Mechanism and stem-cell activity of 5-carboxycytosine decarboxylation determined by isotope tracing

Article abstract of DOI:10.1002/anie.201202583

Eraserhead: Stem cells seem to erase epigenetic information by decarboxylation of the newly discovered epigenetic base 5-carboxycytosine (caC; see picture). This reaction is likely to involve a nucleophilic attack of the C5-C6 double bond. Copyright

Full text of DOI:10.1002/anie.201202583

Angewandte
Chemie
DOI: 10.1002/anie.201202583
Epigenetics
Mechanism and Stem-Cell Activity of 5-Carboxycytosine
Decarboxylation Determined by Isotope Tracing**
Stefan Schiesser, Benjamin Hackner, Toni Pfaffeneder, Markus Mꢀller, Christian Hagemeier,
Matthias Truss,* and Thomas Carell*
5-Methylcytosine (mC) is an important, well-known nucleo-
base modification that is involved in many biological pro-
cesses, including gene expression, genomic imprinting, X-
chromosome inactivation, and suppression of transposable
elements.[1–3] Recently it was discovered that mC can be
further processed in neurons and in embryonic stem cells by
TET enzymes, which utilize 2-ketoglutarate, to oxidize mC
first to 5-hydroxymethylcytosine (hmC)[4,5] and further to 5-
formylcytosine (fC)[6] and 5-carboxycytosine (caC)[7,8]
(Scheme 1).[9,10] It is
special glycosylases,[12–15] such as the thymine–DNA glycosy-
lase (TDG), which was shown to also cleave the glycosidic
bonds of fC and caC, as well as of 5-hydroxymethyluridine
(hmU).[15,16] The latter is formed after deamination of hmC.[15]
This base cleavage reaction first generates an abasic site,
which is repaired by the base excision repair system (BER) by
insertion of dC (Scheme 1, lower part). This whole process
leads in summary to an exchange of mC by dC. The second
mechanism is thought to involve retro-Aldol-type chemistry
currently
believed
that this oxidation
chemistry is involved
in a process of active
demethylation, which
allows
cells
to
exchange mC with
unmodified 2’-deoxy-
cytidine
(dC)
to
revert the biological
effects caused by
cytosine
methyla-
tion.[11] Two possible
active demethylation
mechanisms are cur-
rently being inten-
sively
investigated
Scheme 1. Active demethylation pathways involving elimination (mechanism 2) of formaldehyde from hmC, formic
(Scheme 1). Mecha-
acid from fC, or decarboxylation of caC. In contrast, mechanism 1 involves the base excision repair (BER)-based
nism 1 is based on replacement of fC and caC by dC, or of hmC by dC after prior deamination of hmC to hmU.
[*] S. Schiesser,[+] B. Hackner,[+] T. Pfaffeneder, M. Mꢀller,
starting with hmC[17,18] or fC. Alternatively, decarboxylation
Prof. Dr. T. Carell
of caC (Scheme 1) is a highly attractive alternative, because
this mechanism allows exchange of mC by dC without
formation of intermediate strand breaks, which are DNA
lesions that are known to cause genome instability, and
without formation of potentially harmful side products, such
as formaldehyde. Although decarboxylation of caC has never
been observed, similar transformations are known for orotate
and isoorotate, which are decarboxylated to uracil.[19]
Center for Integrated Protein Science at the Department of
Chemistry, Ludwig-Maximilians Universitꢁt Mꢀnchen
Butenandtstrasse 5–13, 81377 Mꢀnchen (Germany)
E-mail: matthias.truss@charite.de
thomas.carell@lmu.de
Dr. C. Hagemeier, Dr. M. Truss
Charitꢂ Universitꢁtsklinikum, Otto-Heubner-Centrum fꢀr Kinder
und Jugendmedizin, Klinik fꢀr Allgemeine Pꢁdiatrie, Labor fꢀr
Pꢁdiatrische Molekularbiologie
To gain deeper insight into the putative decarboxylation
of caC in stem cells, we developed a sensitive isotope tracing
experiment. A double 15N-labeled caC phosphoramidite
reagent [15N2]-caC was developed, which we incorporated
into the 30 mer oligonucleotide D1 with a sequence from the
Ecat1 promoter[23] that is known to be the subject of active
demethylation. The natural occurrence of the double 15N
isotopologue of dC ([15N2]-dC) is extremely low, which allows
us to monitor the chemistry that operates on the caC
Ziegelstrasse 5–9, 10098 Berlin (Germany)
[+] These authors contributed equally to this work.
