Engineered telomere degradation models dyskeratosis congenita.
ABSTRACT Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism. However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4-5 mo of age. These results provide experimental support for the notion that DC is caused by telomere dysfunction, and demonstrate that key aspects of a human telomere-based disease can be modeled in the mouse.
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ABSTRACT: The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders.Genes & Development 10/2014; · 12.64 Impact Factor
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ABSTRACT: Telomeres have evolved to protect the ends of linear chromosomes from the myriad of threats posed by the cellular DNA damage signaling and repair pathways. Mammalian telomeres have to block nonhomologous end joining (NHEJ), thus preventing chromosome fusions; they need to control homologous recombination (HR), which could change telomere lengths; they have to avoid activating the ATM (ataxia telangiectasia mutated) and ATR (ATM- and RAD3-related) kinase pathways, which could induce cell cycle arrest; and they have to protect chromosome ends from hyperresection. Recent studies of telomeres have provided insights into the mechanisms of NHEJ and HR, how these double-strand break (DSB) repair pathways can be thwarted, and how telomeres have co-opted DNA repair factors to help in the protection of chromosome ends. These aspects of telomere biology are reviewed here with particular emphasis on recombination, the main focus of this collection.Cold Spring Harbor perspectives in biology 09/2014; · 8.23 Impact Factor
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ABSTRACT: The single-stranded DNA binding proteins in mouse shelterin, POT1a and POT1b, accumulate at telomeres as heterodimers with TPP1, which binds TIN2 and thus links the TPP1/POT1 dimers with TRF1 and TRF2/Rap1. When TPP1 is tethered to TIN2/TRF1/TRF2, POT1a is thought to block RPA binding to the ss telomeric DNA and prevent ATR kinase activation, Similarly, TPP1/POT1b tethered to TIN2 can control the formation of the correct single-stranded telomeric overhang. Consistent with this view, the telomeric phenotypes following deletion of POT1a/b or TPP1 are phenocopied in TIN2- deficient cells. However, the loading of TRF1 and TRF2/Rap1 are additionally compromised in TIN2 KO cells, leading to added phenotypes. Therefore, it was not excluded that in addition to TIN2, other components of shelterin contribute to the recruitment of TPP1/POT1a,b as suggested by prior reports. To test whether TIN2 is the sole link between TPP1/POT1a,b and telomeres, we defined the TPP1-interaction domain of TIN2 and generated a TIN2 allele that is unable to interact with TPP1 but retains its interaction with TRF1 and TRF2. We demonstrate that cells expressing TIN2ΔTPP1 instead of wild type TIN2 phenocopy the POT1a,b knockout setting without showing additional phenotypes. Thus, these results are consistent with TIN2 being the only mechanism by which TPP1/POT1 heterodimers bind to shelterin and function in telomere protection.Journal of Biological Chemistry 07/2014; · 4.60 Impact Factor
Engineered telomere degradation models
Dirk Hockemeyer,1,4Wilhelm Palm,1,5Richard C. Wang,2Suzana S. Couto,3and Titia de Lange1,6
1Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, New York 10065, USA;2Department of
Dermatology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA;3Pathology and
Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous
symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in
telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism.
However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the
characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere
dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the
shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits
several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4–5 mo of age.
These results provide experimental support for the notion that DC is caused by telomere dysfunction, and
demonstrate that key aspects of a human telomere-based disease can be modeled in the mouse.
[Keywords: Bone marrow failure; dyskeratosis congenita; POT1; shelterin; telomere; telomerase]
Supplemental material is available at http://www.genesdev.org.
Received March 28, 2008; revised version accepted April 29, 2008.
DC is a rare hereditary multisystem disorder (for review,
see Dokal and Vulliamy 2005). In addition to fatal bone
marrow failure in the first or second decade, the charac-
teristic symptoms of DC are nail dystrophy, reticulated
hyperpigmentation of the skin, and leukoplakia of the
buccal mucosa. DC patients can present with various
other features including testicular atrophy, pulmonary
fibrosis, liver cirrhosis, predisposition to cancer, osteo-
porosis, abnormal dentition, and mental retardation. DC
has been proposed recently to be part of a spectrum of
related syndromes with diverse clinical manifestations
(Yaghmai et al. 2000; Yamaguchi et al. 2003; Yamaguchi
et al. 2005; Armanios et al. 2007). DC-like syndromes
may therefore be more frequent than previously inferred.
Autosomal forms of DC can be caused by mutations in
the genes encoding the RNA (hTR) or reverse transcrip-
tase (hTERT) components of telomerase (Vulliamy et al.
2001; Armanios et al. 2005; Marrone et al. 2007). In
mammals, telomerase is required to counteract the at-
trition of telomeric DNA, which has been argued to re-
sult primarily from nucleolytic processing of the C-rich
telomeric DNA strand (Lingner et al. 1995; Makarov et
al. 1997). Whereas telomerase is absent from most hu-
man somatic cells, its activity is detectable in certain
stem cell compartments, including the bone marrow.
Telomerase is inferred to extend the replicative life span
of hematopoietic stem cells (Allsopp et al. 2003), most
likely by diminishing the net rate of telomere shortening
rather than fully counteracting telomere attrition (Vaziri
et al. 1994). Reduced telomerase activity and exception-
ally short telomeres have been documented in peripheral
blood lymphocytes of DC patients with mutations in
hTR or hTERT (Yamaguchi et al. 2003; Marrone et al.
