Telomere length heterogeneity in ALT
cells is maintained by PML-dependent
localization of the BTR complex
to telomeres
Taylor K. Loe,
1
Julia Su Zhou Li,
2
Yuxiang Zhang,
1
Benura Azeroglu,
3
Michael Nicholas Boddy,
1
and Eros Lazzerini Denchi
3
1
Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037, USA;
2
Ludwig Institute for Cancer
Research, Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA;
3
Laboratory of Genome Integrity, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear
chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are
some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative
lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML
nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with
telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can
occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used
CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in
ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs’ role in lo-
calizing the BLM–TOP3A–RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex
to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR
localization to telomeres is sufficient to sustain ALT activity.
[Keywords: telomere; ALT; telomere length; BLM; PML]
Supplemental material is available for this article.
Received October 18, 2019; revised version accepted February 26, 2020.
Telomeres are nucleoprotein structures that act to pro-
tect chromosome ends from being inappropriately recog-
nized as sites of DNA damage (Palm and de Lange 2008).
Due to the “end replication problem,” terminal telo-
meric DNA repeats are progressively lost during cellular
division. As a result, telomere length determines the pro-
liferation potential of cells that lack telomere mainte-
nance mechanisms (Denchi 2009). The majority of
cancers overcome this proliferation barrier by expressing
telomerase, the RNA-templated reverse transcriptase ca-
pable of extending telomeric ends (Kim et al. 1994).
However, ∼10%–15% of cancers, in particular those of
mesenchymal origin, rely on a different mechanism for
telomere extension, coined alternative lengthening of
telomeres (ALT) (Bryan et al. 1997; Dunham et al.
2000). ALT relies on a recombination-based mechanism
to elongate telomeres using homologous telomeric
DNA sequences as a template for synthesis, though
how ALT is initiated and sustained remains largely
unclear.
The bulk of our understanding of the ALT pathway de-
rives from the analysis of the pathways that allow Saccha-
romyces cerevisiae to survive in the absence of
telomerase. This work revealed that cells could survive
by engaging either a Rad51-dependent recombination
pathway (type I survivors), or a Rad51-independent
break-induced replication process (type II survivors)
(Lundblad and Szostak 1989; Teng and Zakian 1999).
Both pathways are dependent on RAD52 and the Pol32
subunit of polymerase δ (Lundblad and Szostak 1989;
Lydeard et al. 2007). Recent work in mammalian cells
has paralleled the work done in yeast, revealing that
ALT-positive cancer cells display a type I-like ALT mech-
anism that is RAD51-dependent and characterized by
Corresponding authors: eros.lazzerinidenchi@nih.gov; nboddy@scripps.edu
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.333963.119.
© 2020 Loe et al. This article is distributed exclusively by Cold Spring
Harbor Laboratory Press for the first six months after the full-issue publi-
cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After
six months, it is available under a Creative Commons License (Attribu-
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telomere clustering and recombination-mediated telo-
mere synthesis (Cho et al. 2014; Ramamoorthy and Smith
2015). In addition, ALT-positive mammalian cells also
display RAD51-independent type II-like mechanisms of
telomere elongation characterized by telomere synthesis
in the G2/M phase of the cell cycle (Dilley et al. 2016;
Min et al. 2017; Pan et al. 2017).
Furthermore, work in S. cerevisiae has revealed that the
RecQ-like helicase Sgs1 is required for telomere mainte-
nance in type II survivors (Cohen and Sinclair 2001;
Huang et al. 2001; Johnson et al. 2001). Like its counter-
part in yeast, the mammalian ortholog of Sgs1, the Bloom
syndrome helicase (BLM), has been implicated in the
mammalian ALT pathway. Depletion by siRNA in ALT-
positive cells results in the reduction of ALT-associated
phenotypes such as the accumulation of extrachromo-
somal telomeric repeats in the form of partially single-
stranded C-rich circles, termed C-circles, and G2/M
telomere synthesis (O’Sullivan et al. 2014; Sobinoff et al.
