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Cell Stem Cell
Short Article
p27
Kip1
Directly Represses Sox2
during Embryonic Stem Cell Differentiation
Han Li,
1,5
Manuel Collado,
1,5,6,
*Aranzazu Villasante,
1
Ander Matheu,
3
Cian J. Lynch,
1
Marta Can
˜amero,
2
Karine Rizzoti,
3
Carmen Carneiro,
4
Gloria Martı
´nez,
4
Anxo Vidal,
4
Robin Lovell-Badge,
3
and Manuel Serrano
1,
*
1
Tumor Suppression Group
2
Histopathology Unit
Spanish National Cancer Research Centre (CNIO), Madrid, E28029, Spain
3
Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
4
Departamento de Fisioloxia, Facultade de Medicina, Universidade de Santiago de Compostela, Instituto de Investigaciones Sanitarias (IDIS),
Santiago de Compostela, E15782, Spain
5
These authors contributed equally to this work
6
Present address: Instituto de Investigacio
´n Sanitaria de Santiago de Compostela (IDIS), Complexo Hospitalario Universitario de Santiago de
Compostela (CHUS), SERGAS, Santiago de Compostela, E15706, Spain
*Correspondence: manuel.collado.rodriguez@sergas.es (M.C.), mserrano@cnio.es (M.S.)
http://dx.doi.org/10.1016/j.stem.2012.09.014
SUMMARY
The mechanisms responsible for the transcriptional
silencing of pluripotency genes in differentiated cells
are poorly understood. We have observed that cells
lacking the tumor suppressor p27 can be reprog-
rammed into induced pluripotent stem cells (iPSCs)
in the absence of ectopic Sox2. Interestingly, cells
and tissues from p27 null mice, including brain, lung,
and retina, present an elevated basal expression of
Sox2, suggesting that p27 contributes to the repres-
sion of Sox2. Furthermore, p27 null iPSCs fail to fully
repress Sox2 upon differentiation. Mechanistically,
we have found that upon differentiation p27 associ-
ates to the SRR2 enhancer of the Sox2 gene together
with a p130-E2F4-SIN3A repressive complex. Finally,
Sox2 haploinsufficiency genetically rescues some of
the phenotypes characteristic of p27 null mice,
including gigantism, pituitary hyperplasia, pituitary
tumors, and retinal defects. Collectively, these results
demonstrate an unprecedented connection between
p27 and Sox2 relevant for reprogramming and cancer
and for understanding human pathologies associated
with p27 germline mutations.
INTRODUCTION
Differentiated cells can be converted into induced pluripotent
stem cells (iPSCs) through the combined action of transcription
factors, most notably OCT4, KLF4, and SOX2 (Takahashi and
Yamanaka, 2006). Importantly, the mechanisms involved in this
process might provide clues about the molecular mechanisms
governing stem cell biology and cancer. Recently, we and others
have shown that tumor suppressors, such as those encoded by
the p53 gene and the Ink4a/Arf locus, oppose reprogramming
and limit the efficiency of the process (Banito et al., 2009;
Hong et al., 2009;Kawamura et al., 2009;Li et al., 2009;Mario
´n
et al., 2009;Utikal et al., 2009; Zhao et al., 2008).
The tumor suppressor p27
Kip1
binds and inhibits multiple
cyclin-dependent kinases (Besson et al., 2008). Importantly,
low protein levels of p27 constitute a poor prognosis marker for
several types of cancer (Chu et al., 2008) and germline mutations
of the p27 gene (also known as CDKN1B) are responsible for
a subset of human multiple endocrine neoplasia (MEN)
syndromes, notably characterized by pituitary tumors (Marinoni
and Pellegata, 2011;Vandeva et al., 2010). The cyclin-dependent
kinase 2 (CDK2) is one of the main CDKs inhibited by p27 (Besson
et al., 2008). Paradoxically, however, the main phenotypes of
p27 null mice, namely, increased body size, organ hyperplasia,
pituitary tumors, and retinal dysplasia (Fero et al., 1996;Kiyokawa
et al., 1996;Nakayama et al., 1996), are not rescued by con-
comitant deletion of Cdk2, thus suggesting that these p27 null
phenotypes are not primarily caused by uncontrolled CDK2
activity (Aleem et al., 2005;Martı
´n et al., 2005).
In the context of investigating the role of tumor suppressors
during reprogramming, we studied p27 null cells and we noticed
that these cells can be reprogrammed into iPSCs without
ectopic expression of Sox2. This observation led us to explore
the potential link between these two previously unrelated
proteins, p27 and SOX2.