2004; Vulliamy et al. 2004; Armanios et al. 2005; Cerone
et al. 2005; Alter et al. 2007; Westin et al. 2007). A telo-
mere-based disease mechanism is consistent with the
generational anticipation observed for DC (Vulliamy et
al. 2004; Armanios et al. 2005), since affected parents are
expected to contribute shorter telomeres to the next gen-
eration. The dominant inheritance pattern of DC caused
by hTR and hTERT mutations can be explained from
haploinsufficiency of these telomerase components
(Marrone et al. 2004; Vulliamy et al. 2004; Armanios et
The more severe X-linked recessive form of DC is due
to mutations in the dyskerin gene (DKC1) (Heiss et al.
1998), and an autosomal recessive form of DC has been
shown recently to be due to mutations in NOP10 (Walne
et al. 2007). Dyskerin and NOP10 bind H/ACA RNAs,
which contribute to a wide variety of cellular processes,
including ribosome biogenesis, pre-mRNA splicing, and
telomerase biogenesis (Meier 2006). Observations on
Present addresses:4Whitehead Institute for Biomedical Research, 9 Cam-
bridge Center, Boston, MA 02142, USA;5Max Planck Institute of Mo-
lecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307
E-MAIL email@example.com; FAX (212) 327-7147.
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.1679208.
GENES & DEVELOPMENT 22:1773–1785 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org1773
mice with a lesion in the DKC1 locus have been inter-
preted as evidence that DC is due to a defect in ribosome
biogenesis rather than telomere dysfunction (Ruggero et
al. 2003). On the other hand, dyskerin binds hTR, pro-
motes telomerase biogenesis, and is associated with ac-
tive human telomerase (Mitchell et al. 1999; Mochizuki
et al. 2004; Cohen et al. 2007). Furthermore, diminished
telomerase activity and telomere length defects have
been demonstrated for DC cases due to mutations in
dyskerin and NOP10 (Mitchell et al. 1999; Wong et al.
2004; Wong and Collins 2006; Walne et al. 2007).
An additional complication in the context of a telo-
mere-based etiology of DC is that telomerase has been
proposed to have functions independent of telomere
maintenance, including in stem cell regulation (Zhu et
al. 1999; Stewart et al. 2002; Sarin et al. 2005). So far,
mouse models have not yielded a decisive answer to the
question whether DC is a disease of telomere dysfunc-
tion because mice lacking components of telomerase
have failed to show the phenotypes typical of DC. Due to
the large telomere reserve of Mus musculus musculus,
mTR and mTERT null mice have no discernable pheno-
types in the first generations (Blasco et al. 1997; Nikaido
et al. 1999; Liu et al. 2000). When the telomeres become
shortened in the later generations, the telomerase KO
mice show prominent premature aging phenotypes con-
sistent with stem cell depletion in highly proliferative
organs (Lee et al. 1998; Rudolph et al. 1999, 2000). The
phenotypes include testicular and ovarian atrophy, alo-
pecia, hairgraying, ulcerative skin lesions, and atrophic
villi in the duodenum of older mice. Late generation
telomerase KO mice are also less tolerant to exogenously
induced stress, such as treatment with 5-FU, skin lac-
eration, and liver injury. Nonetheless, the hallmark of
DC, progressive bone marrow failure, is not observed in
late generation telomerase KO mice. Although the mice
have slightly reduced levels of hematopoietic progeni-
tors, they do not show histological signs of defects in the
bone marrow, spleen, or thymus; they are not immuno-
deficient or anemic; and they have normal peripheral red
and white blood cell counts and blood chemistry. Late
generation telomerase knockout mice also lack the hy-
perpigmentation and nail dystrophy that are typical of
DC, and their life span is only moderately reduced (18
mo vs. 24 mo). Even when the initial telomere length is
curbed by using M. m. castaneus, telomerase deficiency
did not give rise to overt DC phenotypes (Hao et al.
Here we report on a mouse model in which key char-
acteristics of DC are induced by enhanced telomere
through nucleolytic attack on the 5? end of the chromo-
some, represented by the [CCCTAA]n telomeric strand
(referred to as the C-strand). C-strand processing nor-
mally takes place with each cell division and serves to
generate the 3? overhang, which provides binding sites
for essential telomeric proteins (Baumann and Cech
2001) and contributes to the formation of the t-loop
structure (Griffith et al. 1999). We reported previously
that the generation of the 3? overhang is controlled by a
member of the POT1 family of proteins, which are
single-stranded telomeric DNA-binding proteins within
shelterin (Hockemeyer et al. 2006), the complex that pro-
tects telomeres from the DNA damage response (de
Lange 2005; Palm and de Lange 2008). Among the two
mouse POT1 proteins, POT1a is essential, probably be-
cause in its absence, telomeres induce an ATR-depen-
dent DNA damage response (Hockemeyer et al. 2006;
Wu et al. 2006; Lazzerini Denchi and de Lange 2007). In
contrast, mice lacking POT1b are viable but have telo-
meres with unusually long 3? overhangs, suggesting ex-
cessive C-strand degradation (Hockemeyer et al. 2006).
We argued that if POT1b protects the 5? ends of telo-
meres from degradation, POT1b-deficient cells and mice
might experience accelerated telomere shortening and
could thus provide an alternative approach to modulat-
ing telomere length in the mouse. Here we show that
loss of POT1b results in telomere degradation in mouse
cells and use POT1b KO mice to study the phenotypic
consequences of this type of telomere length modula-
POT1b protects telomeres from C-strand degradation
To test whether the extended overhangs in POT1b-defi-
cient mouse cells are caused by excessive nucleolytic
degradation of the C-rich telomeric strand, we deter-
mined the effect of POT1b loss on telomere shortening.
As reported previously, POT1b was inactivated either
through insertion of a STOP cassette before exon 3
(POT1bS/S) or through deletion of exon 3 (POT1b−/−)
(Hockemeyer et al. 2006). Both genotypes represent ap-
parent null alleles of POT1b and the phenotypes of the
cells and mice were indistinguishable. The alleles used
in this study are identified in each experiment.
