Article

Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2

Department of Pathology, Taub Institute for Aging, Columbia University, New York, New York 10032, USA.
Nature (Impact Factor: 41.46). 08/2012; 488(7413):652-5. DOI: 10.1038/nature11333
Source: PubMed

ABSTRACT

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.

Full-text

Available from: Keiichi Inoue
LETTER
doi:10.1038/nature11333
Early-stage epigenetic modification during somatic
cell reprogramming by Parp1 and Tet2
Claudia A. Doege
1
, Keiichi Inoue
1
, Toru Yamashita
1
, David B. Rhee
1
, Skylar Travis
1
, Ryousuke Fujita
1
, Paolo Guarnieri
2,3
,
Govind Bhagat
1,3
, William B. Vanti
1
, Alan Shih
4
, Ross L. Levine
4
,SaraNik
5
, Emily I. Chen
5,6
& Asa Abeliovich
1
Somatic cells can be reprogrammed into induced pluripotent stem
cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and
c-Myc (together referred to as OSKM)
1
. iPSC reprogramming
erases somatic epigenetic signatures—as typified by DNA methyla-
tion or histone modification at silent pluripotency loci—and
establishes alternative epigenetic marks of embryonic stem cells
(ESCs)
2
. Here we describe an early and essential stage of somatic
cell reprogramming, preceding the induction of transcription
at endogenous pluripotency loci such as
Nanog
and
Esrrb
.By
day 4 after transduction with OSKM, two epigenetic modification
factors necessary for iPSC generation, namely poly(ADP-ribose)
polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are
recruited to the
Nanog
and
Esrrb
loci. These epigenetic modifica-
tion factors seem to have complementary roles in the establishment
of early epigenetic marks during somatic cell reprogramming:
Parp1 functions in the regulation of 5-methylcytosine (5mC)
modification, whereas Tet2 is essential for the early generation
of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs
3,4). Although 5hmC has been proposed to serve primarily as an
intermediate in 5mC demethylation to cytosine in certain con-
texts
5–7
, our data, and also studies of Tet2-mutant human tumour
cells
8
, argue in favour of a role for 5hmC as an epigenetic mark
distinct from 5mC. Consistent with this, Parp1 and Tet2 are
each needed for the early establishment of histone modifications
that typify an activated chromatin state at pluripotency loci,
whereas Parp1 induction further promotes accessibility to the
Oct4 reprogramming factor. These findings suggest that Parp1
and Tet2 contribute to an epigenetic program that directs
subsequent transcriptional induction at pluripotency loci during
somatic cell reprogramming.
We performed a functional screen for epigenetic modification
factors that promote OSKM-mediated somatic cell reprogramming.
Overexpression of a single pool of 29 candidate epigenetic modifica-
tion factors, selected on the basis of a proteomic analysis of iPSCs
(Fig. 1a–c and Supplementary Tables 1 and 2), promoted iPSC colony
production in mouse embryonic fibroblast (MEF) cultures transduced
with OSKM (OSKM-MEFs; relative to green fluorescent protein vector
control transduced MEF; Fig. 1d). Candidate epigenetic modifica-
tion factors were retested in successive subpools, and Parp1 was iden-
tified as a potent inducer of OSKM-MEF reprogramming (Fig. 1d).
iPSCs generated by OSKM-MEFs with Parp1 were confirmed as
pluripotent by immunocytochemistry (Supplementary Fig. 2a),
gene expression (Supplementary Fig. 2b–f and Supplementary Table 3)
and pyrosequencing (Supplementary Fig. 2g–i and Supplementary
Table 4) analyses at multiple pluripotency loci. Parp1 overexpression
did not alter the proliferation rate of transduced cultures (as
determined by bromodeoxyuridine (BrdU) incorporation; Supplemen-
tary Fig. 2j).
Parp1 is a broadly expressed nuclear protein involved in the detec-
tion and repair of DNA damage, the remodelling of chromatin and the
regulation of transcription
9
. A time course of endogenous Parp1
expression revealed detectable levels even in wild-type (WT) MEFs
and a peak at day 4 (d4) after transduction with OSKM (Fig. 1e, f
and Supplementary Figs 2k and 3a), thus preceding expression of the
endogenous pluripotency loci including Nanog, Oct4 and Esrrb (Fig. 1f
and Supplementary Fig. 3b). Immunocytochemistry analysis at d4
after transduction of WT MEFs with OSKM (WT d4-OSKM-MEFs)
demonstrated Parp1 accumulation in a majority of nuclei in compar-
ison with d4 control vector-transduced MEFs (WT d4-CONT-MEFs;
Fig. 1g). The increased accumulation of Parp1 holoprotein during
reprogramming was not paralleled by a corresponding accumulation
of cleaved Parp1, a marker of apoptosis (Supplementary Fig. 3a).