2017; Pan et al. 2019; Zhang et al. 2019). Notably, BLM
also plays an important role at telomeres in cells that do
not use ALT to maintain their telomeres, acting to facili-
tate telomere replication and suppressing rapid telomere
deletions (Stavropoulos et al. 2002; Sfeir et al. 2009; Bare-
field and Karlseder 2012; Zimmermann et al. 2014 ; Dro-
sopoulos et al. 2015; Pan et al. 2017). BLM is part of the
BTR complex that also includes the topoisomerase
TOP3α, and the OB-fold containing structural compo-
nents RMI1 and RMI2 (Johnson et al. 2000; Wu et al.
2000; Yin et al. 2005; Xu et al. 2008). Interestingly, overex-
pression of BLM or dysregulation of the BTR complex in-
duced by the loss of FANCM in ALT-positive cells has
been shown to induce up-regulation of ALT-associated
phenotypes, suggesting that this factor is limiting for the
ALT pathway and led to the proposal that the BTR com-
plex acts in ALT to dissolve recombination intermediates
into noncrossover products, which results in telomere
lengthening (Sobinoff et al. 2017; Lu et al. 2019; Min
et al. 2019; Pan et al. 2019; Silva et al. 2019).
Cancer cells that maintain telomeres using the ALT
pathway harbor unique features that are used as ALT bio-
markers such as large promyelocytic leukemia (PML)
nuclear bodies that contain telomeric DNA, termed
ALT-associated PML bodies (APBs), extrachromosomal
telomeric DNA in the form of C-circles, elevated levels
of telomere–sister chromatid exchanges (T-SCEs) and
highly heterogenous telomere lengths (Ogino et al. 1998;
Tokutake et al. 1998; Yeager et al. 1999; Henson et al.
2002; Cesare and Griffith 2004; Londoño-Vallejo et al.
2004; Wang et al. 2004; Henson et al. 2009; Nabetani
and Ishikawa 2009; Min et al. 2017). Interestingly, al-
though many of these characteristics are conserved in
yeast, APBs are a feature of ALT not found in S. cerevisiae
yet are suggested to have a functional role in the ALT
pathway in mammalian cells. PML bodies are mem-
brane-less compartments formed by liquid–liquid phase
separation (LLPS) organized by the intramolecular
interactions between SUMO (small ubiquitin-like modifi-
cation) posttranslational modifications and SUMO-inter-
acting motifs (SIM) (Chung et al. 2012; Banani et al.
2016). APBs consist of a PML and Sp100 shell bound
together by SUMO–SIM interactions and contain, in addi-
tion to telomeric DNA, the long noncoding RNA
(lncRNA) telomeric repeat-containing RNA (TERRA),
telomere-associated proteins and several DNA damage
factors (Yeager et al. 1999; Arora et al. 2014). APBs have
been proposed to play a critical role in ALT by clustering
telomeres and DNA repair factors together, thus concen-
trating substrates and enzymes required for recombina-
tion-based telomere elongation (Henson et al. 2002;
Cesare and Reddel 2010; Chung et al. 2011, 2012; Min
et al. 2019; Verma et al. 2019). In agreement with this hy-
pothesis, it has recently been shown that telomeres can be
elongated during mitosis in APB-like foci in a process
termed mitotic DNA synthesis MiDAS (Özer et al. 2018;
Min et al. 2019). Moreover, depletion of PML by siRNA
has been shown to reduce telomere elongation (Osterwald
et al. 2015). Still, how telomeres assemble within APBs
and the role of APBs in the ALT process remains unknown.
Moreover, certain ALT-positive cells have been shown to
lack APBs, yet continue to maintain their telomeres in
the absence of telomerase, calling into question the neces-
sity of APBs for the ALT process (Cerone et al. 2005; Fasch-
ing et al. 2005; Marciniak et al. 2005).