Telomere dynamics were studied in SV40-LT immor-
talized mouse embryo fibroblasts (MEFs) carrying condi-
tional alleles of POT1b (POT1bF/−or POT1bF/F). As ex-
pected, Cre-mediated deletion of POT1b induced an in-
crease in single-stranded telomeric DNA in telomerase-
proficient (mTR+/+) and telomerase-deficient (mTR−/−)
cells (Fig. 1A; Supplemental Fig. 1A). In addition, POT1b
deletion induced telomere shortening. Telomere short-
ening was apparent from examination of the bulk telo-
meres as well as the higher-molecular-weight (MW) spe-
cies and occurred in cells with and without telomerase
(Fig. 1A; Supplemental Fig. 1A). In contrast, deletion of
POT1a did not affect telomere length (Supplemental Fig.
1A). These results are consistent with the idea that
POT1b has a specific function in protecting telomeres
from C-strand degradation.
Exo1 has been implicated in C-strand degradation in
Saccharomyces cerevisiae (Maringele and Lydall 2002).
We therefore tested whether Exo1 modulated the effects
of POT1b deletion using POT1bF/FExo1−/−MEFs gener-
ated by crossing the pertinent genetically altered mice.
Regardless of whether Exo1 was present, deletion of
POT1b resulted in C-strand degradation as evidenced by
Hockemeyer et al.
1774GENES & DEVELOPMENT
an increase in the G-strand signal (Supplemental Fig. 1B).
Therefore, the nuclease(s) responsible for C-strand deg-
radation in POT1b-deficient cells remains to be deter-
SV40LT-immortalized mTR-proficient and -deficient MEFs before and after deletion of POT1b with Cre. (Top) Size-fractionated
MboI-digested DNA hybridized in gel under native conditions with a radiolabeled [CCCTAA]4-oligo detecting the 3? overhang.
(Bottom) The same gels after in situ denaturation of the DNA and rehybridization with the same probe. Numbers above the lanes
reflect population doublings after introduction of pWZL-hygro-Cre (or the empty vector). MWs are indicated in kilobases on the left.
(B) Telomere FISH analysis on metaphase chromosome spreads of the cells analyzed in A. PDs are given in C. DAPI-stained chro-
mosomes are false colored the in red, and the telomere hybridization signal is shown in green. Arrows point at chromosome fusions.
(C) Quantification of chromosome fusions detected in metaphases as shown in B.
Accelerated telomere shortening in POT1b-deficient cells. (A) Telomere length dynamics and telomeric overhangs of
POT1b KO DC mouse model
GENES & DEVELOPMENT1775
The telomere shortening associated with POT1b defi-
ciency was obviously exacerbated in mTR−/−cells com-
pared with their telomerase positive counterparts (Fig.
1A–C). However, even when telomerase was present, the
telomeres shortened upon deletion of POT1b. Such fail-
ure in telomerase-mediated telomere maintenance in
POT1b−/−cells could be explained if POT1b played a role
in the telomerase pathway. Alternatively, the telomere
shortening rate in POT1b−/−cells might simply exceed
the maximal rate of telomere elongation by the telom-
erase present in these cells. The shortening rate of the
largest class of telomeric fragments (MW > 60 kb) was
increased from 400 to 700 bp per end per PD in POT1b−/−
cells lacking the mTR gene, suggesting that telomerase
adds in the order of 300 bp per end per PD in POT1b−/−
mTR+/+cells. This telomere synthesis rate is close to the
maximal rate of telomere elongation reported for mam-
malian telomerases (Barnett et al. 1993; McChesney et
al. 2000; Loayza and de Lange 2003; Cristofari and Ling-
ner 2006), suggesting that the rate of degradation of telo-
meres in POT1b−/−cells may be too great to be counter-
acted by telomerase.
In the absence of healing by telomerase, the degrada-
tion of the telomeric DNA in POT1b−/−mTR−/−cells is
expected to eventually disable the protective function of
telomeres and elicit chromosome end fusions. Indeed,
after the cultures had been propagated for ∼60 PD, the
frequency of fused chromosomes was elevated in the
cells compared with mTR−/−
POT1b−/−cells (Fig. 1C). As expected, most of the fusions
in the POT1b−/−mTR−/−cells were due to short arm
(Robertsonian) fusions that are stable in mitosis and
therefore persist longer than fusions involving long-arm
In summary, the data on telomere dynamics in MEFs
indicate that POT1b deficiency leads to unusually rapid
telomere shortening as a consequence of C-strand degra-
dation, and that telomerase can counteract some, but not
all, of this terminal sequence loss.
Telomerase is essential in the context
of POT1b deficiency
Mice lacking either POT1b or telomerase are viable,
have a normal life span, and are fertile (Blasco et al. 1997;
Liu et al. 2000; Hockemeyer et al. 2006). The enhanced
telomere shortening in the POT1b−/−mTR−/−cells raised
the possibility that diminished telomerase activity
might affect the phenotype of POT1b deficiency and vice
versa. To determine the effect of combined deficiency of
POT1b and mTR, we analyzed the offspring of inter-
crosses of POT1b+/−mTR+/−mice (Fig. 2A). All genotypes
were represented at frequencies consistent with Mende-
lian inheritance of unlinked genes, with the exception of
the POT1b−/−mTR−/−genotype, which was grossly un-
derrepresented (one in 281 offspring; 15-fold less fre-
quent than expected). Similarly, POT1b−/−mTR−/−mice
were not recovered from an intercross of POT1b+/−
mTR−/−breeding pairs that yielded 42 mice (expected
number of doubly deficient mice ∼12). Thus, the
POT1b−/−mTR−/−genotype is associated with dimin-
ished viability. The single POT1b−/−mTR−/−pup born
was runted, remained hairless, failed to thrive, and died
3 wk after birth (Fig. 2B). In contrast, POT1b−/−mTR+/−
mice survived to adulthood.
