LETTERS
Puma and p21 represent cooperating checkpoints
limiting self-renewal and chromosomal instability of
somatic stem cells in response to telomere dysfunction
Tobias Sperka1 , Zhangfa Song1,5 , Yohei Morita1 , Kodandaramireddy Nalapareddy1 , Luis Miguel Guachalla1 ,
André Lechel1 , Yvonne Begus-Nahrmann1 , Martin D. Burkhalter1 , Monika Mach2 , Falk Schlaudraff3 , Birgit Liss3 ,
Zhenyu Ju4 , Michael R. Speicher2 and K. Lenhard Rudolph1,6
The tumour suppressor p53 activates Puma-dependent
apoptosis and p21-dependent cell-cycle arrest in response to
DNA damage. Deletion of p21 improved stem-cell function and
organ maintenance in progeroid mice with dysfunctional
telomeres, but the function of Puma has not been investigated
in this context. Here we show that deletion of Puma improves
stem- and progenitor-cell function, organ maintenance and
lifespan of telomere-dysfunctional mice. Puma deletion
impairs the clearance of stem and progenitor cells that have
accumulated DNA damage as a consequence of critically short
telomeres. However, further accumulation of DNA damage in
these rescued progenitor cells leads to increasing activation of
p21. RNA interference experiments show that upregulation of
p21 limits proliferation and evolution of chromosomal
imbalances of Puma-deficient stem and progenitor cells with
dysfunctional telomeres. These results provide experimental
evidence that p53-dependent apoptosis and cell-cycle arrest
act in cooperating checkpoints limiting tissue maintenance and
evolution of chromosomal instability at stem- and
progenitor-cell levels in response to telomere dysfunction.
Selective inhibition of Puma-dependent apoptosis can result in
temporary improvements in maintenance of
telomere-dysfunctional organs.
Increased rates of apoptosis have been associated with impairments
in organ maintenance and lifespan in response to DNA damage,
telomere dysfunction or mitochondrial dysfunction1–3 . Vice versa,
reduction in cellular stress responses were associated with reduced
apoptosis and increased longevity4 . However, functional studies
on the role of apoptosis pathways in mammalian ageing are
lacking at present. Experiments on fungal ageing have provided
genetic evidence that deletion of apoptosis checkpoints can increase
the lifespan of multicellular organisms5 . The functional role of
apoptosis in mammalian ageing remains under debate. Inhibition
of BCL2-associated X protein (Bax)-dependent apoptosis improved
the maintenance of germ cells in ageing ovaries6 . Deletion of Trp53
rescued apoptosis and spermatogenesis in the testis of mice harbouring
dysfunctional telomeres7 , but in somatic tissues Trp53 deletion
accelerated tissue atrophy by impairing the clearance of genetically
unstable stem cells8 . Trp53 deletion affects various checkpoints,
including cell-cycle arrest, apoptosis and autophagy. In vivo studies on
the selective role of p53-dependent apoptosis checkpoints in telomeredysfunction-induced ageing of somatic tissues have not been reported.
Puma (p53 upregulated modulator of apoptosis) is one of the
BH3-only proteins inducing p53-dependent apoptosis in response
to acute DNA damage in various tissues and cell types including
stem and progenitor cells9–14 . Here we investigated consequences of
Puma deletion on telomere-dysfunction-induced ageing by crossing
Puma knockout mice (Puma−/− ; ref. 10) with telomerase knockout
mice (mTerc −/− ; refs 2,15) to generate Puma+/+ and Puma−/− , thirdgeneration telomerase knockout mice with dysfunctional telomeres
(G3 mTerc −/− ) as well as the respective groups of telomerase wild-type
mice with long telomere reserves (mTerc +/+ ).
In agreement with previous studies, G3 mTerc −/− mice exhibited a
significantly shortened lifespan when compared with mTerc +/+ mice
(Fig. 1a). Puma deletion had no significant influence on the lifespan of
mTerc +/+ mice during the follow-up period. In contrast, Puma deletion
significantly increased the lifespan of G3 mTerc −/− , Puma−/− mice
1
Institute of Molecular Medicine and Max-Planck-Research Department on Stem Cell Aging, University of Ulm, 89081 Ulm, Germany. 2 Institute of Human Genetics,
Medical University of Graz, Harrachgasse 21/8, A-8010 Graz, Austria. 3 Institute of Applied Physiology, University of Ulm, 89081 Ulm, Germany. 4 Institute of Aging
Research, Hangzhou Normal University College of Medicine, 16 Xuelin Road, 310036, Hangzhou, China. 5 Present address: Department of Colorectal Surgery, Sir Run
Run Shaw Hospital, 3 East Qingchun Road, 310016, Hangzhou, China.
6
Correspondence should be addressed to K.L.R. (e-mail: [email protected])
Received 26 July 2011; accepted 27 October 2011; published online 4 December 2011; DOI: 10.1038/ncb2388
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION
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LETTERS
b
P < 0.0001
P < 0.0001
50
mTerc +/+ Puma +/+, n = 32
mTerc +/+ Puma –/–, n = 29
25
G3 mTerc–/– Puma +/+, n = 50
G3 mTerc –/– Puma –/– , n = 44
0
100
mTerc+/+ Puma+/+
200
300
Age (days)
400
mTerc+/+ Puma–/–
500
300
NS
200
P < 0.0001
100
0
G3
Puma+/+
G3
mTerc–/–
e
7.5
50 µm
mTerc+/+ Puma–/–
G3 mTerc–/– Puma +/+
P < 0.0001
5.0
2.5
0
f
mTerc+/+ mTerc+/+ G3 mTerc–/– G3 mTerc–/–
Puma +/+ Puma –/– Puma +/+
Puma–/–
150
P < 0.0001
NS
100
50
0
Puma–/–
Olfm4
10.0
mTerc+/+ mTerc+/+ G3 mTerc–/– G3 mTerc–/–
Puma–/–
Puma +/+ Puma–/– Puma+/+
mTerc+/+ Puma +/+
mTerc–/–
12.5 P < 0.0001
Organoids/well
75
0
c
d
Colonic crypts (mm2 )
Percentage of survival
100
Olfm4+ cells/100 µm
a
P = 0.0327
mTerc+/+ mTerc+/+ G3 mTerc–/– G3 mTerc–/–
Puma+/+ Puma–/– Puma+/+
Puma –/–
G3 mTerc–/– Puma –/–
Figure 1 Puma deletion prolongs lifespan and improves stem- and
progenitor-cell-based organ maintenance of telomere-dysfunctional mice.
(a) Kaplan–Meier survival curves of the indicated mouse cohorts. Puma gene
status did not affect the lifespan of mTerc +/+ mice during the experimental
period but significantly prolonged survival of G3 mTerc −/− Puma −/−
mice when compared with G3 mTerc −/− Puma +/+ mice (median survival:
333 days versus 283 days). (b) Quantification of colon crypt density of
10- to 12-month-old mice of the indicated genotypes (n = 5 mice per
group). Note that the number of crypts is significantly reduced in G3
mTerc −/− ,Puma +/+ mice but partially rescued in G3 mTerc −/− ,Puma −/−
mice. (c) Representative images of small-intestine sections labelled for the
intestinal stem-cell marker olfactomedin-4 (Olfm4) through RNA in situ
hybridization (scale bar, 100 µm). (d) Quantification of Olfm4-labelled stem
cells in the intestine of 10- to 12-month-old mice of the indicated genotypes.
Note that G3 mTerc −/− mice show a significant reduction in intestinal
stem-cell numbers, which is partially rescued by Puma deletion. (e) In vitro
cultures of single freshly isolated crypts from 10- to 12-month-old mice
of the indicated genotypes. Pictures were taken at day 7 in culture. Note
that de novo growth and budding of crypts containing granulated Paneth
cells (arrows) was reduced in crypts from G3 mTerc −/− Puma +/+ mice but
partially rescued in crypts from G3 mTerc −/− Puma −/− mice (scale bar,
50 µm). (f) Quantification of organoids containing newly formed crypts after
seven days in culture (n = 3 independent cultures per group). Error bars
represent s.e.m.
