A DNA Repair Complex Functions
as an Oct4/Sox2 Coactivator
in Embryonic Stem Cells
Yick W. Fong,1Carla Inouye,1Teppei Yamaguchi,1Claudia Cattoglio,1Ivan Grubisic,2and Robert Tjian1,3,*
1Howard Hughes Medical Institute, Department of Molecular and Cell Biology
2UC Berkeley-UCSF Graduate Program in Bioengineering
3Li Ka Shing Center for Biomedical and Health Sciences
University of California, Berkeley, Berkeley, CA 94720, USA
The transcriptional activators Oct4, Sox2, and Nanog
cooperate with a wide array of cofactors to orches-
trate an embryonic stem (ES) cell-specific gene
expression program that forms the molecular basis
of pluripotency. Here, we report using an unbiased
in vitro transcription-biochemical complementation
assay to discover a multisubunit stem cell coactiva-
tor complex (SCC) that is selectively required for
the synergistic activation of the Nanog gene by
Oct4 and Sox2. Purification, identification, and re-
constitution of SCC revealed this coactivator to be
the trimeric XPC-nucleotide excision repair complex.
SCC interacts directly with Oct4 and Sox2 and is
recruited to the Nanog and Oct4 promoters as well
as a majority of genomic regions that are occupied
by Oct4 and Sox2. Depletion of SCC/XPC com-
promised both pluripotency in ES cells and somatic
cell reprogrammingoffibroblasts to inducedpluripo-
tent stem (iPS) cells. This study identifies a tran-
scriptional coactivator with diversified functions in
maintaining ES cell pluripotency and safeguarding
The molecularevents leadingto themaintenance of pluripotency
in embryonic stem (ES) cells and reacquisition of a stem-like
state in induced pluripotent stem (iPS) cells during somatic re-
programming represent mechanistically distinct processes that
converge on a set of remarkably similar transcriptional events
on fundamental transcription frameworks that are governed by
a common set of ‘‘core’’ stem cell-specific transcription factors,
namely Oct4, Sox2, and Nanog (Jaenisch and Young, 2008).
These activators, in turn, collaborate with both ubiquitous and
cell type-specific transcription factors to orchestrate complex
gene expression programs that confer upon stem cells the
unique ability to safeguard stemness while remaining poised to
execute a broad range of developmental programs that drive
lineage specification (Boyer et al., 2005; Chen et al., 2008; Kim
et al., 2008; Marson et al., 2008).
Proper execution of these highly regulated processes by
sequence-specific transcription factors often requires the coor-
dinated recruitment of coactivator proteins to their cognate
promoters. For example, transcriptional activators direct histone
modifiers (e.g., CBP/p300) and chromatin remodelers (e.g.,
PBAF/BAF) to gene promoters to alter chromatin structure
toward a state that is more permissive to transcriptional activa-
tion (Na ¨a ¨r et al., 2001). Independent of chromatin, a variety of
activators recruit other classes of coactivators, such as the
multisubunit Mediator, various TBP/TAF complexes, SRC, etc.,
via direct protein-protein interactions to execute specific tran-
scriptional programs. This class of coactivators often serves as
molecular ‘‘adaptors’’ by bridging activators to the general tran-
scription machinery, thereby mediating the synergistic response
by these activators (Na ¨a ¨r et al., 1999). Interestingly, subunits of
Mediator have also been shown to interact with cohesin possibly
to promote DNA looping and thereby facilitate long-distance
interactions between enhancers and core promoters in vivo (Ka-
gey et al., 2010). Indeed, such coactivators are often multifunc-
tional and can activate transcription through chromatin-depen-
dent as well as independent mechanisms. Further expanding
the transcriptional repertoire of coactivator complexes, their
protein levels and subunit compositions are frequently modu-
lated in a developmental stage and cell type-specific manner
(Roeder, 2005; Taatjes et al., 2004). Additionally, these protein-
protein-driven coactivator-activator transactions are often crit-
cues, thereby coupling gene networks with specific cellular
responses to produce complex biological programs of gene
expression (Rosenfeld et al., 2006).
Totipotent ES cells employ these same sets of coactivators in
conjunction with special activators such as Oct4 and Sox2 to
regulate transcription of a large number of genes, including
Nanog, that form the molecular basis of pluripotency (Gao
et al., 2008; Kagey et al., 2010; Kidder et al., 2009; Tutter et al.,
2009). The transcription of Nanog is exquisitely dependent on
120 Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc.
Oct4 and Sox2 (Kuroda et al., 2005; Rodda et al., 2005).
However, coexpression of Oct4 and Sox2 failed to robustly
activate a Nanog promoter reporter construct in differentiated
cells like 293 or NIH 3T3 cells, even though Mediator, p300/
CBP, and PBAF/BAF complexes remain abundantly expressed
and active (Rodda et al., 2005). This led us to speculate that
one or more as yet unidentified stem cell-specific cofactors
may be required to activate the transcription of Nanog and other
Oct4/Sox2 target genes in ES cells. Indeed, recent studies of
germ cells and differentiated somatic cells revealed that even
parts of the general transcriptional machinery may be radically
altered in a tissue- or cell-specific context (Goodrich and Tjian,
2010; Mu ¨ller et al., 2010). Diversification of the transcriptional
apparatus may therefore represent a fundamental strategy,
particularly in ES cells, to cope with the multidimensional nature
of transcription programs that must be precisely tuned to both
maintain pluripotency and, at the same time, allow for lineage-
specific programs of differentiation (Liu et al., 2011).
The human Nanog promoter contains a prototypic composite
oct-sox cis-acting regulatory element located immediately
upstream of the transcription start site that is conserved across
several mammalian species (Kuroda et al., 2005; Rodda et al.,
2005). A Nanog promoter-GFP reporter construct containing a
DNA fragment encompassing this promoter-proximal oct-sox
of endogenous Nanog in ES cells in an Oct4-, Sox2-dependent
manner (Kuroda et al., 2005; Rodda et al., 2005). Unbiased
genome-wide motif searching analyses of Oct4 in both mouse
and human ES cells identified an oct-sox composite consensus
sequence element, confirming that Oct4 likely orchestrates an
ES-specific gene expression program primarily through cooper-
ation with Sox2 (Chen et al., 2008; Loh et al., 2006). Because the
oct-sox cis-control element in the Nanog promoter represents a
common configuration that is present in the promoters of many
other Oct4- and Sox2-activated genes in ES cells, the well-char-
acterized Nanog proximal promoter provided us with a useful
model template for identifying uncharacterized transcriptional
cofactors required for Oct4- and Sox2-directed activation.
Therefore, we took advantage of a fully reconstituted in vitro
transcription system in which one can unambiguously and
systematically test and identify transcriptional cofactors that
may be directly required to potentiate Oct4- and Sox2-depen-
dent gene activation of Nanog. Here, we report the biochemical
purification and identification of a multisubunit stem cell coacti-
vator (SCC) that is required for the synergistic activation of
Nanog by Oct4 and Sox2 in vitro. After extensive biochemical
characterization, we surprisingly found that SCC is none other
than the XPC-RAD23B-CETN2 (XPC) nucleotide excision repair
(NER) complex. SCC/XPC interacts directly with Oct4 and
Sox2 and co-occupies a majority of Oct4 and Sox2 targets
genome-wide in mouse ES cells. Importantly, SCC/XPC is
required for stem cell self-renewal and efficient somatic cell
reprogramming. Thus, our findings unmask an unanticipated
selective coactivator role of an NER complex in transcription in
the context of ES cells and may provide a previously unknown
molecular link that couples stem cell-specific transcription to
DNA damage response with potential implications for enhanced
ES cell genome stability.
Detection of an Oct4- and Sox2-Dependent Coactivator
Activity in EC and ES Cells
Having chosen the Nanog promoter as our model template, we
next set out to develop an in vitro reconstituted transcription
assay that could recapitulate the Oct4- and Sox2-dependent
transactivation at the Nanog promoter observed in vivo. To
enhance the sensitivity of the assay, we inserted four copies of
the Nanog oct-sox-binding sites immediately upstream of the
native oct-sox element found in the human Nanog promoter.