Heterozygosity for mTR exacerbates POT1b−/−
Comparison of POT1b−/−mice to wild-type and hetero-
zygous littermates showed that both males and females
were smaller and had a reduced body weight (Fig. 2C).
The body weight of the POT1b−/−mice was further re-
duced in the context of mTR heterozygosity. An effect of
mTR heterozygosity is consistent with previous reports
indicating haploinsufficiency for the core telomerase
components in mice and humans (Hathcock et al. 2002;
Erdmann et al. 2004; Marrone et al. 2004; Armanios et al.
2005; Yamaguchi et al. 2005).
In addition to their reduced body weight, POT1b−/−
males showed a marked (approximately threefold) reduc-
tion in the size of the testis, and histological analysis
revealed depletion of cells from the testicular lumen,
including a reduction in spermatids (Fig. 2E,F). As ex-
pected from this phenotype, POT1b-deficient males be-
came prematurely infertile, but when allowed to mate at
a young age (<6 mo), homozygous intercrosses of POT1b-
deficient animals yielded offspring for five generations.
The testicular atrophy was both more frequent and more
severe in the POT1b−/−mTR+/−setting (Fig. 2D–F).
POT1b−/−mice also showed a defect in the small intes-
tine as evidenced by apoptotic cells in a large percentage
of the crypts (Fig. 2G,H) and this phenotype was also
significantly worse in mice that lacked POT1b in the
context of mTR heterozygosity.
All POT1b-deficient mice displayed striking hyperpig-
mentation of the paws, snout, ears, and tail (Fig. 3A,B).
Hyperpigmentation developed when the mice were 3–4
mo old and appeared progressive with age (data not
shown). The hyperpigmentation of the paws was more
prominent in the POT1b−/−mTR+/−mice, and became
visible at an earlier age (Fig. 3B). Histological analysis of
hyperpigmented tails from two 8-mo-old POT1b−/−
mTR+/+mice demonstrated a marked increase in the
density of the melanosomes of the keratinocytes com-
pared with age-matched wild-type controls (Fig. 3C). In
addition, higher amounts of melanin were present in the
stratum corneum (upper layers) of the epidermis suggest-
ing higher melanin content in the melanosomes (Fig. 3C,
arrowheads). Using both H&E and Fontana-Masson
(melanin) stains, mutant mouse tails were found to have
a markedly higher melanin content than the normal tails
(Fig. 3C). After UV radiation of human skin, melano-
somes become more densely distributed in an umbrella
or “microparasol” over keratinocyte nuclei presumably
Hockemeyer et al.
1776GENES & DEVELOPMENT
in an effort to shield DNA from further damage (Gates
and Zimmermann 1953; Byers et al. 2003). The increased
melanosome content of the mutant mice share this “mi-
croparasol” distribution, suggesting a role for DNA dam-
age in the generation of the increased pigmentation.
In addition to hyperpigmentation, DC patients present
with nail dystrophy. Nails were examined in detail in a
cohort of 10-mo-old POT1b KO mice representing the
fifth generation of POT1bS/Sintercrosses and compared
with age-matched wild-type controls (Fig. 3D). Among
the POT1b KO mice, ∼25% had developed nail abnor-
malities on one or more digits while none of the control
animals (n = 25) displayed nail malformations. Further
analysis will be needed to assess the condition of the
nails in younger POT1b-deficient animals. In particular,
the POT1b−/−mTR+/−mice may have died before nail
dystrophy became an overt phenotype (see below).
Progressive bone marrow failure and reduced life span
All POT1b−/−mTR+/−mice developed a severe defect in
the bone marrow (Fig. 4). Peripheral blood cell counts
showed significant pancytopenia with severe leukocyto-
penia (lymphopenia, neutropenia, and monocytopenia),
thrombocytopenia, and mild anemia (Fig. 4A). Bone mar-
row from the femur and vertebrae was examined histo-
POT1b+/−mTR+/−breeding pairs. (B) Photograph showing the single POT1b−/−mTR−/−animal born and its littermates. (C) Scatter plot
of the bodyweights of adult (>3 mo) female and male mice with the indicated genotypes. All animals were age-matched and derived
from the intercross described in A. Error bars indicate the standard error of the mean. (D) Quantification of testis size of age-matched
males of the indicated genotypes. Error bars indicate the standard error of the mean. (E) H&E stained testis sections at two magnifi-
cations. Mice as in A. (F) Incidence of testicular atrophy in mice of the indicated genotypes. Grading is based on H&E staining as shown
in E. (G) TUNEL assay on H&E-stained sections from the small intestine of age-matched mice of the indicated genotypes. (H)
Quantification of apoptotic cells in the small intestines based on TUNEL staining of age-matched mice with the indicated genotypes
generated as in A. (POT1b+/+mTR+/+, POT1b+/+mTR+/−, POT1b+/−mTR+/−n = 4; POT1b−/−mTR+/+and POT1b−/−mTR+/−n = 5). For each
animal >60 crypts were analyzed. Error bars indicate the standard error of the mean.