In view of the effect of Parp1 overexpression in somatic cell
reprogramming, we next tested the impact of Parp1 deficiency.
Reprogramming of iPSCs was suppressed in the context of
Parp1
2/2
OSKM-MEFs relative to WT OSKM-MEFs (Fig. 2a and
Supplementary Fig. 2k, l). Resupplying WT Parp1 partly rescued
iPSC generation in Parp1
2/2
OSKM-MEFs. In contrast, expression
of Parp1 mutants
10,11
, compromising either the catalytic activity or the
DNA-binding activity, failed to rescue iPSC generation, indicating that
both activities are required for iPSC reprogramming (Fig. 2a and
Supplementary Fig. 2l).
The early expression and functional role of endogenous Parp1 in the
reprogramming process suggested the possibility of a direct interaction
at pluripotency loci. Consistent with this notion, chromatin immuno-
precipitation (ChIP) at the Nanog and Esrrb pluripotency loci, in the
transcription start site regions (Supplementary Table 5), demonstrated
increased Parp1 binding in d4-OSKM-MEFs and iPSCs (compared
with d4-CONT-MEFs; Fig. 2b, c and Supplementary Fig. 3g, h, k).
Parp1 has been broadly implicated in the regulation of epigenetic
remodelling events
7,12
, but its role during the reprogramming of
pluripotency loci is unclear. We therefore investigated the impact of
modified Parp1 expression on two distinct forms of cytosine methyla-
tion at pluripotency loci, 5mC and 5hmC, during somatic cell repro-
gramming. 5hmC is a more recently described DNA modification that
has been suggested to participate in the maintenance of pluripotency at
regulatory elements
5,13–17
in ESCs, but its role during somatic cell
reprogramming has not previously been described. We quantified total
cytosine methylation (5mC plus 5hmC; either by HpaII digestion
sensitivity assays or pyrosequencing of bisulphite-treated DNA) or
5hmC alone (either by MspI sensitivity assay (glucosylation-coupled
methylation-sensitive quantitative polymerase chain reaction;
GlucMS-qPCR)
16,18
or hydroxymethylated DNA immunoprecipita-
tion (hMeDIP); see Supplementary Tables 6 and 7 and Supplemen-
tary Methods) at regulatory regions of the Nanog or Esrrb loci. Both
d4-OSKM-MEFs and iPSCs showed a significant and consistent
1
Departments of Pathology and Cell Biology and Neurology, Taub Institute for Aging, Columbia University, New York, New York 10032, USA.
2
Biomedical Informatics Shared Resources, Bioinformatics
Division, Columbia University, New York, New York 10032, USA.
3
Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York 10032, USA.
4
Human Oncology and Pathogenesis
Program, Memorial Sloan Kettering Cancer Center, New York, New York 10016, USA.
5
Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794, USA.
6
Stony Brook
University Proteomics Center, School Of Medicine, Stony Brook, New York 11794, USA.
652 | NATURE | VOL 488 | 30 AUGUST 2012
Macmillan Publishers Limited. All rights reserved
©2012
Page 1
increase in 5hmC relative to d4-CONT-MEFs (Fig. 2d and Sup-
plementary Fig. 3c, d, n–q) at the pluripotency loci. In contrast to
5hmC, 5mC was not accumulated at the early time point (in d4-
OSKM-MEFs, relative to d4-CONT-MEFs) at either locus (Fig. 2e
and Supplementary Fig. 3j, m). The two pluripotency loci differed with
respect to the overall pattern of 5mC during reprogramming, as previ-
ously described
2,19
:theNanog locus showed a canonical pattern of
hypermethylation (elevated 5mC) in MEFs and became demethylated
(low 5mC) in reprogrammed iPSCs (Fig. 2e and Supplementary
Fig. 3e, f), whereas the Esrrb locus showed a relatively low level of
methylation even in MEFs, and remained hypomethylated in fully
reprogrammed iPSCs (Supplementary Fig. 3i, j, l, m). Thus among
these pluripotency loci, 5hmC rather than 5mC seemed to be an early
and predictive epigenetic mark for subsequent activation. Immuno-
cytochemistry analysis with an antibody against 5hmC showed
increased nuclear staining in most cells in d4-OSKM-MEFs (relative
to d4-CONT-MEF cultures; Fig. 3a).