(n = 44) when compared with G3 mTerc −/− , Puma+/+ mice (n = 50;
18% median lifespan extension, P < 0.0001; Fig. 1a). These effects
of Puma deletion were similar to previous studies showing that p21
deletion increases the lifespan of telomere-dysfunctional mice16 .
In line with previous studies2,8,16–18 , telomere-dysfunctional mice
developed a strong atrophy of intestinal crypts at 10–12 months
of age (Fig. 1b and Supplementary Fig. S1a–c) associated with a
premature loss in body weight (data not shown). Puma deletion
rescued these phenotypes in age-matched G3 mTerc −/− , Puma−/−
mice, but did not influence crypt numbers of mTerc +/+ mice (Fig. 1b
and Supplementary Fig. S1a–c).
To determine Puma-dependent effects on maintenance and
functional capacity of stem cells in response to telomere dysfunction,
we focused on the analysis of intestinal stem cells (ISCs). The number
of ISCs was quantified using an in situ hybridization against Olfm4
messenger RNA—a specific marker for ISCs (ref. 19). In line with the
data on crypt numbers (Fig. 1b and Supplementary Fig. S1a–c), this
analysis revealed a decreased number of ISCs in the small intestine of 10to 12-month-old G3 mTerc −/− mice when compared with mTerc +/+
mice (Fig. 1c,d; P < 0.0001). Puma deletion increased the number of
ISCs in the small intestine of telomere-dysfunctional mice, indicating
that Puma contributed to limitations in ISC maintenance in response
to telomere dysfunction. The formation of organoid crypt structures
in cell culture, especially the budding of new, Paneth-cell-containing
crypts, has recently been identified as a functional characteristic of
purified ISCs (ref. 20). Therefore, we carried out in vitro cultures
of freshly isolated intestinal crypt cells. Intestinal crypts of 10- to
12-month-old G3 mTerc −/− , Puma+/+ mice showed a significantly
reduced crypt-forming capacity when compared with age-matched
mTerc +/+ mice (Fig. 1e,f; P < 0.0001). Puma deletion improved
the growth capacity of telomere-dysfunctional, intestinal stem and
progenitor cells in culture (Fig. 1e,f; P = 0.0327). Puma gene status did
not influence the crypt-forming capacity of mTerc +/+ crypts.
Together, these experiments suggested that activation of Pumadependent apoptosis contributed to impairments in stem- and
progenitor-cell maintenance and function in telomere-dysfunctional
mice. To substantiate this interpretation, we analysed Puma expression
and the influence of Puma gene status on apoptosis rates in intestinal
stem and progenitor cells. TdT-mediated dUTP nick end labelling
(TUNEL) revealed elevated rates of apoptosis in intestinal basal crypts
of 10- to 12-month-old G3 mTerc −/− mice when compared with
mTerc +/+ mice (Fig. 2a,b). Telomere dysfunction was associated with
2
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LETTERS
a
b
mTerc+/+ Puma –/–
P = 0.0005
TUNEL+ cells/crypt
mTerc +/+ Puma+/+
P = 0.0011
NS
4
2
0
mTerc+/+ mTerc+/+ G3 mTerc–/– G3 mTerc–/–
Puma+/+ Puma–/–
Puma+/+
Puma–/–
Puma–/–
d
e
Mr (K)
25
Puma
Unspec.
20
mTerc+/+
Puma+/+
mTerc+/+
Puma–/–
mTerc–/–
G3
Puma+/+
mTerc–/–
G3
Puma–/–
Relative expression to HMBS
TUNEL
0.02
80
G3 mTerc–/–
Puma +/+
60
40
20
0
0
4
8
Cell position
f
Puma mRNA
P = 0.0263
0.01
0
Not
detectable
mTerc+/+ G3 mTerc–/–
Puma+/+
Puma+/+
G3 mTerc–/–
Puma–/–
12
P < 0.0001
PCNA+ cells/100 µm
c
Apoptotic index (%)
G3 mTerc–/–
G3 mTerc–/– Puma +/+
50
P < 0.0001
NS
40
30
20
10
0
mTerc+/+ mTerc+/+ G3 mTerc–/– G3 mTerc–/–
Puma+/+
Puma–/–
Puma+/+ Puma–/–
Figure 2 Puma deletion reduces apoptosis in stem and progenitor cells with
dysfunctional telomeres. (a,b) Apoptosis detection through TUNEL staining
in basal crypts of the small intestine. (a) Representative staining of 10- to
12-month-old mice of the indicated genotypes, scale bar 30 µm. (b) Number
of TUNEL-positive cells per crypt from 10- to 12-month-old mice of the
indicated genotypes (n = 6–9 mice per group). (c) Apoptosis index in basal
crypt cells of 10- to 12-month-old G3 mTerc −/− , Puma +/+ mice. Apoptosis
was quantified for the indicated position of crypt cells relative to the middle
cell on the crypt base. Highest rates of apoptosis occurred at stem-cell
position +4 and in transient amplifying progenitor cells (position >4; n = 3
mice). (d) Western blot analysis of Puma expression in whole-intestine
lysates from 10- to 12-month-old mice of the indicated genotypes. The
unspecific band at a relative molecular mass of 20,000 (Mr 20K) serves
as a loading control. (e) qPCR analysis of Puma mRNA expression relative
to hydroxymethylbilane synthase (HMBS) in small intestine of 10- to
12-month-old mice of the indicated genotypes (n = 3 mice per group).
(f) The number of proliferating-cell nuclear antigen (PCNA)-positive cells in
basal crypts of 10- to 12-month-old mice of the indicated genotypes. Error
bars represent s.e.m., n = 5 mice per group. A full scan of the Western blot
is provided in Supplementary Fig. S7.
increased apoptosis in transient amplifying progenitor cells as well as
in putative ISCs located in between the Paneth cells at positions 1–4
of the crypt base (Fig. 2c). Puma deletion significantly reduced the
rates of apoptosis in the intestine of G3 mTerc −/− mice (n = 6–9 mice
per group, P = 0.0011, Fig. 2a,b). Western blot and quantitative PCR
(qPCR) analysis confirmed an upregulation of Puma in whole-tissue
lysates of the small intestine of 10- to 12-month-old G3 mTerc −/− mice
when compared with mTerc +/+ mice (Fig. 2d,e). The partial rescue
in apoptosis (Fig. 2a,b) and the improved maintenance of ISCs in
G3 mTerc −/− mice in response to Puma deletion (Fig. 1d) correlated
with an increase in the number of proliferative cells in the intestinal
epithelium (Fig. 2f and Supplementary Fig. S2a,b). Puma deletion
did not lead to a complete rescue in apoptosis, indicating that other
apoptosis pathways contributed to telomere-dysfunction-induced
apoptosis. In agreement with this interpretation, qPCR analysis
revealed increased expression of Bax and Noxa (also known as Pmaip1)
in the intestine of G3 mTerc −/− , Puma−/− mice when compared with
age-matched wild-type controls (Supplementary Fig. S2c,d).
Together, these results indicated that Puma deletion reduced organ
atrophy in telomere-dysfunctional intestine by impairing apoptosis and
improving the functional reserve of somatic stem and progenitor cells.
Of note, this improvement in maintenance of telomere-dysfunctional
organs in response Puma deletion did not result in tumour formation
(Supplementary Fig. S2e,f).
To analyse whether prolonged maintenance of telomeredysfunctional progenitor cells in response to Puma deletion would lead
to an accumulation of genetic alterations, the number of DNA damage
foci was determined in intestinal basal crypts using phosphorylated
histone variant H2AX (γH2AX) staining. In agreement with previous
studies, the number of DNA damage foci in intestinal crypts was
elevated in 10- to 12-month-old G3 mTerc −/− mice when compared
with age-matched mTerc +/+ mice (Fig. 3a and Supplementary Fig.
S3a). This accumulation of DNA damage was significantly accelerated
in G3 mTerc −/− , Puma−/− mice when compared with G3 mTerc −/− ,
Puma+/+ mice (n = 5, P < 0.0001; Fig. 3a and Supplementary Fig. S3a).