Curtailed telomerase exacerbates POT1b KO phenotypes. (A) Genotypes of 281 offspring from intercrosses between 15
POT1b KO DC mouse model
GENES & DEVELOPMENT1777
logically, and revealed marked attrition with replace-
ment of hematopoietic precursor cells of all recogniz-
able hematopoietic lineages (myeloid, erythroid, and
megakaryocytic) by stromal adipose tissue (Fig. 4B; Sup-
plemental Fig. 2A). This bone marrow defect was also ob-
served in a cohort of POT1bS/SmTR+/−mice (Supplemental
Fig. 2A,B). When examined at 3 mo of age, POT1b−/−
mTR+/−mice also had abnormal spleen architecture with
decreased lymphoid population in the white pulp, as
demonstrated by immunohistochemical staining for the
lymphoid marker B220, accompanied by increased eryth-
ropoiesis as indicated by expansion of the Ter-119-
marked red pulp (Fig. 4C). Such extramedullary erythro-
poiesis may explain why the anemia in these mice is not
In comparison with their POT1b+/−mTR+/−littermates
who thrived, POT1b−/−mTR+/−mice progressively dete-
riorated and died ∼4–5 mo of age. The median survival of
the POT1b−/−mTR+/−mice was 135 d, a significant re-
duction in life span compared with telomerase-deficient
mice or mice lacking POT1b (Fig. 4D). Similarly,
POT1bS/SmTR+/−mice had a severely shortened life span
of 147 d (Supplemental Fig. 2C). The significant reduc-
tion in life span of POT1b−/−mTR+/−and POT1bS/S
mTR+/−mice is likely due to the pancytopenia associ-
ated with bone marrow failure.
snout, ears, tail, and paws of POT1bS/Sanimals. (B) Hyperpigmentation of the paws of mice with the indicated genotypes and ages. (C)
H&E and Fontana-Masson staining of tail sections of 8-mo-old mice with the indicated genotypes. Arrows indicate the increased
deposition of melanosomes over the nuclei of the hyperpigmented mouse tail sections. Arrowheads indicate retention of the melanin
in the stratum corneum. Enlarged images of the Fontana-Masson staining of tail sections of POT1b−/−and wild-type mice are shown
at right. (D) Photographs showing abnormal nails on one paw of an 11-mo-old G5 POT1S/Smouse. (Top) The wild-type control is
Cutanous phenotypes. (A) Photographs of POT1bS/Sand age-matched control animals showing hyperpigmentation on the
Hockemeyer et al.
1778GENES & DEVELOPMENT
Because of the severe bone marrow phenotype in the
POT1b−/−mTR+/−mice, we analyzed the status of the
telomeres in this tissue by Q-FISH (Fig. 5A). The bone
marrow of 3-mo-old POT1b proficient mice, either with
or without mTR, showed the considerable telomere re-
serve typical of the genetic background of the mice
(C57BL/6). POT1b-deficient mice containing normal lev-
els of telomerase had a significant reduction of telomere
length, consistent with the accelerated telomere short-
ening observed in MEFs (Fig. 1). The telomeres in the
bone marrow of the POT1b−/−mTR+/−mice were further
reduced in length. The shortening of the telomeres in
these mice was also obvious from the occurrence of sig-
nal free ends in metaphase spreads (Fig. 5A,B). Further-
more, bone marrow chromosome spreads showed an in-
crease in the frequency of chromosome end fusions (Fig.
5C), indicating considerable telomere dysfunction in this
Our data indicate that key aspects of DC can be modeled
in mice by inducing telomere degradation. Mouse mod-
els for DC are needed to gain insight into the etiology of
the disease and to evaluate new treatment strategies.
Mouse telomeres recapitulate most aspects of human
telomere biology, although differences in telomerase
regulation, the telomere damage response pathway, and
the composition of shelterin have been noted (Prowse
and Greider 1995; Smogorzewska and de Lange 2002; Ja-
cobs and de Lange 2004; Hockemeyer et al. 2006, 2007).
By far the greatest challenge in generating mouse models
for human telomere biology is the large telomere reserve
of standard laboratory mice (subspecies M. m. muscu-
lus), which can sustain several generations in absence of
telomerase (Blasco et al. 1997). In later generations,
telomerase knockout mice show the impact of telomere
dysfunction on several organs, yet the mice do not de-
velop most of the characteristic symptoms of DC. The
engineered telomere degradation in POT1b-deficient
mice provides a first step toward a genuine DC model
(Fig. 6). POT1b-deficient mice, in particular in the con-
text of limiting telomerase activity, represent the most
severe and lethal aspect of DC: progressive bone marrow
failure. In addition, the POT1b-deficient mice show two
features typical of DC: hyperpigmentation and nail ab-
the indicated genotypes (POT1b+/+mTR+/+, n = 6, average age: 149 d; POT1b+/+mTR+/−, n = 5, average age: 132 d; POT1b+/−mTR+/−,
n = 6, average age: 137 d; POT1b−/−mTR+/+, n = 9, average age: 138 d; POT1b−/−mTR+/−, n = 8, average age: 110 d). Error bars indicate
the standard error of the mean. Differences between POT1b−/−mTR+/+animals and POT1b−/−mTR+/−mice are significant (P < 0.05,
upaired t-test) for all parameters except for the red blood cells counts. (B) H&E staining of bone marrow section of a 121-d-old
POT1b+/−mTR+/−mouse (left panels) and an 83-d-old POT1b−/−mTR+/−mouse (right panels). (C) Immunohistochemical staining of
B220 and Ter119 in spleen sections of mice with the indicated genotypes. (age: POT1b+/+mTR+/+and POT1b−/−mTR+/+animals 179 d,
POT1+/−mTR+/−and POT1b−/−mTR+/−animals 121 d). (D) Kaplan-Meyer survival plot of mice derived from a heterozygous intercross
of POT1b+/−mTR+/−mice (see Fig. 2A). POT1b−/−mTR+/−mice die prematurely compared with the controls (P < 0.0001, based on Log
Bone marrow failure and premature death in POT1b−/−mTR+/−mice. (A) Peripheral blood counts of age-matched mice with
POT1b KO DC mouse model
GENES & DEVELOPMENT1779
normalities. Importantly, several DC phenotypes arise in
a setting with normal telomerase activity. This provides
a strong argument that DC is due to telomere dysfunc-
tion rather than a defect in ribosome biogenesis or a de-
ficiency in of one of the nontelomeric functions ascribed
Engineering telomere degradation
A general attribute of telomeres in unicellular organisms
is that they render chromosome ends resistant to nucleo-
lytic attack. A POT1 ortholog from ciliates protects telo-
mere termini from nucleases in vitro (Gottschling and
marrow of POT1b−/−mTR+/−mice. (A) Q-
FISH analysis of the metaphases isolated
from bone marrow of 3-mo-old littermates
with the indicated genotypes. The X axis
shows telomere fluorescence hybridiza-
tion intensity (TFU), the Y axis shows the
frequency of chromosomes ends with the
indicated TFUs. Average TFU values are
indicated in each panel. (B) Telomere FISH
analysis of metaphases isolated from the
bone marrow of mice (3-mo-old litter-
mates) with the indicated
DAPI stained chromosomes are false col-
ored in red and the telomere hybridization
signal is shown in green. (C) Quantifica-
tion of chromosome abnormalities de-
tected in metaphases as shown in B. Con-
trols represent pooled data obtained from
bone marrow samples of two POT1b+/−
mTR+/−, two POT1b+/−mTR−/−, and two
POT1b−/−mTR+/+mice. tel+and tel−indi-
cate fusions with and without telomeric
FISH signals at the fusions sites, respec-
Telomere dysfunction in bone
Hockemeyer et al.