RESULTS
Cells Lacking p27 Express Higher Levels of Sox2 and
Can Be Reprogrammed without Ectopic Sox2
While investigating the effect of tumor suppressor genes on the
process of reprogramming to induced pluripotent stem cells
(iPSCs) by the three Yamanaka factors (Oct4,Klf4, and Sox2)
(Takahashi and Yamanaka, 2006), we tested the three possible
combinations of two factors (abbreviated as 2F-OK, 2F-OS,
and 2F-KS in reference to Oct4, Klf4, and Sox2) in a series of
primary mouse embryo fibroblasts (MEFs) lacking cell cycle
regulators and tumor suppressors. After repeated attempts, we
were unable to obtain alkaline-phosphatase-positive (AP
+
)
colonies in any of the tested MEFs using 2F-OS or 2F-KS.
Interestingly, however, p27 null MEFs and, to a lesser extent,
p130 null MEFs gave rise to AP
+
colonies with 2F-OK (Figures
1A and 1B). Absence of the p27-related protein p21 also
Cell Stem Cell 11, 845–852, December 7, 2012 ª2012 Elsevier Inc. 845
produced AP
+
colonies and further increased the number of AP
+
colonies when combined with p27 deficiency, thus suggesting
some degree of functional redundancy between p27 and p21.
In all these MEFs, the emergence of visible AP
+
colonies was
delayed compared to the standard three-factor cocktail (3F-
OKS) (4 weeks versus 2 weeks) and the average efficiency was
about 100-fold lower (9 310
!5
in p27 null/2F-OK versus 8 3
10
!3
in WT/3F-OKS). In contrast to this, WT MEFs or MEFs defi-
cient in p53,Arf,orInk4a/Arf could not be reprogrammed by
2F-OK despite the fact that these cells are reprogrammed with
very high efficiency by 3F-OKS (Banito et al., 2009;Hong et al.,
2009;Kawamura et al., 2009;Li et al., 2009;Mario
´n et al.,
2009;Utikal et al., 2009;Zhao et al., 2008). Also, absence of
p27 had a modest stimulatory effect on 3F-OKS reprogramming
(Figure S1A available online). Together, these observations
suggest that the absence of p27 selectively renders cells
susceptible to reprogramming in the absence of ectopic Sox2.
The p27 null/2F-OK AP
+
colonies were confirmed to be bona
fide iPSCs based on their expression of endogenous pluripo-
tency genes (Nanog,Sox2, and Oct4;Figure S1B), production
CB
wt
p27-null
genetic
-5)
10
20
30
25
15
5
2F-OK
n=9
n=9
n=2
n=2 n=2 n=2
n=9
n=9
A
F
0
10
20
30
40
50
60
**
*
retina brain lun
g
levels (relative to Gapdh x10-3)
wt
p27-null !
Sox2 mRNA
*
DSOX2nuclei
10
50
40
30
20
eventsevents
10
50
40
30
20
0
100
200
wt
p27-null
0
100
200
p27-null
+ empty
SOX2nuclei/p27/SOX2
events events
0
100
200
10
50
40
30
20
0
100
200
10
50
40
30
20
*
**
p27-null
+ p27
E
Sox2 mRNA
-2
-1
0
1
2
MEFs : ESCs
*
levels (relative to Gapdh x10-3)
log10
***
450x
7x
64x
Figure 1. Absence of p27 Allows Two-
Factor (Oct4 and Klf4) Reprogramming
(A) Two-factor (Oct4 and Klf4, 2F-OK) re-
programming of primary MEFs of the indicated
genotype. Efficiency is measured as the number
of alkaline-phosphatase-positive (AP
+
) colonies
relative to the total number of infected cells.
n values correspond to independent MEF isolates.
(B) Representative picture of AP stained plates.
(C) Picture of chimeric mice generated from p27
null/2F-OK iPSCs (black-C57BL6 genetic back-
ground) after microinjection into albino-C57BL6
blastocysts.
(D) Sox2 mRNA levels in WT (n = 3) and p27 null (n =
5) MEFs and ESCs. mRNA levels were determined
by qRT-PCR.
(E) Upper panel: representative picture of SOX2
immunofluorescence in WT and p27 null MEFs and
quantification of the immunofluorescence corre-
sponding to one experiment. A total of two
experiments were performed, each with different
MEF isolates, with similar results obtained in both
of them. Lower panel: representative picture of
p27 and SOX2 immunofluorescence in p27 null
MEFs infected with empty vector or with pBabe-
p27. A total of three independent experiments
were performed, each with different MEF isolates,
and similar results were obtained in the three
of them. The average ± SD of each distribution
was compared with its corresponding control
using the Student’s t test.
(F) Sox2 mRNA levels in WT (n = 6) and p27 null
(n = 10) mice ("1 year old).
All data correspond to the average ± SD. Statistical
significance was assessed by two-tailed Student’s
t test: ***p < 0.001; **p < 0.01; *p < 0.05. See also
Figure S1.
of teratomas (Figure S1C), and efficient
contribution to chimeric mice (Figure 1C).