Telomere dysfunction can accelerate the evolution of chromosomal
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LETTERS
P < 0.0001
20
10
0
d
NS
mTerc+/+ mTerc+/+ G3 mTerc–/– G3 mTerc–/–
Puma+/+ Puma–/–
Puma+/+
Puma–/–
30
c
P = 0.0067
3.5
P = 0.0351
20
NS
10
G3 mTerc–/– Puma+/+
G3 mTerc–/– Puma+/+
NS
P = 0.0188
mTerc +/+ Puma–/–
mTerc +/+ Puma+/+
P = 0.0094
Aberrations /crypt
30
40
0
0
e
P < 0.0001
40
30
20
10
0
mTerc +/+
Puma +/+
P = 0.0035
NS
G3 mTerc–/– G3 mTerc–/– iG4 mTerc–/– iG4 mTerc–/–
Puma+/+
Puma–/– Puma+/+
p53–/–
f
P < 0.0001
50
P = 0.0115
3.0
2.5
2.0
1.5
1.0
0.5
mTerc+/+ mTerc+/+ G3 mTerc–/– G3 mTerc–/– iG4 mTerc–/– iG4 mTerc–/– iG4 mTerc–/–
Puma+/+ Puma–/–
Puma+/+
Puma–/–
Puma+/+
p53–/–
p21–/–
p21+ CBC cells/crypt
40
NS
Percentage of p21+ cells/crypt
P < 0.0001
P = 0.0104
Percentage of anaphase bridges
b
Percentage of γH2AX + cells/crypt
a
1.25
P = 0.0139
1.00
0.75
P = 0.0236
0.50
0.25
0
mTerc+/+ G3 mTerc –/– G3 mTerc –/– G3 mTerc –/–
Puma–/– Puma +/+
Puma –/–
Puma –/–
G3 mTerc–/–
Puma+/+
g
p21
Rel. expression p21 in LT-HSCs
low weight high weight
loss
loss
0.125
G3 mTerc–/–
Puma–/–
G3 mTerc–/–
Puma–/–
low weight
loss
high weight
loss
P = 0.0151
0.100
P = 0.0266
0.075
P = 0.0237
0.050
0.025
0
mTerc+/+
Puma+/+
mTerc+/+
Puma–/–
G3mTerc–/–
Puma+/+
G3mTerc–/–
Puma–/–
Figure 3 Puma deletion accelerates the accumulation of DNA damage and
the upregulation of p21 in telomere-dysfunctional stem and progenitor
cells. (a) Quantification of γH2AX-positive cells (containing three or more
nuclear γH2AX foci) per crypt in small intestine of 10- to 12-month-old
mice (n = 5 mice per group). (b) Anaphase bridges were quantified in
haematoxylin and eosin stained sections of the small intestine of 10to 12-month-old mice (percentage of total number of anaphases; inset,
representative anaphase bridge). G3 mTerc −/− mice show an increase in
anaphase bridges when compared with mTerc +/+ mice. Note that Puma
deletion leads to a significant increase in G3 mTerc −/− crypts whereas p21
deletion has no significant effect (G3 mTerc −/− , Puma −/− group, n = 12;
G3 mTerc −/− , Puma +/+ and iG4 mTerc −/− , Puma +/+ groups, n = 5; other
groups, n = 4). Note that there is no significant difference between G3
and iG4 mTerc −/− mice. (c) aCGH analysis was carried out on individual
laser-captured crypts of the small intestine of 10- to 12-month-old mice
(n = 7–9 crypts per group). The histogram depicts the total number of
aberrations per crypt for the indicated genotypes. (d–f) Immunostaining
of p21 in crypts of the small intestine of 10- to 12-month-old mice.
The group of G3 mTerc −/− , Puma −/− mice was subdivided into animals
exhibiting low average weight loss (10–15%) or high average weight loss
(>15%) indicating an end of the rescue period mediated by Puma deletion.
(d) Representative p21 staining in basal crypts (arrows point to p21-positive
cells; scale bar, 20 µm). (e) Quantification of p21-positive cells per crypt
(n = 6 mice per group). (f) Quantification of p21-positive stem cells at
the crypt base (n = 6). Inset: Morphological appearance of a stem cell
between granular Paneth cells at the crypt base. Note that Puma deletion
led to a significant increase in the accumulation of p21-positive cells
in G3 mTerc −/− , Puma −/− crypts when compared with G3 mTerc −/− ,
Puma +/+ crypts, which is further enhanced on increasing weight loss.
(g) qPCR analysis of p21 mRNA expression in freshly isolated long-term
haematopoietic stem cells (LT-HSCs; CD34lo/− LSK) of 10- to 12-month-old
mice (n = 3 mice per group). Error bars represent s.e.m.
instability by induction of chromosomal fusions resulting in
fusion–bridge–breakage cycles. Anaphase bridges represent a hallmark
feature of this type of chromosomal instability21,22 . In line with previous
studies, intestinal basal crypts of G3 mTerc −/− mice exhibited increased
numbers of anaphase bridges when compared with mTerc +/+ mice
(Fig. 3b). The number of anaphase bridges was significantly increased
in G3 mTerc −/− , Puma−/− mice (n = 12) when compared with G3
mTerc −/− , Puma+/+ mice (n = 5, P = 0.0094, Fig. 3b). A similar
increase of anaphase bridges was seen in telomere-dysfunctional
mice carrying a homozygous deletion of p53 but not in telomeredysfunctional mice carrying a homozygous deletion of p21 (Fig. 3b).
Previous studies revealed that the p53 deletion leads to an
accumulation of chromosomal unstable ISCs and accelerated tissue
atrophy in telomere-dysfunctional mice8 . To determine whether Puma
deletion would also affect chromosomal stability at stem-cell level,
single crypt sections of 10- to 12-month-old mice were analysed
by array-based comparative genomic hybridization (aCGH). The
crypt is a stem-cell-based self-renewing unit populated by dividing
transit-amplifier cells and differentiated cells. Chromosomal gains
and losses acquired at stem-cell level will clonally expand, and can
be detected by aCGH. In contrast, sporadic instabilities at singleprogenitor-cell level will be averaged out in the analysis and therefore
4
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40
p2
1
sh
RN
A
0
G3 mTerc–/– G3 mTerc–/–
Puma –/–
Puma +/+
mTerc+/+ mTerc+/+ G3 mTerc–/– G3 mTerc–/–
Puma –/–
Puma +/+
Puma +/+ Puma –/–
p21 shRNA 2
P = 0.0156
20
l
0
P = 0.0032
RN
A
20
60
sh
P < 0.0001
P < 0.0001
40
80
on
to
P = 0.0022
C
p21 shRNA 1
d
NS
NS
60
p2
1
b
G3 mTerc–/– Puma+/+ G3 mTerc–/–Puma–/–
Organoid/cyst index (%)
mTerc+/+Puma–/–
Control
mTerc+/+Puma+/+
p2 Co
nt
1
r
s
p2 hR ol
N
1
sh A 1
RN
A
2
p2 Co
nt
1
ro
s
p2 hR l
1 NA
sh
RN 1
A
2
p2 Co
1 ntr
sh ol
p2 R
1 NA
sh
RN 1
A
2
p2 Co
nt
1
s
o
p2 hR l
N
1
sh A 1
RN
A
2
a
Percentage of organoids
with CIN/ total organoids
LETTERS
c ISC organoids: G3 mTerc Puma p21 shRNA, n = 13
5
6
7
8
9
10
11
12 13
14 15 16 17 18 19
–/–
3
4
6
7
8
9
10
p53
TelDys-induced
DNA damage
P = 0.0007
11
12 13
TelDys-induced
chromosomal
fusions
p21
Puma
Apoptosis
Cell-cycle arrest
G3 mTerc –/– G3 mTerc –/–
Puma+/+
Puma–/–
14 15 16 17 18 19
Telomere dysfunction (TelDys)
P = 0.0004
50
0
5
A
sh tro
RN l
sh A 1
RN
A
2
p2 Co
n
1
sh trol
p2 RN
1
sh A 1
RN
A
2
p2 Co
nt
1
sh rol
p2 RN
1
A
sh
1
RN
A
2
1
100
p2
p2
mTerc+/+ G3 mTerc–/– G3 mTerc–/–
Puma+/+
Puma–/–
Puma–/–
2
i
1
on
0
h
chr:1
A
P = 0.1071
10
4
sh
RN
20
P = 0.0067
P = 0.0185
60
100
Time (d)
p2
1
P = 0.0408
P = 0.0004
0
ro
l
NS
30
3
–/–
sh
RN
60
100
Time (d)
2
1
on
t
0
chr:1
–/–
g HSC colonies: G3 mTerc Puma p21 shRNA, n = 9
P = 0.009
C
P = 0.082
P = 0.126
1
P = 0.005
2
p2
1
G3W p21 shRNA 2
2
G3P control
G3P p21 shRNA 1
G3P p21 shRNA 2
3
Percentage of HSC colonies
with CIN/total colonies
G3W control
G3W p21 shRNA 1
Total GFP+ chimaerism
in peripheral blood
3
C
f
HSC colony formation (n)
e
Total GFP+ chimaerism
in peripheral blood
–/–
Tissue atrophy
Puma
deletion
Accumulation of DNA damage by
fusion–bridge–breakage cycles
Figure 4 p21 and Puma represent synergistic checkpoints preventing
proliferation and the evolution of chromosomal instability of telomeredysfunctional stem and progenitor cells. (a–d) Freshly isolated intestinal
basal crypt cells of 10-month-old mice were infected with lentivirus expressing control or two different shRNAs targeting p21. (a) Representative pictures
of primary organoid cultures on day 10 after lentivirus transduction (scale bar,
200 µm). (b) A quantification of organoid development. Note that deletion
of Puma and knockdown of p21 synergistically rescue the organoid-forming
capacity of telomere-dysfunctional stem and progenitor cells. (c,d) Organoids
were cultured over a period of 70 days, collected and subjected to aCGH
analysis. (c) Superimposed aCGH profiles showing chromosomal gains and
losses (peaks above and below baseline) in G3 mTerc −/− , Puma −/− p21
shRNA organoids. chr, chromosome. (d) Quantification of chromosomal gains
and losses in epithelial organoids (n = 9–13 organoids per genotype). CIN,
chromosomal instability. (e–h) Freshly isolated haematopoietic stem and
progenitor cells (LSK) of 10-month-old mice were infected with lentivirus
and transplanted along with non-infected cells into lethally irradiated mice.