[**] We thank the Excellence cluster CiPSM and the collaborative
research center SFB749 as well as the DFG, grant CA275/8-4, the
Volkswagen Foundation and NGFNplus (01GS0870) for financial
support. T.P. and S.S. thank the Fonds der Chemischen Industrie for
pre-doctoral fellowships.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202583.
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compound by high-resolution mass spectrometry. The syn-
thesis of the [15N2]-caC phosphoramidite is depicted in
Scheme 2 (see also the Supporting Information). The starting
point is [15N2]-uracil 1, which was prepared from [15N2]-urea
and propiolic acid.[20] Vorbrꢀggen nucleosidation with the
bis(toluoyl)-protected chlororibofuranoside[21] furnished the
b-configured nucleoside 2 in 60% yield. Electrophilic iodi-
nation to 3,[22] conversion of the C4-keto function into the
amine as needed for the cytosine base (4), and protecting-
group exchange gave the key intermediate 5. Pd-catalyzed
CO insertion and quenching of the Pd–CO intermediate with
methanol provided the [15N2]-caC precursor 6 in about 65%
yield. Double benzoyl protection of the C4-amino group (7)
and cleavage of the TBS groups (8) was followed by
protection of the 5’OH group with dimethoxytritylchloride
(DMT-Cl; 9). We subsequently converted the 3’OH group
into the final phosphoramidite 10 using standard procedures.
The phosphoramidite was used to synthesize the 30 mer
oligonucleotides ODN1 and ODN2. The two strands were
finally hybridized to give the DNA duplex D1 with the
sequence of the Ecat1 promoter containing two [15N2]-labeled
caC derivatives.
In one of the DNA strands (ODN1), we also inserted the
base 5-octadienyl-dU (Scheme 2, bottom) and subsequently
used mild click chemistry with the depicted biotin azide
(Scheme 2, bottom; Supporting Information) to attach
a biotin label to 5-octadienyl-dU, giving the biotinylated
uridine Y.[23] All of the DNA strands needed for the experi-
ment were carefully purified by HPLC to obtain the labeled
DNA material in extremely high purity (> 99.9%). After
successful click chemistry, we purified the biotinylated
oligonucleotide again using a second HPLC purification
step. The identity of the oligonucleotides was confirmed in all
cases by MALDI-TOF mass spectrometry (Scheme 2; Sup-
porting Information).
For the isotope tracing experiment depicted in Figure 1a,
we added the DNA duplex D1 (ODN1 + ODN2) containing
the two [15N2]-labeled caC bases and the biotin tag to nuclear
extracts of mouse embryonic stem cells (mESC). After 1 h of
incubation at 378C (for details see Supporting Information),
we re-isolated the biotinylated oligonucleotide with the help
of streptavidin-bound magnetic beads. The isolated DNA was
subsequently totally digested (Supporting Information) and
the digest was analyzed by HPLC-MS using a high-resolution
mass spectrometer (Orbitrap XL; for details, see the Sup-
porting Information). The obtained data are depicted in
Figure 1b–e. We first investigated the results of the control
experiments. To this end we treated D1 with non-mESC
nuclear extracts and we incubated D1 with buffer only. The
mass-spectrometric analysis of the control experiments (Fig-
ure 1d,e) provided two distinct signals with m/z = 230.1014
and 230.0976 in the mass range of the dC nucleoside
(Figure 1e). These m/z signals correspond to the naturally
occurring isotopologues [13C2]- and [18O]-dC, respectively, and
[15N,13C]-dC. The exact mass expected for double 15N-
containing [15N2]-dC was not detected in all of these control
experiments, even when we digested and analyzed higher
amounts of DNA (2.5 nmol dC vs 0.9 nmol dC; Figure 1d,e).