To further validate the use of alkaline
phosphatase as a marker of reprogram-
ming under our experimental conditions (most notably charac-
terized by the absence of ectopic c-Myc and by the use of
serum-free medium), we obtained WT and p27 null MEFs
carrying a transgenic GFP reporter under the Sox2 promoter
(D’Amour and Gage, 2003), and we observed that >90% of the
AP
+
colonies were GFP
+
. All together, these results indicate
that the absence of p27 eliminates the absolute requirement
for ectopic Sox2 in reprogramming.
Mouse fibroblasts express low, but detectable, levels of Sox2
(Eminli et al., 2008), and therefore, we wondered whether p27
deficiency affected Sox2 expression. Indeed, p27 null MEFs
had a significant increase in Sox2 mRNA levels compared
to WT controls (7-fold), although these levels were still about
64-fold lower than in embryonic stem cells (ESCs) (Figure 1D).
We also detected SOX2 protein by immunofluorescence.
Consistent with the mRNA data, quantitative image analyses
indicated that SOX2 protein levels were globally increased in
p27 null MEFs (Figure 1E). Of note, the distribution of SOX2 fluo-
rescence intensity in p27 null MEFs is broad, and therefore, it is
conceivable that only those p27 null cells with the highest SOX2
Cell Stem Cell
p27
Kip1
Represses Sox2
846 Cell Stem Cell 11, 845–852, December 7, 2012 ª2012 Elsevier Inc.
levels are the ones susceptible of 2F-OK reprogramming. It is
also worth mentioning that WT and p27 null MEFs have the
same proliferative rate (Coats et al., 1999), thus implying that
their different SOX2 levels are not secondary to a different prolif-
erative activity. We tested whether the increased levels of SOX2
observed in p27 null MEFs could be reverted by ectopic overex-
pression of p27. Interestingly, quantitative immunofluorescence
of p27-overexpressing p27 null MEFs indicated a downregulation
of SOX2 levels (Figure 1E). Finally, we also observed higher Sox2
mRNA levels in the retina, brain, and lung of adult p27 null mice
(Figure 1F). Together, these data indicate that p27 contributes to
the silencing of Sox2 in differentiated cells and tissues.
Sox2 Expression Is Repressed by p27
To further define the repressive role of p27 on Sox2,we
analyzed the differentiation of pluripotent stem cells upon treat-
ment with retinoic acid (RA). This differentiation protocol
efficiently reduces SOX2 protein and mRNA concomitantly
with a dramatic upregulation of p27 (Figure S2A). The upregula-
tion of p27 during RA-induced differentiation is in agreement
with a previous report on differentiating human embryonic carci-
noma cells (Bahrami et al., 2005). We wondered whether the
absence of p27 would affect the kinetics of Sox2 repression.
For this, we generated iPSCs derived from WT or p27 null
MEFs after reprogramming them with Oct4,Klf4, and Sox2.
The levels of Sox2 mRNA were similar in undifferentiated WT
or p27 null iPSCs (Figure 2A). However, upon RA-induced differ-
entiation, Sox2 levels in p27 null iPSCs were not reduced as
efficiently as in WT iPSCs (Figure 2A). Similarly, we tested
Sox2 expression during iPSC differentiation into embryoid
bodies (EBs). Again, Sox2 mRNA levels were abnormally high
in p27-deficient EBs compared to WT ones (Figure 2B). These
observations are in agreement with a previous study reporting
numerous abnormalities in EBs from p27 null ESCs (Bryja
et al., 2005). We considered the possibility that the increased
Sox2 levels in p27 null EBs could reflect a skewed neural differ-
entiation, but the levels of Nestin mRNA were similar in WT and
p27 null EBs (Figure S2B). To directly test the repressive activity
of p27, we retrovirally transduced ESCs with a pMSCV vector
expressing p27, and interestingly, p27 overexpression was
able to reduce the levels of Sox2 to an extent comparable to
RA (Figure 2C). Previous studies have demonstrated that Sox2
null ESCs spontaneously differentiate into trophectoderm-like
cells (Masui et al., 2007). We asked whether p27 overexpression
in ESCs promotes the trophectoderm-like differentiation char-
acteristic of Sox2 null ESCs. Indeed, this was the case and
p27 overexpression selectively induced trophectoderm markers
to a similar extent as RA differentiation, whereas ectoderm,
endoderm, and mesoderm markers were not induced by p27
but were induced by RA (Figure 2C). Moreover, p27-overex-
pressing ESCs produced cells with giant trophoblast-like
morphology, while this type of cell was absent in RA-
differentiated ESCs (Figure S2C). To extend the generality of
these findings to other pluripotent cells, we overexpressed
p27 in murine P19 embryonal carcinoma (P19EC) cells at levels
that did not affect proliferation and found a significant decrease
of Sox2 mRNA levels (Figure S2D). Together, these results
indicate that p27 exerts a repressive effect on Sox2 that is
relevant during differentiation.