(e) Total chimaerism of green fluorescent protein (GFP)-positive cells in the
peripheral blood was determined. Note that p21 knockdown had a positive
effect on the repopulation capacity of LSK cells from G3 mTerc −/− , Puma +/+
mice (G3W, left) and G3 mTerc −/− , Puma −/− mice (G3P, right, n = 2–3 mice
per group). (f) 100 days after transplantation single-sorted GFP+ , long-term
HSCs (CD34lo/− LSK) were isolated from primary recipients and cultured for
two weeks. Knockdown of p21 and Puma deletion synergistically rescued the
colony-forming capacity of G3 mTerc −/− HSCs (n = 60 single-sorted HSCs
per genotype). (g) Superimposed aCGH profiles showing chromosomal gains
and losses in colonies derived from single-cell-sorted G3 mTerc −/− , Puma −/−
p21 shRNA HSCs. (h) Quantification of chromosomal gains and losses in
HSC colonies (n = 8–9 colonies per genotype). (i) Model of cooperating
checkpoints in response to telomere dysfunction. Puma-dependent apoptosis
represents the primary checkpoint to chromosomal fusions, whereas p21
is activated in response to DNA damage. Prolonged survival and organ
maintenance of cells with fused chromosomes leads to accumulation of DNA
breaks through fusion–bridge–breakage cycles in G3 Terc −/− Puma −/− mice.
Accumulating DNA damage leads to increased p21 activation, limiting the
rescue period of improved organ maintenance. Error bars represent s.e.m.
escape detection by this technique. In contrast to previous data on p53
(ref. 8), deletion of Puma did not lead to a significant increase in clonal
chromosomal imbalances in basal crypts of G3 mTerc −/− , Puma−/−
mice, indicating that ISCs maintained a stable genome (Fig. 3c and
Supplementary Fig. S3b). One possible explanation for the evolution
of chromosomal instability in telomere-dysfunctional stem cells in
response to p53 deletion but not in response to Puma deletion could be
that p21 compensated for loss of Puma and prevented clonal growth of
chromosomal-unstable tissue stem cells.
Co-staining of p21 and TUNEL revealed that induction of p21
and Puma-dependent apoptosis predominantly occurred in separate
cell populations in intestinal basal crypts of G3 mTerc −/− , Puma+/+
mice (Supplementary Fig. S4a,b). These data indicated that Pumadependent apoptosis and p21-dependent cell-cycle arrest might act in
parallel checkpoints in response to telomere dysfunction. However, a
significant accumulation of p53-positive (Supplementary Fig. S4c,d)
and p21-positive cells (Fig. 3d,f) was evident in basal intestinal crypts
of 10–12 month old G3 mTerc −/− , Puma−/− mice when compared
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5
LETTERS
with G3 mTerc −/− , Puma+/+ mice. Accumulation of p21-expressing
cells also occurred at the level of ISCs at the crypt base23–25 (Fig. 3f
and Supplementary Fig. S4e). Similar to the results on the intestinal
epithelium, a significant overexpression of p21 mRNA was detected
in haematopoietic stem cells (HSCs) of 10- to 12-month-old G3
mTerc −/− , Puma−/− mice when compared with age-matched G3
mTerc −/− , Puma+/+ mice (Fig. 3g). Co-staining of p21 and the
proliferation marker phospho-histone H3 revealed that p21-expressing
intestinal crypt cells were cell-cycle arrested (data not shown). The
highest numbers of p21-positive stem and progenitor cells occurred
in basal crypts of G3 mTerc −/− , Puma−/− mice that developed
progressive weight loss (Fig. 3e,f) and intestinal atrophy (data not
shown), indicating that these mice had reached the end stage of the
rescue period mediated by Puma deletion.
Together, these data indicated that p21-mediated cell-cycle arrest
could represent a secondary checkpoint impairing proliferation and
tissue contribution of telomere-dysfunctional stem and progenitor
cells with unstable chromosomes when Puma-dependent apoptosis was
abrogated. To directly test this hypothesis, we analysed consequences of
a stable, lentiviral-mediated short hairpin RNA (shRNA) knockdown
of p21 (Supplementary Fig. S5a) on growth capacity and chromosomal
instability in Puma−/− and Puma+/+ intestinal and haematopoietic
stem cells harbouring dysfunctional telomeres.
The analysis of organoid-formation capacity of intestinal crypt cells
revealed that shRNA-mediated knockdown of p21 cooperated with
Puma deletion to improve proliferation of telomere-dysfunctional
ISCs (Fig. 4a,b). aCGH analysis on individual organoids showed
that p21 knockdown in G3 mTerc −/− , Puma−/− ISCs resulted in
chromosomal gains and losses after long-term culture (Fig. 4c) that
were not present in organoids of G3 mTerc −/− , Puma−/− infected
with a scrambled shRNA control (Fig. 4d, Supplementary Fig. S5b
and Table S1). Similar to the results on ISCs, the shRNA-mediated
knockdown of p21 cooperated with Puma deletion to improve both
the in vivo repopulation capacity and the in vitro colony-formation
capacity of G3 mTerc −/− HSCs (Fig. 4e,f and Supplementary Fig. S6).
Furthermore, the knockdown of p21 also resulted in a significant
increase in chromosomal imbalances in G3 mTerc −/− , Puma−/− HSCs
when compared with scrambled-shRNA-infected HSCs (Fig. 4g,h,
Supplementary Fig. S5c and Table S2). In contrast to the data on
p21–Puma co-deletion, the knockdown of p21 in G3 mTerc −/− ,
Puma+/+ stem cells resulted in partial improvements of stem-cell
function (Fig. 4a,b,f) but not in a significant increase in chromosomal
imbalances (Fig. 4d,h, Supplementary Fig. S5d and Table S3).
