Current Biology 18, 183–187, February 12, 2008 ª2008 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2007.12.059
Telomeres Acquire Distinct Heterochromatin
Characteristics during siRNA-Induced
RNA Interference in Mouse Cells
Cecilia Yuen Sze Ho,1John Patrick Murnane,2
Ava Kit Ying Yeung,1Ho Keung Ng,1
and Anthony Wing Ip Lo1,*
1Department of Anatomical and Cellular Pathology
The Chinese University of Hong Kong
Prince of Wales Hospital
Shatin, New Territories
Hong Kong SAR
The People’s Republic of China
2Department of Radiation Oncology
University of California
San Francisco, California 94103
ear chromosomes and consist of simple repeating-DNA
sequences and specialized proteins [1, 2]. Integrity of the
telomeres is important in maintaining genome stability
RNA (21–23 nucleotides long), termed short interference RNA
(siRNA), resulting in the downregulation of genes with cog-
nate sequences [7–9]. During transient siRNA-induced RNAi
in mouse fibroblast cultures, we found significant reversible
heterochromatin features. There were increased bindings
of Argonaute-1 (AGO1), telomeric repeat-binding factor 1
(TERF1), and heterochromatin protein 1b (HP1b) on the
telomeres. Histone H3 (lysine 9) was hypermethylated at the
telomeres. The chromosome ends also were associated
gene inserted adjacent to the telomere was downregulated.
In addition, the concentration of a group of heterogeneous
high-molecular-weight RNA containing telomeric repeat se-
of transient, discrete nuclear foci. Our findings suggest that
telomeres participate actively in the siRNA-induced RNAi
process. These responses of telomeres to the RNAi process
might partially account for the off-target effects of RNAi.
Results and Discussion
Heterochromatin Was Formed at the Telomeres
during siRNA-Induced RNAi
The chromatin of telomeres was examined with chromatin im-
munoprecipitation assays in transient transfection of siRNA
against green fluorescent protein (GFP-siRNA) in the mouse
tem to investigate the effect of siRNA in telomere biology. This
mouse cell line does not contain GFP genes or homologous
sequences in the genome. The amount of telomeric repeat
sequences coprecipitated with antibodies against different
DNA-binding proteins was measured with quantitative PCR
. After transient transfection of GFP-siRNA, the binding of
3-phosphate dehydrogenase (Gapdh) or b-actin (Figure 1A).
AGO1 is an important component of the RNAi machinery
[7, 8, 11–13]. The bindings of AGO2 and RNA polymerase II
(RPII) showed only minor changes after GFP-siRNA transfec-
tion. These results suggested that the protein of the RNAi
machinery was associated with the telomeres during siRNA-
Consistent withtheassociation oftheRNAimachinerytothe
telomeres during transient transfection of GFP-siRNA, corre-
sponding changes in the chemical modifications of histones
also were detected. The amount of trimethylated histone H3
(lysine 9) at the telomeres was increased by a difference of
138% (Figure 1B). Dimethylated and monomethylated histone
H3 (lysine 9) remained unchanged. Acetylation of histone H3
(lysine 9) appeared marginally reduced. This histone modifica-
tion profile is similar to that observed in the RNAi-dependent
heterochromatin assembly on the outer repeat sequences of
the fission yeast centromeres [7, 14]. In fission yeast, this
modomain protein Swi6. In our transient GFP-siRNA mouse
model, we alsoobserved amarked increase ofthe heterochro-
matin protein 1b (HP1b, an ortholog of Swi6), but not the other
There also were changes in the binding of telomere-specific
proteins to the ends of the chromosomes during the transient
siRNA-induced RNAi in mouse fibroblasts (Figure 1D). During
RNAi, association of the telomeres and the double-stranded
telomeric repeat-binding proteins, telomeric repeat-binding
factor1(TERF1), was increasedbyadifference of376%. Other
telomere-specific proteins, including telomeric repeat-binding
factor 2 (TERF2), protection of telomeres 1 (POT1), telomerase
(TERT), and tankyrase (TNKS), showed only minor changes.
This observation suggested that TERF1 might be involved in
the organization of the chromatin of the telomeres in addition
to the better known telomerase-dependent telomere-length
maintenance function .
To investigate the functional significance of chromatin
changes at the telomeres during transient siRNA-induced
in the mouse fibroblast cell line RBP. This cell line contains
a tagged telomere in which a neomycin resistant gene (neo)
was inserted into an interstitial site of the long arm of chromo-
the q arm and seeded the new stable telomeric repeat se-
quences immediately distal to the transgene [4, 15–17]. Tran-
sient transfection of GFP-siRNA in RBP resulted in 70% inhibi-
tion (percentage difference) of the subtelomeric neo gene
transient RNAi effect.