We then analyzed the digests obtained from D1 after
Scheme 2. Synthesis of the [15N2]-labeled caC phosphoramidite and of
the DNA strands ODN1 and ODN2 containing one [15N2]-labeled caC
nucleobases each. The biotin label was inserted using click chemistry.
Reagents and conditions: a) polyphosphoric acid, 958C, 19 h; b) 1.
HMDS, TMS-Cl, 1208C, 1 h; 2. Hoffer’s chlorosugar, CHCl3, RT, 2.5 h;
c) CAN, LiI, MeCN, 808C, 2 h; d) 1. 1,2,4-triazole, POCl3, TEA, 308C,
overnight; 2. NH4OH, 1,4-dioxane, RT, 10 min; e) 1. K2CO3, MeOH,
RT, 19 h; 2. TBS-Cl, imidazole, DMF, RT, 4 days; f) [Pd(MeCN)2Cl2],
CO, MeOH, 608C, 17 h; g) BzCl, pyridine, 08C to RT, 19 h; h) HF-
pyridine, EtOAc, RT, 14 h; i) DMT-Cl, pyridine, RT, 16.5 h; j) Diisopro-
pylammonium tetrazolide, 2-cyanoethyl-N,N,N’,N’-tetraisopropylphos-
phordiamidite, CH2Cl2, RT, 17 h. HMDS=Hexamethyldisilazane,
TMS=trimethylsilyl, CAN=cerium ammonium nitrate, TEA=triethyl-
amine, TBS=tert-butyldimethylsilyl, DMT=dimethoxytrityl.
incubation with mESC nuclear extract. Surprisingly we
noted that after 1 h of incubation with mESC nuclear extracts,
the [15N2]-labeled dC species was indeed detected in the total
digest of the re-isolated duplex D1. The compound [15N2]-dC
eluted with a retention time of 7.18 min. The obtained mass
2
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provided no evidence for decarboxylation (data
not shown). This is not surprising because decar-
boxylation would furnish a high-energy cytosine
À
carbanion. In contrast, saturation of the C5 C6
double bond followed by tautomerization of the
C4-amino group would give an imine intermediate
with the substructure of a b-iminocarboxylic acid,
which would allow efficient decarboxylation
(Figure 2).[10,24] To investigate this idea, we re-
duced the 5-carboxy-dC methyl ester 11 with
NaBH4. The reduced compound 12 is stable and
was isolated and fully characterized (Figure 2a;
Supporting Information). However, upon saponi-
fication of the methyl ester, rapid decarboxylation
to compound 13 is observed. Subsequent treat-
ment of the dihydro-dC derivative with DDQ
furnishes the TBS-protected compound dC. This
À
result shows that saturation of the C5 C6 double
bond is critical for efficient decarboxylation, as
suggested recently.[10] As reaction with a HÀ spe-
cies is unlikely to occur naturally, we then treated
TBS-protected caC with a thiol/imidazole mixture,
hoping for a more temporary 1,4-addition fol-
lowed by decarboxylation and elimination. This
chemical cascade is indeed observed (data not
shown). Treatment of the caC nucleoside with
octane thiol in presence of imidazole gave rise to
a new spot on the TLC plate. This spot was not
observed in the absence of imidazole, showing that
some proton catalysis is required for the reaction.
Isolation of the compound and full characteriza-
tion (Supporting Information) showed that the
new compound is indeed the TBS-protected dC.