The present study provides experimental evidence that p21dependent cell-cycle arrest and Puma-dependent apoptosis act in
cooperating checkpoints that limit the proliferative capacity and
the evolution of chromosomal instability of somatic stem and
progenitor cells in response to telomere dysfunction. Significant
increases in anaphase bridges in telomere-dysfunctional tissues
occur in response to Puma deletion but not in response to
p21 deletion, indicating that Puma is an important p53 target
in the setting of breakage of fused chromosomes in telomeredysfunctional tissues. These data suggest a model indicating that
Puma deletion prolongs survival of telomere dysfunctional cells with
fused chromosomes. However, this prolonged cellular survival leads
to an accumulation of DNA damage and enhanced expression of p21
6
ultimately halting cell-cycle progression of genetically unstable stem
and progenitor cells (Fig. 4i). This model of secondary checkpoint
activation provides a plausible explanation for why Puma deletion
can result in temporary improvements in the maintenance of
telomere-dysfunctional tissues without resulting in chromosomal
instability at stem-cell level.
The present study provides proof of principle that a selective
inhibition of apoptosis can improve the maintenance of somatic tissues
in the context of telomere dysfunction. These results could be relevant
for human ageing, which is characterized by telomere shortening26 , an
accumulation of DNA damage27 and elevated rates of apoptosis28–30 .
On the basis of these results, apoptosis inhibitors31 could have beneficial
effects on organ maintenance in aged tissues or disease tissues exhibiting
telomere shortening and increased rates of apoptosis, for example liver
cirrhosis32 , lung fibrosis33 and myelodysplastic syndromes34 .
METHODS
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturecellbiology
Note: Supplementary Information is available on the Nature Cell Biology website
ACKNOWLEDGEMENTS
We thank G. Zambetti for providing Puma−/− mice, C. Kuo for the R-spondin
construct and H. Clevers for discussions. The Deutsche Forschungsgemeinschaft
(Klinische Forschergruppe 142 & 167 and Ru745/10) and the European Union
(GENINCA) supported this work.
AUTHOR CONTRIBUTIONS
T.S., Z.S., Y.M., K.N., Y.B-N., M.D.B. and Z.J. carried out, designed and analysed
experiments; T.S., A.L., Y.B-N., M.M. and M.R.S. carried out and analysed aCGH
experiments; F.S. and B.L. carried out microdissection; Z.S. and L.M.G. generated
mouse crosses; K.L.R. and T.S. wrote the manuscript; K.L.R. conceived the study.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturecellbiology
Reprints and permissions information is available online at http://www.nature.com/
reprints
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NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION
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7
METHODS
DOI: 10.1038/ncb2388
METHODS
Preparation of high-titre vector stocks. Virus with lentiviral vectors carrying
Mice. Puma−/− mice10 were backcrossed seven generations with C57BL/6 mice and
an expression cassette for GFP and Mir30-based shRNA (pGIPZ based), packaged
with attenuated human immunodeficiency virus-derived gag-pol (pCMV1R8.91),
pseudotyped with VSV-G envelope (pMD.G), was prepared by transient transfection
of HEK 293T cells. Virus-containing supernatant was collected every 24 h and
passed through a 0. 45 µm filter, and virus was pelleted by centrifugation and
resuspended in PBS. Viral titre was immediately determined on NIH-3T3 cells
by applying serial virus dilutions and FACS analysis for GFP expression on
day three after transduction. Concentrated virus was stored short term at 4 ◦ C
until use on haematopoietic or intestinal cells. The target sequences for p21
and the control are: p21_1, 50 -ACCGGAACATCTCAGGGCCGAA-30 ; p21_2, 50 ACAGCCTGACAGATTTCTATCA-30 ; scrambled, 50 -ACCTCCACCCTCACTCTGCCAT-30 .
crossed with mTerc +/− mice2,15,35 to generate mTerc +/− Puma+/− mice. These were
crossed successively to produce third-generation G3 mTerc −/− , Puma−/− (n = 44),
G3 mTerc −/− , Puma+/− (n = 51) and G3 mTerc −/− , Puma+/+ (n = 50) mice.
Mice were maintained on C57BL/6J background. Mice were monitored by weekly
inspection and killed at ages from 10 to 12 months when losing 10–15% of body
weight on average or when mice were moribund. Peripheral blood was routinely
analysed with VetABC (Scilvet). The state government of Baden Württemberg
approved mouse experiments. iG4 mTerc −/− and iG4 mTerc −/− , p21−/− mice are
the offspring from crossing G3 mTerc −/− , p21+/− × mTerc +/− , p21+/− . The same
strategy was used for iG4 mTerc −/− , Trp53−/− mice.
Histology. Organs were fixed in 4% buffered formalin overnight. Colon whole
mounts were prepared as previously described2 . Haematoxylin and eosin sections
were prepared according to standard protocols and the anaphase bridge index was
determined by counting 100 anaphases per mouse.
Microscopy. Microscopy was carried out on Leica and Zeiss systems with
magnification ranging from ×4 to ×100 and equipped with proprietary software;
pictures from individual experiments were recorded with identical settings and
identically processed with Adobe Photoshop and Illustrator.
In situ hybridization. 8 µm sections were rehydrated, treated with 0.2 M
NaCl and proteinase K and postfixed with 4% paraformaldehyde. Sections
were demethylated with acetic anhydride and prehybridized. Hybridization was
done in a humid chamber with 500 ng ml−1 freshly prepared digoxigeninlabelled RNA probe of OLFM4 (>48 h, 68 ◦ C). After washing and incubation
with secondary anti-digoxigenin antibody (overnight, 4 ◦ C, Roche) sections
were washed and developed using nitroblue tetrazolium/5-bromo-4-chloro-3indolyl phosphate.
Apoptosis . An in situ cell death detection kit (Roche, no 11684817910) was
employed for TUNEL-based apoptosis detection on 3 µm paraffin sections. The
number of apoptotic cells per crypt was counted in 30 crypts per mouse.
Immunohistochemistry. 5 µm sections were de-paraffinized, rehydrated and
antigen was retrieved in antigen unmasking solution (pressure cooker, Vector, H3300) or citrate buffer (microwave at pH 6.0). Sections were incubated with primary
antibody overnight at 4 ◦ C: anti-PCNA (Calbiochem, no NA03, 1 µg ml−1 ), anti-p53
(Vector, CM5, 1:500), anti-p21 (Santa Cruz, sc-6264, 2 µg ml−1 ), anti-γH2AX
(Millipore, no 05-636, 2 µgml−1 ; Cell Signalling Technology, no 9718, 1:120), antipSer histone H3 (Santa Cruz, sc-12927, sc-8656, 1 µg ml−1 ), musashi-1 (eBioscience,
14H1, 0. 5 µg ml−1 ). p21 and γH2AX were developed with an Vectastain ABC Elite
kit and Nova Red (Vector Laboratories). p21, PCNA, musashi-1, p53, pH3 and
γH2AX were visualized with appropriate secondary antibodies labelled with Cy3 or
Alexa488 (2 µg ml−1 ). Staining was analysed in at least 30 crypts per mouse. p21
and TUNEL costain was carried out in the order primary (Santa Cruz, sc-6264,
2 µg ml−1 ), secondary antibody (anti-mouse Cy3, Zymed, 1:200), TUNEL mix
(90 min, 37 ◦ C).
Western blot. Cell extracts of whole frozen intestine or NIH-3T3 fibroblasts
were prepared in Laemmli sample buffer (50 mM Tris at pH 6.8, 4% SDS, 10%
glycerol). Protein was resolved in 12% SDS–polyacrylamide gel electrophoresis,
transferred to polyvinylidene difluoride membrane and detected using antibodies
against Puma (Abcam, ab9643, 2 µg ml−1 ), p21 (Santa Cruz, sc-6264, 0.4 µg ml−1 )
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Bethyl Laboratories,
A300-641A, 0.05 µg ml−1 ).