Pattern of Telomere FISH Signals Changed
during siRNA-Induced RNAi
Besides suppression of subtelomeric transgene, the chroma-
tin changes at the telomeres during transient siRNA-induced
RNAi in mouse fibroblasts also were associated with changes
in the pattern of telomere fluorescence in situ hybridization
(FISH) signals. Telomeres normally can be visualized as small,
double-dotted signals at the ends of metaphase chromo-
somes. In the interphase nuclei, telomere FISH signals are dis-
tributed diffusely as numerous, dot-like signals in the nuclei
(Figure 2A) [4, 16, 18]. During transient transfection of GFP-
siRNA, this normal pattern of the telomere FISH signals was
lost (Figure 2B). No telomere signals were detected at the
ends of metaphase chromosomes. The small, dot-like FISH
signals in interphase nuclei also were gone. The loss of the
normal telomere FISH signals pattern after transient siRNA-
transfection followed the amount of GFP-siRNA transfected
in a dose-dependent manner. At 50 pmol of GFP-siRNA (as
shown in Figure 2B), this loss of the normal telomere FISH sig-
nal pattern was detected in 97 out of 100 interphase nuclei
(Figure S1 available online). These changes in the telomere
mere FISH signals returned as the effect of GFP-siRNA waned
off after 7 days posttransfection. Other heterochromatin
blocks, such as pericentromeric and centromeric regions,
were not affected during GFP-siRNA transfection (Figures S2
and S3, respectively).
In addition, we also observed that the normal pattern of telo-
RNA nucleases RNase A or RNase Iffrom methanol-acetic
acid-fixed GFP-siRNA-transfectedcells priorto theapplication
ofthetelomere probes(Figure2C). This RNAwas not the exog-
enous siRNA, as indicated in tracking experiments with fluoro-
chrome-labeled siRNA (Figure S4). The normal telomere FISH
signals pattern also could be revealed in RNAi-induced cells
when the genomic DNA was denatured by stronger agents,
such as formamide, suggesting that only the accessibility of
the FISH probe to the telomeres was changed during RNAi.
This RNA present at the chromosome ends during siRNA-
induced RNAi could be considered as one of the components
of the telomeric heterochromatin. The identity of this telo-
mere-associated RNA remains to be determined. In fission
yeast, one of the AGO1-containing RNAi machinery com-
plexes—called RNA-induced initiation of transcriptional gene
silencing (RITS)—is proposed to mediate transcription-depen-
dent gene silencing [11, 13]. One of the hypotheses on the
mechanisms of RNAi-related heterochromatin formation sug-
gests transcripts of site-specific RNA being an intermediate of
heterochromatin formation. The orthologs of the components
heterochromatin formation exists in mammalian cells and
whether a similar complex would assemble at locations other
than the target sequence of the siRNA remain to be explored.
Similar changes in the telomere FISH signals pattern in
mouse fibroblast NIH 3T3 and suppression of subtelomeric
transgene neo in RBP were observed in siRNA-induced RNAi
experiments involving different genes including Gapdh, a con-
mere maintenance in yeast ; and scrambled sequences,
which contain no homology to any known mammalian gene
FISH signal pattern also were made when GFP-siRNA experi-
ments were performed in human cancer cell lines A549
(lung adenocarcinoma), SNU-1 (gastric adenocarcinoma),
Figure 1. Chromatin Changes of the Telomeres
during RNAi in Mouse Fibroblast NIH 3T3
The changes in protein binding at the telomeres
in mouse fibroblast NIH 3T3 transfected with
GFP-siRNA were measured by chromatin immu-
noprecipitation. The abundances of telomeric
repeat sequences in the input and immunopre-
cipitated portions weremeasured by quantitative
PCR . The percentages of changes with and
of the euchromatin of the genes Gapdh or b-ac-
tin, are shown on the y axes. Antibodies against
various groups of proteins are listed as follows:
(A) proteins related to RNAi machineries, RNA
polymerase II (RP II), AGO1, and AGO2; (B) his-
tone H3 modifications atlysine 9,including trime-
thylated (triMe), dimethylated (diMe), monome-
thylated (monoMe) and acetylated (acetyl); (C)
heterochromatin protein homologs HP1a, b, and
g; and (D) telomere-specific proteins showing
TERF1, TERF2, POT1, TERT, and TNKS. The
expressions of the neo transgene, located at
the subtelomeric region of chromosome 15 in
the mouse fibroblast cell line RBP, were followed