We next sought to determine the role of Parp1 in the regulation of
5hmC and 5mC epigenetic marks at the pluripotency loci. Parp1 defi-
ciency (which suppressed iPSC reprogramming) led to a consistent,
large increase in 5mC accumulation in Parp1
2/2
d4-OSKM-MEFs,
relative to WT d4-OSKM-MEF cultures at both the Nanog and Esrrb
loci (Fig. 2e and Supplementary Fig. 3j, m). The increased 5hmC in
WT d4-OSKM-MEFs was not suppressed in Parp1
2/2
d4-OSKM-
MEFs; rather, in the context of Parp1 deficiency, 5hmC induction
seemed similar to that of WT cells, for example at the Nanog locus,
or modestly further increased, for example at the Esrrb locus (Fig. 2d
and Supplementary Fig. 3d, n–q). Parp1 overexpression (which pro-
moted iPSC reprogramming) did not consistently modify 5mC or
5hmC in d4-OSKM-MEFs, although a modest increase in 5hmC levels
was observed at the Esrrb locus but not at the Nanog locus (Fig. 2d and
Supplementary Fig. 3o, q). Taken together, these data implicate Parp1
in the regulation of 5mC; in contrast, we speculate that the variable
impact of Parp1 on 5hmC may be indirect.
+ OSKM
iPSC clone
a
b
cd
MEF 1
MEF 2
MEF 3
ESC
iPSC 1
iPSC 2
iPSC 3
0.2
0.7
0.6
0.5
0.4
0.3
MEF
iPSC colony count
Parp1
29 factors
GFP
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Rcc1
Rcc2
Top2a
Smc3
0
25
20
15
5
10
ESC clone
0 15.0
MEF
1
MEF 3
MEF 2
iPSC 3
iPSC 2
iPSC 1
ESC
Lbr
Dek
Dnmt1
Dnmt3a
Dnmt3l
Dnmt3b
Nup35
Lmnb1
Nup93
Lamc1
Smarcc1
L1td1
Smarca5
Cbx3
Rbbp4
Chd4
Supt16h
Ssrp1
Hist1H1a
Ruvbl1
Nolc1
Uhrf1
Pdcd11
Nid1
Rcc1
Top2a
Rcc2
Smc3
Parp1
Group 6 Group 4 Group 3 Group 2 Group 1
Nuclear
extract
Mass spectrometry
Nuclear
extract
Nuclear
extract
Group 5
14 d OSKM
Relative expression
0
0.07
0.06
0.03
MEF
ESC
iPSC
∗∗∗
Endogenous Nanog
Endogenous Parp1
Endogenous Oct4
0.5 d
4 d
3 d
2 d
1.5 d
1 d
5 d
∗∗∗
∗∗∗
0.05
0.04
0.01
0.02
OSKM
Pick colonies
MEF
iPSC
clone
0.5 d
28 d
14 d
4 d
3 d
2 d
1.5 d
1 d
5 d
Collect samples
f
e
g
100 μm
Parp1Sytox
Parp1
–/–
d4-OSKM-MEF
WT
d4-CONT-MEF
WT
Parp1
HI
(%)
Parp1
–/
d4-OSKM-MEF
WT d4-OSKM-MEF
0
40
20
60
80
100
WT d4-CONT-MEF
Figure 1
|
Parp1 promotes OSKM-mediated iPSC generation. a, Diagram of
proteomic strategy to identify candidate epigenetic modification (EM) factors.
b, Unsupervisedhierarchical clustering analysis (Spearman rank correlation) of
mass spectrometry data from nuclear extracts of MEFs (n 5 3), iPSCs (n 5 3)
and ESCs (n 5 1). The scale bar represents the correlation height
(5 1 2 Abs[correlation]). c, Dual-colour heat map for expression levels of 29
proteins highly enriched in both the iPSC and ESC samples (relative to MEFs).
The colour scale bar represents the spectral count. Candidate EM factors were
divided into six groups for further functional testing. d, Functional screen of
candidate EM factors for promotion of somatic cell reprogramming in OSKM-
MEFs. EM candidates were transduced together as a single pool of 29 genes, as 6
subpools, or as individual factors from group 6 (as in c). Alkaline phosphatase-
positive (AP
1
) iPSC colonies were counted at day 14 after transduction with
OSKM. e, Diagram of time-course analyses of iPSC reprogramming. f,Gene
expression time course of endogenous Parp1, Nanog and Oct4.
g, Immunocytochemistry analysis of WT or Parp1
2/2
d4-OSKM-MEFs and
d4-CONT-MEFs with an antibody against Parp1 (upper panels; red), and
counterstained with Sytox nuclear marker (lower panels; green). Increased
Parp1 expression in d4-OSKM-MEFs is quantified on the right (Parp1
HI
;
defined as mean plus 2 s.d. or greater than the expression level in d4-CONT-
MEFs); modified nuclear morphology apparent in d4-OSKM-MEFs is as
described previously
30
. Results in d, f and g are shown as means and s.d. for
three independent experiments. Asterisk, P , 0.05; three asterisks, P , 0.001.