Here we set out to test whether PML and its associated
APBs are critical for the ALT pathway by generating PML-
null cell lines to assess the long-term consequences on
telomere length maintenance by ALT. Our results indi-
cate the PML is required for ALT telomere maintenance,
although not required for long-term cell viability. Using
these cells, we next interrogated the presence of other
ALT hallmarks, finding that PML-null cells display a
marked decrease in C-circle levels. Moreover, by estab-
lishing a native FISH protocol to assay for single-strand
telomeric DNA on a single-cell basis, we determined
that PML was required for the formation of C-circles. As
expected, PML was found to be required for the localiza-
tion of APB components to telomeric ends, including
BLM. To assess the requirement of the BTR complex in
ALT and its interactions with the functions of PML, we
generated BLM and RMI1 knockouts of the same ALT
cell line, observing that the BTR complex was, similar
to PML, required for ALT telomere maintenance and
C-circle formation. Overexpression of PML in the BTR
knockouts did not rescue ALT phenotypes, indicating
that PML and BTR were acting within the same pathway.
However, by tethering the BTR component RMI1 to telo-
meres, it was possible to induce C-circle formation and G2
telomeric synthesis in a PML-independent fashion. To-
gether, these data show that PML functions in ALT by re-
cruiting the BTR complex to telomeric ends.
Results
PML is required for APB formation, telomere
heterogeneity, and telomere length maintenance
in ALT cells
To test whether PML is required for the ALT pathway, we
used CRISPR/Cas9 to delete PML in the ALT-positive cell
Interplay between PML and BLM at ALT telomeres
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lines U2OS and GM847. Guide RNAs targeting exon 1 of
the PML gene were used to create a mutation at the begin-
ning of the PML gene that led to a downstream, early
STOP codon within the first three PML exons. Since all
known isoforms of PML share the same first three exons,
a STOP codon within this region leads to a knockout of all
known isoforms of PML (Nisole et al. 2013). Using this ap-
proach, we established three PML-null U2OS clones (2C,
9H2, and 15G4) and two PML-null GM847 clones (5A
and 4E). We confirmed that these clones did not harbor
any PML wild-type alleles by Sanger sequencing (Supple-
mental Fig. S1A,B), as well as verified the lack of PML pro-
tein products by Western blot and immunofluorescence
analysis (Fig. 1A,B; Supplemental Fig. S1C,D). Remark-
ably, loss of PML did not affect proliferation or cell cycle
progression (Supplemental Fig. S1E,F).
Next, we assessed whether other APB components
could still localize to telomeres in the absence of PML.
To this end, we performed immunostaining for the telo-
mere-associated protein TRF2 and three well-established
APB components: Bloom helicase (BLM), the single-
stranded binding protein RPA and the APB scaffold pro-
tein Sp100 (Yeager et al. 1999). This experiment showed
that, in the absence of PML, localization of all three
APB components to telomeres drastically diminished, in-
dicating a loss of APB structures (Fig. 1C–F; Supplemental
Fig. S1F). Despite the lack of APBs, all of the PML-null cell
lines had no significant change in long-term viability and
were able to be propagated for >100 d in culture. Impor-
tantly, complementation of PML-null cells with wild-
type PML-IV, an isoform of PML known to interact
with the SUMOylated form of the telomeric protein
TRF1 that is important for APB formation (Potts and Yu
2007; Hsu et al. 2012), restored APB formation but had
no significant impact on cell viability ( Supplemental Fig.
S1H–L).