for 6 days after GFP-siRNA transfection (E). The
relative expression levels were measured by
quantitative RT-PCR and were calculated by the
comparative CTmethod (DDCT), with the expres-
sion of Gapdh as a reference. Results are shown
compared to control experiments (6SEM of
Current Biology Vol 18 No 3
and SK-N-DZ (neuroblastoma) (Figure S6). There were no
changes in the telomere FISH signal pattern in control experi-
ments in mouse fibroblast NIH 3T3 when GFP-siRNAs were re-
the sense or the anti-sense strands), short single-stranded
DNA (21–23 nucleotides, either the plus or the minus strands),
or short double-stranded DNA, with the same target sequence
of GFP in each case (Figure S7). However, RNAi experiments
induced in mouse fibroblast NIH 3T3 by short hairpin RNA
(shRNA) containing scrambled sequences or sequences
mere FISH signals pattern. This latter result suggested that
although either siRNA or shRNA are both capable of inducing
downregulation of gene in a sequence-specific manner, these
RNA species might be handled differently inside the cells and
might have diverse off-target effects. The difference in mecha-
be clarified. At the present moment, we do not have good
mechanistic explanations for the differences of these two
ways of inducing RNAi.
A Heterogeneous Group of RNA Containing Telomeric
Repeat Sequences Was Enhanced during RNAi
In control mouse fibroblast NIH 3T3, northern blot analyses of
total RNA probed with telomere-specific sequences revealed
a novel group of heterogeneous RNA with both strands of
the telomeric repeat sequences (Figures 3A and 3B). These
RNAs formed a smear of molecular weight from 1 to >9 kb in
the northern blot, indicating their heterogeneous nature. After
the transfection of GFP-siRNA, the signals of both strands of
these telomeric RNAs in the northern blot were increased (Fig-
ures 3A and 3B). Quantitative RT-PCR assay estimated an
RNAs after the induction of RNAi. Cloning and sequence anal-
yses confirmed that these RNA contained perfect telomeric
repeats (GenBank accession number EF685181). No unique
sequences or low-copy-number repeat sequences were
detected in all the clones we obtained.
Active transcripts from the telomeres, termed telomere re-
peat-containing RNA (TERRA), were observed in human and
mouse cell lines . Similar to the siRNA-induced RNA de-
scribed in this study, TERRA also is a group of heterogeneous
high-molecular-weight RNA. TERRA also contains UUAGGG
repeats. There was little, if any, CCCUAA repeat sequence, as
telomeric unique sequences. In our GFP-siRNA RBP model,
subtelomeric sequences were not detected. In fact, the neo
gene in RBP is located immediately adjacent to the telomere
telomeric repeat-sequence RNA was increased. Within the
Figure 2. Telomere FISH on siRNA-Transfected Mouse Fibroblasts NIH 3T3
(A)Telomeres are shown as green signals in the merged figure of the top row and grayscale images in the bottom row. DNA were counterstained by 40,
6-diamidino-2-phenylindole and are shown as blue signals. In control experiments of telomere FISH in cytogenetic preparation of mouse fibroblast NIH
3T3 cells, the normal telomere FISH signals pattern consisted of small, dot-like signals at the ends of the metaphase chromosomes, and numerous small
dot-like signals in the interphase nuclei.
(B) After transfection with GFP-siRNA, the normal pattern of telomere FISH signals was lost. Only a few strong telomere FISH nuclear foci were detected at
the interphase nuclei (arrows).
(C) After removing RNA by RNase Ifor RNase A prior to application of telomere FISH probe, the strong nuclear foci were removed and the normal pattern of
telomere FISH signals could be detected again.
Telomeres Respond Actively during RNAi
technical limitations of plasmid-based molecular cloning, all
our clones of GFP-siRNA-induced RNA did not contain any
subtelomeric sequences. Functionally, TERRA is shown to be
associated with the telomeres and protects chromosome
ends from telomere loss. In shRNA experiments, TERRA is
found to be negatively regulated by a group of proteins called
Suppressors with Morphogenetic Defects in Genitalia (SMG)
. In the present study, we did not observe changes in the
protective functions of the telomeres during transient siRNA-
ging chromosomes, dicentric chromosomes, and other gross
chromosomal abnormalities were not increased in transient
GFP-siRNA experiments (see Figure 2B and Figures S2–S7).