b
c
2
0
4
Relative enrichment
(to GAPDH)
Parp1
Nanog
+46
6
+659
+325
+48
0
TSS
100 bp
Intron 1Exon 1/intron 1
+515
+
5
52
iPSC
de
0.2
0
0.4
5hmC (%)
i
P
SC
WT d
4
-OSKM-ME
F
+ Parp1
5hmC
5mC (%)
20
0
40
100
60
80
5mC
Parp1
–/
d4-OS
K
M-M
EF
W
T
d4-
O
SKM
-MEF
WT d4-
CONT-MEF
Parp1
–/–
d4-OS
K
M-ME
F
W
T d4-
O
SKM-MEF
WT d4-
CONT-MEF
i
P
SC
WT
d
4
-OSKM-ME
F + Parp1
Parp1
–/–
d4-OS
K
M-ME
F
W
T d4-O
SKM
-MEF
WT d4-
CONT-MEF
a
510150
WT
Parp1
–/–
iPSC colony count (14 d OSKM)
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗
∗∗
∗∗
1
L139P
E988K
C21G
C125G
xxx
x
NLS 1014
Zn1 Zn3Zn2
Catalytic DNA binding
Auto-
modication
BRCT
ΔCAT
CAT-only
WT Parp1
DBD mutant
CAT mutant
GFP
GFP
653
NAD
+
binding
Figure 2
|
Parp1 activities during iPSC reprogramming. a, Functional
analysis of Parp1 mutants for rescue of iPSC colony formation in Parp1
2/2
OSKM-MEFs. Cultures were transduced with green fluorescent protein (GFP),
WT Parp1, or Parp1 mutants encoding a catalytic domain missense mutation
(CAT mutant; E988K), deletion of the catalytic domain (DCAT), deletion of the
DNA-binding and automodification domains (CAT-only), or triple missense
mutation of the DNA-binding domain (DBD mutant; C21G/C125G/L139P).
Zn, zinc fingers; BRCT, BRCA1 carboxy terminus. b, Schematic representation
of the Nanog locus transcription start site (TSS) region. Indicated are HpaII/
MspI sites (green bars) and amplicons for ‘exon 1/intron 1’ and ‘intron 1’
regions(thickgrey lines). bp, base pairs. c, Parp1 ChIP analyses of the culturesas
indicated, presented as the relative enrichment to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). d, Content of 5hmC assessed by GlucMS-qPCR (as a
percentage of total cytosine). e, Content of 5mC, quantified by subtraction of
5hmC content (as in d) from the total methylated cytosine (5mC 1 5hmC, as
determined by HpaII digestion insensitivity; see Supplementary Fig. 3f). Results
in a and ce are shown as means and s.d. for three independent experiments.
Asterisk, P , 0.05; two asterisks, P , 0.01; three asterisks, P , 0.001.
LETTER RESEARCH
30 AUGUST 2012 | VOL 488 | NATURE | 653
Macmillan Publishers Limited. All rights reserved
©2012
Page 2
Given the potential role of 5hmC early in somatic cell reprogram-
ming, we obtained an expression time-course analysis of the TET
enzymes, a family of Fe
II and 2-oxoglutarate-dependent enzymes that
generate 5hmC from 5mC (ref. 4). Expression of Tet2, but not Tet1 or
Tet3, was significantly induced in WT d4-OSKM-MEFs and remained
elevated in iPSCs (Fig. 3b and Supplementary Fig. 4a). Consistent with
a functional role for Tet2, short hairpin RNA (shRNA)-mediated Tet2
knockdown (KD; Supplementary Table 8) abolished iPSC colony
formation (Fig. 3c and Supplementary Fig. 4b, c). ChIP analysis with
an antibody against Tet2 showed a direct interaction with the Nanog
and Esrrb pluripotency loci in WT d4-OSKM-MEFs; this was not
altered in Parp1
2/2
d4-OSKM-MEFs (Fig. 3d, e and Supplementary
Fig. 4d, e, l, m). Furthermore, Tet2 KD in d4-OSKM-MEFs suppressed
the typical induction of 5hmC at both the Nanog and Esrrb pluripo-
tency loci (Fig. 3f, g and Supplementary Fig. 4f, g). In contrast, the
effect of Tet2 KD in d4-OSKM-MEFs on 5mC seemed variable: 5mC
seemed to be decreased at the Nanog locus but mildly increased at the
Esrrb locus (which is typically hypomethylated even in MEFs
19
; Fig. 3h
and Supplementary Fig. 4h–k). Given the early induction of 5hmC but
not 5mC at the pluripotency loci, as well as the consequences of Tet2
deficiency in preventing transcriptional activation and 5hmC induc-
tion even in the absence of 5mC reduction, these data support a
primary role for 5hmC as a distinct epigenetic mark in the somatic
cell reprogramming process, and argue against an alternative model in
which 5hmC functions simply as an intermediate in the 5mC
demethylation process
20
. Epigenetic marks with 5hmC may recruit
select chromatin modification factors to the pluripotency loci
21
.