Our basal in vitro transcription assay consisted of purified re-
combinant TFIIA, -B, -E and -F together with immunoaffinity-
purified native RNA polymerase II, TFIID, and TFIIH (Figure S1A
available online). When purified Oct4 and Sox2 were added to
this reconstituted transcription system, only a very weak activa-
tion of the Nanog promoter was detected (Figure 1A, lanes 1 and
2). As a control, we could show that the same complement of
general transcription factors (GTFs) was able to support strong
Sp1-dependent activation from a GC box-containing ‘‘generic’’
transcription template (G3BCAT) (Figure 1A, lanes 5 and 6).
This initial result suggested that efficient activation of Nanog
by Oct4 and Sox2 may require additional cofactors to potentiate
a full activator-dependent response.
We reasoned that such a putative coactivator ought to be
selectively active in pluripotent cell types that express Nanog
under the control of Oct4 and Sox2. For example, NTERA-2
(NT2) is a pluripotent human embryonal carcinoma (EC) cell
line that expresses Oct4, Sox2, and Nanog and shares with ES
cells core molecular mechanisms that govern self-renewal (Pal
and Ravindran, 2006). Detailed expression profiling of NT2 and
bona fide human ES cell lines revealed many similarities,
including robust expression of Nanog (Schwartz et al., 2005;
Sperger et al., 2003). However, unlike human ES cells, NT2 cell
culture can be more readily scaled up, a prerequisite to gener-
ating sufficient quantities of starting materials for the biochem-
ical purification of putative Oct4/Sox2 coactivators. We there-
fore chose extracts derived from NT2 cells as our starting
material in our efforts to develop a ‘‘biochemical complementa-
tion’’ assay to hunt for pluripotent stem cell-selective cofactors.
We first fractionated NT2 nuclear extracts by conventional
phosphocellulose ion exchange chromatography. Next, we
supplemented our ‘‘basal’’ reconstituted transcription reactions
with various salt-eluted fractions from the phosphocellulose
column to see whether there was any activity that could restore
Oct4/Sox2-dependent activation of our Nanog promoter. This
strategy allowed us to unmask an activity in the high salt phos-
phocellulose fraction (P1M) prepared from NT2 nuclear extracts
(but not HeLa extracts) (Figure S1B) that strongly potentiated
transcription of the Nanog promoter in an Oct4- and Sox2-
dependent manner using either a naked (Figure 1A, lanes 3
and 4) or a Nanog chromatin template assembled with a crude
Drosophila cytosolic extract (data not shown). This new cofactor
no effect on either basal- or Sp1-activated transcription from
a control G3BCAT template (Figure 1A, lanes 5–8). Importantly,
this P1M fraction also stimulated the Oct4/Sox2-dependent
transcription fromanative Nanog promoter template (Figure1B),
Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc. 121
as well as two other Oct4/Sox2-dependent templates derived
from the mouse Fbxo15 promoter (Tokuzawa et al., 2003)
(mFbxo15CAT) (Figure S1C, lanes 1–4) and the human HESX1
promoter (Chakravarthy et al., 2008) (HESX1CAT) (Figure S1C,
lanes 5–8). Thus, our in vitro complementation assay pro-
grammed with naked DNA templates revealed at least one
potential coactivator activity that directs Oct4/Sox2-dependent
activation of Nanog. We decided to pursue characterization of
this cofactor that does not appear to require chromatin-based
functions. Tothebestofourknowledge, thisfindingalsodemon-
strates for the first time a fully reconstituted, in vitro transcription
system that can faithfully recapitulate stem cell-specific gene
Wenextinvestigatedtherelative requirementsforother cofac-
tors in our assay system. Consistent with previous studies dem-
onstrating that TAFs in the TFIID complex are often required for
transcriptional activation by a variety of activators, including
nuclear receptors (Lemon et al., 2001), Sp1 (Ryu et al., 1999),
and SREBP-1 (Na ¨a ¨r et al., 1998), substituting holo-TFIID with
recombinant human TBP resulted in a near complete loss of
activation by Oct4 and Sox2 (Figure 1C). The very weak residual
activation that we see using TBP (Figure 1C, lanes 2 and 4) is
most likely due to trace amounts of TFIID present in the NT2
P1M fraction (data not shown). These findings suggest that
TAFs/holo-TFIID and the putative cofactor detected in the NT2
P1Mfraction areboth requiredforoptimaltranscription ofNanog
elicited by Oct4 and Sox2. Interestingly, in this reconstituted
system, the addition of CRSP/Mediator complex was not
required to obtain robust Oct4/Sox2 activation at the Nanog
promoter. However, it is likely that some CRSP/Mediator is
present in the P1M fraction, and it remains possible that some
other component of the reconstituted system (i.e., Pol II) may
have some residual amount of CRSP/Mediator contamination
(Na ¨a ¨r et al., 2002). We found, however, that adding purified
completely failed to enhance Oct4/Sox2-dependent activation
of Nanog transcription (Figure S1D). This finding indicates that
the NT2 cofactor must be distinct from Mediator. Furthermore,
addition of other transcriptional activators implicated in Nanog
expression (i.e., Nanog, Sall4 [Zhang et al., 2006], Klf4 [Jiang
et al., 2008] and Esrrb [van den Berg et al., 2008; Zhang et al.,
2008]) also did not replace or enhance Oct4/Sox2-dependent
transcription of Nanog in vitro (Figure S1E).
To confirm that this newly detected cofactor activity in NT2
cells is also present in bona fide ES cells, P1M fractions were
for transcription. We found that the D3 P1M fraction was as
active as the NT2 P1M fraction in potentiating Oct4/Sox2-acti-
vated transcription of Nanog (Figure 1D, compare lane 2 to 6
and 10). Interestingly, the highest levels of transactivation by
the NT2 or D3 P1M fractions were observed only when both
activators were added to the transcription reaction, whereas
no activation was detected with Oct4 alone and a moderate level
of activation was seen with Sox2 alone (Figure 1D, lanes 3–10).
Apparently, this cofactor mediates the synergistic activation of
Nanog by Oct4 and Sox2. If, as we postulated, this new coacti-
vator functions selectively in pluripotent cells, one might expect
that its presence or activity would need to be downregulated
- - - -
NT2 P1M- - ++
NT2 P1M- - ++
- - - -
O S O S
NT2 P1M-- ++
1x 1x 2x 2x
+ NT2 P1M
+--+- +- +
+ D3 P1M
+--+- +- +
Figure 1. Transcriptional Activation of Nanog by Oct4 and Sox2
Requires a Stem Cell-Specific Cofactor
(A) Reconstituted in vitro transcription reactions supplemented with Oct4 and
with either a Nanog template engineered with four extra copies of the oct-sox
composite element (NanogCAT, lanes 1–4), or a GC box-containing template
(G3BCAT, lanes 5–8). Oct4/Sox2, NT2 P1M-dependent transcripts are indicated
by filled arrowheads and Sp1-dependent transcriptions by open arrowheads.
(B) Transcription of the native Nanog promoter requires Oct4, Sox2, and NT2
P1M fraction (lane 4).
(C) TFIID and NT2 P1M fraction are needed to potentiate Oct4/Sox2-depen-
dent activation. Transcription reactions contain Oct4 and Sox2 (lanes 1–6),
NT2 P1M fraction (lanes 2, 4, and 6) with increasing amounts of recombinant
TBP (13 or 23, lanes 1–4), or TFIID (lanes 5 and 6).
(D) Synergistic activation of Nanog by Oct4 and Sox2 requires P1M fractions
prepared from NT2 or mouse ES cell line D3 nuclear extracts. In vitro tran-
scription reactions contain equal amounts (?0.7 mg) of NT2 (lanes 3–6) or D3
P1M fractions (lanes 7–10),withOct4 alone (lanes 4and 8), Sox2alone (lanes 5
and 9), or both activators (lanes 2, 6, and 10).
(E) Immunoblotting analysis of Oct4 levels in whole-cell extracts (WCE)
prepared from pluripotent D3 cells (D3, lane 1) and cells treated with retinoic
acid for 6 days (RA, lane 2).
(F) P1M fractions prepared from pluripotent (D3, lanes 1 and 2) and differen-
tiated (RA, lanes 3 and 4) D3 nuclear extracts were added to transcription
reactions with or without Oct4 and Sox2.