1780GENES & DEVELOPMENT
Zakian 1986). Similarly, Cdc13, the single-stranded telo-
meric DNA-binding protein of budding yeast, prevents
degradation of the 5?-ended telomeric DNA strand by
ExoI (Maringele and Lydall 2002), and fission yeast lack-
ing Pot1 show rapid loss of the telomeric DNA (Bau-
mann and Cech 2001).
Our data suggest that mammalian telomeres conform
in this regard since deletion of POT1b results in resec-
tion of the C-rich telomeric strand and accompanying
telomere shortening. Previous data indicates that POT1b
does not control other aspects of telomere protection.
For instance, POT1b-deficient cells do not show an im-
mediate DNA damage response at telomeres nor do they
display inappropriate DNA repair reactions at chromo-
some ends (Hockemeyer et al. 2006). These protective
aspects depend on other components of shelterin, includ-
ing POT1a (Hockemeyer et al. 2006; Lazzerini Denchi
and de Lange 2007). Thus, POT1b provides a specific tool
to induce telomere degradation without immediate ef-
fects on the essential aspects of telomere function.
Manipulating telomere length in the mouse
The length of mouse telomeres has been primarily ma-
nipulated by enforcing telomere attrition through telom-
erase withdrawal. In the absence of telomerase, telo-
meres shorten at ∼4 kb per generation, reflecting attri-
tion during the ∼20 cell divisions separating the germline
of one generation from the next (Blasco et al. 1997).
Within each generation, additional attrition will occur
during the cell divisions required to form and maintain
adult tissues. This process is likely to shorten telomeres
to variable extents in different somatic cell types de-
pending on their replicative history. However, the actual
extent of telomere attrition in the murine soma is largely
unknown. Telomere dysfunction is predicted to only oc-
cur in tissues in which sufficient cell divisions have oc-
curred to deplete the telomere reserve provided in that
generation. Therefore, the telomerase KO approach cre-
ates a window for telomere dysfunction to arise in a sub-
set of tissues in the last viable generation. An improved
version of this approach was recently developed using M.
m. castaneus, a Mus musculus subspecies whose telo-
mere length is substantially shorter and therefore accel-
erates the onset of telomere dysfunction by several gen-
erations (Hao et al. 2005). These approaches have pro-
vided important insights into the impact of telomere
attrition on organ function and tumorigenesis.
The accelerated telomere shortening resulting from
POT1b deficiency adds two important new aspects to
manipulating telomere length in the mouse. First, the
degradation of telomeres in POT1b−/−mTR+/−mice is so
extensive with each cell division that phenotypes arise
in the first generation even within the context of the
long telomeres typical of standard laboratory mice. This
rapid onset of the phenotype represents a considerable
gain in terms of experimental effort. Second, the degra-
dation of telomeres in the POT1b−/−setting has shifted
the spectrum of the tissues that are affected. Impor-
tantly, the POT1b−/−mTR+/−mice develop progressive
bone marrow failure, a phenotype not seen in the telo-
merase knockout systems, presumably because their
telomere reserve in the bone marrow is sufficient to sus-
tain a minimal level of hematopoiesis, even in the last vi-
able generation. Conversely, the POT1b−/−mTR+/−mice do
the mouse and comparison with DC Com-
parison of the symptoms found in DC pa-
tients with the phenotypes observed in the
late generation of telomerase knockout mice
and POT1b−/−and POT1b−/−mTR+/−mice.
Telomere length manipulation in
POT1b KO DC mouse model
GENES & DEVELOPMENT 1781
not show hair-graying or alopecia, which is observed in
late generation mTR−/−mice. The simplest explanation
for this difference in affected organs in the two experi-
mental systems is to assume that the extent of telomere
degradation in absence of POT1b is affected by both the
number of cell divisions and the level of the nuclease(s)
that resects the 5? end of the telomeres. Thus, the hema-
topoietic system may be more severely affected in the
POT1−/−setting than in the mTR−/−mice due to higher
levels of the culprit nuclease in hematopoietic stem
cells. Conversely, cell types with less nuclease activity
or higher telomerase activity will be protected from telo-
mere dysfunction in the POT1b−/−mTR+/−setting.