To further probe the roles of Tet2 and Parp1 in early epigenetic
events, we evaluated the chromatin state of the Nanog and Esrrb loci in
d4-OSKM-MEFs. ChIP analysis revealed an enrichment in the occu-
pancy of these loci by the activation-associated marker histone H3
lysine 4 dimethylation (H3K4me2)
22,23
and a parallel decrease in the
transcriptional-silencing-associated marker histone H3 lysine 27
trimethylation (relative to d4-CONT-MEFs; H3K27me3 (refs 24–26);
Fig. 4a–d and Supplementary Fig. 5a, b). Deficiency of either Parp1 or
Tet2 diminished the H3K4me2 chromatin mark at the pluripotency
loci of d4-OSKM-MEFs (Fig. 4a, c and Supplementary Fig. 5a). The
effects on H3K27me3 were variable: Parp1 deficiency did not signifi-
cantly alter H3K27me3 at either locus, whereas Tet2 KD led to a
decrease at the Nanog locus but not at the Esrrb locus (Fig. 4b, d and
Supplementary Fig. 5b). We speculated that altered chromatin states in
d4-OSKM-MEFs may affect chromatin accessibility at the pluripo-
tency loci, for example to the transduced Oct4 pluripotency factor.
Oct4 occupancy, as quantified by ChIP of d4-OSKM-MEFs, was sig-
nificantly diminished in the context of Parp1 deficiency at both plur-
ipotency loci, whereas Tet2 KD did not diminish Oct4 occupancy
(Fig. 4e, g and Supplementary Fig. 5c). Consistent with this, Parp1
overexpression potentiated Oct4 binding at both pluripotency loci of
d4-OSKM-MEFs (Fig. 4e, g and Supplementary Fig. 5c). Furthermore,
f
Tet1
Tet2
∗∗∗
3
4
2
0
1
Expression (fold change)
MEF
ESC
iPSC
0.5 d
4 d
3 d
2 d
1.5 d
1 d
5 d
∗∗
∗∗∗
∗∗∗
iPSC colony count
50
40
30
0
20
10
14 28
Mock KD
Tet2 KD
21
Days
0
c
b
g
d
Exon 1/intron 1
Nanog
100 bp
40
10
20
0
30
Intron 1
0
5hmC (%)
0.4
0.2
0
0.3
0.1
5hmCSytox
a
0
40
20
60
80
100
5hmC
h
20
10
40
5mC (%)
30
5mC5hmC
5hmC
HI
(%)
∗∗
100 μm
+515
+552
WT d4-OSKM-MEFWT d4-CONT-MEF
WT d4-OSKM-MEF
WT d4-CONT-MEF
Mock KD d4-OSKM-MEF
Tet2
KD d4-OSKM-MEF
Mock KD d4-OSKM-MEF
Tet2
KD d4-OSKM-MEF
Mock KD d4-OSKM-MEF
Tet2
KD d4-OSKM-MEF
Tet2
e
1.5
1
0
0.5
2
2.5
Relative enrichment
(to GAPDH)
iPSC
WT d4-OSKM-MEF + Parp1
Parp1
–/–
d4-OSKM-MEF
Mock KD d4-OSKM-MEF
Tet2
KD d4-OSKM-MEF
WT d4-CONT-MEF
Figure 3
|
Tet2 is required for 5hmC formation at the
Nanog
locus.
a, Immunocytochemistry of d4-OSKM-MEFs and d4-CONT-MEFs with an
antibody against 5hmC (ref. 5) (upper panels; red) and counterstained with
Sytox nuclear marker (lower panels; green). Representative images show
increased 5hmC in d4-OSKM-MEFs, as quantified on the right (5hmC
HI
;
defined as mean plus 2 s.d. or greater above the level in d4-CONT-MEFs).
b, Time course of Tet1 and Tet2 gene expression assessed by qPCR (relative to
ESC level). c, OSKM-mediated iPSC colony formation assay (AP
1
) in shRNA-
mediated Tet2 knockdown (Tet2 KD; blue) and non-silencing control shRNA
(mock KD; black)-treated MEFs. d, Diagram of the Nanog locus; regions are the
same as in Fig. 2b. e, Tet2 ChIP-qPCR at the exon 1/intron 1 amplicon.
