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Formation of telomeric repeat-containing RNA(TERRA) foci in
highly proliferating mouse cerebellarneuronal progenitors and
medulloblastoma
Zhong Deng1, Zhuo Wang1, Chaomei Xiang2,3, Aliah Molczan2,
Valérie Baubet2,3, Jose Conejo-Garcia2,Xiaowei Xu4, Paul M.
Lieberman1,* and Nadia Dahmane2,3,*1Gene Expression and Regulation
Program, The Wistar Institute, Philadelphia, PA 19104,
USA2Molecular and Cellular Oncogenesis Program, The Wistar
Institute, Philadelphia, PA 19104, USA3Department of Neurosurgery,
University of Pennsylvania School of Medicine, Philadelphia, PA
19104, USA4Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine, Philadelphia, PA
19104, USA
*Authors for correspondence ([email protected];
[email protected])
Accepted 1 May 2012Journal of Cell Science 125, 4383–4394� 2012.
Published by The Company of Biologists Ltddoi:
10.1242/jcs.108118
SummaryTelomeres play crucial roles in the maintenance of genome
integrity and control of cellular senescence. Most eukaryotic
telomeres canbe transcribed to generate a telomeric
repeat-containing RNA (TERRA) that persists as a heterogeneous
nuclear RNA and can bedevelopmentally regulated. However, the
precise function and regulation of TERRA in normal and cancer cell
development remains
poorly understood. Here, we show that TERRA accumulates in
highly proliferating normal and cancer cells, and forms large
nuclearfoci, which are distinct from previously characterized
markers of DNA damage or replication stress. Using a mouse model
formedulloblastoma driven by chronic Sonic hedgehog (SHH)
signaling, TERRA RNA was detected in tumor, but not adjacent
normal
cells using both RNA fluorescence in situ hybridization (FISH)
and northern blotting. RNA FISH revealed the formation of TERRA
foci(TERFs) in the nuclear regions of rapidly proliferating tumor
cells. In the normal developing cerebellum, TERRA aggregates could
alsobe detected in highly proliferating zones of progenitor
neurons. SHH could enhance TERRA expression in purified granule
progenitorcells in vitro, suggesting that proliferation signals
contribute to TERRA expression in responsive tissue. TERRA foci did
not colocalize
with cH2AX foci, promyelocytic leukemia (PML) or Cajal bodies in
mouse tumor tissue. We also provide evidence that TERRA iselevated
in a variety of human cancers. These findings suggest that elevated
TERRA levels reflect a novel early form of telomereregulation
during replication stress and cancer cell evolution, and the TERRA
RNA aggregates may form a novel nuclear body in highly
proliferating mammalian cells.
Key words: Telomere repeat-containing non-coding RNA, TERRA,
Neuronal progenitors, Cancer, Cerebellum, Medulloblastoma, Sonic
hedgehog
IntroductionTelomeres are repetitive DNA structures at the ends
of linear
chromosomes required for chromosome maintenance and genome
stability (Blackburn et al., 2006; Cech, 2004). Mammalian
telomere DNA consists of variable length double stranded
TTAGGG repeats ending in a single stranded 39 overhang thatcan
form complex higher-order nucleoprotein structures to cap
chromosome ends (de Lange, 2002; de Lange, 2004). The
telomere-associated proteins, termed shelterin or telosome,
are
important for telomere end-protection and length regulation
(de
Lange, 2005a; Liu et al., 2004; Palm and de Lange, 2008). In
proliferating cells, telomere repeat length is maintained by
an
elaborate mechanism involving DNA replication machinery,
telomerase and its associated factors, and nucleolytic end-
processing enzymes (Jain and Cooper, 2010; Ye et al., 2010).
In
the absence of telomerase activity, telomeres shorten by
attrition,
and critically short telomeres elicit a DNA damage signal that
can
lead to cell cycle arrest and replicative senescence (Sahin
and
Depinho, 2010). Loss of telomere capping function leads to
cell
cycle arrest in normal senescing cells, but this protective
mechanism is bypassed in normal proliferating cells and
potentially further deregulated in human cancers (Blasco,
2005;
Blasco, 2007; De Lange, 2005b).
TERRA is a heterogeneous length non-coding RNA transcribed
through the terminal telomere repeats of eukaryotic
chromosomes
(Azzalin et al., 2007; Schoeftner and Blasco, 2008). In
fission
yeast, transcripts can be detected for both sense and
antisense
strands of the telomere repeat tract and adjacent telomere
sequence
(Bah et al., 2012; Greenwood and Cooper, 2012). TERRA RNA
can interact with shelterin proteins to regulate telomere
heterochromatin formation (Deng et al., 2009) and capping by
the telomere single strand DNA binding protein Pot 1 (Flynn et
al.,
2011; López de Silanes et al., 2010). TERRA can also interact
with
the catalytic subunit of telomerase (TERT) and inhibit
telomerase
enzyme activity in vitro (Redon et al., 2010; Schoeftner and
Blasco, 2008). TERRA RNA expression can be regulated by
several mechanisms, including developmental status, cellular
stress, and telomere epigenetic state (Azzalin et al., 2007;
Caslini et al., 2009; Deng et al., 2009; Schoeftner and
Blasco,
2008). In addition, TERRA levels are elevated in both human
and
mouse iPS cells, suggesting that TERRA expression correlates
with proliferative capacity and contributes to nuclear
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reprogramming (Marion et al., 2009; Yehezkel et al., 2011).
However, the expression and function of TERRA in the context
ofcancer remains poorly understood.
To investigate the regulation and function of TERRA innormal and
cancer cell development, we examined TERRA
expression in human cancer biopsies and in a mouse model
formedulloblastoma. Medulloblastoma is a malignancy of
thecerebellum with the highest incidence of all human pediatric
malignant brain tumors (Ellison, 2010). A subset of these
tumorsis thought to arise from deregulation of granule
neuronprogenitors (GNPs) development in the cerebellum
(Ellison,
2002; Ellison, 2010; Gilbertson, 2004; Gilbertson and
Ellison,2008). GNPs are subject to very high rates of proliferation
duringthe early postnatal period. Sonic hedgehog (SHH) is required
forthe rapid proliferation of these neuronal progenitors during
normal development (Dahmane and Ruiz i Altaba, 1999;Wallace,
1999; Wechsler-Reya and Scott, 1999). Patched 1(Ptch1) is a
receptor for the SHH ligand and an important
negative regulator of SHH signaling. Mutations in Ptch1 can
leadto medulloblastoma in human (Hahn et al., 1996; Johnson et
al.,1996) and mouse models (Goodrich et al., 1997) (reviewed by
Corcoran and Scott, 2001; Ruiz i Altaba et al., 2002). In
thiswork, we show that normal and cancer proliferating
granuleneuron progenitors express high level of TERRA and
exhibit
formation of TERRA foci. These foci (TERFs) are distinct
fromcH2AX DNA damage foci, but occur in cells where the
telomererepeat DNA has shortened. TERRA foci can also be found
inhighly proliferating progenitor cells during normal mouse
development. Finally, we show that TERRA is elevated invarious
types of human cancers originating in diverse organs.
