image Analyzer FLA-3000.
Nucleotide markers were generated from synthetic oligonucleotide duplexes containing an
unmodified C (dCMP), a 5mC (5m-dCMP) or 5hmC (5hm-dCTP) at the internal C of the CCGG
sequence by restriction digestion, end-labeling and enzymatic hydrolysis as shown in fig. S1A.
HPLC analysis of DNA nucleoside hydrolysates.
DNA containing >20 ng of 5hmC o r 5caC was heat-denatured and hydrolyzed with 0.1 U of
nuclease P1 (Sigma) at 50°C for at least 1 hr or at 37°C overnight (the reaction volume is 18 μl,
containing 20 mM NaOAc, pH 5.3 and 0.2 mM ZnSO4). 2.1 μl of 10 X CIAP buffer and 0.9 μl
of calf intestinal alkaline phosphatase (TaKaRa) were then added to the reaction followed by
incubation for at least 2 hrs or overnight at 37°C. The reaction products were then analyzed on an
Agilent 1200 HPLC machine with an AQ-C18 column of 5-μm particle size, 25 cm x 4.6 mm.
The mobile phase was 10 mM KH2PO4, pH 3.7, running at 1 ml per min, and the detector was set
at 280 nm. 5hmC nucleoside standard was prepared by dephosphorylation of 5hmdCTP (Bioline)
using CIAP (TaKaRa), and 2’-deoxycytidine and 5-methyl 2’-deoxycytidine were bought from
Sigma.
Mass spectrometry experiments.
For identification of new species in the oxidation products formed by Tet enzymes in vitro and in
vivo, peaks of interest in HPLC analysis were collected using an automatic fraction collector
(Agilent Technologies). After desalting, fractions were resuspended in 20 μl of H2O/CAN (1:1).
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5 μl of the sample solution was used for analysis at Loma Linda University on an LTQ-Orbitrap
Velos mass spectrometer (Thermo Scientific) equipped with a static nano-electrospray interface
in negative ion mode (1.3 kV). The accuracy was 0.4-0.7 ppm (parts per million).
HPLC-ESI-MS/MS detection of 5caC was performed using a triple quadrupole mass
spectrometer (Agilent 6410 QQQ) set to MRM (multiple-reaction-monitoring) mode. MRM
signal was obtained from collision-induced dissociation of 2'-deoxy-5-carboxycytidine
(precursor ion at m/z 270) into three characteristic product ions at m/z 110, 154 and 227
(transitions 270 →110, 270 →154, and 270 →227). Judgment of the occurrence of 5caC in the
sample was based on elution of the three transitions on a reversed-phase HPLC column.
The LC-MS/MS system used at the Institute of Materia Medica, CAS was a Thermo Fisher
TSQ Quantum mass spectrometer (San Jose, CA) that is interfaced with an electrospray
ionization (ESI) probe and coupled with an Agilent 1100 LC system (Waldbronn, Germany).
Chromatographic separations were achieved using a Phenomenex Gemini 5m C18 column
(50×2.0 mm i.d.; 5 μm; Torrance, CA). The mobile phases (delivered at 0.25 ml/min) consisted
of H2O (containing 0.01% HCOOH) for A and acetonitrile (containing 0.01% HCOOH) for B.
An isocratic elution was performed at stop time of 5 min. The MS/MS parameters in the
negative-ion ESI mode were tuned to a maximum generation of deprotonated molecules for the
formate adduct of the analyte and its characteristic product ions. For selected reaction monitoring
of 5caC, the following precursor-to-product ion pairs were used: m/z 270.05→110.1 (the optimal
collision energy, 22 V), 270.05→93.2 (27 V), and 270.05→135.2 (24 V), respectively, with a
scan time of 0.1 s for each ion pair. The overall period was introduced to the mass spectrometer
for data acquisition. Standard solutions were prepared with 5caC diluted in DI water in
concentrations ranging from 0.46 to 333 ng/ml and standard curves were constructed using a
weighted (1/X) linear regression of the peak areas of the analyte (Y) against the corresponding
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concentrations of the analyte (X, ng/ml). The concentration of samples was calculated by
interpolation from the standard curves.
Incorporation of 14C in the 5-methyl group on cytosine within CpG dinucleotides.
5 μg of 101-bp PCR DNA fragments were incubated with 20 units of M.SssI CpG
methylransferase and 8 μl S-[methyl-14C]-adenosyl-L-methionine (14C-SAM, 1.48-2.22
GBq/mmol, PerkinElmer) in a total reaction volume of 200 μl for 2 hrs at 37°C. The DNA was
purified by phenol-chloroform extraction and ethanol precipitation before dissolved into 40 μl
H2O.
Base excision assay.
