Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells
Citation: Cell Discovery
Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells
Hyeung Kim 0
Feng Li 1
Quanyuan He 0
Tingting Deng 1
Jun Xu 2
Feng Jin 3
Cristian Coarfa 3
Nagireddy Putluri 3
Dan Liu 0 2
Zhou Songyang 0 1
0 Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine , Houston, TX , USA
1 Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University , Guangzhou , China
2 Cell-Based Assay Screening Service Core, Baylor College of Medicine , Houston, TX , USA
3 Department of Molecular and Cellular Biology and Advanced Technology Core, Baylor College of Medicine , Houston, TX , USA
CRISPR/Cas9 technology enables efficient loss-of-function analysis of human genes using somatic cells. Studies of essential genes, however, require conditional knockout (KO) cells. Here, we describe the generation of inducible CRISPR KO human cell lines for the subunits of the telosome/shelterin complex, TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, which directly interact with telomeres or can bind to telomeres through association with other subunits. Homozygous inactivation of several subunits is lethal in mice, and most loss-of-function studies of human telomere regulators have relied on RNA interference-mediated gene knockdown, which suffers its own limitations. Our inducible CRISPR approach has allowed us to more expediently obtain large numbers of KO cells in which essential telomere regulators have been inactivated for biochemical and molecular studies. Our systematic analysis revealed functional differences between human and mouse telomeric proteins in DNA damage responses, telomere length and metabolic control, providing new insights into how human telomeres are maintained.
In the past 20 years, we have gained tremendous
insight into how the ends of mammalian chromosomes
or telomeres are maintained and regulated. Together
with the telomerase, which consists of the reverse
transcriptase TERT and RNA template TR/TERC, a
multitude of telomere-binding proteins participate in
telomere maintenance [
]. In particular, six core
telomeric proteins, TRF1, TRF2, RAP1, TPP1, TIN2 and
POT1, dynamically assemble on telomeres as a large
complex called telosome or shelterin and are essential in
telomere length regulation and end protection in
]. Extensive research has revealed the
interactions and functions of telosome components. For
instance, TRF1 and TRF2 bind directly to the telomere
duplex through their myb domains [
POT1 binds 3? single-stranded (ss) telomeric overhangs
]. RAP1 is recruited by TRF2, but apparently does
not directly interact with any of the other subunits .
TIN2 can interact with both TRF1 and TRF2 [
It also binds TPP1 and helps bring to telomeres the
TPP1-POT1 heterodimer that is essential for regulating
telomerase access to telomeres [
]. The core
telomere proteins often act as interaction hubs to recruit
factors of diverse pathways to telomeres and ensure
crosstalk between telomere maintenance pathways and
other cellular processes [
8, 19, 31, 32
]. In fact, several key
telomere regulators have been shown to regulate
metabolism, providing direct evidence of the close ties
between telomere regulation and metabolic control. For
example, the human telomerase reverse transcriptase has
been found to localize to the mitochondria and reduce
intracellular oxidative stress [
]. Our lab has found
that TIN2 can also localize to the mitochondria and
regulate oxidative phosphorylation [
Numerous studies have demonstrated that
dysfunctional telomeres can lead to telomere length defects,
deprotected telomeres, genomic instability and diseases
1, 4, 32, 38
]. Much of our knowledge regarding the
molecular and functional significance of mammalian
telomeric proteins comes from studies using mouse knockout
(KO) mouse embryonic fibroblast (MEF) cells, as genes
are more readily targeted in mouse embryonic stem cells.
However, notable differences exist in telomere regulation
between mouse and human. For instance, human
telomeres are considerably shorter than those of laboratory
mice and human has one POT1 gene, whereas mouse has
two (Pot1a and Pot1b). Such disparities underscore the
need for loss-of-function human cellular models. Majority
of the loss-of-function studies in human cells have relied on
RNA interference (RNAi)-mediated inhibition of
endogenous genes. The limitations of RNAi knockdown (KD)
and the fact that several key telomere regulators including
TRF2 and TIN2 are essential genes have complicated data
analysis and interpretation. Complete inactivation of these
telomere regulatory genes in cells may cause cell death,
precluding further detailed biochemical and molecular
studies, especially experiments that require extended
culturing and/or large numbers of cells.
The advent of the CRISPR/Cas9 genome-editing
technology has afforded investigators unprecedented
opportunities to more efficiently and specifically target genes in
human cells and to explore the consequences of their
]. In this study, we took advantage of the
highly flexible and adaptable CRISPR/Cas9 system and
generated human inducible KO cell lines for each of the
telosome components. This panel of cells has allowed us to
survey the functional significance of each telomeric protein
and probe the impact of individual subunit inhibition on
telomere regulation as well as metabolic control. With this
systematic analysis of the function of human telomere
proteins using inducible KO cell lines, we are able to better
delineate the differences between mouse and human
telomere biology. In addition, our panel of inducible KO cell
lines should prove invaluable to investigators seeking to
further explore the consequences of telomere dysfunction
and to study how diverse cellular functions may be
disrupted upon telomere dysregulation.