(G) Western blots (2-fold titration) of P1M fractions prepared from pluripotent
(D3) and differentiated (RA) D3 nuclear extracts using anti-BRG-1, anti-
MED23, and anti-MED7 antibodies. Asterisk indicates a nonspecific band or
a breakdown product recognized by anti-MED7 antibody.
See also Figure S1.
122 Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc.
upon differentiation, as is the case for Oct4. To investigate
whether the cofactor activity is restricted to the pluripotent state
of ES cells, D3 cells were induced to differentiate by removal of
LIF and treatment with retinoic acid (RA). The extent of differen-
tiation was monitored by the loss of Oct4 expression that was
complete after 6 days (Figure 1E). Nuclear extracts and P1M
fractions were then prepared from D3 cells before and after
differentiation. When compared to pluripotent D3 P1M fractions,
an equivalent amount of P1M fraction prepared from differenti-
ated D3 nuclear extracts showed significantly decreased co-
factor activity in our in vitro transcription assay (Figure 1F,
compare lanes 1 and 3). This decrease is not due to a wholesale
loss of transcription factors and other cofactors during stem cell
differentiation because the levels of PBAF/BAF (BRG-1) and the
Mediator complex (MED23 and MED7) were largely unchanged
in the two extracts (Figure 1G).
Purification andIdentification ofaStem CellCoactivator
Starting with 200–400 L of NT2 cells, we were able to separate
the cofactor activity into two distinct chromatographic fractions.
at ?0.3 M KCl (Q0.3; data not shown), whereas a second distinct
activity eluted at ?0.6 M KCl (stem cell coactivator [SCC]
(Figures 2A and 2B). Full synergistic Oct4/Sox2-dependant
activation of Nanog required both fractions in our in vitro recon-
stituted transcription reactions (Figure S2). Using this biochem-
ical complementation system, we sequentially purified the
more robust activity, SCC, over eight chromatographic columns,
resulting in > 50,000-fold increase in specific activity (Figure 2A).
Because SCC activity migrated with an apparent native molec-
ular mass (Mr) of ?600 kDa during size-exclusion chromatog-
raphy (Figure 2C), it seemed likely that this coactivator was a
multiprotein complex. Accordingly, SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) of the most purified Mono S frac-
with multiple breakdown products) that consistently copurified
with the SCC activity (Figures 2D and 2E). For the remainder of
this report, we focus on the identification and functional charac-
terization of SCC in vitro and in vivo.
To identify polypeptides comprising the SCC complex, peak
Mono S-purified fractions were pooled and separated by SDS-
by high-sensitivity mass spectrometry revealed all detectable
constituents of SCC to be none other than the Xeroderma pig-
mentosum group C (XPC)-RAD23B-Centrin 2 (CETN2) nucleo-
tide excision repair (NER)complex (Araki etal.,2001) (Figure 3A).
We next carried out western blot analysis with antibodies
specific to XPC, RAD23B, and CETN2 to confirm the identities
of the purified SCC subunits (Figure 3B). As expected, these
three polypeptides were highly enriched in the purified SCC
Mono S peak fractions when compared to the crude NT2 P1M
fraction (Figure 3B). Because identification of SCC as being
identical to the XPC-NER complex was so unexpected, particu-
larly as this repair complex has not been associated with any cell
type-specific function nor linked to stem cell transcription, we
next wanted to compare the relative amounts of this factor in
different cell types. Consistent with the notion that SCC may
be functioning in an unusual way in pluripotent stem cells, we
found that these three proteins are highly enriched in ES and
EC cells. For example, the levels of XPC, RAD23B, and CETN2
in the NT2 P1M fraction are much higher than in an equivalent
amount of P1M fraction prepared from HeLa nuclear extracts
(Figure 3B). Accordingly, in in vitro transcription reactions,
Oct4/Sox2-dependent activation of Nanog by HeLa P1M frac-
tion is much lower than that of NT2 P1M fraction (Figure S1B).
differentiation of mouse D3 ES cells, whereas CETN2, compo-
nents of the basal transcription machinery (TBP and TFIIE), and
other NER factors (XPA and XPB) decreased only slightly while
-46 48 50 52 54 56 58 60 62
Superose 6 frac?ons Superose 6 frac?ons
27 29 31 33 35 37 39 41 43 45 47 49
0.3M KCl 0.6M KCl
670 158 44Mr(kDa)
Mono S frac?ons
14 15 16 1718 19 20 Mr(kDa)
Mono S frac?ons
IN 12 13 14 15 16 17 18 19 20 21 22 23 24
Figure 2. Purification of Stem Cell Coactivator
(A) Chromatography scheme for partial purification of Q0.3 and purification of
SCC from NT2 nuclear extracts (NT2 NE). NT2 NE is first subjected to
ammonium sulfate precipitation (55% saturation) followed by a series of
chromatographic columns as indicated.
(B) Buffer (?) and fractions containing SCC eluted from a Poros-HQ anion
exchanger (top) assayed in the presence of Oct4 and Sox2 in in vitro tran-
(C) Coactivator SCC migrates as a large complex. Input (IN), buffer (?), and
Superose 6 fractions (top) assayed as in (B) except that all reactions are
supplemented with Q0.3 (A). Mobilities of peak activity (500–700 kDa) and gel
filtration protein standards are shown (bottom).
(D) Transcription profile of stem cell coactivator (SCC) activity after the final
Mono S chromatography step. Reactions contain input (IN) and Mono S
fractions (top) and are assayed as in (C).
(E) Silver-stained SDS-PAGE gel of the active Mono S fractions. Filled
arrowheads indicate polypeptides that comigrate with SCC activity.
See also Figure S2.
Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc. 123
finding is consistent with our previous observation that the D3
P1M fraction from differentiated cells is significantly less active
than the pluripotent D3 P1M fraction in potentiating Nanog tran-
scription (Figure 1F).
Reconstitution and Mechanism of Coactivation by SCC
While we were in the process of further characterizing the role of
the XPC-RAD23B-CETN2 complex in transcription, Le May et al.
reported that XPC and other components of the NER apparatus
can be recruited to a gene promoter (e.g., RARb2) upon nuclear
hormone induction (Le May et al., 2010). Although the mecha-
nism by which XPC and other NER factors mediate gene
activation remains unclear, these recent studies and our new
findings have unmasked a hitherto unknown and potentially
important role for XPC that is directly linked to transcription. In
our case, the most striking finding was the direct requirement
for the SCC/XPC complex in selectively potentiating the tran-
scriptional activation of Nanog by Oct4 and Sox2 in ES cell
extracts. However, to more firmly establish this exciting new
connection, we first needed to eliminate the possibility that trace
amounts of contaminants present in our purified SCC fraction
were responsible for the coactivator activity detected in our
in vitro transcription assays. Therefore, we set about to recon-
stitute the heterotrimeric XPC-RAD23B-CETN2 complex from
recombinant gene products expressed in insect (Sf9) cells
following co-infection with baculoviruses expressing His-tagged
XPC, FLAG-tagged RAD23B, and untagged CETN2. Using an
efficient two-step affinity purification procedure, we were able
to purify the recombinant heterotrimeric complex to near homo-
geneity (Figure 4A). Our ability to generate pure polypeptide
subunits, as well as various combinations of dimeric and trimeric
complexes, allowed us to address a number of important ques-
tions, such as whether known functional domains of XPC
required for NER are also necessary for the cofactor activity. It
is well established that XPC’s ability to interact nonspecifically
with DNA is essential for its NER function. Indeed, a single point
mutation in the DNA-binding domain (W690S) of XPC, identified
in an XP patient (XP13PV), abolishes binding to damaged (and
undamaged) DNA and is defective in repair in vivo and in vitro
(Maillard et al., 2007; Yasuda et al., 2007). To address whether
XPC’s nonspecific DNA-binding activity is also important for its
coactivator function, a mutant DNA-binding-defective XPC
(W690S) complex (that had been independently confirmed to
be compromised for DNA binding in vitro; Figures S3A and
S3B) was reconstituted in Sf9 cells and tested along with the
wild-type complex for their ability to support Oct4/Sox2-depen-
dent transcriptional activation of Nanog in vitro. Surprisingly,
specific activities for coactivation comparable to that observed
for purified native endogenous SCC from NT2 cells (Figure 4B).