The base excision activity of various glycosylases toward different dsDNA substrates was
analyzed with the “nicking” procedure described previously (30) with slight modifications. The
substrate DNA containing a single G/U mismatch or a chemically modified cytosine at the MspI
site was prepared by annealing a modified oligonucleotide (5’-
GAGCGTGACMGGAGCTGAAA-3’, M= U, 5hmC or 5caC) end-labeled by [γ-32P]-ATP with
an equal molar amount of a G-strand oligonucleotide (5’- TTTCAGCTCCGGTCACGCTC-3’)
or a complementary oligonucleotide containing the corresponding modification at the internal C
at CCGG. The nicking reaction was carried out by incubating 40 nM of glycosylase or 40 μg of
nuclear extract with 10 nM of the substrate DNA in 25 mM HEPES, pH 7.8, 0.5 mM EDTA, 0.5
mM DTT and 0.5 mg/ml BSA for 30 min at 30oC. To convert the AP sites in the DNA duplex
into single-strand breaks, NaOH and EDTA were added to a final concentration of 90 mM and
10 mM, respectively, and the reaction was heated for 5 min at 100oC. The samples were then
resolved on a 20% denaturing polyacrylamide gel followed by autoradiography. Radioactivity of
the cleavage product band and the uncleaved substrate band was quantified using the Storm
instrument and ImageQuant software (Molecular Dynamics).
7
Detection of 5caC in Tet2-transfected HEK 293T cells.
The expression plasmid encoding FLAG-Tet2 full-length was transfected into HEK-293T cells
with Lipofectamine Reagent 2000 (Invitrogen). 48 hrs later, cells were collected for extraction of
genomic DNA using phenol/chloroform method. The genomic DNA was then treated with
RNase A/T1 twice (50 μg RNase A and 1000 U RNase T1 per 100 μg DNA), at least 4 hrs each
time. The DNA was precipitated in 70% ethanol, dissolved in TE buffer, and hydrolyzed with
nuclease P1 (1 U per 100 μg DNA) overnight. CIAP (TaKaRa) was used to remove the
5’-phosphate from the nucleotides. Samples were concentrated to 20-30 μl for HPLC-MS/MS
detection.
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Fig. S1. Detection of a 5mC-modifying activity in the nuclear extract of HEK 293T cells
transfected with Tet2.
(A) Procedure for the detection of 5mC-modifying activity in nuclear extracts.
(B) Detection of 5mC-modifying activity in HEK 293T nuclear extract by TLC. A 32P spot on a
TLC plate with unknown identity is indicated with ‘X’. Lanes 6-8 are 32P end-labeled
nucleotide standards, 2'-deoxycytidine-5'-monophosphate (dCMP), 5-methyl
2'-deoxycytidine-5'-monophosphate (5m-dCMP) and 5-hydroxymethyl
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2'-deoxycytidine-5'-monophosphate (5hm-dCMP) which were derived from enzymatic
hydrolysis of synthetic DNA oligonucleotide duplexes with the same sequence containing a
corresponding modified nucleotide 3’ of the EcoNI cleavage site (refer to A). Note that a
compound of a higher polarity has a smaller Rf value because it interacts more strongly with
the polar adsorbent on the TLC plate.
Fig. S2. Coomassie staining of Flag-tagged full-length Tet proteins purified from transfected
HEK 293T cells.
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Fig. S3. DNA dioxygenases Tet1 and Tet3 also oxidize 5mC and 5hmC to 5caC. The two upper
HPLC profiles show peaks of the standard nucleosides (indicated with downward arrows).
11
Fig. S4. The C-terminal domains of all three Tet enzymes are capable to catalyze oxidation of
5mC to 5caC.
(A) Flag-tagged Tet1, Tet2 and Tet3 CD proteins purified from transfected 293T cells. The gel
was stained with Commassie Blue.
(B) HPLC profiles of nucleosides derived from enzymatic hydrolysis of DNA samples
containing 5mC upon treatment with the catalytic domain of three Tet proteins. The bottom
HPLC profile shows nucleoside standards derived from synthetic DNA.
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Fig. S5. 5mC and 5hmC oxidation activities of the Tet1 catalytic domain purified from Sf9
insect cells. The Tet1 CD was added with an N-terminal Flag-tag and a C-terminal 6xHis-tag to
facilitate the purification.
(A) Coomassie-stained SDS-PAGE gel of purified Tet1CD protein.
(B) HPLC profiles showing the generation of 5caC from 5mC and 5hmC.
13
Fig. S6. Cofactor dependencies of Tet2 for the oxidation of 5mC to 5caC in vitro.
