TELOMERES AND CANCER
LABORATORY (A. Londoño)
« Résultats et Autoévaluation »
During the last five years, the “Telomeres
and Cancer” laboratory continued its
research work on human telomere
metabolism and its involvement in cancer
and other pathologies. We had proposed to
study several basic aspects of this
metabolism, including the timing of
replication of single telomeres in humans
and other primates, the mechanism of
telomere
recombination
in
human
immortalized cells without telomerase and
the contribution of telomere instability to the
oncogenic process. Several new themes
were also initiated during this period,
particularly motivated by interactions with
other laboratories, including the role of
telomeres during cell reprogramming, the
function of telomere maintenance in
idiopathic pulmonary hypertension and the
involvement of the helicase RTEL1 in cases
of severe short telomere syndrome in
humans. In 2011, the laboratory was
“Labellisé” by the Ligue Nationale Contre le
Cancer.
Figure 1. Timing of replication of telomeres in a
human primary fibroblast cell line. Left: The
percentage of replicating telomeres for every
chromosome arm detected during pulses 1+2 (early
S), 3+4 (middle S) and 5+6 (late S) is represented in
horizontal bars. The total sum of partial percentages
is normalized to 100% with horizontal lines inside
bars representing confidence intervals (c.i.)
(a=0.05). For the sake of simplicity, only upper limits
for early S and lower limits for late S are
represented. Chromosome arms are listed (left) from
the lowest to the highest mean replication timing
(right). Acrocentric chromosomes are grouped
according to the following convention: group D for
chromosomes 13, 14 and 15 and group G for
chromosomes 21 and 22. Right: Examples of early
(19q), middle (6p) and late (4q) replicating
telomeres.
I. The timing of telomere replication in
human cells
We have used a CO-FISH-based approach,
described previously by Zou et al for the
muntjac1, to determine whether replication
timing for individual human telomeres was
spatially or temporally controlled. We
showed that, in human primary cells,
telomeres located at specific chromosome
ends tend to preferentially replicate during a
defined window of the S-phase (Figure 1).
We showed that telomere length or
telomerase activity did not affect this
pattern.
We observed that telomeres located on
4qter, 10qter and the short arms of
acrocentric
chromosomes
consistently
replicated late during S-phase in all cells
examined. We examined the impact of βsatellite and D4Z4 sequences present in
these extremities. Because D4Z4-carrying
extremities had been described as being
associated with the nuclear periphery2, we
tested both the replication timing and the
nuclear localization of newly created
telomeres carrying a defined subtelomeric
composition with D4Z4 and/or β-satellite
sequences. While a single D4Z4 repeat
conferred to a chromosome extremity a
more peripheral position within the nucleus,
multiple copies of D4Z4 repeats did not. We
found that extremities carrying only one
D4Z4 repeat (and bearing a more
peripheral localization in the nucleus)
replicated later than the others. On the
other hand, telomeres connected to βsatellite sequences alone replicated later
and this effect was independent of nuclear
localization. Finally, native chromosome
ends carrying telomeres that replicated late
had a clear tendency to localize at or near
the nuclear periphery, whereas early
replicating extremities were found in the
inner part of the nuclear volume. Together,
our data showed, for the first time, a strong
association between telomere replication
timing and nuclear localization.
II. Mechanisms of telomere replication in
human cells
11
fragment spanning the two OB fold domains
in cells with long telomeres in which WRN
expression had been abolished was able to
completely rescue the replication of the Cstrand, but not that of the G-strand.
This work showed that WRN plays a much
more important role at telomeres than
previously assumed, and unveiled an
unanticipated role for POT1 during the
replication of human telomeres (Figure 2).
III.