There also was no gross change in the average and interchro-
mosomal variability of telomere lengths during the transient
siRNA transfection (Figure S8). Hence, increase in the RNA-
containing telomeric repeat sequences induced by siRNA
transfection, in the absence of suppression of SMG, did not
result in chromosomal instability.
In the GFP-siRNA mouse fibroblast model, a few strong nu-
and Figures S2–S7). These interphase nuclear foci could be
removed with prior RNase treatments (Figure 2C), suggesting
that single-stranded RNA, with telomeric repeat sequences,
was one of the components of these nuclear foci. Like the
loss in normal patterns of telomere FISH signals, these nuclear
foci disappeared 6 days after GFP-siRNA transfection as the
effect of RNAi subsided. Such nuclear foci have not been
observed in TERRA experiments .
with the telomeres during siRNA-induced RNAi. The telomeres
reversibly acquired distinct heterochromatin characteristics.
The chromosome ends were associated with an unidentified
RNA. There were increases in histone H3 (lysine 9) methylation
and increased binding of HP1b, TERF1, and AGO1 at the telo-
meric repeat sequences. We also have identified a group of
high-molecular-weight RNAs, which contained telomeric re-
peat sequences. During transient siRNA-induced RNAi, the
concentration of this RNA with telomeric repeat sequences
was markedly increased and formed discrete nuclear foci.
siently, uncovering one possible mechanism of the off-target
effects of RNAi. Our findings provided evidence for the
changes of the telomeres in the process of RNAi, suggesting
active participation of the telomeres in the process.
Mouse fibroblast NIH 3T3, A549, SNU-1, and SK-N-DZ were obtained from
the American Type Culture Collection. The mouse embryonic fibroblast with
tagged telomere (RBP) was obtained from the transgenic mouse derived
from the embryonal stem cell line A405 [4, 16].
RNAi by siRNA
siRNAs specific for GFP and the DNA helicase Pif1 were constructed with
the Silencer siRNA Construction Kit (Ambion) by using the sense sequence
50-AAAGTGAAAAGTTCTTCTCCTCCTGTCTC-30and the anti-sense se-
quence 50-AAAGGAGAAGAACTTTTCACTCCTGTCTC-30(for GFP) and the
sense sequence 50-AAACTCAGATCTGGAGAACATCCTGTCTC-30and the
anti-sense sequence 50-AAATGTTCTCCAGATCTGAGTCCTGTCTC-30(for
Pif1). siRNAs for Gapdh and scrambled sequences (Silencer Negative Con-
trol #1 siRNA) were obtained from a commercial source (Ambion). Labeling
of siRNA with Cy3 was performed by using Silencer siRNA Labeling Kit-Cy3
(Ambion). Typical transfection experiments consisted of 50 pmol siRNA in
Lipofectamine 2000 (Invitrogen) into 6 3 104mouse fibroblasts, which
were seeded for 24 hr in 24-well dishes. For the telomere assay, chromatin
immunoprecipitation, or cytogenetic studies, transfected cells were har-
vested after incubation for 48 hr. Control experiments were performed by
blank lipofection, involving only Lipofectamine without any siRNA, plus or
minus Cy3-labeled nucleotides where appropriate.
RNAi by shRNA
In each transfection experiment, 0.8 mg of the pSilencer 4.1-CMV neo plas-
mids, expressing shRNA with either (1) limited homology to any known
sequences in the human, mouse, and rat genomes or (2) a Gapdh-specific
hairpin RNA (Ambion), was transfected to 6 3 104mouse fibroblasts by us-
ing Lipofectamine 2000 (Invitrogen). G418-containing medium was added
24 hr posttransfection. Transfected cells were collected for future studies
after incubation for 48 hr. In case of the vector expressing Gapdh-specific
hairpin RNA, suppression of the Gapdh gene was monitored by quantitative
Chromatin immunoprecipitation was performed after crosslinking DNA and
protein in 0.75% formaldehyde. Antibodies were obtained from commercial
sources. Quantitative PCR wasused tomeasurethe amount of telomericre-
peat sequences relative to the abundance of the euchormatic genomic DNA
of Gapdh or b-actin, calculated by the comparative CTmethod (DDCT) .
of telomeric repeat sequences in siRNA-transfected cells against control
Figure 3. Increase in High-Molecular Weight RNA with Telomeric Repeat
Sequences after siRNA Transfection in Mouse Fibroblast NIH 3T3
Northern blot analyses on total RNA extracted from mouse fibroblasts NIH
3T3 transfected with GFP-siRNA and in blank controls were performed con-
secutively with (A) 50-(CCCTAA)3-30telomeric repeat-specific sequences,
(B) 50-(TTAGGG)3-30telomeric repeat sequences, and (C) b-actin as probes.
bridization of different probes. A smear of telomeric repeat sequences was
seen at the high molecular range, from 1 kb to >9 kb in the control (third lane
in [A] and [B]). A marked increase in the amount of this smear of telomeric
repeat sequences was noted in GFP-siRNA-transfected cells. Molecular
weight markers for single-stranded RNA are shown on the left (first lane).