C
A
iPSC differentiation into EBs
10
20
30
40
levels (relative to Gapdh x10-3)
Sox2 mRNA Nanog mRNA
10
20
30
40
D0
00
D0D10 D10
*** wt
p27-null
days :
B
ectopic p27 in ESCs
+ p27
+ RA
- 1
0
1
2
levels (relative to undifferentiated ESCs)
log10
trophectoderm ectoderm
endoderm
mesoderm
levels (relative to Gapdh x10-3)
RA-differentiation of iPSCs
0
5
10
15
20
25
30
35
40
45
50
wt
p27-null !
Sox2 mRNA
D0 D1 D2 D3 D4
***
days :
SOX2
-ACTIN
wt p27-null
D0 D1 D3 D4D2 D0 D1 D3 D4D2
Figure 2. p27 Represses Sox2 Expression
(A) Sox2 mRNA and protein levels in iPSCs undergoing in vitro differentiation
by the addition of retinoic acid (RA) in the absence of LIF for the indicated
number of days. mRNA values correspond to the average ± SD. (n = 6 inde-
pendent clones per genotype.)
(B) Sox2 and Nanog mRNA levels in iPSCs before and after 10 days of
induction of embryoid bodies (EBs). mRNA values correspond to the average ±
SD. (n = 6 independent clones per genotype.)
(C) Sox2 and p27 mRNA levels in ESCs after infection (3 days) with an empty
vector or with a plasmid overexpressing p27 (pMSCV-p27). ESCs at day 4 of
the RA-differentiation protocol were used as a differentiation control. Tro-
phectoderm markers and markers for other lineages were tested. In the case of
ESCs infected with pMSCV-p27, values are relative to ESCs infected with
empty vector. In the case of RA-differentiated ESCs, values are relative to
nondifferentiated ESCs. Three independent assays were performed for over-
expression of p27 (n = 3).
mRNA levels were determined by qRT-PCR. All data correspond to the
average ± SD. Statistical significance was assessed by the two-tailed
Student’s t test: ***p < 0.01; *p < 0.05. See also Figure S2.
Cell Stem Cell
p27
Kip1
Represses Sox2
Cell Stem Cell 11, 845–852, December 7, 2012 ª2012 Elsevier Inc. 847
p27 Associates to the Sox2-SRR2 Enhancer Together
with Repressive Complex p130-E2F4-SIN3A
The main regulatory element responsible for the expression of
Sox2 in pluripotent stem cells is located "4 kb downstream of
the single Sox2 coding exon and it is named SRR2 (Sikorska
et al., 2008;Tomioka et al., 2002). Based on our above observa-
tions in MEFs and in differentiating pluripotent cells, we asked
whether the presence or absence of p27 had an effect on the
repressive epigenetic marks on the Sox2-SRR2 enhancer. Inter-
estingly, we observed that p27 null MEFs present lower levels of
the H3K9me3 and H3K27me3 repressive marks at the Sox2-
SRR2 enhancer as evidenced by chromatin immunoprecipitation
(ChIP) analysis (Figure 3A). Similarly, RA-induced differentiation
of WT iPSCs dramatically increased the levels of H3K9me3
and H3K27me3 at the Sox2-SRR2 enhancer, while these epige-
netic marks were modestly increased in p27 null iPSCs (Fig-
ure 3B). These results indicate that the absence of p27 leads
to a defective epigenetic remodeling of the Sox2-SRR2 enhancer
both in differentiated cells (MEFs) and during the differentiation
of pluripotent cells.
F
A
D
B
5
10
15
relative to input (%)
H3K9me3 H3K27me3
ChIP – Sox2 enhancer
p27-nullwtMEFs: p27-nullwt
*** ***
ChIP:
E
EV
sh:
2
4
6
1
3
5
levels (relative to empty vector)
E2f4
*
p130
***
Sox2 mRNA
G
p27 ChIP in ESCs
--++
relative to input (%)
0.020
0.015
0.010
0.005
IgG
p27 Ab
***
region: Sox2 enhancer
RA:
Nanog promoter
p27 ChIP in ESCs
0.025
0.050
0.075
0.100
relative to input (%)
controlDNA: control
***
***
region: Sox2 enhancer Nanog promoter
IgG (mouse)
p27 Ab
Flag Ab
IgG (rabbit)
2
4
6
relative to input (%)
2
3
1
wt p27-null wt p27-null
ChIP:
iPSCs:
Sox2 enhancer
H3K9me3 H3K27me3
*** *** ***
-RA
+RA
***
***
relative to input (%)
p27 Sox2 enhance
r
0.5
**
**
iPSCs
wt p27-null
1.0
-RA
+RA
wt p27-null
MEFs
0.2
0.4
Sox2 enhancer
ChIP:
relative to input (%)
p130 SIN3AE2F4
0.1
0.2
0.4
wt p27-null wt p27-nullwt p27-null
***
***
***
***
*** ***
-RA
+RA
0.3
iPSCs:
CFigure 3. p27 Directly Binds to the Sox2-
SRR2 Enhancer
(A) Chromatin immunoprecipitation (ChIP) of
H3K9me3 and H3K27me3 in the Sox2-SRR2
enhancer of WT and p27 null MEFs. Data corre-
spond to one representative assay from a total of
three independent assays, each of them with
different MEF isolates.