ResultsTERRA form foci in a mouse model for medulloblastoma
To analyze the expression of TERRA in a mouse model of
humancancer, we employed Ptch1+/2 mice, a widely used genetic
model
for human SHH-positive subtype medulloblastoma (Ellison,
2010;Goodrich et al., 1997). These tumors are composed of
proliferatingGNPs marked by Math1 (also known as Atoh1) (Oliver et
al.,2005), and express Gli1, a target and mediator of the SHH
signaling
pathway (Goodrich et al., 1997) (Fig. 1A; supplementary
materialFig. S1). Tumor can be readily distinguished from
normal/non-tumor cerebellar tissue based on histology and in situ
expression
analysis of various markers (Fig. 1A; supplementary material
Fig.S1). To examine TERRA expression in mouse normal and
cancertissue, we first employed RNA fluorescence in situ
hybridization
(FISH) using methods that have been optimized for detection
ofrare and unstable RNA (Deng et al., 2009; Flynn et al., 2011).
ATAMRA-conjugated PNA probe was used under non-denaturingconditions
to selectively distinguish telomere RNA from telomere
DNA. RNA-FISH revealed that TERRA forms discrete foci(TERFs) in
the tumor cells, but not in the adjacent non-tumorcells of the same
cerebellum (Fig. 1B). TERFs were not detected in
sections pre-treated with RNase A, indicating that the
signaldetected with the TERRA probe indeed corresponds to
RNAexpression (Fig. 1B, lower panels; supplementary material
Fig.
S1B). As an additional specificity control, a FAM-conjugated
PNAprobe for antisense TERRA failed to detect any distinct
foci(supplementary material Fig. S2B). Quantification of
multiple
RNA FISH experiments using computer imaging softwareindicated
that ,80% of tumor cells have a ,7.5-fold greatermean fluorescence
intensity relative to normal cells in adjacent
non-tumor tissue (Fig. 1C–E). These findings were further
confirmed by RNA FISH using a DNA oligonucleotide
probe(TAACCC)7, which unlike the PNA probe, has very low
capacityfor binding duplex DNA. The (TAACCC)7 DNA
oligonucleotide
probe also revealed elevated TERFs in tumor cells with
nodetectable signal in the normal part of the same cerebellum(Fig.
1F). No signal for TERRA expression was observed with amutated
(TAACAC)7 version of this DNA oligo probe (Fig. 1F),
further indicating that these foci are TERRA-specific and
thatTERRA levels are selectively elevated in tumor cells.
Elevated TERRA expression in mouse medulloblastomatissue
To confirm and validate RNA FISH results with other
independenttechniques, we next examined TERRA expression by
northern blot
and quantitative (q) RT-PCR using RNA isolated from
dissectednormal (non visible tumor) and tumor regions of Ptch1+/2
cerebella(Fig. 2; supplementary material Fig. S3). In agreement
with the in
situ data (Fig. 1; supplementary material Fig. S1), northern
blotanalysis indicated that the tumor contained significantly
higherlevels (,4-fold; P50.0301) of TERRA RNA than the
non-tumorcounterpart (Fig. 2A,B; supplementary material Fig. S3).
RNase A
treatment eliminated TERRA detection, indicating that
TERRAsignal consists of RNA, and not fragmented telomere DNA(Fig.
2A, right lanes). Comparable results were observed with three
other matched non-tumor and tumor cerebella from different
mice(supplementary material Fig. S3A). Quantitative RT-PCR
analysisof TERRA expression from individual subtelomeres revealed
an
increase of TERRA expression from various chromosomes(Fig. 2C;
supplementary material Fig. S3B,C). For example, oneof the tumors
presented a large increase at 11q (,6-fold), but amore moderate
increase at 2q (1.8-fold; Fig. 2C, upper panel).Marker mRNAs for
medulloblastoma tumor identity (Math1 andGli1) and post-mitotic
neuronal marker (Map2) were used toconfirm the accuracy of
dissection (Fig. 2C, bottom panel;
supplementary material Fig. S3B,C). We also observed thatmRNA
for the telomerase subunit TERT was not increased intumor samples
(Fig. 2C, lower panel; supplementary material Fig.
S3). These results validate findings by RNA FISH, indicating
thatTERRA is elevated in mouse cancer tissue, and that some, but
notall telomeres express high TERRA levels (Fig. 2B,C).
TERRA is elevated in highly proliferating progenitor cellsin the
developing mouse brain
Transcriptional profiling analysis have shown that mouse
postnatal
(P1–P10) GNPs resemble a subtype of both mouse and
humanSHH-dependent medulloblastoma (Kho et al., 2004).
Theexpression of TERRA in primary SHH-subtype
medulloblastomasuggested that it might also be expressed in the
rapidly dividing
postnatal GNPs. To investigate whether TERRA
expressioncorrelates with normal proliferating GNPs, we first used
RNAFISH on mouse 5-day postnatal (P5) cerebellar sections; this
stage
corresponds to a peak of proliferation during the amplification
ofthe GNPs pool, and a time when GNPs respond to mitogenic SHH(Fig.
3A) (Dahmane and Ruiz i Altaba, 1999). We detected
elevated TERRA levels in the outer external germinal layer
(oEGL)containing the proliferating GNPs, while TERRA
expressiondecreases as GNPs become SHH unresponsive and postmitotic
in
the inner EGL (iEGL; Fig. 3B, top panel). In the adult
cerebellumonly very few mature granule neurons in the internal
granule layer(IGL) show low levels of TERRA expression (Fig. 3B,
lower
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panel). TERRA foci were not observed in RNase A-treated
cells,indicating that these are indeed RNA foci (data not
shown).
Northern blot and qRT-PCR analyses confirmed the RNA FISHresults
and showed that TERRA RNA levels are highest during
theproliferative expansion phases of GNPs development (P0 and
P5;
Fig. 3C–E) and then decrease as GNPs differentiate (P15 and
adult;Fig. 3C–E), in a similar manner to Gli1 and Math1 (Fig.
3F).
SHH growth factor stimulation increases TERRA levels
To determine whether the growth factor SHH contributes
toelevated TERRA expression, we assayed the effect of
recombinant
SHH on purified primary P5 mouse GNPs in vitro (Fig. 4).
Using
northern blot analysis, we found that high level of SHH
activation
(indicated by a large increase in Gli1 expression) following
GNPs
treatment with recombinant SHH, results in a moderate
(,1.3-fold) increase in bulk TERRA levels in GNP cells, as measured
by
northern blotting (Fig. 4A,B; supplementary material Fig. S4).