Using CRISPR/Cas9 to generate inducible KO human cell lines
Trf1, Trf2 and Tin2 have been reported to be
essential genes in mouse [
]. To determine the roles
of their human orthologs, we first turned to RNAi KD
in human cells through stable expression of short
hairpin RNAs (Supplementary Figure S1A). Even with
effective KD (480%) of TRF2, for example, we could
only observe minor DNA damage responses (DDRs)
at telomeres (data not shown), rarely more severe
phenotypes such as chromosome end-to-end fusions
found in Trf2 KO MEF cells [
], suggesting that
residual TRF2 proteins in the KD cells may have been
sufficient to prevent severe and sustained telomere
DNA damages. We next attempted straight KO of
these genes by CRISPR/Cas9, but failed to isolate any
clones of TRF2, TIN2 or POT1 KO cells. Given such
findings, we decided that human cells conditionally
knocked out for telosome subunits would be more
Traditionally, conditional KO alleles are generated
by inserting into some particular locus recombination
sequences such as loxp sites, which can mediate the
deletion of intervening sequences upon the expression
of recombinases such as Cre [
]. The insertion of
such exogenous sequences may alter gene regulation
and the entire process is often time consuming. To
generate conditional telosome subunit KO cells, we
modified a lentivirus-based inducible CRISPR/Cas9
KO system [
], and constructed separate vectors for
inducible Cas9 and constitutive single guide RNA
(sgRNA) expression (Supplementary Figure S1B).
Hela cells were transduced with lentiviruses encoding
inducible Cas9 and a clone in which Cas9 expression
could be reproducibly activated with doxycycline in a
dose-dependent manner was selected (Supplementary
The double-strand breaks resulting from Cas9
cleavage trigger the non-homologous end joining DNA
repair pathway in the absence of a donor template
]. Non-homologous end joining-mediated DNA
repair may generate small insertions and/or deletions
(indels) at the target site, and compromise gene
function if cleavage occurs within protein coding sequences.
Repair of a single Cas9 cleavage site has a 1/3 chance of
in-frame ligation of the coding sequences, which may
not completely disrupt gene function. We reasoned that
simultaneous targeting with two sgRNAs should
improve the odds of larger deletions and more
complete inhibition of endogenous genes. To test this
strategy, the inducible Cas9 cells were infected with two
viruses encoding two separate TIN2-specific sgRNAs
either singly or together, selected with appropriate
antibiotics, and then cultured in
doxycyclinecontaining media to induce Cas9 expression
(Figure 1a). At different time points following
doxycycline treatment, cells were collected for analysis
of TIN2 protein expression (Figure 1b). As we
predicted, targeting with two sgRNAs appeared to KO
gene expression more efficiently than using a single
sgRNA. Furthermore, lengthier doxycycline treatment
was able to improve KO efficiency (Figure 1b).
Notably, the TIN2 KO cells exhibited proliferative
defects during culturing (Figure 1c). Although all of the
cell lines showed similar growth patterns in the absence
of doxycycline, differences in growth rates became
apparent between doxycycline-induced TIN2 KO cells
after 4-day treatment. Growth of the single sgRNA
TIN2 KO cells was hampered initially, but appeared to
recover with continued culturing, likely due to the
presence of cells with incomplete TIN2 inhibition.
Indeed, the severity of proliferation defects correlated
with the degree of TIN2 ablation, with the dual sgRNA
TIN2 KO cells being more severely affected than the
single sgRNA KO cells (Figure 1c). When we
ectopically expressed sgRNA-resistant TIN2 in the dual
sgRNA KO cells, growth and proliferation were
restored, indicating that TIN2 was critical for cell
growth and that dual sgRNAs more completely
knocked out TIN2.
Although the inducible TIN2 KO cells were
polyclonal, independent inductions of Cas9 led to highly
reproducible results, indicating that the inducible
strategy reliably produces populations of cells with
comparable genotypes and phenotypes. When we
sampled the TIN2 alleles from the induced dual
sgRNA TIN2 KO cells by TOPO cloning and Sanger
sequencing of the sgRNA target region
(Supplementary Figure S2A), we found most (480%)
to contain deletions because of simultaneous Cas9
cleavage at both sgRNA target sites, and the remaining
alleles containing indels at both target sites without
deleting the intervening sequences. Importantly, all of
the alleles are predicted to have impaired TIN2
function, corroborating that dual sgRNA design helped
ensure complete inactivation of endogenous genes.