Taken together, these results confirm that the XPC-RAD23B-
CETN2 complex is indeed SCC and suggest thatits DNA binding
(and repair) activity is dispensable and functionally separable
from its transcriptional cofactor activity at least in vitro. It has
also been reported that XPC can interact directly with TFIIH
(Uchida et al., 2002) and thus might provide a DNA-independent
mechanism by which SCC can be recruited to gene promoters.
To test this possibility, a C-terminal truncation of XPC that abol-
ishes TFIIH (and CETN2) but retains RAD23B binding (amino
acids 1–813, C814St) (Bernardes de Jesus et al., 2008) was
used in our in vitro assay and was found to have no adverse
affect on the ability of a XPC (C814St)-RAD23B heterodimer to
mediate Oct4/Sox2-activated transcription of Nanog (Figures
S3C and S3D). We therefore speculate that SCC/XPC is most
likely targeted to its cognate promoters via potential interactions
with specific activators such as Oct4 and Sox2.
To probe for a potential direct interaction between SCC and
Oct4 and/or Sox2, mouse SCC subunits were overexpressed
with Oct4, Sox2, Klf4, and c-Myc (STEMCCA) (Sommer et al.,
2009) in 293T cells. SCC coimmunoprecipitated with Oct4, but
not with control IgG (Figure 4C). To examine whether the DNA-
binding property of SCC is required for its interaction with
Oct4 and other activators, both the wild-type (WT) and DNA-
binding-defective (W683S in mouse) XPC/SCC complexes
were coexpressed with STEMCCA. Immunoprecipitation of WT
and mutant SCC complexes using an anti-RAD23B antibody
These data indicate a direct and specific protein-protein binding
and Sox2 (but not Klf4; see Figure S1E) in potentiating Nanog
transcription. These findings may also explain why the DNA-
binding activity of the XPC subunit of SCC is dispensable for
transcription in vitro. However, we were unable to reproducibly
detect a stable interaction between SCC and Oct4/Sox2 in D3
ES cell extracts. It is worth noting, though, that other coactiva-
tors implicated in Oct4/Sox2-directed transcriptional activation
0 1 3 7 10
0 1 3 7 10
Figure 3. SCC Is the XPC-RAD23B-CETN2 Nucleotide Excision
(A) Mass spectrometry analysis of Mono S peak activity fractions (16–18) in
Figure 2E with protein identities indicated.
(B) SCC is highly enriched in NT2 P1M fraction. Comparative western blot
analysis of HeLa and NT2 P1M fractions (1.5 mg each) and purified Mono S
SCC fraction (Purif, ?30 ng) using anti-XPC, anti-RAD23B, and anti-CETN2
(C) Downregulation of XPC and RAD23B upon RA-induced differentiation of
mouse D3 ES cells. Western blot analysis of whole-cell extracts prepared
from D3 cells (D3 WCE) collected at indicated days post-RA treatment using
antibodies against XPC, RAD23B, CETN2, OCT4, XPB, XPA, TFIIEb, TBP, and
loading control b-actin (ACTB).
124 Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc.
(e.g., Mediator and p300/CBP) have not been identified in recent
‘‘interactome’’ studies on Oct4-, Sox2-, or Nanog-associating
factors (Engelen et al., 2011; van den Berg et al., 2010; Wang
et al., 2006), supporting the idea that functional coactivator-acti-
vator interactions can often be weak and transient.
The ability to reconstitute active SCC from purified recombi-
nant subunits also provided us with a unique opportunity to
examine the contribution of individual subunits, as well as dif-
ferent dimeric combinations, in supporting Oct4/Sox2 transcrip-
tional activation. Purified individual subunits (XPC or RAD23B),
partial dimeric complexes (XPC-RAD23B or XPC-CETN2), and
holo-SCC complexes (Figure 4E) were assayed over a 4-fold
dose-response range in our fully reconstituted in vitro transcrip-
tion reactions containing Oct4, Sox2, and a partially purified
Q0.3 fraction (Figure 4F). The large XPC subunit alone only
slightly activated transcription above background at the highest
concentrations tested (Figure 4F, compare lanes 1 and 4),
NT2 WT W690S
12345 6 7
Input α αRAD23B
- -+ +
XPC RAD23B RAD23B
1 2 3 456789 10 11 12 13 14 15 16
Figure 4. Reconstitution of Recombinant SCC
(A) Silver-stained SDS-PAGE gel of purified NT2 SCC
(NT2), recombinant wild-type (WT), and DNA-binding-
defective mutant (W690S) XPC-containing SCC com-
plexes reconstituted in insect Sf9 cells by coinfection with
baculoviruses expressing His-tagged XPC, FLAG-tagged
RAD23B, and untagged-CETN2. Major proteolytic frag-
ments of mutant XPC are indicated by asterisks.
(B) Recombinant SCC complex enhances Oct4/Sox2-
activated transcription of Nanog independent of DNA
binding. Buffer (?), NT2 (Mono S peak activity fractions;
lanes 2 and 3), recombinant WT (lanes 4 and 5), and
W690S mutant (lanes 6 and 7) SCC complexes are as-
sayed (over a 3-fold concentration range). All transcription
reactions contain Oct4, Sox2, and Q0.3 (lanes 1–7).
(C) Oct4 interacts with SCC. Western blot analysis of
input lysates (2%) and coimmunoprecipitated proteins
from extracts of 293T cells transfected with a polycistronic
expression plasmid encoding all three subunits of mouse
SCC (mSCC) with or without a polycistronic plasmid ex-
pressing mouse Oct4, Sox2, Klf4, and c-Myc (STEMCCA)
using normal IgG or anti-Oct4 antibody. See also Fig-
(D) SCC-B interacts directly with Oct4 and Sox2 inde-
pendent of DNA binding. Control vector (?), plasmids
expressing wild-type (WT), or mutant (W683S) XPC-
containing mSCC complexes were cotransfected with
STEMCCA into 293T cells and immunoprecipitated with
anti-RAD23B antibody. Input lysates (2%) and RAD23B-
bound proteins were detected by immunoblotting.
(E) Coomassie-stained SDS-PAGE gel of purified re-
combinant XPC, RAD23B, dimeric (XPC-RAD23B and
(F) Titrations (over a 4-fold concentration range) of XPC
(lanes 2–4), RAD23B (lanes 5–7), XPC-RAD23B (lanes
8–10), XPC-CETN2 (lanes 11–13), and XPC-RAD23B-
CETN2 (lanes 14–16) in in vitro transcription reactions
See also Figure S3.
whereas RAD23B alone was essentially inac-
tive. The XPC-CETN2 dimer was slightly more
active than XPC alone. By contrast, a marked
gain in specific activity was observed with the XPC-RAD23B
dimeric complex that was nearly as active as the holo-complex
(Figure 4F). These results suggest that the minimal active
complex likely consists of XPC and RAD23B, whereas CETN2
may enhance the activity of the complex by providing structural
support or stability.
SCC Coactivator Function in ES Cell Self-Renewal
and Somatic Cell Reprogramming
We next set out to determine the role of the SCC/XPC complex
on gene expression and Nanog transcription by loss-of-function
studies in ES cells. Lentiviruses containing two independent
short hairpin RNAs (shRNAs) specifically targeting XPC,
RAD23B, and CETN2 were used to infect mouse D3 ES cells to
selectively depleteSCC (Figures 5A,S4A,andS4B).Knockdown
of SCC subunits resulted in pronounced cellular morphological
abnormalities and decreased alkaline phosphatase (AP) activity
Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc. 125
(Figures 5B and S4C). These knockdown cells also showed
reduced proliferation rates when compared to control ES cells
infected with nontarget viruses, indicating that the self-renewal
capacity of ES cells depleted of SCC may also be compromised
the apoptosis of flattened, fibroblastic AP-negative cells sur-
rounding the collapsing ES cell colonies (Figure 5B and data
not shown). Therefore, knockdown of SCC in ES cells likely
promotes differentiation followed by rapid apoptosis, two
processes that are often coupled. Quantification of colony
assays revealed that ES cells depleted of SCC formed fewer
undifferentiated colonies, with a corresponding increase in
partially and fully differentiated colonies (Figure 5C). Consistent
with the observed morphological changes associated with com-
promised stem cell identity, double and triple knockdown of
XPC, RAD23B, and CETN2 resulted in a 2- to 3-fold reduction
in the mRNA level of Nanog (Figures 5D and S4D) as well as
several other stem cell markers (Fgf4, Zfp42, and Utf1) (Fig-
ure 5D). Knockdown of individual subunits of SCC resulted in
we did not observe overt defects in self-renewal in these single-
subunit knockdown ES cells (data not shown).