By
qRT-PCR analysis, we found that TERRA from several different
chromosomes increase ,2- to 3-fold (Fig. 4D). To control forSHH
activity, we show that SHH treatment of GNPs leads to ,30-fold
increase in Gli1 and ,2.5-fold increase in Math1 RNA levels,two
bona fide targets of SHH activation in these cells (Fig. 4C).
Fig. 1. TERRA foci formation in mouse medulloblastoma. (A) (Top
panel) Hematoxylin and Eosin staining of a region of a Ptch1+/2
mouse cerebellum with non-
tumor (left panel; indicated by an arrow) and tumor tissue
(right panel; no. 6850). The normal morphology of cerebellar folia
with the IGL (containing the mature granule
neurons) and the Purkinje layer containing the Purkinje neurons
is seen in left panel. Scale bar: 200 mm. (Lower panel) Sections of
cerebellum containing both normal andtumor tissue showing the
results of in situ hybridizations with specific digoxigenin-labeled
RNA probes for Gli1 and Math1. Note that Gli1 and Math1 are
highly
expressed in the tumor part of the cerebellum. Asterisk in left
panel denotes the normal expression of Gli1 in the normal Bergmann
glial cells that form a layer in this, still
organized, non-tumor part of the cerebellum. Scale bars: 100 mm.
(B) Confocal images of FISH analyses of TERRA expression in
sections of the cerebellum of the samemouse as above containing
both normal and tumor tissue. Note the presence of TERRA expression
(strong red labeling) in the tumor as compared to the low or
undetectable expression in the non-tumor cerebellar tissue.
Asterisk denote non-specific TERRA signal in the Purkinje layer.
RNase A treatment leads to an elimination
of the tumor-specific TERRA signal (middle panels). Images were
taken with 406lens (lower panels) to reveal details of TERRA
localization in the nuclei. Scale bar:20 mm. Other images were
taken with 206lens. (C) Histogram comparing relative TERRA
fluorescence signal intensity (FU) in non-tumor (black) vs tumor
(red) tissue,analyzed by RNA FISH and ImageProPlus software. (D)
TERRA expression measured as total mean fluorescence intensity
(FU). Values are means 6 standard error
from three independent experiments. (E) Quantification of cells
with §1 TERRA signals of mean intensity .40 FU. More than 800
nuclei from at least three
independent experiments were counted for quantification. The
P-value was calculated using a two-tailed Student’s t-test in all
cases. (F) Confocal images of FISH
analyses of TERRA expression in tumor (top panel) or non-tumor
(middle panel) using an Alexa-Fluor-488-conjugated DNA
oligonucleotide probe (TAACCC)7 (top
two panels) or a control mutant probe (TAACAC)7 as a negative
control (lower panel). Sections of the cerebellum of the same mouse
(no. 7040) as in supplementary
material Fig. S1 containing both non-tumor and tumor tissue were
used. Scale bar: 20 mm.
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These findings indicate that TERRA expression can be elevated
by
high level of SHH signaling in normal GNPs derived from the
developing cerebellum.
TERFs form in cells with shortened telomeres
Telomere shortening has been reported to occur in highly
proliferative normal and cancer cells (Raynaud et al., 2010;
Wentzensen et al., 2011; Xu and Blackburn, 2007). To compare
the average telomere DNA length in tumor relative to normal
cerebellar tissue, we compared the mean average fluorescence
intensity of telomere DNA FISH signals. This method can be
used to assess the average length of telomere repeat DNA
(Baerlocher et al., 2006). Computer analyzed images of FISH
signals showed that in medulloblastoma, telomere repeat
signal
intensity in tumor tissue was significantly reduced relative
to
normal tissue (supplementary material Fig. S5). These
results
indicate that TERRA foci form in tumor cells where telomere
length has shortened.
TERFs are distinct from cH2AX DNA damage foci
DNA damage foci are commonly observed in rapidly dividingand
precancerous tissue (Sedelnikova and Bonner, 2006).
Telomere dysfunction-induced foci (TIFs) are thought to
arisefrom the critical short length and/or uncapping of telomere
ends,and can be examined by colocalization of cH2AX foci
withtelomeric DNA (TelDNA) (d’Adda di Fagagna et al., 2003; Takaiet
al., 2003). To determine if TERRA expression correlates withthe
appearance of TIFs, we examined the immunofluorescence
staining pattern of cH2AX and in situ hybridization signals
forTelDNA (Fig. 5A; supplementary material Fig. S6A, Fig. S7A)or
TERRA RNA (Fig. 5B; supplementary material Fig. S6B,C,
Fig. S7B,C) in mouse medulloblastoma. Medulloblastoma
tumorsaccumulate heterogeneous staining for cH2AX (with ,2.5%
ofcells forming pan-nuclear foci, referred as large cH2AX
foci),while non-tumor sections of the adult cerebellum show
essentially no cH2AX signals. Pan-nuclear cH2AX foci havebeen
observed in certain types of DNA damage conditions,including viral
infection (Fragkos et al., 2009) and inhibition of
ATR signaling (Fragkos et al., 2009; Toledo et al., 2011).
TERFswere also detected in most tumor cells but not in normal
adjacenttissue (Fig. 5B and Fig. 1). Remarkably, TERFs did not
appear in
cells with pan-nuclear cH2AX staining (Fig. 5B;
supplementarymaterial Figs S6,S7). Quantification of at least three
independentexperiments indicated that ,2.5% of tumor cells contain
pan-nuclear cH2AX staining (Fig. 5C). This is in contrast to
the,80% of tumor cells that contain TERFs (Fig. 1E). Among the2.5%
of cH2AX positive cells, we found 80% of thesecolocalized with
strong telomere DNA FISH signals (Fig. 5D).
While small cH2AX foci could also be detected in tumorsections,
these foci did not overlap with TERRA RNA or TelDNA to a
significant extent (supplementary material Figs S6, S7,
S12). Similarly, TERFs did not overlap with smaller cH2AX
fociobserved in rapidly dividing normal neurons of
developingcerebellum in P6 mice (supplementary material Fig. S8).
Taken
together, these findings indicate that TERFs are distinct
fromTIFs and other cH2AX signals, yet form among the samepopulation
of highly proliferative normal and tumor tissue.
TERFs are nuclear foci distinct from PML and Cajal bodies
To further investigate the subcellular localization of TERFs
weexamined at higher magnification the confocal images of mouse
medulloblastoma tumors (Fig. 6A; supplementary material Fig.S9A)
or normal P5 cerebellar (Fig. 6B; supplementary materialFig. S9B)
sections after in situ hybridization with a TERRA PNA
probe. We observed that TERRA foci typically appear as one
tothree bright foci in the nuclear region of tumor tissue (Fig.
6A).Fewer and less bright foci were observed in the nucleus of
normalP5 cerebellar tissues known to be rapidly dividing (Fig. 6B).