POT1b−/−mTR+/−mice as a model for DC
The findings with the POT1b−/−mTR+/−and POT1bS/S
mice strongly suggest that several of the DC phenotypes,
including bone marrow failure, hyperpigmentation, and
nail dystrophy, are indeed due to telomere failure, rather
than a defect in ribosomal biogenesis. The hyperpigmen-
tation in the POT1b−/−mice could be explained from the
induction of a DNA damage response after depletion of
the telomere reserve in a subset of cells. Recent data
have shown that in addition to UV, other forms of DNA
damage can elicit a p53-dependent tanning response in
the skin (Cui et al. 2007). Although we did not test the
requirement for p53 in the hyperpigmentation in the
POT1b−/−mice, the formation of melanosome “micro-
parasols” that cap over the nuclei suggests that the phe-
notype is similar to the tanning response. With regard to
the nail abnormalities, we imagine that one possibility is
that a stem cell population required for normal nail
growth is affected by the accelerated telomere loss.
It will be of interest to study the occurrence of addi-
tional DC characteristics, including oral leukoplakia,
cancer predisposition, and lung fibrosis in this setting,
since this could clarify the contribution of telomere dys-
function to these symptoms. In order to study these and
other late-onset symptoms, it may be necessary to de-
velop a system in which the premature death due to bone
marrow failure is prevented (e.g., by bone marrow trans-
An obvious concern with the POT1−/−mTR+/−model
for DC is that the phenotypes are due to a mutation in a
gene that only occurs as such in rodents (Hockemeyer et
al. 2006). However, our recent data indicate that the
critical protein domains of POT1b required to block telo-
mere degradation are functionally conserved in human
POT1, suggesting that human POT1 can similarly block
telomere degradation in human cells (W. Palm, D. Hocke-
meyer, and T. de Lange, unpubl.). Given this functional
conservation, POT1 should be considered in the genetic
analysis of DC patients lacking mutations in telomerase,
dyskerin, or NOP-10. Our data predicts the possibility of
DC due to a dissociation of function mutation in POT1
that unleashes C-strand resection without affecting the
repression of ATR. Excessive C-strand resection is ex-
pected to result in inappropriate telomere shortening
with accompanying phenotypic consequences. Interest-
ingly, a recent report described mutations in TIN2 in
several DC cases, suggesting that alterations in shelterin
can indeed contribute to this disease (Savage et al. 2008).
Materials and methods
Mouse strains and MEFs
POT1bS/Smice and the crosses of POT1bF/Fto the mTR-/+mice
were described previously (Hockemeyer et al. 2006). As no sig-
nificant differences between POT1b+/+and POT1bS/+mice were
detectable, the data from these cohorts were pooled and used as
controls. POT1b−/−mice were generated by crossing mice car-
rying the POT1b FLOX allele to the germline deleting B6.FVB-
Tg(EIIa-cre)C5379Lmgd/J mouse strain (Jackson Laboratories).
Chimeric mice with germline deletion of the Floxed allele were
crossed to C57BL/6J mice to remove the Cre transgene. The
resulting POT1b+/−mice were crossed to mTR+/−mice to gen-
erate POT1b+/−mTR+/−mice. All analysis was performed exclu-
sively on mice obtained from heterozygous intercrosses of
POT1b+/−mTR+/−mice. Offspring of >15 breeding pairs were
used in the analysis. Exo1−/−mice were provided by W. Edel-
mann and crossed to the POT1bF/Fmice to introduce the con-
ditional POT1b allele. MEFs were derived and cultured as de-
scribed previously (Hockemeyer et al. 2006). POT1b mice and
MEFs were genotyped by PCR as described previously (Hock-
emeyer et al. 2006). mTR-deficient mice and MEFs were geno-
typed by PCR with the following primers: 5?-TTCTGACCAC
AG-3?, and 5?-GGGGCTGCTAAAGCGCAT-3?. Exo1−/−mice
and MEFs were genotyped by PCR with primers 5?-CTCTT
GATA-3?, and 5?-AGGAGTAGAAGTGGCGCGAAGG-3?.
Analysis of telomeric DNA
Telomere fluorescence in situ hybridization, in-gel telomere
overhang assays, and telomere length analyses were performed
as described (Hockemeyer et al. 2006). Bone marrow cells for
telomere length analysis were isolated from the femur by flush-
ing with PBS. Cells were cultured in medium supplemented
with 15% fetal calf serum and 200 ng/mL demecolcine (Sigma)
for 2 h. Cells were harvested and prepared for metaphase spreads
and FISH analysis as described previously (van Steensel et al.
1998). Q-FISH analysis was performed as a blind study as de-
scribed (Hao et al. 2005).
Histology and immunohistochemistry
For H&E staining, 4–6 µm paraffin sections were rehydrated in
water and placed in Harris Hematoxylin solution (Poly Scien-
tific) for 2 min. Slides were rinsed in water and in Clarifier 1
solution (Richard-Allan Scientific) for 10 sec. After another
wash with water, slides were stained with Bluing Reagent (Rich-
ard-Allan Scientific) for 8 sec and washed with tap water. After
dipping the slides once in 95% ethanol, slides were incubated
for 1 min in Eosin Y Alcoholic Working solution (Poly Scien-
tific). Samples were dehydrated with serial washes of 70%, 95%,
and 100% ethanol, and sealed with a coverslip using Cytoseal
XYL solution (Richard-Allan Scientific). All steps were per-
formed at room temperature.
For detection of B220 and Ter-119, slides with tissue sections
were heated for 30 min at 58°C to 60°C and afterward deparaf-
finized by sequential incubations in 100%, 95%, and 70% etha-
Hockemeyer et al.