(B) ChIP of the indicated proteins in the Sox2-
SRR2 enhancer of WT and p27 null iPSCs before
and after RA differentiation. Data correspond to
one representative assay from a total of two
independent assays, each of them with different
iPSC clones.
(C) ChIP of p27 in the Sox2-SRR2 enhancer of WT
and p27 null MEFs, and in WT and p27 null iPSCs
before and after RA differentiation. Data corre-
spond to one representative assay from a total of
three independent assays, each of them with
different MEF isolates and iPSCs clones.
(D) ChIP of p27 on the Sox2-SRR2 enhancer of
ESCs before and after RA differentiation. Data
correspond to one representative assay from
a total of two independent assays.
(E) ChIP of p27 in ESCs 2 days after transfection
with empty vector (control) or a plasmid express-
ing flag-tagged p27 (p27-flag).
(F) ChIP of the indicated proteins in the Sox2-
SRR2 enhancer before and after RA differentiation
of WT and p27 null iPSCs.
(G) Sox2 mRNA levels in WT MEFs 48 hr after
retroviral transduction with shp130,shE2f4,or
empty vector (EV). Data correspond to two inde-
pendent assays (n = 2).
All data correspond to the average ± SD. Statis-
tical significance was assessed by the two-tailed
Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05.
See also Figure S3.
Based on the above data and the
recent report that p27 can associate to
gene promoters in association with the
repressive complex p130-E2F4-SIN3A (Pippa et al., 2012), we
hypothesized that p27 might be recruited in this manner to
the Sox2-SRR2 enhancer. To directly test this, we performed
ChIP with anti-p27 antibodies in MEFs and we detected
p27 associated to the Sox2-SRR2 enhancer (Figure 3C). As
before, we wondered whether this was also the case in dif-
ferentiating pluripotent cells. Interestingly, RA-induced differen-
tiation of iPSCs was accompanied by a strong recruitment of
p27 to the Sox2-SRR2 enhancer (Figure 3C). To further extend
these observations, we used ESCs and, as in the case of
iPSCs, p27 was immunoprecipated at the Sox2-SRR2 en-
hancer upon RA-induced differentiation, but not at the Nanog
promoter used here as a control (Figure 3D). We sought addi-
tional proof by performing ChIP from ESCs transfected with
flag-tagged p27 and we also found p27 bound to the Sox2-
SRR2 enhancer after immunoprecipitation with antibodies
against p27 or against the flag tag (Figure 3E). Similar results
were obtained in P19EC cells, both upon RA-induced differen-
tiation (Figure S3A) and upon transfection of flag-tagged p27
(Figure S3B).
Cell Stem Cell
p27
Kip1
Represses Sox2
848 Cell Stem Cell 11, 845–852, December 7, 2012 ª2012 Elsevier Inc.
To examine the presence of a p130-E2F4-SIN3A repressive
complex at the Sox2-SRR2 enhancer, we performed ChIP
assays using antibodies against p130, E2F4, and SIN3A in WT
or p27 null iPSCs undergoing RA-induced differentiation.
Interestingly, the three proteins were detected in the Sox2-
SRR2 enhancer of RA-differentiated cells regardless of the
presence or absence of p27 (Figure 3F). We also detected
binding of p130, E2F4, and SIN3A to the Sox2-SRR2
enhancer in WT and p27 null MEFs (Figure S3C) and RA-
differentiated P19EC cells (Figure S3D). These results indicate
that a repressive p130-E2F4-SIN3A complex is assembled at
the Sox2-SRR2 enhancer upon differentiation and independently
of p27.