(A) The bar graph shows the rates of 5caC generation in the presence or absence of cofactors
indicated. ‘all’ indicates that ATP, 2-oxoglutarate (2-OG), 100 μM Fe(NH4)2(SO4)2(Fe2+),
and DTT were added in the reaction buffer. The DNA substrate used in the oxidation reaction
was a 101-bp fragment containing 42 methylcytosines incorporated by PCR using 5-methyl
dCTP. 300 ng of the substrate (4.5 pmol, corresponding to 189 pmol of 5mC) were incubated
with 4 μg of purified full-length Flag-Tet2 protein (18 pmol) for 1 hour. Activities were
14
determined by HPLC measurement of the 5caC product (see Methods). With regard to the
ATP requirement, Tet2 is similar to thymine-7-hydroxylase (31). The latter enzyme catalyzes
sequential oxidation of the methyl group of thymine, generating 5-carboxyl uracil.
(B) Differential stimulations of ATP on the conversion of 5mC to 5hmC or 5caC by Tet2. ATP
concentrations tested were 0, 0.1, 0.3, 0.5, 1 mM. 300 ng of the substrate (as above) were
incubated with 8 μg of purified Flag-Tet2 full-length protein (36 pmol) for 1 hour. The
amounts of 5hmC and 5caC generated from 5mC in each reaction were determined by
HPLC. A similar observation was made with a different batch of Flag-Tet2 preparation. The
distinctive response of 5hmC and 5caC generation to fluctuations of ATP and 2-OG
concentrations (data not shown) implies that the oxidation outcomes of 5mC could be
regulated by factors including those reflecting a certain cellular metabolic status.
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Fig. S7. Behavior of 5caC in restriction digestion and bisulfite sequencing analyses.
(A) Resistance of 5caC DNA against digestion by HpaII and MspI. A synthetic 20-mer
oligonucleotide duplex analyzed (with the sequence given in B) contained a 5caC, C or
5hmC at the internal C of a CCGG sequence. Shown is an autoradiogram of 32P end-labeled
oligoduplexes resolved on PAGE upon restriction digestion. Note that MspI is insensitive to
5hmC as previously reported (8).
(B) 5caC is displayed as C in conventional bisulfite sequencing analysis. Presence of 5caC in
DNA did not cause biased PCR amplification (data not shown). Note that 5hmC appears as
16
5mC as reported (32). ‘X’ in the original sequence is either C, 5caC or 5hmC.
Fig. S8. Processive activity of purified Flag-Tet2 in the conversion of 5mC to 5caC. Shown is
bisulfite sequencing data of methylated DNA before and after incubation with Flag-Tet2. A
101-bp DNA region subjected to the analysis contained 21 CpG dinucleotides but no other C.
The DNA was methylated in vitro with bacterial CpG methyltransferase M.SssI. 100 ng of the
methylated DNA (1.5 pmol) was then incubated with 2 μg of purified Flag-Tet2 full-length
protein (10 pmol) for 1 hour. Open circles represent unmethylated cytosine or 5caC, and filled
circles represent mC or 5hmC, respectively. Each line represents a unique template strand
analyzed. Since the fraction of unmethylated cytosines remaining in the substrate after in vitro
M.SssI methylation was only 2.6% (upper panel), most of open circles in the product profile
(lower panel) should represent 5caC. Note that the percentage of 5caC (61.8%) is close to the
5caC level (64.2%) detected in HPLC (data not shown).
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Fig. S9. Confirmation of Flag-Tet2 expression in transfected HEK 293T cells. Immunostaining
of Tet2 full-length (FL), catalytic domain (CD) and enzymatically inactive mutant was
performed using anti-Flag antibody.
18
Fig. S10. DNA substrates and enzymes used in glycosylase assay.
(A) The DNA substrates. They were prepared by annealing two oligonucleotides, one of which
was end-labeled with 32P.
(B) Commassie blue staining of purified glycosylases. The Flag-tagged glycosylases were
purified from transfected 293T cells using FLAG M2 affinity beads. GST-SMUG1 was
purified from E. coli cells. N151A is a catalytically inactive mutant of TDG.
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Fig. S11. Preference of TDG for hemi-carboxylated DNA substrate. A hemi-hydroxymethylated
(upper panel) or hemi-carboxylated substrate (lower panel) was used in all lanes except for lane
1 in which a fully hydroxylmethylated or carboxylated substrate was incubated with the
wild-type TDG (labeled with ‘*’). TDG displayed stronger base excision activity toward
hemi-carboxylated substrate than fully carboxylated one (compare lane 3 to 1).
20
Fig. S12. Depletion of TDG in mouse ES cells by siRNA. Stable cell lines transduced with a
lentiviral siRNA knockdown construct were established. Shown is Western analysis of extracts
from two independent cell lines (a and b) expressing each shRNA. Antibodies used are indicated
at the left.