1782GENES & DEVELOPMENT
nol and water. Afterward, sections were incubated in 1% hy-
drogen peroxide in phosphate-buffered saline (PBS) for 15 min
and rinsed in PBS. For Ter-119 staining the slides were micro-
waved in 10 mM Sodium Citrate (pH 6.0) at a high-power set-
ting for 15 min. Slides were incubated for 30 min in 10% normal
rabbit serum (MP Biomedicals) diluted in 2% BSA in PBS in a
humid chamber. Next, sections were incubated with a 1:200
dilution of primary antibody (B220: CD45R/B220; Ter-119: Lys-
76; BD Pharmingen) in 2% BSA in PBS overnight at 4°C in a
humid chamber. Sections were rinsed three times with PBS and
incubated with a 1:100 dilution in PBS of a biotinylated mouse-
absorbed anti-Rat IgG (Vector Labs) for 30 min at room tem-
perature. Slides were rinsed three times in PBS and incubated
for 30 min using the Avidin-Biotin Complex Elite kit (Vector
Laboratories) using conditions provided by the manufacturer.
After washing the slides three times with PBS, sections were
stained with 3,3?-Diaminobenzidine (Sigma-Aldrich). Slides
were washed with water and counterstained with hematoxylin.
Afterward slides were dehydrated with three changes of 95%
ethanol and 100% ethanol and mounted with coverslips.
For H&E staining of skin sections, paraffin sections were pro-
cessed using a Leica Autostainer XL. Sections were dried for 10
min and rehydrated with three changes of xylene (3 min, 3 min,
and 1 min), two changes of absolute alcohol (30 sec, 20 sec), 95%
alcohol (30 sec), and deionized water (30 sec). Sections were
stained with Harris Hematoxylin solution for 8 min. Samples
were washed with running water (1 min) and Clarifier solution
(30 sec). Samples were washed with running water (30 sec) and
stained with Bluing Solution (30 sec). Samples were washed
with running water (30 sec), dehydrated with 95% alcohol (30
sec), and stained with Eosin-Y (1 min). Slides were dehydrated
with three changes of absolute alcohol (30 sec) and cleared with
three changes of xylene (20 sec). Finally, slides were mounted
with synthetic resin.
For Fontana-Masson staining of skin sections, paraffin tail
sections were deparaffinized and hydrated in distilled water.
The slides were immersed 10% (w/v) silver nitrate solution in a
56°C waterbath for 1–2 h. The reaction was stopped when gran-
ules were dark brown and the background colorless. The sec-
tions were rinsed in distilled water and then immersed in 0.2%
(w/v) gold chloride solution for 10 min. The sections were
rinsed in distilled water and then placed in 5% (w/v) sodium
thiosulfate solution for 5 min. The sections were rinsed in dis-
tilled water and then counterstained in nuclear-fast red for 5
min. The slides were washed for 1 min in running water. The
slides were dehydrated with two changes each of 95% and ab-
solute alcohol and cleared with xylene. Finally, slides were
mounted with synthetic resin.
TUNEL assays were performed essentially as described (Gavri-
eli et al. 1992). Slides with paraffin sections were heated for 30
min at 58°C–60°C and deparaffinized by three washes with xy-
lene and rehydrated by three washes of 100% ethanol for 10 min
each, three 3-min washes with 95% ethanol, and three 3-min
washes with water. Sections were immersed in 10 mM Tris-HCl
(pH 8.0) for 5 min and incubated with 20 µg/mL proteinase K in
10 mM Tris-HCl (pH 8.0) for 15 min in a humid chamber. Next,
slides were washed in water four4 times for 2 min each and
incubated in 3% H2O2in PBS for 5 min at room temperature.
Afterward, slides were washed three times with distilled water
for 2 min each. Sections were incubated in 30 mM Tris-HCl,
140 mM Na-Cacodylate, 1 mM CoCl2(pH 7.2), and terminal
transferase reactions were performed in 0.1 M Na-Cacodylate,
0.1 mM DTT, 0.05 mg/mL BSA, 2.0 U/µL Terminal Transferase
(Roche), 0.1 nmol Biotin-16-dUTP, and 2.5 mM CoCl2(pH 7.2)
at room temperature for 1 h. Reactions were terminated with
300 mM NaCl and 30 mM Na-citrate (pH 6.0), and slides were
washed three times for 2 min with PBS. Slides were blocked
with 2% BSA (Sigma-Aldrich) in PBS for 10 min in a humid
chamber and washed three times 2 min each in PBS. Sections
were incubated in Avidin-Biotin Complex Elite kit (Vector Labs)
using conditions provided by the manufacturer for 30 min and
washed with PBS three times for 5 min each. Afterward, sec-
tions were stained with 3,3?-Diaminobenzidine for 3 min and
washed with water, and subsequently counterstained with he-
matoxylin and dehydrated before sealing with a coverslip.
We thank Margaret Strong and Carol Greider for performing the
Q-FISH analysis and for critical discussion of this work. We are
grateful to Devon White for expert mouse husbandry and tech-
nical help with mouse procedures. Diana Argibay is thanked for
assistance with genotyping. Sean Rooney is thanked for help
with isolation of bone marrow cells. Joe Susa and Dermpath
Diagnostics are thanked for tissue processing. Winfried Edel-
man is thanked for a generous gift of the Exo1−/−mice. We
thank Monika Bessler, Phil Mason, Ronald DePinho, and mem-
bers of the de Lange laboratory for helpful discussion. Nadya
Dimitrova, Kristina Hoke, Eros Lazzerini Denchi, and Hiro
Takai are thanked for technical assistance. D.H. was supported
by a Cancer Research Institute Predoctoral Emphasis Pathway
in Tumor Immunology Grant and Rockefeller University
Graduate Program Funds. W.P. was supported by the Studien-
stiftung des deutschen Volkes. This work was supported by a
grant from the NCI to T.d.L. (CA076027).
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