To establish whether PML is required to maintain telo-
mere length in ALT-positive cells, we monitored telomere
E
F
B
A
C
D
G
Figure 1. PML is required for APB formation
and ALT activity in U2OS cells. (A) Western
blot analysis of PML expression in PML
−/−
clones (2C, 9H2, and 15G4) and parental
U2OS cells. Vinculin is used as a loading
control. Representative immunofluorescence
images (B–E) and relative quantifications
(F) of parental U2OS cells and PML
−/−
clones
showing the localization of APB components
PML, BLM, SP100 and RPA (all red) and telo-
meric protein TRF2 (green). All stainings
done in triplicate with a minimum of 300 to-
tal cells counted percondition. (
∗
) P < 0.05; (
∗∗
)
P < 0.005; (
∗∗∗
) P < 0.0005; (n.s.) P > 0.05. (G,
top) Telomere restriction fragment analysis
of parental U2OS cells and PML
−/−
clones at
the indicated population doubling (PD). (Bot-
tom) Quantification of basepairs lost per dou-
bling and an enlarged image of a section of the
blot (indicated by red dashed box) showing
migration of telomere bands over time.
Loe et al.
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length in parental wild-type U2OS cells and PML-null
cells over the course of ∼100 d in culture. Telomere re-
striction fragment (TRF) analysis showed that, in contrast
to parental U2OS cells, PML-null cells presented progres-
sive telomere shortening at a rate of ∼30–80 bp per divi-
sion, which is consistent with an estimated rate of
erosion of ∼50–200 bp per division caused by the end rep-
lication problem (Allsopp et al. 1995; Allsopp and Harley
1995). Strikingly, all of the PML-null cells showed a dis-
tinct banding pattern by TRF, indicating that upon PML
depletion, each clone analyzed lost telomere length het-
erogeneity, a unique feature of ALT cells (Fig. 1G). Exoge-
nous expression of PML-IV was able to restore both
telomere length maintenance and telomere heterogenei-
ty, erasing the distinct banding pattern evident in PML-
null clones (Supplemental Fig. S1M). Together these
data show that PML is required for telomere maintenance
in ALT cells.
PML-deficient cells have reduced levels of telomeric
C-circles
Following validation of the PML-null cells as APB-free and
ALT-negative, we asked whether PML-deficient cells re-
tained other ALT hallmarks, such as T-SCEs. Chromo-
some orientation-FISH (CO-FISH) on the PML-null cells
showed no significant difference in T-SCE rates between
PML-null clones and the parental U2OS cell line (Fig.
2A,B; Supplemental Fig. S2A). This result indicates that
PML, although required for telomere maintenance by
ALT, is not required for T-SCEs.
Next, we assessed whether, in the absence of PML,
cells can still accumulate extrachromosomal telomeric
DNA in the form of C-circles, an established hallmark
of ALT (Henson et al. 2009). C-circles are partially sin-
gle-stranded C-rich telomeric circles, with unknown
function and origin that have been postulated to be a
byproduct of telomeric recombination or to play an ac-
tive role in the ALT pathway as a template for telomeric
extension (Tokutake et al. 1998; Cesare and Griffith
2004; Wang et al. 2004; Henson et al. 2009; Nabetani
and Ishikawa 2009; Cesare and Reddel 2010). The C-cir-
cle assay (CCA) showed that, consistent with C-circles
being an indicator of ALT activity, PML-null cells had
an approximately fivefold reduction in C-circle levels
compared with parental U2OS cells (Fig. 2C,D). Howev-
er, this assay does not discriminate between uniformly
decreased C-circle levels or a smaller fraction of cells
E
F
BA
CD
I
J
GH
Figure 2. Loss of ALT features in PML-null cells.
(A) Representative images of metaphase telomeres
stained by CO-FISH with T-SCEs indicated by ar-
rows. (B) Quantification of T-SCEs from CO-FISH
staining, shown as weighted average and standard
deviation from four stainings with a minimum of
1400 chromosomes counted per genotype. Signifi-
cance was determined as P < 0.05. (C) C-circle
analysis (CCA) of parental U2OS cells and three
PML-null clones showing a significant decrease
of C-circle levels in PML
−/−
cells. (D) Quantifica-
tion relative to parental U2OS cells from three
CCA experiments. (
∗∗
) P < 0.005; (
∗∗∗
) P < 0.0005.