To
determine whether TERFs colocalized with other
well-characterized nuclear structures, we performed immuno-RNA-FISH
with antibodies to either promyeolocytic leukemia (PML)
protein (Fig. 6C), coilin (Fig. 6D), or phospho-histone
H3(H3pS10; Fig. 6E). While TERRA can colocalize with PML insome ALT
cell lines (data not shown), we found only few TERFs
that colocalize with PML in mouse medulloblastoma tumor
tissue(Fig. 6C). Similarly, TERFs rarely colocalized with coilin,
whichis a marker of Cajal bodies where the telomerase holoenzyme
is
assembled (Fig. 6D). Additionally, TERFs were not observed
inH3pS10 positive cells, which is a marker for mitotic cells.
Thus,TERFs do not colocalize with several well-characterized
nuclear
Fig. 2. TERRA is highly expressed in mouse primary
medulloblastoma.
(A) Total RNA from dissected mouse medulloblastoma tumor and
non-tumor
tissue (no. 7040) were assayed by northern blot and the blot was
first probed with32P-labeled (TAACCC)4 for TERRA RNA expression.
18S RNA expression is
shown as a quantification control. Numbers on the left show the
position of RNA
markers (in kb). (B) Quantification of TERRA levels from at
least three
independent northern blot analyses using RNA isolated from
matched non-
tumor and tumor cerebella in different mice, one of which is
shown in A. Bar
graph represents TERRA signal intensity relative to 18S signal,
and relative
intensity for non-tumor cerebella was set at 100. The P-value
was calculated
using a two-tailed Student’s t-test. (C) Top panel: quantitative
RT-PCR analysis
of TERRA using primers specific for subtelomeres of chromosome
2q, 11q, 5q
and telocentric chromosomes (Telocen). Lower panel: expression
of Map2, Tert,
Gli1 and Math1 in non-tumor and tumor cerebellum (no. 7040) to
validate the
accuracy of dissection. DDCT method relative to non-T cerebellum
and Gapdh
were used to calculate relative RT-PCR between non-tumor and
tumor
cerebellum. Bar graph represents the average value from at least
three
independent PCR reactions (means 6 s.d.).
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structures, and may represent a unique nuclear structure
that
forms during the interphase of rapidly dividing cells.
TERRA expression is elevated in many different humancancer
types
To explore whether TERRA levels are elevated in human
cancers, we first examined RNA derived from human ovarian
tissue diagnosed as either normal, advanced primary tumor,
or
metastatic tumor (Fig. 7A–D). Northern blot analysis
indicated
that most of the ovarian primary and metastatic tumors
expressed
higher levels of TERRA than otherwise normal ovarian tissue
(Fig. 7A,B). Chromosome-specific qRT-PCR analysis also
confirmed that primary and metastatic tumors had higher
levels
of TERRA at most, but not all subtelomeres tested relative to
the
normal tissue (Fig. 7C,D). In one sample derived from normal
ovarian tissue, we observed very high levels of TERRA by
northern blotting (supplementary material Fig. S10A).
However,
upon further molecular characterization of these tissues,
this
sample (HR406) was found to express levels of the
proliferation
marker Ki67 that were .50-fold relative to two other
normalovarian tissue, and 3- to 5-fold greater than advanced
primary or
metastatic tumors (supplementary material Fig. S10B),
indicating
that this ovarian tissue was highly proliferative. This
further
supports the correlation between cell proliferation state
and
elevated TERRA levels. To further investigate whether TERRA
was elevated in other human cancer tissues, we compared
TERRA expression in primary human tumors and matched
normal tissue controls derived from various cancer biopsies,
including those derived from stomach, lung and colon (Fig.
7E–
H). Northern blot analyses showed that TERRA is expressed at
higher levels in tumor-derived tissues relative to matched
control
tissues (Fig. 7E; supplementary material Fig. S10A).
Antisense
TERRA was not detected when the same northern blot was
stripped and reprobed with G-rich probe, indicating that the
telomere RNA species is strand specific (Fig. 7E, right
panel;
supplementary material Fig. S11). TERRA expression was also
analyzed by chromosome-specific qRT-PCR for several
different
telomeres (Fig. 7F–H). The cell proliferation marker Ki67
was
used as a control for tumor dissection, and was elevated ,6-
to100-fold relative to normal matched tissue RNA. Consistent
with
northern blotting data, we found that TERRA RNA was elevated
in tumor tissue relative to matched control tissue for most
subtelomeres tested. However, only a few subtelomeres showed
significantly (.4-fold) higher TERRA levels in tumor
tissuerelative to normal, and no particular subtelomere showed
elevated TERRA consistently for all the tumor samples. For
example, only telomere 13q expressed high (.4-fold) TERRAlevels
in one stomach cancer biopsy, while 17q and 2q were
elevated in lung, and 10q and 17q elevated in a colon
carcinoma
biopsy. No one subtelomere was consistently elevated for
Fig. 3. TERRA is elevated in highly proliferating
progenitor cells. (A) Schematic representation of the mouse
cerebellar cortex during the first postnatal week. Granule
neurons, their progenitors (GNPs) and Purkinje neurons
(red) are shown. Proliferation of GNPs occurs in the outer
external germinal layer (oEGL). Postmitotic GNPs
accumulate in the inner external germinal layer (iEGL) and
migrate to the internal granular layer (IGL) through the
Purkinje layer. Cycling GNPs in the oEGL respond to SHH
secreted form Purkinje neurons. (B) RNA FISH analysis of
TERRA expression on wild-type mouse P5 and adult
cerebella sagittal sections. The iEGL and outer oEGL are
indicated. Note the expression of TERRA in the P5 EGL,
with a stronger expression in the oEGL where most cells are
cycling. All nuclei are counterstained with DAPI (blue).
TERRA expression is low in mature granule neurons in the
adult IGL. Scale bar: 20 mm. (C) Northern blot analysis ofTERRA
levels in normal mouse cerebellum at various stages
of development. 18S RNA expression is shown as
quantification control. Numbers on the left show the
position of markers in kb. Highest TERRA levels are
observed at the highest peak of GNPs proliferation at P5.
Levels of TERRA are downregulated as GNPs start their
differentiation program. (D) Quantification of northern blot
analyses for TERRA levels for each cerebellar stages from
three independent experiments, one of which is shown in C.
(E) Quantitative RT-PCR of TERRA RNA from different
cerebellar stages using TERRA-specific primers for
subtelomeres of chromosomes 2q, 11q, and telocentric
chromosomes (TeloCen). DDCT methods relative to P0
cerebellum and Gapdh were used to calculate relative RT-
PCR between different cerebellar stages. Bar graph
represents the average value from two independent
experiments and at least three independent PCR reactions
(means 6 s.d.). (F) Quantitative RT-PCR analysis of Gli1
and Math1 expression in the developing and adult
cerebellum as described in E.