To further probe the molecular mechanism underpinning the
function of SCC as a transcriptional coactivator for Oct4 and
Sox2 in vivo, we investigated whether regulatory regions of
Nanog and Oct4 might serve as direct SCC targets by perform-
ing chromatin immunoprecipitation (ChIP) assays in D3 cells
using a RAD23B antibody. ChIP-qPCR analysis revealed that
RAD23B (and presumably XPC/SCC) occupancy sites coincide
with those of Oct4 (Boyer et al., 2005; Chen et al., 2008; Kim
et al., 2008) and Sox2 (Figures 6A and S5A). By contrast, we
failed to detect any significant enrichment of RAD23B at house-
keeping genes b-actin (Actb) (Figure 6A) and dihydrofolate
reductase (Dhfr) (Figure S5B) or an intergenic region on chromo-
some 1 (Figure S5B).
To evaluate the extent to which Oct4 and Sox2 target sites
RAD23B ChIP assays followed by high-throughput sequencing
(ChIP-seq) to identify an entire range of RAD23B/SCC-bound
genomic regions in D3 cells. RAD23B ChIP-seq results were
then compared with published Oct4 and Sox2 ChIP-seq data,
along with those of Nanog and Tcf3 (Marson et al., 2008), to
assess any potential bias in RAD23B occupancy in relation to
these transcription factors. This analysis revealed a striking
binding preference of RAD23B/SCC to genomic sites that are
also co-occupied by Oct4 and Sox2, but not Nanog or Tcf3 only
(?70% versus ?28%, p < 10?15, ANOVA). This strong bias is
maintained whether the ChIP-seq data sets are analyzed by the
degree of peak overlap (defined by any two peaks with at least
one nucleotide of overlap) (Figure 6B) or base pair coverage (Fig-
ure 6C), indicating that the majority of RAD23B/SCC-binding
sites align with those of Oct4 and Sox2. Importantly, the same
analyses performed on ChIP-seq samples obtained from control
IgG immunoprecipitations yielded only background correlation
(between 4% and 8%), confirming the specificity of the
between overlapping RAD23B/SCC and Oct4/Sox2 peaks (see
Extended Experimental Procedures). The majority of them
(76%) lie within close proximity (% 200 base pairs) of each other
(Figure 6D). Even though most of RAD23B/SCC-bound regions
overlap poorly with those bound by Nanog/Tcf3 (?28%), those
that do are still largely (64%) positioned within 200 base pairs
than that of Oct4/Sox2 (p < 10?15, ANOVA, Figure 6D). However,
that overlap with RAD23B-bound sites, we found that many of
them (?40%) could, in fact, contain Oct4 and/or Sox2 when an
alternative peak calling strategy (MACS) was used. Taken
together, these data strongly suggest a classical coactivator
function rather than a purely NER function of SCC both in vitro
with naked DNA and in the context of chromatin in ES cells, as
XPC/RAD23B-mediated DNA damage repair generally involves
transient interactions with DNA (Camenisch et al., 2009) that
would not show either sequence or promoter specificity.
Rel. to AC
NT NTSCC KD SCC KD
Figure 5. SCC Is Required for ES Cell Maintenance
(A) Efficiency of shRNA-mediated depletion of SCC in mouse ES cell line D3.
Whole-cell extracts of mouse D3 cells infected with nontarget (NT) lentiviruses
(MOI of 300) or with an equal mixture of three lentiviruses (MOI of 100 each)
targeting XPC, RAD23B, and CETN2 (SCC KD) are analyzed by western
blotting. Specific bands recognized by their respective antibodies are indi-
cated by filled arrowheads. Asterisks denote nonspecific signals.
(B) ES cell colony morphology and alkaline phosphatase (AP) activity (red) are
maintained in control D3 cells (NT, top) but are compromised in SCC-depleted
D3 cells (SCC KD, bottom). See also Figure S4C.
(C) Clonal assays on SCC-depleted D3 ES cells. Stable nontarget (NT) and
SCC-depleted (SCC KD) D3 cell pools were plated at 300 cells per well in
6-well plates, and emerging colonies were stained for AP activity. Differenti-
ation status was scored based on AP staining intensity, ES cell morphology,
and colony integrity after 6 days.
(D) Two nonoverlapping sets of shRNAs targeting SCC (SCC #1 and SCC #2)
are used to deplete SCC. Quantification of Nanog, Utf1, Fgf4, and Zfp42
mRNA levels are analyzed by real-time quantitative PCR (qPCR) and normal-
ized to Actb. Data from representative experiments are shown; error bars
represent standard deviations. n = 3.
See also Figure S4.
126 Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc.
Given the importance of SCC in stem cell maintenance, we
next asked whether it might also play a role in the reacquisition
of pluripotency during somatic cell reprogramming. Downregu-
lation of either XPC or RAD23B in Oct4-GFP mouse embryonic
fibroblasts (MEFs)—which express some SCC, albeit at signifi-
cantly lower levels than ES cells—led to a dramatic reduction
in the reprogramming efficiency. We observed a significant de-
(SSEA-1+, GFP+) reprogrammed cells, as determined by FACS
sorting (Figures 7A, 7B, and S6A). Consistent with our in vitro
reconstitution result showing that the CETN2 subunit may not
be essential for the transcriptional activity of SCC (Figure 4F),
knockdown of CETN2 had minor effects on iPS cell derivation
efficiency. As expected, reprogramming efficiency using MEFs
derived from XPC and RAD23B knockout (KO) mice (Ng et al.,
2003) was also highly compromised. Surprisingly, RAD23A KO
MEFs were nearly as efficient as wild-type or RAD23A and B
double-heterozygous MEFs in generating AP-positive colonies
enh-950TSSintron -5000 enhTSS intronTSSintron
OCT4/SOX2 (5,030,849 bp)
NANOG/TCF3 (1,151,570 bp)
75.1% (3,776,285 bp)
28.5% (327,992 bp)
8.3% (420,442 bp)
3.7% (42,672 bp)
Figure 6. SCC Is Recruited to the Nanog and Oct4
Promoters and Genomic Regions Occupied by
Oct4 and Sox2
(A) Co-occupancy of SCC, Oct4, and Sox2 on the
promoters of Nanog and Oct4. ChIP analysis of RAD23B
occupancy on distal enhancers (enh), proximal promoter
(transcription start site, TSS), and upstream (positions
indicated by numbers) and downstream intronic regions of
the Nanog (left), Oct4 (middle), and Actb (right) gene loci.
Representative data (n > 5) showing the enrichment of
RAD23B (black bars) compared to normal IgGs (white
bars) are analyzed by qPCR and expressed as percentage
of input chromatin. Schematic diagrams of Oct4- and
Sox2-binding sites on the Nanog and Oct4 regulatory
regions (TSS and enhancers; see also Figure S5A) are
indicated at the bottom. Error bars represent standard
deviations. n = 3.
(B) Percent peak overlap between RAD23B and control
IgG ChIP-seq data relative to published Oct4/Sox2 and
Nanog/Tcf3 peak data.
(C) Percent base pair overlap between RAD23B and
control IgG ChIP-seq data relative to Oct4/Sox2 and
Nanog/Tcf3 ChIP-seq data sets.
(D) Distribution of distance (in base pair) of RAD23B
and control IgG peaks from Oct4/Sox2 and Nanog/Tcf3
See also Figure S5.
upon iPS cell induction (Figures 7C and S6B).
Thisresult maypointtoanonredundant function
of RAD23B in somatic reprogramming indepen-
dent of its role in DNA repair, as RAD23B KO
(and RAD23A KO) MEFs are NER proficient
(Ng et al., 2003). Importantly, depletion of XPC
(knockdown and knockout) and CETN2 in
MEFs did not affect proliferation rates when
compared to nontarget or Oct4 knockdown
MEFs. However, RAD23B-depleted MEFs dis-
played noticeable changes in growth rates,
which may partially account for the marked
reduction in reprogramming efficiency (data not shown). These
data suggest that efficient reprogramming may require SCC/
XPC in conjunction with Oct4 and Sox2 to re-establish ES-
specific gene expression programs.