Quantitative real-time PCR. 3,000 LT-HSCs per mouse (CD34lo LSK) were
sorted into Trizol LS (Invitrogen) for RNA isolation. Transcription into complementary DNA was carried out using SuperScript III (Invitrogen). All qPCRs were
carried out in duplicate for each cDNA sample with TaqMan probes for Cdkn1a
or GAPDH (Applied Biosystems, Mm00432448_m1 and 4308313), Absolute QPCR
ROX Mix (ABgene) and primers from Applied Biosystems. Small-intestine total
RNA was isolated after pulverizing 3 cm of organ in liquid nitrogen in a mortar with
RNAzol B (WAK-Chemie). Reverse transcription was carried out using the GoScript
Reverse Transcription System (Promega). qPCR was carried out in triplicate for each
cDNA sample and employed the Universal ProbeLibrary (Roche), Absolute QPCR
ROX Mix (ABgene) and the following primers: Puma (fw, 50 -ttctccggagtgttcatgc-30 ;
rv, 50 -tacagcggagggcatcag-30 ; probe no 79), Noxa (fw, 50 -cagatgcctgggaagtcg-30 ;
rv, 50 -tgagcacactcgtccttcaa-30 ; probe no 79), Bax (fw, 50 -gtgagcggctgcttgtct-30 ; rv,
50 ggtcccgaagtaggagagga-30 ; probe no 83), HMBS (fw, 50 -tccctgaaggatgtgcctac-30 ; rv,
50 -aagggttttcccgtttgc-30 ; probe no 79). All PCRs were run on the ABI PRISM 7300
Sequence Detection System (Applied Biosystems).
FACS. Bone-marrow cells from tibias, femurs and spine were crushed with
sterile PBS. Suspensions were filtered and cells counted. Antibody (eBiosciences)
combinations were as follows: HSC–Sca1, c-Kit, CD34, Flt3 and lineage (CD3, CD4,
CD8, Mac-1, B220, Gr-1 and Ter119). For cell sorting on FACS Aria (BD) total bone
marrow was lineage depleted (magnetic activated cell separation column) and cells
were stained for Sca1, c-Kit, CD34 and Flt3.
LSK cell isolation and viral transduction. Bone-marrow cells from 10-monthold mice (Ly5.2) were c-Kit enriched by magnetic activated cell separation and
200,000 c-Kit+ cells per group were put into culture with SFEM medium (Stem Cell
Technologies), 50 ng µl−1 and 50 ng µl−1 murine thrombopoietin. The remaining
c-Kit+ cells were labelled for FACS, and 150,000 LSK cells (Sca1+ , c-Kit+ , lineage− )
were FACS isolated per group and cultured in SFEM medium with 50 ng µl−1 murine
stem cell factor and 50 ng µL−1 murine thrombopoietin. The next day cells were
transduced with concentrated virus at a multiplicity of infection MOI = 10 in the
presence of 8 µg ml−1 polybrene, and medium was exchanged another day later. On
day four cells were washed with PBS to remove remaining virus, and LSK and c-Kit+
cells were mixed and transplanted into three lethally irradiated Ly5.1/Ly5.2 recipient
mice (12 Gy) per group. GFP chimaerism in peripheral blood was determined at
regular intervals by detection of Ly5.1, Ly5.2 and GFP.
In vitro HSC culture. GFP-positive LT-HSCs (Lin− , c-Kit+ , Sca1+ , CD34lo )
from control and two p21 shRNA groups were isolated from bone marrow of
transplanted primary recipient mice 15 weeks after transplantation. 60 single HSCs
were sorted into individual 96-well plates and cultured over two weeks in Roswell
Park Memorial Institute medium (RPMI), 10% fetal bovine serum, 20 ng ml−1
murine thrombopoietin, 20 ng ml−1 stem cell factor, 20 ng ml−1 interleukin3, 10 ng ml−1 erythropoietin, 50 µM β-mercaptoethanol, penicillin/streptomycin.
Colony formation was assessed after two weeks and colonies were collected for
aCGH analysis.
In vitro crypt culture. Crypt culture was done as previously described20 . A total
of 500 crypts was mixed with 50 µ l of Matrigel (BD) and plated in 24-well
plates. After gelling of the Matrigel, 500 µl of crypt culture medium (Advanced
DMEM/F12, B27 and N2 supplement (Invitrogen), 50 ng µl − 1 epidermal growth
factor, 100 ng ml−1 Noggin (Peprotech), 500 ng µl − 1 R-spondin-1, 1.25 mM N acetylcystein) was added. Organoid growth was quantified after seven days. For
transduction in vitro-grown organoids were collected from near-confluent 24-well
plates, enzymatically digested with TrypLE (Invitrogen) and passed through a
40 µm mesh to remove undigested fragments (BD). Remaining single cells and
oligomers were stained with Trypan blue to quantify viable cells, and were
transduced with lentivirus (37 ◦ C, MOI ≈ 30). After transduction cells were
spun down and resuspended in Matrigel. On day 3 after seeding growth of
thin-walled GFP-positive cysts was quantified, indicating the presence of active
transit-amplifying and stem cells20 . On day 10 after seeding growth of fully
developed GFP-positive organoids (indicative of stem cells only20 ) was quantified
and normalized to the number of cysts to correct for variations at the start
of culture (norganoids /ncysts = proliferation index). Organoids were passaged every
14 days and whole organoids were collected for aCGH analysis after 70 days
of culture.
aCGH. Each intestinal crypt represents a self-renewing unit generated from a
few ISCs. We reasoned that genomic instability at the single-crypt level (20–30
cells) should only be detectable when genetically unstable stem cells regenerated
the crypt and not when random instability occurred at the level of progenitor
cells. Whole-genome amplification of the DNA of the microdissected crypts
was carried out as described previously36 . Briefly, the GenomePlex Single Cell
Whole Genome Amplification Kit (no WGA4; Sigma-Aldrich) was employed
according to the manufacturer’s instructions. In brief, after tissue lysis and
fragmentation of the genomic DNA an amplification of the genomic library
was carried out. DNA was purified using the GenElute PCR Clean-up Kit
NATURE CELL BIOLOGY
© 2011 Macmillan Publishers Limited. All rights reserved.
METHODS
DOI: 10.1038/ncb2388
(no NA1020; Sigma-Aldrich). The quality of the amplification was evaluated
using a multiplex PCR approach as previously described37 . aCGH was carried
out using a whole-genome oligonucleotide microarray platform (no G4426B,
Mouse Genome CGH 44K Microarray Kit, was used for in vivo-derived material;
no G4839, Mouse Genome CGH 180K Microarray Kit, was used for in vitroderived material; Agilent Technologies). This array consists of approximately
43,000 (44K) or 170,000 (180K) 60-mer-oligonucleotide probes with a spatial
resolution of about 22 kilobases (kb) (44K) or 10.9 kb (180K). As reference DNA
we used amplified male DNA from the same mouse strain. 500 ng test DNA
and reference DNA were enzymatically labelled with dUTP-Cy5 or dUTP-Cy3
according to the manufacturer’s instructions (Agilent Genomic DNA Enzymatic
Labelling Kit). Slides were scanned using a microarray scanner (no G2505B)
and images were analysed using DNA Analytics software 4.0 (both from Agilent
Technologies) with the statistical algorithm ADM-2; the sensitivity threshold was
4.0. At least ten consecutive clones had to be aberrant to be scored by the software.
Statistics. Statistical analysis was done using Microsoft Excel and Graph Pad Prism
software. The unpaired two-tailed Student’s t -test, Logrank test and χ 2 test were
used to calculate P values.
35. Herrera, E. et al. Disease states associated with telomerase deficiency appear earlier
in mice with short telomeres. EMBO J. 18, 2950–2960 (1999).
36. Geigl, J. B. & Speicher, M. R. Single-cell isolation from cell suspensions and whole
genome amplification from single cells to provide templates for CGH analysis.
Nat. Protoc. 2, 3173–3184 (2007).
37. van Beers, E. H. et al. A multiplex PCR predictor for aCGH success of FFPE samples.
Br. J. Cancer 94, 333–337 (2006).
NATURE CELL BIOLOGY
© 2011 Macmillan Publishers Limited. All rights reserved.