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TERRA expression, even when tumors were derived from the
same organ (data not shown). These results indicate that
TERRA
expression is elevated in various human cancer tissues, and
can
be expressed from a few chromosomes, which may vary among
cell and cancer types.
DiscussionWe show here that in mammalian cells in vivo,
TERRA
expression is linked to the proliferative and tumorigenic
state
of the cell. We found that TERRA levels were elevated in
highly
proliferating normal and cancer cells in both mouse tissue
sections and human cancer biopsies. Using a well-established
mouse model of brain cancer (i.e. medulloblastoma), we found
that TERRA RNA was elevated in tumor, but not in normal
cerebellar tissue (Figs 1, 2; supplementary material Figs
S1–S3).
RNA FISH revealed pronounced TERRA foci, termed TERFs, in
cells overexpressing TERRA RNA. TERFs were sensitive to
RNase A and not detected with control probes containing a
mutation in the telomere repeat sequence, indicating that
these
were indeed TERRA-containing foci. Furthermore, TERFs
appeared only in tissues where high level TERRA expression
was validated by northern blotting and qRT-PCR (Fig. 2;
supplementary material Fig. S3). TERFs generally formed as
one or few major foci in the nuclear region of proliferating
cells.
TERRA and TERFs were also elevated in rapidly dividingprogenitor
cells of the developing cerebellum, indicating that
normal proliferating cells can also form TERFs (Fig. 3).
TERRAlevels were also enhanced by addition of recombinant SHHgrowth
factor to purified progenitor cells in vitro, suggesting thatstrong
and chronic growth factor stimulation promotes TERRA
accumulation (Fig. 4; supplementary material Fig. S4).
TERFsformed in the same cell populations where cH2AX DNA damagefoci
form (Fig. 5; supplementary material Figs S6, S7, S12) and
telomere DNA shortening has occurred (supplementary materialFig.
S5), but TERFs did not colocalize with cH2AX foci (Fig. 5),nor with
PML or Cajal nuclear bodies (Fig. 6). Finally, we
provide evidence that TERRA is expressed at higher levels inmany
human cancer tissues relative to matched controls (Fig.
7;supplementary material Fig. S10). These findings support themodel
that TERRA expression is coupled to cellular proliferation
and that TERFs are a novel indicator of proliferative
stress.
Our results are different from several previous reports
thatdescribed either a decrease in TERRA expression between
normal and colon cancer (Schoeftner and Blasco, 2008),diminished
TERRA in advanced astrocytoma (Sampl et al.,2012) or a lack of
expression in cancer cell lines (Ng et al., 2009;
Zhang et al., 2009). The differences may reside in the use
oftumor tissue compared to cancer cell lines (Ng et al., 2009;
Zhanget al., 2009) as well as in the techniques used to detect
TERRAexpression [a comprehensive use of RNA FISH, northern
blotting
and qRT-PCR in our study as opposed to dot blotting inSchoeftner
and Blasco or quantitative RT-PCR from fewchromosomes (supplemental
material Figs. S13-15) (Schoeftner
and Blasco, 2008; Sampl et al., 2012)]. It is also likely
thatTERRA expression in human cancers may be regulated in a
morecomplex manner corresponding to the patient history, cancer
type, and tumor tissue integrity. In our experiments, human
tumorRNA was purified from freshly isolated unfixed biopsies to
avoidRNA degradation. Our use of a mouse cancer model has also
enabled us to examine adjacent non-tumor and tumor tissue onthe
same section. In mouse model of medulloblastoma, we wereable to
utilize methods developed for RNA FISH that reduce thetime of
fixation and denaturation that commonly degrade or
modify RNA. Together, these methods have allowed us toobserve
TERRA expression and localization in tissue sectionsand isolates
that better preserve the in vivo physiology of cellular
proliferation and cancer.
The function and regulation of TERRA has been explored inseveral
previous studies (Azzalin et al., 2007; Deng et al., 2009;
Redon et al., 2010; Schoeftner and Blasco, 2008). We
havepreviously shown that TERRA can protect telomere ends fromDNA
damage signaling by promoting telomeric heterochromatinformation
(Deng et al., 2009). TERRA has also been shown to
inhibit telomerase activity in vitro (Redon et al., 2010;
Schoeftnerand Blasco, 2008). TERRA levels can be elevated in cell
lineslacking DNA methyltransferase 3b (DNMT3b) (Yehezkel et
al.,
2008), as well as in response to developmental and stress
signals(Schoeftner and Blasco, 2008). Our data suggests that
TERRAexpression can be enhanced by growth factor signaling
(e.g.
SHH) and cell proliferation in vivo. Moreover,
chronicproliferation signals associated with carcinogenic
mutations(like Ptch1+/2) lead to elevated TERRA and TERF
formation.
We also observed that telomere DNA signal
decreases(supplementary material Fig. S5) and that TERT mRNA
levelsare not elevated (Fig. 2; supplementary material Fig. S3)
in
Fig. 4. TERRA expression can be induced in purified progenitor
cells
stimulated with high SHH signaling activation in vitro. (A) GNPs
were
purified from wild-type mouse P5 cerebella and cultured for 12
hours with or
without SHH (600 ng/ml). TERRA RNA was analyzed by northern
blotting
using a 32P-labeled (TAACCC)4 oligonucleotide probe. 18S RNA
expression
is shown as an internal control for RNA loading. RNase A
treatment is shown
in the right panel. (B) Quantification of TERRA levels from at
least three
independent northern blot analyses using RNA isolated from GNPs
treated
with SHH for 12 hours or left untreated, one of which is shown
in A. Bar
graph represents TERRA signal intensity relative to 18S signal,
and relative
intensity for SHH (2) was set at 100. The P-value was calculated
using a two-
tailed Student’s t-test. (C) qRT-PCR analysis for expression of
Gli1 and
Math1 is shown as a control for SHH activity. DDCT methods
relative to
untreated GNPs and Gapdh were used to calculate relative RT-PCR
by SHH
treatment. Bar graph represents mean 6 standard deviations from
three
independent experiments. (D) qRT-PCR analysis of individual
TERRA
expression using subtelomere-specific primers for chromosomes
2q, 11q,
TeloCen, 18q or 5q as described in C.
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tumor tissue. We propose that TERRA is required for telomere
end processing and protection during normal cell
proliferation,
and that elevated TERRA and TERF protects telomeres from
eliciting a DNA damage response during conditions of
proliferative stress (Fig. 8).
cH2AX-positive DNA damage foci have been shown to beelevated in
highly proliferating cells and precancerous lesions
(Bartkova et al., 2005; Gorgoulis et al., 2005; Jackson and
Bartek,
2009). We found that small cH2AX foci accumulate in
mousemedulloblastoma, with ,2.5% of the tumor cells containing
pan-nuclear cH2AX foci (Fig. 5). In contrast, ,80% of these
cellswere TERF positive. Importantly, most cH2AX foci did
notoverlap with TERFs, but did colocalize with telomere DNA
foci
(TIFs). Others have shown that telomere DNA forms aggregates
in highly proliferating cancer cells (Mai and Garini, 2006).