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Fig. S13. Detection of 5caC in TDG-deficient mouse iPS cells.
(A) Characterization of TDG-deficient iPS cell lines. Shown is real-time PCR data indicating
that expression of Oct4 and Tet1 has been reactivated during reprogramming. All iPS cell
lines were able to form colonies with typical ES morphology (data not shown). For each Tdg
genotype, two independently established cell lines (1 and 2) were validated.
(B) Lack of 5caC base excision activity in TDG-deficient (-/-) iPS cells. Nuclear extracts were
tested as in Fig. 4A.
(C) Detection of 5caC in Tdg-null iPS cells by triple quadrupole mass spectrometry. Genomic
DNA from TDG-proficient (red line) and TDG-deficient iPS cells (pink line) were analyzed.
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Synthetic 5caC nucleoside was used as a reference (blue line). At least two independent iPS
cell lines of each genotype (Tdg -/-, f/- and f/f) were analyzed and 5caC was only detected in
Tdg-null (-/-) cell lines.
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Fig. S14. Model for DNA demethylation promoted by Tet and TDG. Consecutive hydroxylation
of 5mC (and 5hmC) generates further oxidized 5caC that is recognized and excised by TDG. The
resulting abasic site in turn induces the base excision repair pathway, leading to the incorporation
of unmethylated cytosines.
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References
1. R. Jaenisch, A. Bird, Nat Genet 33 Suppl, 245 (Mar, 2003).
2. S. Simonsson, J. Gurdon, Nat Cell Biol 6, 984 (Oct, 2004).
3. X. J. He, T. Chen, J. K. Zhu, Cell Res 21, 442 (Mar, 2011).
4. Z. Liutkeviciute, G. Lukinavicius, V. Masevicius, D. Daujotyte, S. Klimasauskas, Nat Chem Biol 5, 400 (Jun, 2009).
5. C. P. Walsh, G. L. Xu, Curr Top Microbiol Immunol 301, 283 (2006).
6. S. C. Wu, Y. Zhang, Nat Rev Mol Cell Biol 11, 607 (Sep, 2010).
7. C. Dahl, K. Gronbaek, P. Guldberg, Clin Chim Acta 412, 831 (May 12, 2011).
8. M. Tahiliani et al., Science 324, 930 (May 15, 2009).
9. T. Pfaffeneder et al., Angew Chem Int Ed Engl, (Jun 30, 2011).
10. D. Globisch et al., PLoS One 5, e15367 (2010).
11. T. Lindahl, R. D. Wood, Science 286, 1897 (Dec 3, 1999).
12. D. Cortazar et al., Nature 470, 419 (Feb 17, 2011).
13. M. T. Bennett et al., J Am Chem Soc 128, 12510 (Sep 27, 2006).
14. B. Hendrich, U. Hardeland, H. H. Ng, J. Jiricny, A. Bird, Nature 401, 301 (Sep 16, 1999).
15. H. E. Krokan, R. Standal, G. Slupphaug, Biochem J 325 ( Pt 1), 1 (Jul 1, 1997).
16. R. J. Boorstein et al., J Biol Chem 276, 41991 (Nov 9, 2001).
17. R. Metivier et al., Nature 452, 45 (Mar 6, 2008).
18. B. Zhu et al., Proc Natl Acad Sci U S A 97, 5135 (May 9, 2000).
19. S. Cortellino et al., Cell 146, 67 (Jul 8, 2011).
20. W. A. Pastor et al., Nature 473, 394 (May 19, 2011).
21. G. Ficz et al., Nature 473, 398 (May 19, 2011).
22. C. X. Song et al., Nat Biotechnol 29, 68 (Jan, 2011).
23. H. Wu et al., Genes Dev 25, 679 (Apr 1, 2011).
24. K. Williams et al., Nature 473, 343 (May 19, 2011).
25. Y. Xu et al., Mol Cell 42, 451 (May 20, 2011).
26. H. Wu et al., Nature 473, 389 (May 19, 2011).
27. Q. Dai, C. He, Org Lett 13, 3446 (Jul 1, 2011).
25
28. J. Moffat et al., Cell 124, 1283 (Mar 24, 2006).
29. P. Liu, N. A. Jenkins, N. G. Copeland, Genome Res 13, 476 (Mar, 2003).
30. P. Neddermann, J. Jiricny, J Biol Chem 268, 21218 (Oct 5, 1993).
31. B. J. Warn-Cramer, L. A. Macrander, M. T. Abbott, J Biol Chem 258, 10551 (Sep 10, 1983).
32. Y. Huang et al., PLoS One 5, e8888 (2010).