Finally, we asked whether the direct inhibition of the
p130-E2F4-SIN3A complex would also result in derepression
of Sox2. In agreement with previous reports (Dannenberg
et al., 2005), depletion of SIN3A had a dramatic effect on cell
viability that precluded us from further examining the effect on
Sox2 expression. Interestingly, however, knockdown of p130
F
INL
ONL
rods&cones
p27-null Sox2-het/p27-null
wt
pituitary mass (mg)
2
4
6
8
B
Sox2 :
p27 :
+/+ +/- +/+ +/-
+/+ +/+ -/- -/-
pituitary mass
***
***
***
***
10
C
Sox2 :
progenitor layer
of the pituitary
4
2
1
3
5
thickness of Sox2
+
layer (cells)
*** ***
p27 :
+/+ +/- +/+ +/-
+/+ +/+ -/- -/-
E
pituitary tumors (%)
20
40
60
80
100 8/8
3/10
**
pituitary tumor
Sox2 :
p27 :
+/+ +/-
-/- -/-
G
10
20
30
40
50
p27 :
Sox2 :
+
nuclei (%)
**
***
retina
+/+ +/- +/+ +/-
+/+ +/+ -/- -/-
A
body mass (g)
body mass
30
20
10
Sox2 :
p27 :
+/+ +/- +/+ +/-
+/+ +/+ -/- -/-
*** *
n.s
*
40
DH
retina
**
Sox2 :
p27 :
+/+ +/-
-/- -/-
+/+
+/+
protrusions free (%)
***
wt
Sox2-het
p27-null
Sox2-het/p27-null
20
40
60
80
100 3/3
2/7
5/5
SOX2
Figure 4. p27 Null Phenotypes Are Rescued
by Sox2 Haploinsufficiency
(A) Body mass of 2-month-old males of the indi-
cated genotypes (n = 3 for WT; n = 6 for Sox2-het;
n = 5 for p27-null; and n = 3 for Sox2-het/p27-null).
(B) Pituitary mass (n = 9 for WT; n = 12 for Sox2-het;
n = 7 for p27-null; and n = 10 for Sox2-het/p27-null,
males and females pooled, 3–6 months old).
(C) Representative pictures of the progenitor layer
of the pituitary cleft stained with SOX2. Bars
correspond to 50 mm.
(D) Thickness of the progenitor layer of the
pituitary cleft expressed as number of cells (n = 3
for each genotype, males and females pooled,
3–6 months old).
(E) Incidence of pituitary adenomas in 3- to
6-month-old mice.
(F) Representative pictures of the retina (H&E
staining). A focal protrusion is apparent in the p27
null retina. ONL = outer nuclear layer; INL = inner
nuclear layer.
(G) Incidence of retinas free of protrusions (n = 3
for WT; n = 7 for p27-null; and n = 5 for Sox2-het/
p27-null, males and females pooled, 1 year old).
(H) Relative number of SOX2
+
nuclei in the retina
(n = 3 for each genotype, males and females
pooled, 1 year old).
Data in (A), (B), (D), and (H) correspond to the
average ± SD and statistical significance was
assessed by the two-tailed Student’s t test. Data
in (E) and (G) correspond to ratios and statistical
significance was assessed by the Fisher’s test.
***p < 0.001; **p < 0.01; *p < 0.05; n.s., not
significant. See also Figure S4.
or E2f4 with shRNAs resulted in a severe
reduction of their expression (Figure S3E)
and, importantly, this was accompanied
in both cases by a significant upregula-
tion of Sox2 expression (Figure 3G).
In summary, we conclude that p27
associates to the Sox2-SRR2 enhancer
together with the repressive complex
p130-E2F4-SIN3A, and together contribute to the repression of
Sox2 upon cell differentiation.
Sox2 Heterozygosity Rescues p27 Deficiency in Mice
Based on our above data, we wondered whether the incomplete
repression of Sox2 observed in p27-deficient cells and tissues
could mediate some of the phenotypes characteristic of p27
null mice. To evaluate this in a genetic manner, we generated
compound Sox2 heterozygous (Sox2-het) and p27 null mice.
Sox2 null mice are not viable, but Sox2-het are viable and
display a moderate reduction in body size and hypopituitarism
(Kelberman et al., 2006). Interestingly, the characteristic gigan-
tism of p27 null mice was normalized by deletion of one Sox2
allele (Figure 4A; Figure S4A), as was also the case for the pitu-
itary mass in young 3- to 6-month-old mice (at this age, p27
null mice present pituitary hyperplasia, but do not have pituitary
tumors yet) (Figure 4B). However, given the fact that Sox2-het
mice have a modest, but detectable, defect in growth, the above
results leave open the possibility that p27 and Sox2 simply have
Cell Stem Cell
p27
Kip1
Represses Sox2
Cell Stem Cell 11, 845–852, December 7, 2012 ª2012 Elsevier Inc. 849
opposite effects on growth that balance each other. For this
reason, we decided to focus in the progenitor cell layer that
surrounds the pituitary cleft and which is formed by SOX2-
positive (SOX2
+
) cells (Fauquier et al., 2008;Garcia-Lavandeira
et al., 2009;Gleiberman et al., 2008). The progenitor layer in
Sox2-het pituitaries had the same thickness as in WT pituitaries
(Figures 4C and 4D; Figure S4B). Interestingly, the thickness of
the progenitor layer was significantly increased in p27 null
mice compared to WT or to Sox2-het mice, and this defect
was absent in Sox2-het/p27-null mice (Figures 4C and 4D).