f, g, Content of 5hmC in the cultures indicated, assessed by hMeDIP of the
exon 1/intron 1 region (f, relative to GAPDH) or GlucMS-qPCR intron 1
amplicon(g, as a percentage of total cytosine). h, Content of 5mC at the intron 1
amplicon, quantified by subtraction of 5hmC (as in g) from the total
methylated cytosine levels (as in Supplementary Fig. 4k; determined by HpaII
sensitivity assay; as a percentage of total cytosine). Results in ac and eh are
shown as means and s.d. for three independent experiments. Asterisk, P , 0.05;
two asterisks, P , 0.01; three asterisks, P , 0.001.
TSS_MspI_AExon 1/intron 1
d
Relative enrichment
(to GAPDH)
H3K27me3
∗∗∗
∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗
∗∗
H3K27me3
6
2
4
0
3
2
4
0
1
ca
e
Oct4
15
5
10
0
20
Relative enrichment
(to GAPDH)
0
25
3
1
2
7.5
5.5
Oct4
0
2.0
1.0
1.5
0.5
0
1.0
1.5
0.5
∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗
∗∗
∗∗∗
Oct4 Oct4
hgf
b
0
1.7
0.10
0.15
0.05
0.7
1.2
H3K4me2 H3K4me2
∗∗∗
∗∗∗
0
0.10
0.25
0.05
0.15
0.20
∗∗∗
∗∗∗
Nanog Esrrb
∗∗∗
∗∗
∗∗
∗∗
∗∗∗
∗∗
i
P
SC
WT d4-OSKM-MEF +
Parp1
Parp1
–/–
d
4
-OSKM-MEF
Moc
k
KD d4
-OSKM-
MEF
Te
t
2
KD d4-OSKM
-
MEF
W
T d4-CONT-MEF
i
P
SC
Parp1
–/–
d
4
-OSKM-MEF
Moc
k
KD d4
-OSKM-
MEF
Te
t
2
KD d4-OSKM
-
MEF
W
T d4-CONT-MEF
i
P
SC
Parp1
–/–
d
4
-OSKM-M
EF
Moc
k
KD d4
-OSKM-
MEF
Te
t
2
KD d4-OSKM
-
MEF
WT d4-CONT-MEF
i
P
SC
Parp1
–/–
d
4
-OSKM-MEF
Moc
k
KD d4
-OSKM-
MEF
Te
t
2
KD d4-OSKM
-
MEF
WT d4-CONT-MEF
i
P
SC
Parp1
–/–
d
4
-OSKM-MEF
Moc
k KD d4
-OSKM
-
MEF
Te
t
2
KD d4-OSKM
-
MEF
W
T d4-CO
NT
-MEF
WT d4-OSKM-MEF +
Parp1
i
P
SC
Parp1
–/–
d
4
-OSKM-MEF
Moc
k
KD d4
-OSKM-
MEF
Te
t
2
KD d4-OSKM
-
MEF
WT d4-CONT-MEF
WT d
4
-O-MEF
+
Parp
1
WT d4-O-MEF
WT d4-CO
N
T-MEF
WT d4-O-MEF +
Parp1
WT d4
-
O
-
MEF
WT d4-CONT-MEF
Figure 4
|
Impact of Parp1 and Tet2 on chromatin state and Oct4
accessibility at the
Nanog
and
Esrrb
loci. ad, H3K4me2 (a, c) and
H3K27me3 (b, d) ChIP-qPCR at Nanog or Esrrb amplicons in cultures as
indicated. eh, Oct4 ChIP-qPCR. Results are shown as means and s.d. for three
independent experiments. Asterisk, P , 0.05; two asterisks, P , 0.01; three
asterisks, P , 0.001.
RESEARCH LETTER
654 | NATURE | VOL 488 | 30 AUGUST 2012
Macmillan Publishers Limited. All rights reserved
©2012
Page 3
Parp1 overexpression robustly promoted exogenous Oct4 binding to
the pluripotency loci even in the absence of transduction of the other
pluripotency factors necessary for somatic cell reprogramming (d4-O-
MEFs without SKM; Fig. 4f, h and Supplementary Fig. 5d).