Unfortunately, we were unable to colocalize TERFs with
telomere
DNA signals, so can not exclude the possibility that TERFs
are
expressed at telomere aggregates in highly proliferating cells.
We
did find that telomeres were generally shorter in mouse
cancer
cells that express TERRA and form TERFs (supplementary
material Fig. S5), suggesting that short telomeres may
promote
TERRA transcription activation. Elevated TERRA expression in
highly proliferating cancer and normal progenitor cells may
stabilize these abnormally short telomeres, perhaps through
inhibition of the DNA damage response. This is consistent
with
the observed lack of DNA damage associated cH2AX
focicolocalizing with TERFs (Fig. 5; supplementary material
Figs
S6 and S7). TERRA may prevent DNA damage response at short
telomeres by stabilizing the shelterin complex,
heterochromatin
formation, or protection of single stranded telomeric DNA
(Deng
et al., 2009; Flynn et al., 2011).
TERRA foci and telomeric transcript accumulations (Tacs)
have
been described previously (Azzalin et al., 2007; Marion et
al.,
2009; Schoeftner and Blasco, 2008; Zhang et al., 2009). In
human
cell lines, TERRA can colocalizes to metaphase telomeres and
form variable numbered foci that can be upregulated by
depleting
components of the nonsense-mediated decay (NMD) pathway
(Azzalin et al., 2007). In undifferentiated mouse ES cells,
Tacs
associate with both sex chromosomes, but upon ES cell
differentiation, Tacs relocalize to the inactive X-chromosome
in
females or the Y-chromosome in males (Schoeftner and Blasco,
2008). In telomerase-deficient TRF2-overexpressing (K5TRF2/
TERC2/2) mouse models of telomere dysfunction, Tacs
delocalize
from the inactive X-chromosome, reflecting a novel form of
telomere dysfunction (Schoeftner et al., 2009). While we did
not
directly examine the localization of TERFs with respect to
the
inactive X chromosome, the TERFs that we observe in rapidly
proliferating cells are likely to be very similar to Tacs.
However,
since Tacs have not been reported to be elevated in rapidly
proliferating neuronal progenitor or medulloblastoma, we
refrain
from concluding that these TERFs and Tacs are identical
structures.
RNA mediated nuclear body formation has been observed for a
variety of other RNA species (Shevtsov and Dundr, 2011).
Fig. 5. Tumor-associated TERRA foci do not
colocalize with cH2AX. (A) Confocal microscopy
images of immunofluorescence of cH2AX foci
(green) combined with DNA FISH for telomere
repeat DNA (TelDNA; red) in non-tumor (non-T) or
tumor sections. DAPI stain is in blue. Arrowheads
indicate large cH2AX foci. Arrows indicate nuclei
containing intense telomere DNA signals
colocalizing with large cH2AX foci. Enlarged (56)images of the
regions in the yellow boxes are shown
in the right panels. (B) Confocal microscopy images
of immunofluorescence of cH2AX foci (green)
combined with RNA FISH for TERRA (red) in non-
tumor or tumor sections of the cerebellum.
Arrowheads indicate large cH2AX foci. Enlarged
(56) images of the regions in the yellow boxes areshown in the
right panels. (C) Quantification of cells
containing large cH2AX foci. More than 1000 nuclei
from at least three independent experiments were
counted for quantification. The P-value was
calculated using a two-tailed Student’s t-test.
(D) Quantification of large cH2AX foci that
colocalize with intense telomere DNA FISH signals
to form TIFs. More than 1000 nuclei from at least
three independent experiments were counted for
quantification. The P-value was calculated using a
two-tailed Student’s t-test.
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Transcription of the tandem arrays of satellite III (sat III)
repetitive
DNA is known to form nuclear stress bodies located at
pericentromeric DNA (Biamonti and Vourc’h, 2010). Recent
genome-wide sequencing studies have found that satellite
repeat
transcripts were aberrantly overexpressed in various human
and
mouse epithelial cancers (Ting et al., 2011). While, Sat III
repeat
DNA and RNA have some intriguing similarities with TERRA
DNA and RNA, we did not observe that TERFs had similar
patterns
of granulation commonly seen with SatIII nuclear stress bodies
in
tissue culture cell lines. However, it will be important to
determine
if TERF positive tumor tissue is also enriched in Sat III
nuclear
bodies, and whether these structures share common features
with
TERFs. TERFs may also share features with other
RNA-nucleated
bodies, including the miRNA processing P-bodies (Parker and
Sheth, 2007) and cytoprotective aggresomes (Wileman, 2007).
In
some cancer cells, telomere DNA has been shown to aggregate
in
conjunction with nuclear lamina (Mai and Garini, 2006; Raz et
al.,
2008). In contrast to TERFs, telomere aggregates colocalize
with
cH2AX DNA damage foci, suggesting that these are distinctnuclear
structures (Raz et al., 2008). Nevertheless, it is possible
that
TERFs reflect a cluster of several highly transcribed telomeres
that
correspond to the telomere aggregates observed in some
cancer
types. TERFs did not show significant colocalization with
other
well-characterized nuclear structures, including
coilin-containing
Cajal bodies or PML-containing ND10 bodies (Fig. 6). Thus,
TERFs appear to represent a unique nuclear body formed by
TERRA accumulation from one or more telomeres in highly
proliferating cells.
An important unanswered question is how TERRA expression,
processing, and localization may differ in normal
differentiated,
normal proliferating, and abnormal cancer cells. TERRA
levels
and TERFs can be detected in both proliferating cell types,
although we did observe greater TERRA processing to several
smaller RNA species (Fig. 7; supplementary material Fig.
S10)
and an average greater density of TERFs in cancer tissue
relative
to progenitor cells (Figs 2,6). It may not be surprising
that
progenitor cells have similar TERRA expression patterns as
cancer cells, since they are known to share common gene
expression profile as in the case of medulloblastoma (Kho et
al.,
2004). We suggest that the major difference between cancer
and
Fig. 6. TERFs are nuclear, interphase-associated foci
distinct from PML and Cajal bodies. (A) Confocal
microscopy images of TERRA RNA FISH on tissue
sections derived from mouse medulloblastoma (no. 6850).
(B) Same as in A, except in normal cerebellar tissue
derived from a stage P5 mouse. (C) TERRA RNA FISH
(red) combined with immunofluorescence with antibody
to PML (green) on tissue sections derived from mouse
medulloblastoma (no. 7171). All nuclei were
counterstained with DAPI (blue). Enlarged (zoomed)
merged images are shown in the right panels. (D) Same as
in C, except TERRA RNA FISH (red) combined with
coilin immunofluorescence (green).