Establishment of ground state pluripotency in embryonic stem
cells represents one of the most remarkable events in develop-
ment. Stem cells have evolved a subset of cell type-specific
activators among a constellation of previously identified tran-
scription factors and cofactors to resolve the dichotomy
between self-renewal versus differentiation. Our de novo purifi-
cation of the SCC/XPC complex as a potent coactivator for
Oct4 and Sox2 was unanticipated but may, in part, reflect the
need for stem cells to robustly expand and diversify their tran-
scriptional repertoire while also maintaining genome integrity.
Indeed, other NER factors have been shown to participate in
transcriptional regulation both at the basal and activated levels.
Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc. 127
For instance, the general transcription factor TFIIH is a classic
example with established roles in both transcription initiation
and NER (Schaeffer et al., 1993). Interestingly, it has recently
been reported that, in HeLa cells, the entire NER complex can
be assembled onto promoters of activated genes in an XPC-
dependent manner. However, XPC alone is not sufficient, as
other NER components appear to be responsible for RA-acti-
vated transcription (Le May et al., 2010). This finding in HeLa
cells is distinct from our observation that the XPC-NER (SCC)
complex plays a direct and critical role in Nanog transcription in-
vitro and in ES cells. In our studies, optimal activation of Nanog
by Oct4/Sox2 potentiated by SCC requires a second activity
present in the Q0.3 fraction. However, preliminary mass spec-
trometry analyses of the partially purified Q0.3 fraction failed to
detect any other XP or NER factors or factors previously identi-
fied to copurify with Nanog or Oct4 in ES cells (van den Berg
et al., 2010; Wang et al., 2006) (data not shown). Therefore, the
SCC/XPC complex can potentiate Nanog transcription and likely
other Oct4/Sox2-directed promoters in the absence of addi-
tional XP and NER factors in vitro. Taken together, these results
suggest that the mechanism by which the SCC/XPC complex
coactivates transcription in ES cells may be distinct from its
function in HeLa cells.
Although XPC plays a critical role in DNA lesion recognition,
XPC is not universally required for NER, as certain types of bulky
DNA lesions (e.g., cholesterol-DNA adducts) can be repaired
Figure 7. SCC Is Required for Efficient Somatic
(A) Depletion of SCC blocks somatic cell reprogramming.
Oct4-GFP mouse embryonic fibroblasts infected with
lentiviruses expressing STEMCCA and rtTA together with
nontarget shRNA (NT), shRNAs against Oct4, individual
subunits of SCC, or all three subunits simultaneously at
low or high multiplicity of infection (SCC LO or HI) are
plated in 6-well plates for colony counting and FACS or in
24-well plates for AP staining. AP-positive (red) cells are
stained and counted 17 days (14 days + dox, 3 days ?
dox) postinduction (dpi). Results from two separate
experiments are shown.
(B) Single cell suspensions of 17 dpi Oct4-GFP MEFs as
described in (A) are stained with anti-mouse SSEA-1
antibodies and analyzed by FACS.
(C) Wild-type (WT), RAD23A, and RAD23B double-
heterozygous (23A/B d-Het) MEFs, together with XPC,
RAD23A, and RAD23B knockout (KO) MEFs, are induced
with STEMCCA. AP-positive colonies are stained and
counted as in (A).
See also Figure S6.
without XPC (Mu et al., 1996). Intriguingly,
eventhough XPCisrecruited to genepromoters
irrespective of DNA damage signals (Le May
et al., 2010), the XPC-NER complex is the only
factor in the XP family that is dispensable for
transcription-coupled repair (TCR) (Venema
et al., 1990). Indeed, our findings suggest that
the coactivator and NER duties carried out by
SCC are mechanistically distinct processes, as
SCC can function as part of the transcriptional
cofactor apparatus via a direct interaction with Oct4 and Sox2
without requiring either DNA or TFIIH binding mediated by XPC.
It is worth noting that the effect of single knockdown of XPC or
RAD23B was much more pronounced in the reprogramming of
MEFs than in the maintenance of ES cells. We surmise that
perhaps other redundant regulatory mechanisms in established
ES cells can partially compensate for the loss of SCC. Such
robust regulatory circuitries are likely to be less developed
during the early phase of reprogramming in MEFs and are
thus more susceptible to perturbation by SCC depletion. It is
conceivable that SCC/XPC may also contribute to the process
of chromatin reorganization and facilitate changes in the epige-
netic landscape that are conducive to iPS conversion (Le May
et al., 2010).
Also in agreement with our in vitro and cell-based studies,
a mouse double KO of RAD23B and its homolog RAD23A was
found to be early embryonic lethal (Ng et al., 2003). This previ-
ously puzzling phenotype can now be more readily rationalized
revealed here. Taken together, these results strongly suggest
that loss of the SCC/XPC complex may indeed compromise
the transcriptional integrity of pluripotent stem cells, as well as
the ability of somatic cells to re-establish pluripotency. However,
XPC KO mice are UV sensitive but otherwise normal, with no
obvious developmental defects (Sands et al., 1995). It has been
shown that RAD23B is in vast excess relative to XPC (Sugasawa
128 Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc.
et al., 1996), suggesting that RAD23B may exist in other com-
plexes independent of XPC that functionally replace SCC.
Embryonic stem cells are thought to be under strong selective
pressure to maintain genome fidelity because accumulation and
propagation of DNA errors to progenitor cells would be lethal
during development; therefore, DNA damage response factors
and pathways are often upregulated in ES cells (e.g., XPC,
RAD23B, ERCC5, etc.) (Cervantes et al., 2002; Ramalho-Santos
et al., 2002). Should DNA repair fail, UV-damaged ES cells can
be eliminated first by repressing Nanog expression through
p53 upregulation, which in turn promotes spontaneous differen-
tiation and efficient apoptosis (Lin et al., 2005). It is interesting
to note that, upon UV-induced DNA damage in HeLa cells,
recruitment of XPC to non-UV-inducible genes, as well as their
expression, are dramatically delayed (Le May et al., 2010). This
suggests that some sort of redistribution mechanism may redi-
rect XPC from transcription duty at promoter targets to the
NER pathway in response to DNA damage. In light of these
observations, it is tempting to speculate that redistribution of
XPC-RAD23B-CETN2 from Nanog and presumably other Oct4/
Sox2-regulated promoters to DNA damage sites may provide
an efficient sensing mechanism to perturb stem cell-specific
gene expression programs and thus provide a window of oppor-
tunity for ES cells to either repair the lesions or commit to differ-
entiation and apoptosis. The SCC/XPC complex may therefore
act as a molecular link to couple stem cell-specific gene expres-
sion programs and genome surveillance in ES cells.
DNA Constructs, Cell Lines, and Cell Culture
Construction of in vitro transcription templates and protein expression
plasmids are described in Extended Experimental Procedures. HeLa, 293T,
NTERA-2 (NT2), and mouse ES cell line D3 were maintained in standard con-
ditions. Differentiation of D3 ES cells was carried out by LIF removal followed
by retinoic acid treatment (5–10 mM, Sigma).
Purification and Identification of SCC
Nuclear extracts from ?400 l of NT2 cells were purified over eight chromato-
graphic steps to homogeneity. Methods for purification and mass spectrom-
etry analyses of SCC are detailed in Extended Experimental Procedures.
Western Blotting, Immunoprecipitation, and Affinity Purification
Antibodies used are described in Extended Experimental Procedures. Tran-
scriptional activators were purified from transiently transfected HeLa cells
followed by affinity purification using anti-FLAG (M2) agarose (Sigma) as
described in Extended Experimental Procedures. Recombinant SCC com-
plexes were purified from Sf9 cells infected with baculoviruses (BAC-to-BAC
system, Invitrogen) expressing N-terminal His6-tagged or FLAG-tagged
XPC, N-FLAG-tagged RAD23B, and untagged CETN2. Sf9 cells were har-
vested 48 hrafter infection, and protein complexeswere purified by incubating
cell lysates with Ni-NTA resin (QIAGEN), anti-FLAG (M2) agarose (Sigma), and
elution by the FLAG peptides.
shRNA-Mediated Knockdown of SCC by Lentiviral Infection
Control nontarget and pLKO shRNA plasmids targeting XPC, RAD23B, and
CETN2 (Sigma) were transfected with packaging vectors into 293T cells using
FuGENE 6 (Roche). Supernatants were concentrated by ultracentrifugation
and resuspended in PBS. Viral titer was determined by a QuickTiter Lentivirus
Titer Kit (Cell Biolabs). SCC knockdown was performed by incubating lentiviral
concentrates with D3 cells in the presence of 8 mg/ml polybrene followed by
puromycin selection (1.5 mg/ml).