Mechanisms
of
telomere
recombination
In immortal cells that maintain telomeres by
recombination-based
alternative
lengthening mechanisms (ALT), a special
variety of PML bodies (APBs, for ALTassociated PML bodies) is found which
contains telomeric DNA, telomere-specific
proteins and DNA recombination and repair
proteins4. We used an innovative approach
based on the properties of a mutant Herpes
simplex virus protein ICP0, which
accumulates specifically at PML bodies but
is unable to induce the degradation and
destabilization of PML and centromeres5.
The protein accumulates and enlarges
APBs, allowing a detailed visualization of
their content. Using this system, we
demonstrated (Figure 3) that the telomere
DNA associated with a single APB is mainly
if not exclusively contributed by clustered
native chromosome ends. In addition, colocalization studies readily indicated that
recombination proteins RAD51 and RPA
are specifically associated with only one
telomere in a cluster and therefore few such
foci were usually detected per nucleus.
Finally, we showed that infiltration of APBs
Figure 2. POT1 is required for C-strand replication in
the absence of WRN. In the absence of WRN,
polymerization on the lagging strand is compromised
and POT1 (blue circles) binds to the accumulated
single G-strand (solid red line). This prevents the
accumulation of RPA (orange circles) and allows the
uncoupling of the replication fork and the
polymerization on the C-strand (solid green line) by
leading mechanisms. Only sister telomeres
replicated by lagging mechanisms will be shortened.
If POT1 also becomes limiting, RPA accumulates on
the single G-strand and triggers a fork stalling, thus
preventing polymerization on the C-strand. Both
sister telomeres will be shortened.
Replication of telomeres is typically
unidirectional with the G-rich strand being
replicated by lagging mechanisms. It had
been suggested that WRN, a helicase, was
required for telomere replication in a
stochastic way3. We developed a new
approach that measures the efficiency of
telomere replication and detected, upon
WRN downregulation, a substantial loss of
G-strands affecting most extremities while
C-strands remained largely unchanged.
The accumulation of single stranded Gstrands during S/G2 indicated that the DNA
polymerization on lagging and leading
strands had been uncoupled. However, this
replication fork uncoupling did not occur in
human cells carrying abnormally long
telomeres (over 40kb). Furthermore, RPA
significantly accumulated at telomeres in
this context, suggesting that the unreplicated G-strand induced fork stalling,
thus preventing also replication on the Cstrand. We reasoned that, in the absence of
WRN, binding of POT1 to the G-strand at
the lagging telomere may both prevent
excessive RPA accumulation and allow
polymerization on the C-strand to continue.
Indeed, when combined with WRN
depletion, POT1 partial knockdowns led to
the partial loss of both G-and C-strands. On
the other hand, overexpression of a POT1
Figure 3. APBs are
platforms for telomere
recombination.
PML
bodies (top) have the
capacity
to
recruit
chromosome extremities
(bottom) exclusively in
ALT cells, and become
APBs
(ALT-associated
PML
bodies).
The
proximity of telomeres as
well as the passage of
the replication fork both
favor
the
interaction
between
telomeric
strands
on
different
chromosome extremities,
thus
promoting
recombination.
12
by ICP0 significantly increased the number
of telomere bridges connecting different
chromosome ends. Further analyses
showed that the majority of these bridges
correspond to telomeric DNA fibers in which
parental C-rich and G-rich tracts alternate
at
least
once,
suggesting
that
recombination occurred after replication.
This work revealed the potential role of
PML bodies in the formation of
recombination
centers
involving
chromosome domains in somatic cells.
accumulation of mono- ubiquitinated PCNA
(Figure 4). This accumulation is associated
with replication fork stalling, accumulation
of phosphorylated RPA and CHK1, cell
arrest in S-phase and cell death.