(E) Same as in C, except TERRA RNA FISH (red)
combined with immunofluorescence with antibody to
phosphorylated histone H3S10 (green).
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Fig. 7. TERRA is overexpressed in human tumor tissues. (A)
Northern blot analysis of TERRA RNA isolated from normal ovarian
tissue, advanced primary ovarian
cancer and metastatic ovarian cancer tissue. Numbers at the
bottom show the average value of TERRA signals relative to 18S RNA
signals in tumor versus normal tissues
from two independent northern blots. Asterisks indicate that the
sample is not included in the analysis because of the degradation
of the 18S signal. (B) Quantification of
average TERRA levels from many northern blot analyses using RNA
isolated from tissues derived from two normal ovaries (540071 and
709152), eight primary and nine
metastatic ovarian cancers, a representative of which is shown
in A. Bar graph represents TERRA signal intensity relative to the
18S signal, and average relative intensity
for two normal ovary tissues was set at 100. (C) qRT-PCR
analysis of Ki-67 expression and TERRA levels in the indicated
ovarian cancers and normal ovary tissue
(709152). TERRA levels were assayed using primers specific for
TERRA RNA transcribed from subtelomeres of human chromosome 2q,
10q, 13q, as indicated. DDCT
methods relative to normal ovary and Gapdh were used to
calculate relative RT-PCR between normal and tumor samples. Bar
graph represents the average value from
three independent PCR reactions (means 6 s.d.). (D) The same as
in C, except using primers specific for TERRA RNA transcribed from
subtelomeres of human
chromosome XqYq, 15q, 16p, as indicated. (E) TERRA expression in
dissected primary human tumor tissue from carcinoma of the stomach,
lung and colon (normal and
tumor) analyzed by northern blotting. 18S RNA expression is
shown as quantification control. Equal intensity of 18S* signals
indicate that RNA from each sample were
subject to similar levels of degradation during the preparation
process. The same northern blot was stripped, and reprobed with
32P-labeled (TTAGGG)4 probe for anti-
sense TERRA (right panel). Numbers on the left show the position
of markers (in kb). Numbers at the bottom show the value of TERRA
signals relative to the 18S RNA
signals in tumor versus normal tissues. (F) qRT-PCR analysis of
Ki-67 expression (left panel) and TERRA levels (right panel) in the
tumor and patient matched control
tissues from stomach. TERRA levels were assayed using primers
specific for TERRA RNA transcribed from subtelomeres of human
chromosome 2q, 17q, 10q, 13q, 15q,
16q and XqYq, as indicated. Bar graph represents the average
value from at least three independent PCR reactions (means 6 s.d.).
(G) The same as in F, except in the
tumor and patient matched control tissues from lung. (H) The
same as in F, except in the tumor and patient matched control
tissues from the colon.
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progenitor cells is the abnormal prolonged exposure to
proliferation signals and the potential stabilization of
shortened
and damaged telomeres through the action of TERRA. We also
observed TERF expression is highly elevated in human cancer
cell lines with ALT elongated telomeres, as observed in U2OS
cells (supplementary material Fig. S15). It is therefore
possible
that TERRA expression in mouse medulloblastoma correlates
with the ALT phenotype, but additional experiments are
needed
to confirm this possibility. While the precise function,
processing, and localization of TERFs remains unknown, these
details will be essential for understanding the regulation
of
telomeres during normal and cancer cell proliferation. In
summary, our data demonstrates that TERRA accumulates and
forms TERFs in highly proliferating progenitors and cancer
tissue, and suggests that TERFs may provide a novel and
sensitive biomarker for telomere dysfunction in cancer.
Materials and MethodsMouse breeding
Medulloblastoma used in our study formed in the Ptch1+/2 colony
either alone orin combination with p53+/2. The tumor 6850 (Fig. 1A)
originated in aPtch1+/2;RP58fl/fl mouse obtained during the
breeding between Ptch1+/2 andRP58fl/fl;nestinCreER. The RP58fl/fl
does not present any abnormality (Xiang et al.,2012).
All procedures were approved by the Wistar Institute
Institutional Animal Careand Use Committee.
Telomeric RNA FISH on tissue section
Tissue sections (7–12 mm) were prepared as described previously
(Fernandez et al.,2010). Fresh frozen sections were fixed in 4%
paraformaldehyde (PFA) for 10minutes on ice, washed twice with cold
PBS, and twice with RIPA (150 mM NaCl,50 mM Tris-HCl, pH 8.0, 1%
NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1 mMEDTA) buffer for 10
mins each. The sections were fixed again in 4% PFA for 10mins at 25
C̊, washed three times with cold PBS, and acetylated in acetylation
bufferfor 10 mins at 25 C̊. After acetylation, the sections were
washed three times withcold PBS containing 0.05% Tween-20 (PBST),
and prehybridized in hybridizationbuffer (50% formamide, 56SSC,
56Denhardts, 25 mg/ml yeast RNA, 0.5 mg/mlsalmon sperm DNA) for 1
hr at 37 C̊. RNase A treatment was performed in PBSTwith 100 mg/ml
RNase A for 30-60 mins at 37 C̊ before
prehybridization.Hybridization was performed overnight at 37 C̊,
and the following probes were used
in the hybridization: a Tamra-(CCCTAA)3 PNA probe (Panagene
Inc.) or an Alexa-Fluor-488-(TAACCC)7 oligonucleotide probe (IDT)
for TERRA RNA, a Fam-(TTAGGG)3 PNA probe for TERRA antisense, and
an Alexa-Fluor-488-(TAACAC)7 probe as a control for specificity.
After hybridization, slides werewashed as follows: 26SSC, 50%
formamide, three times at 39 C̊ for 5 mins; 26SSC, three times at
39 C̊ for 5 mins each; 16SSC, 10 mins at room temperature; 46SSC
once at room temperature. Slides were counterstained with 0.1 g/ml
DAPI in 46SSC, 0.1% Tween-20, washed in 46SSC, and mounted with
mounting media.Images for H/E staining were taken with a Nikon E600
Upright microscope (NikonInstruments) with ImageProPlus software
(media Cybemetrics) and AdobePhotoShop CS5 for image processing.
Confocal images were taken with ZeissLSM510META NLO laser scanning
confocal system on a Zeiss Axiovert 200Minverted microscope with
Zeiss AIM Ver.4.0 software and Adobe PhotoShop CS5for image
processing. For quantification purpose, the same technical settings
wereapplied to the capture of all images (Ferlicot et al., 2003;
Ourliac-Garnier andLondono-Vallejo, 2011). The mean fluorescence
intensity of TERRA spots (FI/spot)from unmodified black and white
images was automatically quantified byImageProPlus software and
expressed in fluorescence units (FU). The data wasexported into an
Excel worksheet for frequency and statistical analysis.
P-valueswere calculated using two-tailed Student’s t-tests.