Gene Expression Analysis and ChIP
Total RNA from shRNA-mediated knockdown D3 ES cells was isolated using
RNeasy Plus Kit (QIAGEN) and analyzed by qRT-PCR. Chromatin immunopre-
cipitation (ChIP) assays were performed in D3 cells as described in Extended
Experimental Procedures. Precipitated DNA was measured by qPCR or
sequenced using an Illumina HiSeq 2000 sequencing platform. Methods for
gene expression and ChIP analyses are detailed in Extended Experimental
Somatic Cell Reprogramming
Oct4-GFPMEFs(TheJacksonLaboratory) wereinfectedwithlentiviruses con-
taining STEMCCA and rtTA, followed by infection with pLKO shRNA lentiviral
supernatants targeting SCC. Oct4, Sox2, Klf4, and c-Myc expressions were
induced by doxycycline, and SCC knockdown MEFs were selected with puro-
mycin. Reprogrammed cells were either detected by alkaline phosphatase
activity or stained with anti-SSEA-1 antibodies conjugated to Alexa Fluor
647 (BioLegends) and analyzed by FACS. XPC, RAD23A, and RAD23B
knockout MEFs were generous gifts from Dr. Hoeijmakers (Rotterdam, The
Supplemental Information includes Extended Experimental Procedures and
six figures and can be found with this article online at doi:10.1016/j.cell.
The authors wish to thank A. Fischer and M. Richner at the Tissue Culture
Facility (University of California, Berkeley); S. Zheng, G. Dailey, M. Haggart,
and E. Bourbon for technical assistance; S. Chen at National Institute of
Biological Sciences (Beijing, China) for mass spectrometry analysis; S. Zhou
for initial mass spectrometry analysis; J. Hoeijmakers for knockout MEFs; G.
Mostoslavsky for STEMCCA; O. Puig for pGL3-CAT plasmid; S. Ryu for
purified rTBP and G3BCAT plasmid; K. Hochedlinger, M. Stadtfeld, J. de
Wit, and M. Holmes for technical advice; and M. Botchan, M. Holmes, M. Lev-
ine, S. Martin, M. Rape, D. Rio, and all members of our laboratory for critical
reading of the manuscript. Y.W.F. was a California Institute for Regenerative
Medicine Scholar (CIRM training grant T1-00007). T.Y. was supported by the
Swiss National Science Foundation and the Siebel Stem Cell Institute and is
a Jane Coffin Childs fellow.
Received: July 26, 2011
Revised: August 23, 2011
Accepted: August 25, 2011
Published: September 29, 2011
Araki, M., Masutani, C., Takemura, M., Uchida, A., Sugasawa, K., Kondoh, J.,
Ohkuma, Y.,and Hanaoka, F.(2001).Centrosomeproteincentrin2/caltractin1
is part of the xeroderma pigmentosum group C complex that initiates global
genome nucleotide excision repair. J. Biol. Chem. 276, 18665–18672.
Bernardes de Jesus, B.M., Bjøra ˚s, M., Coin, F., and Egly, J.M. (2008). Dissec-
tion of the molecular defects caused by pathogenic mutations in the DNA
repair factor XPC. Mol. Cell. Biol. 28, 7225–7235.
Boyer, L.A., Lee, T.I., Cole, M.F., Johnstone, S.E., Levine, S.S., Zucker, J.P.,
Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G., et al. (2005). Core
transcriptional regulatory circuitry in human embryonic stem cells. Cell 122,
May, E., and Naegeli, H. (2009). Two-stage dynamic DNA quality check by
xeroderma pigmentosum group C protein. EMBO J. 28, 2387–2399.
Cervantes, R.B., Stringer, J.R., Shao, C., Tischfield, J.A., and Stambrook, P.J.
(2002). Embryonic stem cells and somatic cells differ in mutation frequency
and type. Proc. Natl. Acad. Sci. USA 99, 3586–3590.
Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc. 129
Chakravarthy, H., Boer, B., Desler, M., Mallanna, S.K., McKeithan, T.W., and
Rizzino, A. (2008). Identification of DPPA4 and other genes as putative Sox2:
Oct-3/4 target genes using a combination of in silico analysis and transcrip-
tion-based assays. J. Cell. Physiol. 216, 651–662.
Zhang, W., Jiang, J., et al. (2008). Integration of external signaling pathways
with the core transcriptional network in embryonic stem cells. Cell 133,
Engelen, E., Akinci, U., Bryne, J.C., Hou, J., Gontan, C., Moen, M., Szumska,
D., Kockx, C., van Ijcken, W., Dekkers, D.H., et al. (2011). Sox2 cooperates
with Chd7 to regulate genes that are mutated in human syndromes. Nat.
Genet. 43, 607–611.
Gao, X., Tate, P., Hu, P., Tjian, R., Skarnes, W.C., and Wang, Z. (2008). ES cell
pluripotency and germ-layer formation require theSWI/SNFchromatin remod-
eling component BAF250a. Proc. Natl. Acad. Sci. USA 105, 6656–6661.
Goodrich, J.A., and Tjian, R. (2010). Unexpected roles for core promoter
recognition factors in cell-type-specific transcription and gene regulation.
Nat. Rev. Genet. 11, 549–558.
Jaenisch, R., and Young, R. (2008). Stem cells, the molecular circuitry of
pluripotency and nuclear reprogramming. Cell 132, 567–582.
Jiang, J., Chan, Y.S., Loh, Y.H., Cai, J., Tong, G.Q., Lim, C.A., Robson, P.,
Zhong, S., and Ng, H.H. (2008). A core Klf circuitry regulates self-renewal of
embryonic stem cells. Nat. Cell Biol. 10, 353–360.
Kagey, M.H., Newman, J.J., Bilodeau, S., Zhan, Y., Orlando, D.A.,
van Berkum, N.L., Ebmeier, C.C., Goossens, J., Rahl, P.B., Levine, S.S.,
et al. (2010). Mediator and cohesin connect gene expression and chromatin
architecture. Nature 467, 430–435.
Kidder, B.L., Palmer, S., and Knott, J.G. (2009). SWI/SNF-Brg1 regulates self-
renewal and occupies core pluripotency-related genes in embryonic stem
cells. Stem Cells 27, 317–328.
Kim, J., Chu, J., Shen, X., Wang, J., and Orkin, S.H. (2008). An extended
transcriptional network for pluripotency of embryonic stem cells. Cell 132,
Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S.Y., Suemori, H.,
Nakatsuji, N., and Tada, T. (2005). Octamer and Sox elements are required
for transcriptional cis regulation of Nanog gene expression. Mol. Cell. Biol.
Le May, N., Mota-Fernandes, D., Ve ´lez-Cruz, R., Iltis, I., Biard, D., and Egly,
J.M. (2010). NER factors are recruited to active promoters and facilitate chro-
matin modification for transcription in the absence of exogenous genotoxic
attack. Mol. Cell 38, 54–66.
remodelling cofactors for ligand-activated transcription. Nature 414, 924–928.
Lin, T., Chao, C., Saito, S., Mazur, S.J., Murphy, M.E., Appella, E., and Xu, Y.
(2005). p53 induces differentiation of mouse embryonic stem cells by sup-
pressing Nanog expression. Nat. Cell Biol. 7, 165–171.
Liu, Z., Scannell, D.R., Eisen, M.B., and Tijan, R. (2011). Control of embryonic
stem cell lineage commitment by core promoter factor, TAF3. Cell 146,
Loh, Y.H., Wu, Q., Chew, J.L., Vega, V.B., Zhang, W., Chen, X., Bourque, G.,
George, J., Leong, B., Liu, J., et al. (2006). The Oct4 and Nanog transcription
network regulates pluripotency in mouse embryonic stem cells. Nat. Genet.