(E) A panel of ALT-positive (ALT+ve) and ALT-
negative (ALT−ve) cells lines stained under dena-
turing conditions to label double-stranded telo-
meric DNA (TTAGGG; green) or under native
conditions to stain single-stranded C-rich telo-
meric DNA (ss-TeloC; red). (F) CCA of the same
panel of cells used in E.(G) Quantification of the
data shown in C repeated in triplicate with a min-
imum of 300 total cells counted per cell line.
(H) Quantification of data shown in D, repeated
in triplicate. (I) PML
−/−
cells and the parental
U2OS cells stained for single-stranded C-rich telo-
meric DNA (ss-TeloC; red) and double-stranded
G-rich telomeric DNA (TTAGGG; green). (J, left)
Quantification of ss-TeloC data shown in I with
a minimum of 300 total cells counted per condi-
tion, repeated in triplicated with three stars indi-
cating P < 0.0005. (Right) Quantification of the
number of ss-TeloC foci per cell from native
FISH staining in parental U2OS and PML
−/−
cells
from a representative staining of ss-TeloC with a
minimum of 80 cells counted per condition, dem-
onstrating a decrease in number of ss-TeloC foci
per cell in the PML
−/−
clones.
Interplay between PML and BLM at ALT telomeres
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producing C-circles. A uniform decrease in C-circles
would suggest that PML is required for the production
of C-circles and confirm their link with ALT activity,
while a nonuniform decrease might suggest a role for
PML in the stability of these species and that, like
T-SCEs, C-circles may not be as directly connected
with ALT telomere extension as previously thought. To
distinguish between these alternative hypotheses, we de-
veloped a single-cell assay to visualize ALT-specific sin-
gle-stranded telomeric C-rich DNA (ss-TeloC) in situ
(see schematics of approach in Supplemental Fig. S2B).
In this method, fixed cells are stained under nondenatur-
ing condition with a PNA probe complementary to the
C-rich telomeric DNA strand. To verify that ss-TeloC
signal correlates with the presence of extrachromosomal
C-circles and ALT activity, we analyzed a panel of ALT-
negative and ALT-positive cell lines using the estab-
lished C-circle assay (CCA) and ss-TeloC staining. These
experiments showed that ss-TeloC signal is detected
only in ALT-positive cells (Fig. 2E,G) and that the pres-
ence of ss-TeloC signal reflected the levels of C-circles
detected by the standard CCA (Fig. 2F,H). Furthermore,
we were able to combine this ss-TeloC staining protocol
with traditional immunofluorescence to determine that
the ss-TeloC signal was mainly, but not exclusively, lo-
calized to APBs, where ALT activity is thought to occur
(Supplemental Fig. S2C,D). The resulting foci from this
staining may have several possible sources, including
C-circles, C-rich telomeric DNA loops, or telomeric
RNA:DNA hybrids that are enriched in ALT cells. To ex-
clude the possibility that the ss-TeloC signal was coming
mainly from RNA:DNA hybrids, we overexpressed
RNAseH, which dissolves these hybrids, and found that
it had no effect on ss-TeloC staining (Supplemental Fig.
S2E). With this protocol, it is not possible to distinguish
between C-circles and C-rich telomeric DNA loops; how-
ever, the assay was highly specific for cell lines that are
C-circle positive and use ALT to maintain their telo-
meres. Based on this result, we concluded that ss-TeloC
staining can be used as a proxy for the detection of ALT
activity at a single-cell level. Using this technique, we
stained PML-null cells for ss-TeloC and noted an approx-
imately fivefold reduction in the percentage of cells con-
taining ss-TeloC when compared with parental U2OS
cells (Fig. 2I,J), further confirming the correlation be-
tween ss-TeloC signal, C-circle levels detected by CCA,
and ALT activity. Notably, the decrease in ss-TeloC
showed that PML depletion resulted in a broad, popula-
tion-wide depletion when compared with the parental
cell line, suggesting that PML is likely playing an active
role in the generation of ssC-rich telomeric species, rath-
er than a passive role in their stabilization (Fig. 2J).