The progenitor cells of the pituitary have been proposed to
constitute the origin of pituitary adenomas (Gleiberman et al.,
2008). In this regard and in line with our above observations,
the incidence of pituitary tumors was significantly reduced in
Sox2-het/p27-null mice compared to p27 null littermates
(Figure 4E).
Having established that Sox2 heterozygosity rescues the
gigantism and the pituitary phenotypes of p27 null mice, we
wondered whether the same was true for the retinal defects of
p27 null mice. In agreement with previous reports, the retinas
of Sox2-het mice were normal (Taranova et al., 2006)(Fig-
ure S4C), while p27 null retinas presented focal protrusions of
the outer nuclear layer (ONL) (Nakayama et al., 1996)(Figures
4F and 4G). Interestingly, these protrusions were absent in
Sox2-het/p27-null retinas (Figures 4F and 4G). Also, we
observed that p27 null retinas present an increased abundance
of SOX2
+
nuclei at the inner nuclear layer (INL) and mislocalized
SOX2
+
nuclei in the outer plexiform layer (OPL) (Figure S4C). We
quantified the number of SOX2
+
nuclei in complete retinal
sections and confirmed that p27 null retinas present an excess
of SOX2
+
nuclei and, importantly, we found that this defect
was absent in Sox2-het/p27-null retinas (Figure 4H). We wanted
to corroborate the increased abundance of SOX2
+
nuclei in the
absence of p27, and for this we used transgenic mice with
GFP under the control of a Sox2 promoter region that marks
neural multipotent progenitors associated to Sox2 expression
(Sox2-GFP mice) (D’Amour and Gage, 2003). Reinforcing our
above observations, p27-het/Sox2-GFP retinas presented
a significant increase in GFP
+
nuclei when compared with WT/
Sox2-GFP retinas (Figure S4D). Together, we conclude that
a decrease in the gene dosage of Sox2 rescues the main pheno-
types associated with p27 deficiency, namely, gigantism, pitui-
tary hyperplasia, pituitary adenomas, and retinal abnormalities.
In addition to these phenotypes, p27 null mice also have adrenal
gland hyperplasias and tumors (pheochromocytomas) and
female sterility (Fero et al., 1996;Kiyokawa et al., 1996;Na-
kayama et al., 1996). We examined WT adrenal glands and
p27 null pheochromocytomas by immunostaining for SOX2,
but they were negative and for this reason we did not further
pursue this phenotype. Regarding the sterility of p27 null
females, Sox2-het/p27-null females remained sterile, suggesting
that this phenotype is independent of Sox2. Collectively, these
results provide genetic support to the concept that p27 is a nega-
tive regulator of Sox2 in the pituitary and in the retina.
DISCUSSION
The mechanisms responsible for the transcriptional silencing
of pluripotency genes in differentiated cells are poorly under-
stood. The results reported here demonstrate that the tumor
suppressor p27 contributes to the transcriptional repression of
Sox2. We have observed that the absence of p27 leads to
a defective repression of Sox2 in fibroblasts, lung, retina, and
brain, and to a delayed and incomplete silencing of Sox2 during
differentiation of pluripotent cells, including iPSCs, ESCs,
and P19EC cells. These observations led us to identify p27 as
a transcriptional regulator of Sox2 together with a repressive
complex formed by p130, E2F4, and SIN3A at a critical enhancer
responsible for Sox2 expression. These findings are in line with
a recent report describing the capacity of p27 to interact with
the p130/E2F4/SIN3A complex and contribute to its transcrip-
tional repressive activity (Pippa et al., 2012). We have found
that p27 deficiency leads to an expansion of SOX2
+
cells in the
progenitor layer of the pituitary and in the retina, which results
in pituitary hyperplasia and tumors and morphological defects
of the retina. Importantly, these defects are rescued when p27
deficiency is combined with Sox2 heterozygosity. In humans,
germline mutations in p27 and SOX2 also affect the pituitary
and the retina. On one hand, loss-of-function mutations in p27
produce MEN syndrome, notably characterized by pituitary
tumors (Marinoni and Pellegata, 2011;Vandeva et al., 2010).
On the other hand, loss-of-function mutations in SOX2 produce
syndromes characterized by anophthalmia and hypopituitarism
(Engelen et al., 2011;Fantes et al., 2003;Kelberman et al.,
2006;Williamson et al., 2006). Our current findings unveil a mech-
anistic connection between p27 and SOX2, and thereby
contribute to our understanding of the molecular basis of the
human pathologies associated with the deregulation of these
two proteins.