Maillard, O., Solyom, S., and Naegeli, H. (2007). An aromatic sensor with
aversion to damaged strands confers versatility to DNA repair. PLoS Biol.
Marson, A., Levine, S.S., Cole, M.F., Frampton, G.M., Brambrink, T.,
Johnstone, S., Guenther, M.G., Johnston, W.K., Wernig, M., Newman, J.,
circuitry of embryonic stem cells. Cell 134, 521–533.
Mu, D., Hsu, D.S., and Sancar, A. (1996). Reaction mechanism of human DNA
repair excision nuclease. J. Biol. Chem. 271, 8285–8294.
Mu ¨ller, F., Zaucker, A., and Tora, L. (2010). Developmental regulation of
transcription initiation: more than just changing the actors. Curr. Opin. Genet.
Dev. 20, 533–540.
Na ¨a ¨r,A.M.,Beaurang,P.A., Robinson, K.M., Oliner, J.D.,Avizonis, D., Scheek,
S., Zwicker, J., Kadonaga, J.T., and Tjian, R. (1998). Chromatin, TAFs, and
a novel multiprotein coactivator are required for synergistic activation by
Sp1 and SREBP-1a in vitro. Genes Dev. 12, 3020–3031.
Na ¨a ¨r, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W., and Tjian, R.
(1999). Composite co-activator ARC mediates chromatin-directed transcrip-
tional activation. Nature 398, 828–832.
Na ¨a ¨r, A.M., Lemon, B.D., and Tjian, R. (2001). Transcriptional coactivator
complexes. Annu. Rev. Biochem. 70, 475–501.
Na ¨a ¨r, A.M., Taatjes, D.J., Zhai, W., Nogales, E., and Tjian, R. (2002). Human
conformation. Genes Dev. 16, 1339–1344.
Ng, J.M., Vermeulen, W., van der Horst, G.T., Bergink, S., Sugasawa, K.,
Vrieling, H., and Hoeijmakers, J.H. (2003). A novel regulation mechanism of
DNA repair by damage-induced and RAD23-dependent stabilization of xero-
derma pigmentosum group C protein. Genes Dev. 17, 1630–1645.
differentiation potential of NTERA-2 cells as a model for studying human
embryonic stem cells. Cell Prolif. 39, 585–598.
Ramalho-Santos,M.,Yoon,S.,Matsuzaki,Y.,Mulligan, R.C.,and Melton,D.A.
(2002). ‘‘Stemness’’: transcriptional profiling of embryonic and adult stem
cells. Science 298, 597–600.
Rodda,D.J.,Chew,J.L.,Lim,L.H.,Loh,Y.H.,Wang, B.,Ng, H.H.,and Robson,
P. (2005). Transcriptional regulation of nanog by OCT4 and SOX2. J. Biol.
Chem. 280, 24731–24737.
Roeder, R.G. (2005). Transcriptional regulation and the role of diverse coacti-
vators in animal cells. FEBS Lett. 579, 909–915.
Rosenfeld, M.G., Lunyak, V.V., and Glass, C.K. (2006). Sensors and signals:
a coactivator/corepressor/epigenetic code for integrating signal-dependent
programs of transcriptional response. Genes Dev. 20, 1405–1428.
Ryu, S., Zhou, S., Ladurner, A.G., and Tjian, R. (1999). The transcriptional
cofactor complex CRSPis required foractivity of the enhancer-binding protein
Sp1. Nature 397, 446–450.
Sands, A.T., Abuin, A., Sanchez, A., Conti, C.J., and Bradley, A. (1995). High
susceptibility to ultraviolet-induced carcinogenesis in mice lacking XPC.
Nature 377, 162–165.
Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeij-
makers, J.H., Chambon, P., and Egly, J.M. (1993). DNA repair helicase:
a component of BTF2 (TFIIH) basic transcription factor. Science 260, 58–63.
Schwartz, C.M., Spivak, C.E., Baker, S.C., McDaniel, T.K., Loring, J.F.,
Nguyen, C., Chrest, F.J., Wersto, R.,Arenas,E., Zeng, X.,et al. (2005). NTera2:
a model system to study dopaminergic differentiation of human embryonic
stem cells. Stem Cells Dev. 14, 517–534.
Sommer, C.A., Stadtfeld, M., Murphy, G.J., Hochedlinger, K., Kotton, D.N.,
and Mostoslavsky, G. (2009). Induced pluripotent stem cell generation using
a single lentiviral stem cell cassette. Stem Cells 27, 543–549.
Sperger, J.M., Chen, X., Draper, J.S., Antosiewicz, J.E., Chon, C.H., Jones,
S.B., Brooks, J.D., Andrews, P.W., Brown, P.O., and Thomson, J.A. (2003).
Gene expression patterns in human embryonic stem cells and human pluripo-
tent germ cell tumors. Proc. Natl. Acad. Sci. USA 100, 13350–13355.
Sugasawa, K., Masutani, C., Uchida, A., Maekawa, T., van der Spek, P.J.,
Bootsma, D., Hoeijmakers, J.H., and Hanaoka, F. (1996). HHR23B, a human
Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.
Mol. Cell. Biol. 16, 4852–4861.
Taatjes, D.J., Marr, M.T., and Tjian, R. (2004). Regulatory diversity among
metazoan co-activator complexes. Nat. Rev. Mol. Cell Biol. 5, 403–410.
Tokuzawa, Y., Kaiho, E., Maruyama, M., Takahashi, K., Mitsui, K., Maeda, M.,
Niwa, H., and Yamanaka, S. (2003). Fbx15 is a novel target of Oct3/4 but is
dispensable for embryonic stem cell self-renewal and mouse development.
Mol. Cell. Biol. 23, 2699–2708.
130 Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc.
Tutter, A.V., Kowalski, M.P., Baltus, G.A., Iourgenko, V., Labow, M., Li, E., and Download full-text
Kadam, S. (2009). Role for Med12 in regulation of Nanog and Nanog target
genes. J. Biol. Chem. 284, 3709–3718.
Uchida, A., Sugasawa, K., Masutani, C., Dohmae, N., Araki, M., Yokoi, M.,
Ohkuma, Y., and Hanaoka, F. (2002). The carboxy-terminal domain of the
XPC protein plays a crucial role in nucleotide excision repair through interac-
tions with transcription factor IIH. DNA Repair (Amst.) 1, 449–461.
van den Berg, D.L., Snoek, T., Mullin, N.P., Yates, A., Bezstarosti, K.,
Demmers, J., Chambers, I., and Poot, R.A. (2010). An Oct4-centered protein
interaction network in embryonic stem cells. Cell Stem Cell 6, 369–381.
van den Berg, D.L., Zhang, W., Yates, A., Engelen, E., Takacs, K., Bezstarosti,
K., Demmers, J., Chambers, I., and Poot, R.A. (2008). Estrogen-related
receptor beta interacts with Oct4 to positively regulate Nanog gene expres-
sion. Mol. Cell. Biol. 28, 5986–5995.
L.H. (1990). The residual repair capacity of xeroderma pigmentosum comple-
mentation group C fibroblasts is highly specific for transcriptionally active
DNA. Nucleic Acids Res. 18, 443–448.
Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D.N., Theunissen, T.W., and
Orkin, S.H. (2006). A protein interaction network for pluripotency of embryonic
stem cells. Nature 444, 364–368.
Yasuda, G., Nishi, R., Watanabe, E., Mori, T., Iwai, S., Orioli, D., Stefanini, M.,
Hanaoka, F., and Sugasawa, K. (2007). In vivo destabilization and functional
defects of the xeroderma pigmentosum C protein caused by a pathogenic
missense mutation. Mol. Cell. Biol. 27, 6606–6614.
J., Ma, Y., Chai, L., et al. (2006). Sall4 modulates embryonic stem cell pluripo-
tency and early embryonic development by the transcriptional regulation of
Pou5f1. Nat. Cell Biol. 8, 1114–1123.
Zhang, X., Zhang, J., Wang, T., Esteban, M.A., and Pei, D. (2008). Esrrb acti-
vates Oct4 transcription and sustains self-renewal and pluripotency in embry-
onic stem cells. J. Biol. Chem. 283, 35825–35833.
Cell 147, 120–131, September 30, 2011 ª2011 Elsevier Inc. 131