Taken together, these data support necessary but distinct roles for
Tet2 and Parp1 in the regulation of epigenetic marks and local chro-
matin structure at pluripotency loci during an early stage of somatic
cell reprogramming that precedes their transcriptional activation
(Supplementary Fig. 1a, b). The data further suggest that 5hmC,
generated by Tet2, does not simply represent an intermediate in the
5mC demethylation process, but functions as an epigenetic mark,
possibly recruiting trans-acting factors that promote chromatin
remodelling
4,21
. The induction of Parp1 and Tet2 gene expression early
in the course of reprogramming may reflect their direct activation by
OSKM factors (Supplementary Fig. 5e, f)
27
. Finally, loss of Tet2 func-
tion is strongly implicated in human malignancies
8
, and thus Tet2-
mediated chromatin remodelling may affect tumour risk associated
with potential iPSC therapies.
METHODS SUMMARY
Cell culture and generation of iPSCs. MEF were prepared from WT or Parp1
2/2
embryonic day 13.5 embryos (129S-Parp1
tm1Zqw
/J; Jackson Laboratories)
28
. Tail-
tip fibroblasts were prepared from WT and Tet2
2/2
mice
29
. For iPSC generation,
MEFs were transduced by incubation with OSKM retroviral supernatants for 24 h
as described
1
. Subsequently, cells were cultured in ESC medium and samples were
collected at the time points indicated. Epigenetic modification factors were cloned
into lentiviral vectors (pLenti6.3; Invitrogen) for lentiviral production and sub-
sequent transduction.
Tet2
knockdown. Tet2 knockdown was achieved by using a cocktail of five
shRNA lentiviral vectors (Open Biosystems) specific for Tet2, or control non-
silencing shRNA.
Mass spectrometry. Multidimensional protein identification technology
(MudPIT) was performed to analyse nuclear fractions of MEFs, iPSC clones
and ESC clones as detailed in Supplementary Methods.
Chromatin immunoprecipitation. ChIP was performed with the Magnify kit
(492024; Invitrogen) and the following antibodies: anti-dimethyl K4 of H3 (2 mg;
07-030; Millipore), anti-trimethyl K27 of H3 (2 mg; ab6002; Abcam), anti-Oct4
(2 mg; sc-8628 X; Santa Cruz), anti-Parp1 (2 mg; sc-74469 X; Santa Cruz), anti-Tet2
(2 mg; sc-136926; Santa Cruz), normal goat IgG (2 mg; 005-000-003; Jackson
ImmunoResearch), normal rabbit IgG (1 mg; Magnify; Invitrogen) and normal
mouse IgG (1 mg; Magnify; Invitrogen).
Digestion with
Hpa
II and
Msp
I. Detection of 5hmC and 5mC as percentages of
total cytosine species was performed with the EpiMark Kit (E3317S; NEB). The
technique has been described in detail in ref. 16.
Pyrosequencing. Genomic DNA (1 mg) was bisulphite-converted with the EZ
DNA Methylation Kit (D5001; Zymo Research), followed by amplification by
polymerase chain reaction with the PyroMark PCR Kit (978703; Qiagen). PCR
products were sequenced with the PyroMark Q24 instrument (Qiagen).
Received 3 August 2011; accepted 18 June 2012.
Published online 19 August 2012.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank G. Q. Daley, A. P. Feinberg, A. Doi, R. M. Santella and
M. A. Kappil for reagents and for technical assistance with pyrosequencing; A. Califano
and A. Lachmann for assistance with the bioinformatics analyses; E. O. Mazzoni for
assistance with the ChIP analyses; and O. Hobert for critical reading of the manuscript.
This work was supported by New York State Stem Cell Science (NYSTEM) grants
C024402 and C024403 and National Institutes of Health (NIH) grant RO1 NS064433
to A.A., NYSTEM Institution Development Grant N08G-071 to E.I.C., NIH grant RO1
138424 to R.L.L, and a sharedNIH/NationalCenter for Research Resources instrument
grant for mass spectrometry, 1 S10 RR023680-1.
Author Contributions C.A.D. and A.A. designed the experiments and analysed data.
C.A.D., D.B.R., S.T., R.F. and W.B.V. conducted molecular and cellular experiments. T.Y.,
G.B. and K.I. performed and analysed murine in vivo studies. R.L.L. and A.S. supplied
essential reagents. P.G. performed bioinformatics analyses. S.N. and E.I.C. conducted
proteomics. C.A.D. and A.A. wrote the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to A.A. (aa900@columbia.edu).