Interestingly,
the
RTEL1-asscociated
ubiquitination of PCNA leads to recruitment
of translesional (TLS) polymerases upon
UV irradiation, indicating that the PCNA
modification induced by truncated RTEL1 is
compatible with a replication repair
mechanism. Mass spectrometry analyses
using antibodies against RTEL1 and
extracts from RTEL1-overexpressing cells
have revealed a long list of partners, many
IV. Characterization of RTEL1
RTEL1 is a helicase first identified in the
mouse as been required to maintain very
long telomeres6. To explore the function of
RTEL1 in humans we developed a series of
specific tools including high affinity specific
antibodies,
specific
siRNAs
and
overexpressing vectors under constitutive
and inducible promoters. RTEL1 is
expressed at relatively low levels in all
human cells and is mostly located in the
nucleus. IF assays reveal that RTEL1 is
associated with the nucleolus in at least
20% of nuclei and in all phases of the cell
cycle. Importantly, RTEL1 is detected
associated with telomeres only in the APBs
of ALT cells, where the protein
concentrates when it is over-expressed. In
all other contexts, RTEL1 is never seen
associated with telomeres. However, both
knockdown and overexpression of RTEL1
have impacts on telomere metabolism
depending on the context: in telomerase
positive cells with very long telomeres,
RTEL1 KD leads to telomere shortening
while its transient overexpression leads to
increase of telomere length. In ALT cells,
overexpression of RTEL1 leads to both
accumulation of signal free ends and
decrease of telomere fragility, suggesting
roles in preventing recombination and in
favoring replication, as suggested by
observations in the mouse. However, KD of
RTEL1 in ALT cells does not affect
telomere length at least in the short term.
Instead, KD of RTEL1 in these and other
cells leads to chromosome instability. We
have found that RTEL1 interacts with
FANCD2 and FANCI and over-expression
of RTEL1 prevents the formation of
FANCD2/I complexes in response to
mitomycin C. Interestingly, over-expression
of truncated forms of RTEL1 leads to the
Figure 4. RTEL1 impacts PCNA function. A. RTEL1
has a N-terminal helicase domain and a C-terminal
domain carrying two PCNA-interacting motifs (PIP).
Two isoforms are expressed in human cells carrying
one or two PIP boxes (not shown). B. When overexpressed, the C-terminal domain carrying two PIP
boxes (Cter-i2) accumulates at replication forks
(top), leading to fork stalling and cell death in Sphase. This is not observed with the C-terminal
domain carrying only the first PIP (Cter-i1) (bottom).
C. However, both forms induce a strong
accumulation of monoubiquitylated PCNA (left
panel). This effect is lost with fragments with no PIP
box (Cter-ΔPIP), the N terminus of RTEL1 or the fulllength protein with two PIP boxes (FL-i2), indicating
that this activity is somehow restricted by the Nterminus. The levels of monoUb, polyUb and other
modifications of PCNA induced by RTEL1 Cter-i2
are much stronger than those triggered by UV
irradiation (right panel).
13
of which have been validated under
endogenous expression conditions and are
well-known actors of replication-dependent
repair pathways.
Treatment of cells with actinomycin D
(ActD), which causes the redistribution of
nucleolar proteins7, led to a 100% of RTEL1
recruitment to the nucleolus. Importantly,
this recruitment does not require XPO1, a
major RNA/protein trafficking protein. On
the contrary, KD of RTEL1 led to a
complete XPO1 delocalization from the
nucleus and prevented the recruitment of
XPO1 to the nucleolus upon ActD
treatment. Further experiments indicated a
physical interaction between RTEL1 and
XPO1 and the direct implication of RTEL1
in the RNA, but not protein, XPO1dependent export pathway, indicating that
RTEL1 plays a so far unanticipated role in
RNA metabolism. In fact, RTEL1 associates
with several ribonucleotide protein (RNP)
complexes, including telomerase RNP,
suggesting that RTEL1 is a rather wideranging component of RNP biogenesis.