EXPERIMENTAL PROCEDURES
Mice
Mice p27 null (Fero et al., 1996), Sox2-het (Avilion et al., 2003), and Sox2-
promoter/GFP transgenic (D’Amour and Gage, 2003) have been previously
described. All comparisons were made among mice derived from the same
sets of crosses, and they therefore shared the same genetic background.
Animal experimentation at the CNIO, Madrid was performed according to
protocols approved by the CNIO-ISCIII Ethics Committee for Research and
Animal Welfare (CEIyBA) and animal experimentation at the MRC-NIMR, Mill
Hill, London was carried out in accordance with the UK Animals (Scientific
Procedures) Act 1986.
ChIP, RNA Quantification, and Protein Analyses
ChIP and quantitative PCR was performed following standard methods
(detailed in the Supplemental Experimental Procedures). PCR primer se-
quences, shRNA encoding plasmids, and other DNA constructs, as well as
antibodies and other standard molecular biology methods, are all detailed in
Supplemental Experimental Procedures.
Generation of iPSCs
Reprogramming of primary (passage 2–4) MEFs was performed as previously
described by us (Li et al., 2009) using plasmids pMXs-Klf4, pMXs-Sox2, or
pMXs-Oct4 (obtained from Addgene and previously described; Takahashi
and Yamanaka, 2006). For additional details, see Supplemental Experime ntal
Procedures.
Differentiation with RA
Differentiation with RA was performed essentially as described (Savatier et al.,
1996). ESCs or iPSCs were adapted to grow on gelatin-coated plates (and in
the absence of feeder cells). Cells were grown to near confluency in their cor-
responding complete medium (day 0) and then were trypsinized and seeded at
Cell Stem Cell
p27
Kip1
Represses Sox2
850 Cell Stem Cell 11, 845–852, December 7, 2012 ª2012 Elsevier Inc.
lower density in the absence of LIF for 1 day (day 1). During the following 2 days
(days 2 and 3), RA was added at a concentration of 10
!6
M, and day 4 cells
were without LIF and without RA. In the case of P19EC cells, differentiation
was induced by addition of RA (10
!6
M) for 4 days.
Immunohistochemistry and Immunofluorescence
For immunohistochemical stainings, quantifications were performed on
representative fields at the same magnification, on a minimum of three
different areas per sample and a minimum of three different samples per geno-
type. For immunofluorescence, cells were inspected under a Leica TCS-SP5
confocal microscope (AOBS) and analyzed using Definiens Developer XD
1.5 software, under the same exposure conditions. For additional details,
see Supplemental Experimental Procedures.
Statistical Analysis
Unless otherwise specified (Figures 4E and 4G), quantitative data are pre-
sented as mean ± SD and significance was assessed by the two-tailed
Student’s t test.
SUPPLEMENTAL INFORMATION
Supplemental Information for this article includes four figures and Supple-
mental Experimental Procedures and can be found with this article online at
http://dx.doi.org/10.1016/j.stem.2012.09.014.
ACKNOWLEDGMENTS
We are indebted to Diego Megı
´as from the CNIO for technical assistance, and
to the Biological Services staff at NIMR. H.L. has been funded by the Spanish
Association Against Cancer (AECC). H.L. and M. Collado have a ‘‘Ramon y
Cajal’’ contract from the Spanish Ministry of Economy (MINECO). Work in
the laboratory of M.S. is funded by the CNIO and by grants from the MINECO
(SAF and CONSOLIDER), the Regional Government of Madrid, the European
Research Council (ERC), the Botin Foundation, the AXA Foundation, and the
Ramon Areces Foundation. A.M. is supported by a long-term fellowship of
the Human Frontiers Science Program; K.R., R.L.-B., and work in the R.L.-B.
laboratory are funded by the UK Medical Research Council (U117512772).
Work in the A. Vidal laboratory is funded by grants from the MINECO (SAF)
and from the Xunta de Galicia. H.L. and M. Collado performed most of the
experiments and contributed to experimental design, data analysis, discus-
sion, and writing the paper; A. Villasante, C.J.L., and C.C. performed the chro-
matin immunoprecipitations; M. Can
˜amero performed the histological anal-
yses; A.M., K.R., and R.L.-B. provided the Sox2 mouse models, performed
mouse manipulations, and contributed to the analysis of the mouse pheno-
types; C.C., G.M., and A. Vidal provided MEFs and contributed to the analysis
of the pituitary phenotype; M. Collado and M.S. designed and supervised the
study, secured funding, analyzed the data, and wrote the manuscript. All
authors discussed the results and commented on the manuscript. The authors
declare no competing financial interests with this paper.
Received: February 24, 2012
Revised: August 8, 2012
Accepted: September 17, 2012
Published: December 7, 2012
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