This compound is generated by decarboxylation
followed by elimination of the added thiol. To
investigate if this chemistry would be compatible
with the decarboxylation of caC embedded in an
oligonucleotide (Figure 2b,c), we again used the
isotope tracing method. For the experiment, we
incubated the [15N2]-caC-containing DNA strand
ODN2 with different amino acids. Indeed, when
we added the amino acid cysteine or combinations
Figure 1. a) Depiction of the nuclear extract experiment. b,d) HPLC-MS analysis of
D1 treated with mESC nuclear extract (b) and non-mESC extract (d). UV traces of
HPLC runs at the retention time of dC (top), as well as the mass traces for dC
(228.0948–228.1008, middle), and the mass traces for [15N2]-labeled dC (230.0890–
230.0940, bottom). c,e) High-resolution mass spectra (m/z=230.0700–230.1300) of
D1 treated with mESC nuclear extract (c) and non-mESC extract (e).
spectrometry signal has the exact mass of m/zfound = 230.0913
(Figure 1c). The signal is nicely separated from the signals of
the other naturally occurring isotopologues [15N,13C]-dC
(m/z=230.0976) and [18O/13C2]-dC (m/z = 230.1016) and thus
is clearly detectable. Furthermore, the measured high-reso-
lution data for the compound [15N2]-dC agrees very well with
the calculated exact mass of [15N2]-dC (m/zcalcd = 230.0920).
The data show all together that mESC nuclear extract has the
capability to decarboxylate caC to dC. Whether this activity is
caused by a specific enzyme or by other factors present in the
extract requires further investigation.
To investigate the reactions that would allow stem cells to
decarboxylate caC, we first heated the caC nucleoside and an
oligonucleotide containing a caC base. However, under no
circumstances was decarboxylation observable. Even when
the caC nucleoside was heated to reflux in water for several
hours, the mass spectrometric analysis of the reaction solution
of cysteine with histidine/arginine to an aqueous solution of
ODN2, decarboxylation was detectable (Figure 2b). To con-
firm that decarboxylation is occurring, we re-isolated the
DNA after reaction overnight, digested the oligonucleotide
down to the nucleoside level and analyzed the mixture using
high resolution HPLC-MS. Figure 2b (left) shows the data
obtained for the cysteine/histidine combination. The data
depicted in Figure 2b (right) are the results of the cysteine/
arginine experiment. In both cases, the [15N2]-dC signal with
an
m/z value of 230.0922 was clearly detected. The amount of
decarboxylation increases with an increasing concentration of
thiol in solution and was in total around 5% (half-saturated
solution of amino acids, 508C, overnight).
In summary, using isotope tracing with a DNA strand in
which we incorporated double 15N-labeled caC, we could gain
first evidence that stem-cell nuclear extracts have the ability
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hydroxide anion is generated that is thought to
react with the C6 position. This reaction is
followed by a decarboxylation that converts
isoorotate to uracil.[26,27] The chemical reactivity
that we suggest is consequently in agreement
with other known enzymatic transformations. We
believe that the decarboxylation of caC by stem
cells is an important mechanism in the frame-
work of active genome demethylation.
Received: April 3, 2012
Published online: && &&, &&&&
Keywords: 5-carboxycytosine · decarboxylation ·
.
epigenetics · isotopic labeling · stem cells
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Angewandte
Communications
Communications
Epigenetics
Eraserhead: Stem cells seem to erase
epigenetic information by decarboxyla-
tion of the newly discovered epigenetic
base 5-carboxycytosine (caC; see pic-
ture). This reaction is likely to involve
S. Schiesser, B. Hackner, T. Pfaffeneder,
M. Mꢁller, C. Hagemeier, M. Truss,*
T. Carell*
&&&& — &&&&
À
a nucleophilic attack of the C5 C6 double
bond.
Mechanism and Stem-Cell Activity of 5-
Carboxycytosine Decarboxylation
Determined by Isotope Tracing
6
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Angew. Chem. Int. Ed. 2012, 51, 1 – 6
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