Figure 5. Chromosome instability (CIN) due to
telomere shortening induces widespread miR
deregulation in HEK cells. An RNAseq approach
was undertaken to characterize the miR expression
profile of immortalized CIN+ and CIN- HEK cells
derived from the same CIN- clone. Unsupervised
clustering was carried out for both cell lines
(columns) and miRs (rows), then a comparison
between CIN+ and CIN- was performed. Of around
1000 miRs expressed in these cells, half displayed
significantly altered levels in CIN+ cells, with 16%
showing changes >4 fold. Among those, we found
several miRs involved in differentiation (like the miR200 family) and oncogenesis such as family
miR143/145).
V. Telomere instability and cancer.
Telomeric instability is considered a major
force for karyotype evolution during tumor
development. We use a well-defined model
of telomeric instability of ER-SV40tranformed human epithelial cells to explore
the impact of telomere instability on the
acquisition of tumor-related phenotypes. In
this model, cells accumulate chromosome
rearrangements because of repeated
breakage-fusion-bridge
(BFB)
cycles
initiated by telomere shortening. Cells
eventually undergo crisis during which rare
events of telomerase reactivation allows us
to
recover
immortal,
karyotypically
abnormal (CIN+) cells. We determined the
overall microRNA (miR) expression profile
during pre-crisis, crisis and post-crisis. Our
results show that this profile is completely
modified as soon as cells enter the
telomere instability phase and that this
response is directly linked to telomere
instability (Figure 5). The miR expression
changes are highly reproducible and not
explained by genomic gains or losses. The
analysis of differentially expressed miRs in
post-crisis cells revealed a significant
decrease in the expression of certain miRs
implicated in tumor suppression and the
maintenance of epithelial characteristics, in
particular the miR-200 family8. At the same
time, post-crisis cells undergo an epithelialmesenchymal transition that is reversed by
restoring miR-200 expression. Interestingly,
post-crisis cells displayed tumorigenicity
only in senescent microenvironments and
the analysis of cells explanted from these
tumors revealed that they had undergone a
mesenchymal-epithelial
transition,
had
acquired tumor formation capacity by
themselves and responded to senescent
microenvironments by forming spheres.
Taken together, our results indicate that
epithelial cells respond to telomere
instability by dynamically modulating miR
expression,
which
leads
to
transdifferentiation and acquisition of stem-like
and
tumor
behaviors,
which
are
exacerbated
in
senescent
microenvironments.
In 2010, and in collaboration with Yves
Allory, pathologist at the Hôpital Mondor,
Créteil, we initiated a research program on
cancer prostate whose principal objective is
to carry out a comprehensive molecular
analysis of cancer specimens obtained from
untreated patients at different stages of the
disease to try to understand how
14
chromosome instability affects gene
expression and how these aspects relate to
the possibility of developing an aggressive
disease.
pulmonary artery proliferate thus increasing
the resistance to the passage of blood
stream10. In collaboration with Saadia
Eddahibi (INSERM U999) du Centre
Chirurgical Marie Lannelongue (Le PlessisRobinson), we have examined the role of
telomeres and telomerase and its relation to
cell proliferation in this disease. SMCs
obtained from iPH patients present a higher
proliferation capacity in vitro than controls
and do not display overt telomere
shortening with passages while control
SMCs do. iPH-SMCs express higher levels
of TERC and TERT than controls and
TERT expression is required for resistance
to oxidative stress in iPH-SMCs while it is
not in control SMCs. Interestingly, freshly
obtained
iPH-SMCs
display
longer
telomeres than control SMCs and this
length is positively correlated with disease
severity. To directly test the role of telomere
maintenance in iPH, we applied the model
of induced PH in response to chronic
hypoxia to telomerase knockout mice.
While terc+/+ mice developed all the
histopathological and hemodynamic signs
of PH, terc-/- mice were fully protected even
at the earliest generation. Lastly, G3 terc-/;p53+/- mice were fully susceptible to the
hypoxia-induced PH, demonstrating that
short telomeres protect from the disease via
induction of mitotic senescence, thus
providing strong evidence that telomere
maintenance is absolutely required for the
development of pulmonary hypertension.
VI. Telomere biology during cell
reprogramming.