RNA preparation and analysis
Total RNA for normal ovarian tissue samples was purchased from
Biochain(R1234183, lot no. 709152), Agilent Technologies (540071),
and Zyagen(HR406). Total RNA (540045) for normal human breast was
purchased fromAgilent Technologies. All other human tissues were
collected according to theguidelines and policies of the Hospital
of the University of PennsylvaniaInstitutional Review Board. Stage
III–IV human ovarian carcinoma specimenswere procured through
Research Pathology Services at Dartmouth–HitchcockMedical Center
under institutional approval (CPHS17702). For ovarian
cancersamples, primary means from the initial mass in the ovary and
metastatic meansfrom anywhere else in the peritoneal cavity. Total
RNA was purified with Trizolreagent (Invitrogen) as manufacturer’s
instructions. The RNA samples weretreated with DNase I for 45 min
at 37 C̊, followed by DNase I inactivation in thepresence of EDTA
at 65 C̊ for 5 min prior to further analysis. For northern
blotting,about 3–7.5 mg of total RNA were used. Hybridizations were
performed in Churchbuffer (0.5N Na-phosphate, pH 7.2, 7% SDS, 1mM
EDTA, 1% BSA) for 16-18 hrsat 50 C̊. The membrane was washed twice
in 0.2N Na-phosphate, 2% SDS, 1 mMEDTA at room temperature, once in
0.1N Na-phosphate, 2% SDS, 1 mM EDTA at50 C̊, and analyzed by
phosphor-imager (Amersham Biosciences). The blots werefirst
hybridized with a 32P-labeled (TAACCC)4 probe, then stripped, and
probedwith either a 32P-labeled (TTAGGG)4 or 18S probe. When
indicated, RNAsamples were treated with RNase A (Roche) at a final
concentration of 100 mg/mlfor 30–60 mins at 37 C̊. Images were
processed with a Typhoon 9410 Imager (GEHealthcare) and quantified
with ImageQuant 5.2 software (Molecular Dynamics).TERRA RNA levels
were calculated as percentages relative to signals from
controlsamples and 18S internal control. Reverse transcriptions
were performed withSuperScript III (Invitrogen) using 1 mg of total
RNA as per the manufacturer’sinstructions. A specific primer
(CCCTAA)6 was used to obtain TERRA cDNA at55 C̊. Real-time PCR
experiments were performed as described (Deng et al.,2009).
Relative RT-PCR was determined using DDCT methods relative to
controlsamples and internal control Actin and Gapdh for human
samples or Gapdh formouse samples. Primer sequences used for
real-time PCR are listed insupplementary material Table S1.
Telomere DNA FISH
Tissue sections were prepared essentially as described in RNA
FISH methodsection. Telomeric DNA FISH was performed as followings.
After acetylation,sections were subject to RNase A treatment by the
incubation in PBST with100 mg/ml RNase A for 30–60 mins at 37 C̊.
Sections were washed three times inPBS, and dehydrated in cold
ethanol series 5 mins for each (70%, 95%, 100%).Slides with
sections were denatured on a 80–85 C̊ hot plate for 5 mins in
thepresence of about 120 ml of hybridization mix (70% formamide, 10
mM Tris-HCl,pH 7.2, 0.5% blocking reagent) containing telomeric
PNA-Tamra-(CCCTAA)3probe, and hybridization was performed in the
dark for overnight at roomtemperature. The slides were washed two
times for 15 mins each in 70%formamide, 10 mM Tris-HCl, pH 7.2,
0.1% BSA followed by three washes of 5mins each in 0.1 M Tris-HCl,
pH 7.2, 0.15 M NaCl, 0.08% Tween-20, stainedwith 0.1 mg/ml DAPI,
and mounted in mounting medium. Confocal images weretaken with
Zeiss LSM510META NLO laser scanning confocal system on a
ZeissAxiovert 200M inverted microscope with Zeiss AIM Ver.4.0
software and AdobePhotoShop CS5 for image processing. Image capture
and quantification processwere performed using the same methods as
those used for RNA FISH.
For combined cH2AX staining and FISH analysis, we performed RNA
or DNAFISH as described above. After washes, the sections were
fixed again in 4%paraformaldehyde for 15 mins, blocked in blocking
solution (1 mg/ml BSA, 3%goat serum, 0.1% Triton X-100, 1 mM EDTA
in PBS) for at least 30 mins, andincubated with cH2AX monoclonal
antibody (Upstate) diluted in blocking solution
Fig. 8. TERRA foci (TERF) formation during
growth-factor-induced
proliferation in progenitor and cancer cells. Normal progenitor
cells
subjected to high level of proliferation (such as granule neuron
progenitors in
the mammalian postnatal cerebellum) through the activity of
growth factors
(such as SHH) express high level of TERRA RNA (red dots).
Elevated
chronic growth factor signaling, as occurs in the Ptch1+/2 model
of
medulloblastoma, results in high TERRA expression and
stabilization of
shortened telomeres in tumor cells. TERFs form at high level in
early stage
cancer cells in the absence of cH2AX DNA damage foci.
Stabilization of
short telomeres is predicted to promote cancer cell
evolution.
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(1:100) for 1 hr at room temperature. The sections were washed
three times withPBST for 5 mins each, and incubated with
Alexa-Fluor-488-conjugated secondaryantibody (2 mg/ml stock
solution diluted 1:600 in PBST) in blocking solution for30 mins.
The sections were further washed with PBST, counter stained with
DAPI,and mounted in mounting medium before confocal microscope.
Rabbit polyclonalantibodies to Coilin (H-300) and phospho-histone
H3 (Ser10) were purchased fromSanta Cruz and Millipore,
respectively. Monoclonal antibody to mouse PML was agift from Olga
Vladimirova at the Wistar Institute.
Granule neuron progenitor purification
Granule neuron progenitors (GNPs) were purified from P5 mouse
cerebella asdescribed previously (Fernandez et al., 2010). SHH (600
ng/ml, R&D) was addedto the media for 12 hours.
AcknowledgementsWe would like to thank Federico Valdivieso,
Michael Feldman, theTumor tissue bank and the Abramson Cancer
Center for the tumorsamples. We also thank the Department of
Pathology at DartmouthUniversity for ovarian cancer tissue RNA, and
Fred Keeney andJames Hayden in the Wistar Institute Microscopy
Core.
FundingThis work was supported by the Wistar Institute Cancer
Center CoreGrant [grant number P30 CA10815]; the American Cancer
Society[grant number RSG-08-045-01-DDC to N.D.]; the National
BrainTumor Society to N.D., National Institutes of Health [grant
numberRO1CA140652 to P.M.L.); and a Scientist Development grant
fromthe American Heart Association [grant number 11SDG5330017
toZ.D.]. Deposited in PMC for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108118/-/DC1
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/CreateJDFFile false /SyntheticBoldness 1.000000 /Description
>>> setdistillerparams> setpagedevice