S U P P L E M E N TA R Y I N F O R M AT I O N
DOI: 10.1038/ncb2388
a
mTerc+/+ Puma+/+
mTerc+/+ Puma-/-
G3mTerc-/- Puma+/+
G3mTerc-/- Puma-/-
mTerc+/+ Puma+/+
mTerc+/+ Puma-/-
G3mTerc-/- Puma+/+
G3mTerc-/- Puma-/-
b
PCNA DAPI
Intestinal crypts /100µm
c
2.5
2.0
1.5
1.0
P < 0.0001
NS
P = 0.0004
0.5
0.0
mTerc+/+ mTerc+/+ G3mTerc-/- G3mTerc-/Puma+/+ Puma-/- Puma+/+
Puma-/-
Supplementary Figure 1_Rudolph
Figure S1 Puma deletion improves maintenance of the intestinal epithelium
in aging, telomere dysfunctional mice. (a) Representative whole mount
staining of the colonic epithelium of 10-12 month old mice of the indicated
genotypes (scale 200 mm). (b) Representative images of small intestine
stained for the proliferation marker PCNA (red) and counter stained with
DAPI (blue). Proliferating stem and progenitor cells in basal crypt are PCNA
positive. Note that the number of crypts is reduced in G3 mTerc-/-, Puma+/+
mice but partially rescued in G3 mTerc-/-, Puma-/- mice (scale 100 mm).
(c) Quantification of PCNA-positive basal crypts in the small intestine of
10-12 month old mice of the indicated genotypes (n = 5 mice per group
for mTerc+/+ cohorts, n = 12-14 mice per group for G3 mTerc-/- cohorts).
Note that the number of crypts is significantly reduced in G3 mTerc-/-,
Puma+/+ mice but partially rescued in G3 mTerc-/-, Puma-/- mice. (error
bars represent s.e.m.).
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1
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
mTerc+/+ Puma+/+
b
mTerc+/+ Puma-/-
P < 0.0001
NS
pH3+ cells / 100µm
12.5
G3mTerc-/- Puma+/+
G3mTerc-/- Puma-/-
P < 0.0001
10.0
7.5
5.0
2.5
0.0
mTerc+/+ mTerc+/+ G3mTerc-/- G3mTerc-/Puma+/+ Puma-/- Puma+/+
Puma-/-
pS28H3 DAPI
d
Bax mRNA
P = 0.0504
10
0
mTerc+/+ G3mTerc-/- G3mTerc-/Puma+/+ Puma+/+
Puma-/-
100
mice affected [%]
e
P = 0.0215
0.2
rel. expression to HMBS
20
rel. expression to HMBS
c
0.1
P = 0.1607
0.0
mTerc+/+ G3mTerc-/- G3mTerc-/Puma+/+ Puma+/+
Puma-/-
G3mTerc-/- Puma+/+
NS
mTerc+/+ Puma-/50
P = 0.3192
f
mTerc+/+ Puma+/+
Noxa mRNA
G3mTerc-/- Puma+/+
G3mTerc-/- Puma-/not detected
0
macroscopic
tumors
with
ACF
Figure S2 PUMA deletion rescues apoptosis and proliferation of stem and
progenitor cells in aging telomere dysfunctional mice. (a) Representative
images of proliferating cells (H3pSer28-positive) in the epithelium of the
small intestine of 10-12 month old mice of the indicated genotypes (scale
bar 10μm). (b) Quantification of pH3-positive basal crypts in the small
intestine of 10-12 month old mice of the indicated genotypes (n = 5 mice
per group). (c, d) qPCR analysis of Bax and Noxa mRNA expression relative
to HMBS in small intestine of 10-12 month old mice of the indicated
genotypes (n = 3 mice per group). (e) Summary of macroscopic tumours and
colonic aberrant crypt foci (ACF) in 10-12 month old mice of the indicated
genotypes (inspection for macroscopic tumours in n = 16 – 38 mice per
group and for ACF in n = 7 mice per group). (f) Representative image of
methylene blue stained colon epithelium of a G3 mTerc-/-, Puma+/+ mice
revealing ACF (encircled, scale 200mm). (error bars represent s.e.m.).
Supplementary Figure 2_Rudolph
2
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a
mTerc+/+ Puma+/+
mTerc+/+ Puma-/-
G3mTerc-/- Puma+/+
G3mTerc-/- Puma-/-
γH2AX
γH2AX
DNA
b
G3mTerc-/- Puma+/+
G3mTerc-/- Puma-/-
Supplementary Figure 3_Rudolph
Figure S3 Puma deletion causes accumulation of DNA damage but does
not lead to chromosomal imbalances in telomere dysfunctional stem cell
and progenitor cells. (a) Representative immunostainings of gH2AX in small
intestine of 10-12 month old mice of the indicated genotypes (scale bar
30 mm). (b) Array CGH analysis was performed on individual laser-captured
crypts of the small intestine of 10-12 month old mice of the indicated
genotypes (n = 6 – 9 crypts per group). Representative aCGH profiles of
freshly isolated crypts of mice of the indicated genotypes.
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
b
p21 TUNEL DAPI
TUNEL+ cells / crypt [%]
12.5
G3mTerc-/- Puma+/+
P = 0.0134
G3mTerc-/-Puma+/+
G3mTerc-/-Puma-/-
10.0
P = 0.0045
7.5
NS
5.0
2.5
0.0
Total TUNEL+ TUNEL+
cells
p21- cells
TUNEL+
p21+ cells
c
mTerc+/+ Puma+/+
G3mTerc-/- Puma+/+
mTerc+/+ Puma-/-
p53
p53
15
p53+ cells / crypt [%]
d
G3mTerc-/- Puma-/-
e
P = 0.0046
p53
p53
G3mTerc-/- Puma-/-
10
5
0
p21 Msi1 DAPI
mTerc+/+ mTerc+/+ G3mTerc-/- G3mTerc-/Puma+/+ Puma-/- Puma+/+
Puma-/-
Supplementary Figure 4_Rudolph
Figure S4 Deletion of Puma enhances p53/p21 checkpoint activation in
telomere dysfunctional mice. (a, b) Apoptosis and p21 mediated cell cycle
arrest occur mainly in separate cell populations. (a) Immunostaining of
p21 (red) and apoptosis detection via TUNEL (green) in crypts of the small
intestine of 10-12 month old G3 mTerc-/- mice. DNA is labelled with DAPI
(blue). (b) Quantification of TUNEL positive cells and separation in TUNEL
only and TUNEL/p21 double positive populations (n = 4 – 5 mice per
group). (c, d) p53 induction in the intestinal epithelium of 10-12 month old
G3 mTerc-/-, Puma+/+ and G3 mTerc-/-, Puma-/- mice. (c) Representative
pictures of immunostained p53 (red) in crypts of small intestine. (d)
Quantification of p53-positive cells per crypt in small intestine (n = 4 mice
per group). Note the enhanced expression of p53 in G3 mTerc-/-, Puma/- tissue. (e) Increase of CDK inhibitor p21 expression at base of small
intestinal crypts in G3 mTerc-/-, Puma-/- tissue. Co-immunostaining of the
crypt base against p21 (red), the intestine stem cell marker musashi-1
(Msi1, green), and a counter staining with DAPI (blue). Arrow points to Msi1positive cells, p21-positive intestinal stem cell in between Msi-negative
Paneth cells. (error bars represent s.e.m.; scale bar 30 mm).
4
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
shscr
FCS
perm.
shp21_2
FCS refeed after o/n SS
0h
1h
2h
3h
4h
FCS
perm.
shp21_1
FCS refeed after o/n SS
0h
1h
2h
3h
4h
FCS
perm.