In (A) and (B), faint discrete bands are seen at w1.3 and w3.0 kb, and these
RNA were cloned and identified as alternative transcripts of the gene Xkrx
(Kell blood group complex subunit-related X-linked, GeneID: 331524, Gen-
Bank accession number NM_183319).
Current Biology Vol 18 No 3
Fluorescent In Situ Hybridization Download full-text
Telomere FISH was performed on standard cytogenetic preparations or
cells growing in chamber slides by using peptide nucleic acid telomere
probes, 50-(CCCTAA)3-30or 50-(TTAGGG)3-30, labeled with either fluorescein
isothiocyanate (FITC) or Cy3, as previously described [3, 4, 18]. The routine
heat-denaturingprotocol involved directlyheating theprobes andthe slides
at 70?C for 8 min followed by hybridization at room temperatureand washes
at high stringencies. More catastrophic denaturing protocols for FISH
(involving formamide) were performed as previously described [3, 4]. Major
satellite was detected by using FITC-labeled DNA probes (50-ACGTGAAA
TATGGCGAGGAA-30or 50-TCGTCAAGTGGATGTTTCTCA-30). The minor
primers (50-CGTTGGAAACGGGATTTGTA-30and 50-CCAACGAATGTGCTTT
TTCA-30) on mouse genomic DNA. In RNase digestion experiments, FISH
was performed after incubating the slides in RNase A (2 unit/ml) or RNase
If(1 unit/ml) at 37?C for 30 min. In the dose-dependent curve experiments,
telomere FISH experiments were performed on mouse fibroblasts trans-
fected with different concentrations of siRNA against GFP, and 100 inter-
phase nuclei were scored for the absence of the normal, numerous dot-
like patterns of the telomere signals. We used Student’s t test to compare
the mean number of nuclei without a normal pattern of telomere FISH
signals to that in control experiments in which the corresponding amount
of GFP-siRNA was replaced by RNase-free water. Statistical significance
was preset at p < 0.01.
Northern Blot Analysis and Quantitative RT-PCR
Total RNA was isolated from cell lines by using the RNeasy extraction kit
(QIAGEN).Tenmicrogramsofheat-denaturedRNA wasresolved byelectro-
phoresis on 1.2% agarose gel and transferred to Hybond N membrane and,
50-(TTAGGG)3-30in high stringencies. RNA Loading in each lane was moni-
tored by spectroscopic measurement of total RNA concentration, semi-
quantitative assessment of ethidium bromide staining of rRNA bands in
the agarose gel electrophoresis, and northern analysis of the washed blots
with a b-actin probe (Clontech).
of the RNA of the genes Gapdh or b-actin was measured by quantitative
RT-PCR by using random primer for the reverse transcription, followed by
the quantitative PCR as described . Relative expression levels were
calculated by the comparative CTmethod (DDCT).
Cloning of Telomere RNA
RNA was extracted from GFP-siRNA-transfected mouse fibroblast NIH 3T3
by using RNeasy extraction kit (QIAGEN) and resolved in 1% formaldehyde
denaturing gel. The corresponding region of the gel was excised and the
eluted RNA with telomeric repeat sequences was captured by biotinylated
30-mer telomeric repeat oligonucleotides (either plus or minus strands)
with magnetic beads (Streptavidin MagneSphere Paramagnetic Particles,
Promega). The captured RNA was amplified with the primer 50-(CCCTAA)6-
trogen), and analyzed by DNA nucleotide sequencing.
Eight figures are available at http://www.current-biology.com/cgi/content/
We thank Jesse Pang, Raymond Chan, and Raymond Lung for technical as-
sistance. A.W.I.L. was supported by a Competitive Earmarked Research
Grant from the Research Grants Council of Hong Kong (CUHK 4411/03M).
Received: October 6, 2007
Revised: December 10, 2007
Accepted: December 20, 2007
Published online: February 7, 2008
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