LETTER RESEARCH
30 AUGUST 2012 | VOL 488 | NATURE | 655
Macmillan Publishers Limited. All rights reserved
©2012
Page 4
    • "Initial observations in the Wang lab showed that ascorbate enhanced 5hmC generation in cultured cells, most likely by acting as a cofactor for TET to hydroxylate 5mC [26, 101]. This new function of ascorbate was initially discovered in mouse embryonic fibroblasts (MEF) that expressed TETs at low, but detectable levels [27, 73]. Thus, MEFs constituted a convenient and useful tool to analyze TET enzymatic requirements in a cell-based experimental setting. "
    [Show abstract] [Hide abstract] ABSTRACT: Recent advances have uncovered a previously unknown function of vitamin C in epigenetic regulation. Vitamin C exists predominantly as an ascorbate anion under physiological pH conditions. Ascorbate was discovered as a cofactor for methylcytosine dioxygenases that are responsible for DNA demethylation, and also as a likely cofactor for some JmjC domain-containing histone demethylases that catalyze histone demethylation. Variation in ascorbate bioavailability thus can influence the demethylation of both DNA and histone, further leading to different phenotypic presentations. Ascorbate deficiency can be presented systematically, spatially and temporally in different tissues at the different stages of development and aging. Here, we review how ascorbate deficiency could potentially be involved in embryonic and postnatal development, and plays a role in various diseases such as neurodegeneration and cancer through epigenetic dysregulation.
    No preview · Article · Feb 2016 · Cellular and Molecular Life Sciences CMLS
  • Source
    • "ESRRB is a transcription factor that is essential for the maintenance of ESCs (Papp and Plath, 2012; Zwaka, 2012 ); yet to our knowledge, the binding of ESRRB to DNA has not been previously associated with the presence of 5hmC. However, the ESRRB gene locus is known to be strongly enriched in 5hmC in ESCs (Doege et al., 2012), suggesting that 5hmC and ESRRB form a regulatory loop. Gene Ontology analysis carried out with the genes closest to these specific regions (McLean et al., 2010) identified stem cell maintenance and MAPK and Notch cell signaling cascades as the most-enriched functions (Figure 6E ), highlighting the importance of ESRRB for stemness maintenance. "
    [Show abstract] [Hide abstract] ABSTRACT: Epigenetic communication through histone and cytosine modifications is essential for gene regulation and cell identity. Here, we propose a framework that is based on a chromatin communication model to get insight on the function of epigenetic modifications in ESCs. The epigenetic communication network was inferred from genome-wide location data plus extensive manual annotation. Notably, we found that 5-hydroxymethylcytosine (5hmC) is the most-influential hub of this network, connecting DNA demethylation to nucleosome remodeling complexes and to key transcription factors of pluripotency. Moreover, an evolutionary analysis revealed a central role of 5hmC in the co-evolution of chromatin-related proteins. Further analysis of regions where 5hmC co-localizes with specific interactors shows that each interaction points to chromatin remodeling, stemness, differentiation, or metabolism. Our results highlight the importance of cytosine modifications in the epigenetic communication of ESCs.
    Full-text · Article · Jan 2016 · Cell Reports
    • "During ESCs differentiation, POU5F1 and NANOG promoters have been shown to become repressed by DNA methylation [24, 25], while the SOX2 promoter can be inactivated through trimethylation of H3K27 and H3K9 [25]. More recent studies [7, 26, 27] showed the presence of 5hmC in the POU5F1 and NANOG loci in reprogrammed cells expressing these genes. In agreement with previous studies282930, which demonstrated in transcript and protein level the presence of the main pluripotency factors in ADSCs, we observed apparent levels of the POU5F1 and NANOG transcripts, but quite low level of SOX2 in our cell lines. "
    [Show abstract] [Hide abstract] ABSTRACT: Adult stem cells have more restricted differentiation potential than embryonic stem cells (ESCs), but upon appropriate stimulation can differentiate into cells of different germ layers. Epigenetic factors, including DNA modifications, take a significant part in regulation of pluripotency and differentiation of ESCs. Less is known about the epigenetic regulation of these processes in adult stem cells. Gene expression profile and location of DNA modifications in adipose-derived stem cells (ADSCs) and their osteogenically differentiated lineages were analyzed using Agilent microarrays. Methylation-specific PCR and restriction-based quantitative PCR were applied for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) detection in selected loci. The level of DNA modifications in the POU5F1 locus was quantified with deep sequencing. Expression levels of selected genes were assayed by real-time PCR. ADSCs differentiation into osteogenic lineages involved marked changes in both 5mC and 5hmC profiles, but 5hmC changes were more abundant. 5mC losses and 5hmC gains were the main events observed during ADSCs differentiation, and were accompanied by increased expression of TET1 (P = 0.009). In ADSCs, POU5F1 was better expressed than NANOG or SOX2 (P ≤ 0.001). Both 5mC and 5hmC marks were present in the POU5F1 locus, but only hydroxymethylation of specific cytosine showed significant effect on the gene expression. In summary, the data of our study suggest significant involvement of changes in 5hmC profile during the differentiation of human adult stem cells.
    No preview · Article · Aug 2015 · Molecular and Cellular Biochemistry
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