FCS refeed after o/n SS
0h
1h
2h
3h
4h
p21
GAPDH
b
ISC organoids: G3mTerc-/- Puma-/- shCtr, n=9
chr:1
c
3
4
5
6
7
8
9
10
11
12
13
14
15 16
17 18 19
8
9
10
11
12
13
14
15 16
17 18 19
8
9
10
11
12
13
14
15 16
17 18 19
8
9
10
11
12
13
14
15 16
17 18 19
HSC colonies: G3mTerc-/- Puma-/- shCtr, n=9
chr:1
d
2
2
3
4
5
6
7
ISC organoids: G3mTerc-/- Puma+/+ shp21, n=13
chr:1
2
3
4
5
6
7
HSC colonies: G3mTerc-/- Puma+/+ shp21, n=8
chr:1
2
3
4
5
6
7
Supplementary Figure 5_Rudolph
Figure S5 (a) NIH-3T3 mouse fibroblast were transduced with lentiviruses
expressing a scrambled shRNA or 2 different shRNA targeting p21.
Selected, infected cells were analysed after continuous passage in serum
containing medium (FCS) or in response to serum-starvation (over night)
and at the indicated time points after re-stimulation with 10% serum. Note
the knockdown of p21 expression in shRNA-p21 targeted cells compared
to scrambled shRNA infected cells. (b) Small intestine crypt cells of
10-month-old G3 mTerc-/-, Puma-/- mice were infected with lentiviral
constructs expressing a scrambled shRNA control. The infected crypts
were cultured over a period of 70d. Array CGH analysis was performed on
single isolated organoids (G3 mTerc-/-, Puma-/-, shCtr n = 9). Ratio profile
summary of G3 mTerc-/-, Puma-/-, shCtr organoids. All acquired plots of
the same group are superimposed. Chromosomes are staggered along the
x-axis. The ratio of sample to control genomic DNA is plotted along the
y-axis. Note the balanced profile in G3mTerc-/-, Puma-/-, shCtr organoids.
(c) Freshly isolated LSK cells from 10 month old G3 mTerc-/-, Puma/- mice were infected with lentiviral constructs expressing a scrambled
shRNA control. The cells were transplanted into lethally irradiated
recipient mice. Single GFP+ long-term haematopoietic stem cells (linSca1+cKit+CD34loFlt3-) were isolated 15 weeks after transplantation and
cultured for two weeks followed by array CGH analysis (n = 9). Ratio profile
summary of G3mTerc-/-, Puma-/-shCtr cultures. All acquired plots of the
same group are superimposed. Chromosomes are staggered along the x-axis.
The ratio of sample to control genomic DNA is plotted along the y-axis.
Note the balanced profiles in G3 mTerc-/-, Puma-/-, shCtr LT-HSC cultures.
(d) Array-CGH analysis was performed in analogy to (b) and (c) with cells
from G3mTerc-/-, Puma+/+ mice that were lentivirally infected with shRNAs
against p21.
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
GFP+ cells [%]
5.0
peripheral
blood
4.0
P = 0.0104
3.0
2.0
1.0
G3mTerc-/Puma+/+
b
GFP+ cells [%]
12.5
10.0
1
p2
tr
C
sh
sh
1
p2
sh
sh
C
tr
0.0
G3mTerc-/Puma-/-
LSK
cells
P = 0.0408
7.5
5.0
2.5
G3mTerc-/Puma+/+
1
p2
sh
tr
C
sh
1
p2
sh
sh
C
tr
0.0
G3mTerc-/Puma-/-
Supplementary Figure 6_Rudolph
Figure S6 p21 and Puma represent synergistic checkpoints preventing
proliferation of telomere dysfunctional hematopoietic stem and progenitor
cells. Freshly isolated hematopoietic stem and progenitor cells (LSK, Lin-,
Sca1+, cKit+) of 10-month-old mice were infected with lentivirus and
transplanted along with non-infected cells into lethally irradiated primary
recipient mice. 100 days after transplantation GFP+ LSK cells were
isolated and transplanted into lethally irradiated secondary recipients at
a ratio of 6000 LSK cells to 4*105 competitor Ly5.1 total bone marrow
cells. (a) Total chimerism of GFP-positive cells in the peripheral blood
was determined 3 months after transplantation (n = 15 mice in control
groups, n = 11 G3 mTerc-/-, Puma-/-, shp21 mice). Note the synergistic
nature of p21 and Puma checkpoint. (b) Total chimerism of GFP-positive
cells in the bone marrow LSK compartment was determined 6 months after
transplantation (n = 6 mice in control groups, n = 7 G3 mTerc-/-, Puma-/-,
shp21 mice). Note the synergistic nature of p21 and Puma checkpoint
(error bars represent s.e.m.).
6
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
+
+/
ma
-/-
+
+/
a
um
-/-
a
um
+
+/
ma
-/-
ma
+
+/
a
um
-/-
a
um
+
+/
ma
-/-
ma
+
-/-
+/
a
um
a
um
-/- P
-/- P
u + Pu
ma -/- P
-/- P
-/- P
u + Pu
-/- P
+ P
+ P
rc
rc
+/
+/
Pu rc
rc
rc
+/
+/
rc
e
e
e
e
c
c
T
T
e
e
c
c
c
c
m
m
er
er
mT
mT
er
er
mT
mT
er
er
mT G3
G3
mT
mT G3
G3
mT
mT G3
G3
mT
+
+/
Pu
+
+/
25kDa Puma
20kDa -
unspec.
shscr
b
FCS
perm.
shp21_2
FCS refeed after o/n SS
0h
1h
2h
3h
4h
FCS
perm.
FCS refeed after o/n SS
0h
1h
2h
3h
4h
25kDa p21
20kDa -
shp21_1
FCS
perm.
25kDa -
FCS refeed after o/n SS
0h
1h
2h
3h
4h
p21
20kDa -
Supplementary Figure 7_Rudolph
Figure S7 Whole scans of Western blots. (a) Analysis of Puma
expression in intestine of twelve mice with the indicated genotypes.
Whole scan of blot in figure 2. (b) Analysis of p21 expression in
NIH3T3 mouse fibroblasts and verification of shRNA mediated
repression of p21 expression. Whole scan of blot in Supplementary
Figure 5a.
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S U P P L E M E N TA R Y I N F O R M AT I O N
Supplementary Tables
Table S1 p21 knockdown induces chromosomal imbalances in Puma-deficient intestinal stem from G3 Terc-/- mice. Small intestine crypt cells of 10-monthold G3 mTerc-/-,Puma-/- mice were virally equipped with control or p21 shRNA and cultured over a period of 70d. Array CGH analysis was performed on
individual in vitro grown organoids (G3 mTerc-/-, Puma-/-, shCtr n = 9 crypts; G3 mTerc-/-, Puma-/-, shp21 n = 13 crypts). The table contains a summary of
chromosomal imbalances detected in all samples analysed.
Table S2 p21 knockdown induces chromosomal imbalances in Puma-deficient hematopoietic stem cells of G3 Terc-/- mice. LSK cells were isolated from
10 month old G3 mTerc-/-, Puma-/- mice, virally equipped with scrambled shRNA or p21 shRNA and transplanted into recipient mice. Highly purified longterm haematopoietic stem cells (CD34lo, KSL) were isolated 15 weeks after transplantation and cultured as single cells for two weeks followed by array CGH
analysis. (G3 mTerc-/-, Puma-/-, shCtr, n = 9; G3 mTerc-/-, Puma-/-, shp21, n = 9). The table contains a summary of chromosomal imbalances detected in all
samples analysed.
Table S3 p21 knockdown does not induce chromosomal instability in Puma-proficient hematopoietic (HSC) and intestinal stem cells (ISC) of G3 mTerc/- mice. (upper part) LSK cells were isolated from 10 month old G3 mTerc-/-, Puma-/- mice, virally equipped with GFP and control or p21 shRNA and
transplanted into lethally irradiated recipient mice. Highly purified long-term haematopoietic stem cells (CD34lo, LSK) were isolated 15 weeks after
transplantation and cultured as single cells for two weeks followed by array CGH analysis. (lower part) Small intestine crypt cells of 10-month-old G3 mTerc/-, Puma+/+ mice were virally equipped with control or p21 shRNA and cultured over a period of 70d. Array CGH analysis was performed on individual in vitro
grown organoids (HSC: G3 mTerc-/-, Puma+/+, shp21 n = 8 colonies; ISC: G3 mTerc-/-, Puma+/+, shp21 n = 13 organoids). The table contains a summary of
chromosomal imbalances detected in all samples analysed.
8
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