GM847 PML-null clones mirrored the U2OS PML-null
clones with a severe decrease in ss-TeloC signal (Supple-
mental Fig. S2F). Together, these data provide validation
of a novel assay by which to observe ALT activity on a
single-cell level, and determine that PML-null cells,
which no longer maintain their telomeres via ALT and
lack C-circles, yet maintain evidence of telomeric
recombination.
The BTR complex is required for C-circle formation and
ALT-mediated telomere maintenance
A likely mechanism by which PML enables telomere
maintenance and C-circle formation in ALT cells is
through the localization and stabilization of APB factors
to telomeres. To test this hypothesis, we sought to identi-
fy APB components that, when absent, would phenocopy
the requirement of PML for ALT activity. Our focus cen-
tered on components of the BTR complex, which was
seen to be mislocalized from telomeres in PML-null cells
(Fig. 1C) and has been implicated in ALT-associated phe-
notypes (O’Sullivan et al. 2014; Sobinoff et al. 2017; Lu
et al. 2019; Min et al. 2019; Pan et al. 2019). In order to
test whether loss of the BTR complex would affect ALT
activity, we generated U2OS cells deficient in either
BLM helicase or RMI1, which is critical for the recruit-
ment of BLM and the rest of the BTR complex to DNA
(Xu et al. 2008). Two independent BLM and RMI1 clones
were established by CRISPR/Cas9-mediated gene editing
and validated by Sanger sequencing (Supplemental Fig.
S3A), Western blot analysis (Fig. 3A) and immunofluores-
cence staining (Supplemental Fig. S3B,C). The localiza-
tion of BLM to telomeres was drastically reduced in
RMI1-null cells compared with the parental cell line, in
agreement with the role of RMI1 in recruiting BLM to
DNA (Supplemental Fig. S3B). Likewise, there was a re-
duction in RMI1 localization to telomeres in BLM-null
cells, suggesting that BLM is also necessary for the recruit-
ment of other BTR factors (Supplemental Fig. S3C). Addi-
tionally, we found that BTR-null cells display a reduction
in APBs, suggesting a reciprocal relationship between the
BTR complex and APB localization to telomeres (Supple-
mental Fig. S3D,E).
To investigate whether ALT activity is compromised in
BLM- and RMI1-null cells, we first established the levels
of extrachromosomal telomeric C-circles by CCA as
well as ss-TeloC staining (Fig. 3B–D). Both assays revealed
a strong reduction of C-circle levels in BTR-null cells com-
pared with parental U2OS cells, suggesting that the BTR
complex is essential for the production of C-circles and,
likely, ALT activity. Like the PML-null clones, this loss
of C-circles and ss-TeloC signal was paired with no signif-
icant change in the cell cycle profiles of the clones or loss
of cell viability (Supplemental Fig. S3F,G). Analysis of T-
SCEs in BLM- and RMI1-null cells revealed that the
BTR-null clones mirrored the PML-null clones in showing
no significant difference in the incidence of T-SCEs (Sup-
plemental Fig. S3H). Next, we tested whether, similar to
what was observed for PML, the BTR complex is required
for ALT-mediated telomere extension. To test this hy-
pothesis, we took advantage of a recently developed assay,
termed ALT telomere DNA synthesis in APBs (ATSA),
which allows for the direct detection of telomere exten-
sion in ALT cells using EdU incorporation at telomeres
in the G2 phase of the cell cycle (Zhang et al. 2019).
BTR-null cells, as well as PML-null cells, showed a signif-
icant decrease in EdU colocalization with telomeres when
compared with parental U2OS cells, indicating a loss of
ALT telomere synthesis (Fig. 3E,F; Supplemental Fig.
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