Correct maintenance of telomeres has been
shown to be an important aspect of human
cell reprogramming into pluripotent stem
(iPS) cells9. In collaboration with Annelise
Bennaceur-Griscelli (INSERM U935) at the
Stem Cell platform of Hôpital Broussais,
Villejuif, we have initiated a systematic
approach to depict different aspects of
human telomere metabolism during both
reprogramming
and
re-differentiation.
Mesenchymal stem cells (MSC) obtained
from embryonic stem cells (ES cells) are
reprogrammed into iPS cells, thus allowing
the direct comparison of telomere and
shelterin characteristics between ES and
iPS cells. Our results suggest that local
features impacting the elongation efficiency
of single telomeres by telomerase may
affect telomere-length distribution in iPS
cells, perhaps as a consequence of an
insufficient reprogramming of telomeres
and subtelomeres. In fact, preliminary ChIP
analyses suggest an enrichment of
heterochromatin marks in iPS cells with
respect to the parental ES cells. Regarding
the shelterin components, we have
discovered that TPP1, TRF1 and TRF2, on
one side, and POT1, RAP1 and TIN2, on
the other, display opposite dynamics during
reprogramming
and
differentiation.
Interestingly, RAP1 and POT1 accumulate
in the cytoplasm of MSCs. While the
cytoplasmic localization of RAP1 may be
related to its extratelomeric role, the
mislocalization of POT1 is associated to a
decrease of TPP1 levels in MSCs, which
progressively worsens when cells approach
senescence. These observations indicate
that components of shelterin are subjected
to different levels of controls, perhaps in
response to different telomeric and
extratelomeric requirements in MSCs and
iPS cells.
References
1
Zou, Y., Gryaznov, S.M., Shay, J.W., Wright,
W.E., & Cornforth, M.N., Asynchronous
replication timing of telomeres at opposite arms
of mammalian chromosomes. Proc Natl Acad
Sci U S A 101 (35), 12928-12933 (2004).
2
Masny, P.S. et al., Localization of 4q35.2 to the
nuclear periphery: is FSHD a nuclear envelope
disease? Hum Mol Genet 13 (17), 1857-1871
(2004).
3
Crabbe, L., Verdun, R.E., Haggblom, C.I., &
Karlseder, J., Defective telomere lagging strand
synthesis in cells lacking WRN helicase activity.
Science 306 (5703), 1951-1953 (2004).
4
Yeager, T.R. et al., Telomerase-negative
immortalized human cells contain a novel type
of promyelocytic leukemia (PML) body. Cancer
Res 59 (17), 4175-4179. (1999).
5
Lomonte, P. & Morency, E., Centromeric
protein CENP-B proteasomal degradation
induced by the viral protein ICP0. FEBS Lett
581 (4), 658-662 (2007).
VII. Telomere maintenance in idiopathic
pulmonary hypertension.
Idiopathic pulmonary hypertension (iPH) is
a deadly disease of unknown origin. In this
disease, smooth muscle cells (SMCs) of the
15
6
Ding, H. et al., Regulation of murine telomere
length by Rtel: an essential gene encoding a
helicase-like protein. Cell 117 (7), 873-886
(2004).
7
Andersen, J.S. et al., Directed proteomic
analysis of the human nucleolus. Curr Biol 12
(1), 1-11 (2002).
8
Spaderna, S., Brabletz, T., & Opitz, O.G., The
miR-200 family: central player for gain and loss
of the epithelial phenotype. Gastroenterology
136 (5), 1835-1837 (2009).
9
Marion, R.M. et al., Telomeres acquire
embryonic stem cell characteristics in induced
pluripotent stem cells. Cell Stem Cell 4 (2), 141154 (2009).
10
Humbert, M. et al., Endothelial cell dysfunction
and cross talk between endothelium and
smooth muscle cells in pulmonary arterial
hypertension. Vascul Pharmacol 49 (4-6), 113118 (2008).
16