Using the dual sgRNA system, we generated
inducible KO cell lines for all six core telomeric proteins
(Supplementary Figure S2B). Again, we compared
single vs dual sgRNA KO efficiencies. Although some
of the single sgRNAs knocked out endogenous gene
expression quite effectively, using dual sgRNAs to
simultaneously target a single locus consistently proved
more efficient (Supplementary Figure S2C). Again,
longer doxycycline induction led to more effective and
sustained inactivation of endogenous genes
(Supplementary Figure S2C). In the following
experiments, all the cell lines were induced for 6 days with
doxycycline before further analysis and/or treatment
unless otherwise specified.
Profiling the contribution of each subunit to the
telomeric assembly of the telosome complex
The six KO cell lines afforded us the first
opportunity to systematically investigate in detail how
constitutive deletion of one subunit may affect the
Studying telosome subunits using inducible knockout cell lines
telomeric targeting and assembly of the telosome
complex. Each cell line was induced and confirmed for
KO efficiency by western blotting (Figure 2a). The cells
were then harvested for telomere chromatin
immunoprecipitation (ChIP) assays (Supplementary
Of the six proteins, both TRF1 and TRF2 can bind
double-stranded telomeric DNA [
], and as
expected, we found that TRF2 KO had no effect on TRF1
binding to telomeres (Figure 2b). Using ectopically
expressed proteins, we showed previously that TIN2
was essential for telosome assembly [
POT1 can bind ss telomeric DNA [
], targeting of
POT1 to telomeres requires TPP1, which in turn is
tethered to telomeres through TIN2 [
with these previous findings, reductions in telomere
targeting of the remaining subunits were apparent in
TIN2 KO cells, with TPP1 and POT1 being the most
affected (Figure 2b). Similarly, knocking out TPP1 also
led to drastic reductions in telomere ChIP signals for
other telosome subunits, particularly POT1,
underscoring the importance of TPP1 in telosome assembly
and POT1 telomere targeting [
]. Notably, POT1
KO also significantly reduced the targeting of both
TPP1 and TIN2 to telomeres. Taken together, these
data suggest that TIN2, TPP1 and POT1 may form a
tight subcomplex. It is also clear that with the exception
of RAP1, knocking out any of the subunits had a more
global effect on the remaining subunits (Figure 2b),
indicating significant contribution of each protein to
the proper assembly of the telosome complex and that
their roles in maintaining telosome function may be
more complex than previously surmised.
Activation of telomere DDRs in inducible KO cell lines
Considerable efforts have been devoted to
delineating the complex signaling pathways that protect
telomeres and prevent the activation of DDR. Disruption
of the telosome complex can expose telomere ends to
the DDR machinery and eventually lead to
chromosomal abnormalities and cell cycle arrest [
Immunofluorescence (IF) analysis of these inducible
KO cell lines supports previous findings of the
importance of core telomeric proteins to telomere protection.
As evidenced by the recruitment of 53BP1 to telomere
dysfunction induced foci (TIFs) (Figure 3a and b),
upon doxycycline-induced KO, activation of DDRs at
telomeres could be observed. Except for RAP1 KO
cells, which displayed minimal increase in TIFs, all
other KO cell lines exhibited significant increases in
53BP1 foci that co-stained with a telomere DNA
marker. In addition, KO of TRF2 and TIN2 resulted in
a marked increase in telomere fusions (Figure 3c and d,
Supplementary Figure S3B). These results again
reinforce the notion that the six telomeric proteins have
distinct roles in end protection and genomic stability.
Increased DDRs at telomeres can lead to activation
of ataxia-telangiectasia mutated (ATM) and
ATMand Rad3-related (ATR) signaling, and the subsequent
phosphorylation of Chk2 and Chk1, respectively.
TRF2 dysfunction has been shown to activate ATM
], whereas the POT1-TPP1
heterodimer is important for inhibiting ATR activation
]. Indeed, marked induction of phosphorylation
of Chk2 upon TRF2 KO and Chk1 upon POT1/TPP1
KO was observed (Figure 3e and f, Supplementary
Figure S4). In comparison, RAP1 deletion had no
impact, whereas TPP1 and TRF1 appear to be more
specific for ATR-mediated DDR regulation. TIN2 and
POT1 are both important for DDR, and their KO
resulted in robust phosphorylation of both Chk2 and
Chk1. The Chk2 response in POT1 KO cells was
somewhat unexpected, because deletion of mouse
Pot1a mainly induced Chk1 activation and Pot1b
inactivation mostly impacted telomere overhangs
]. Perhaps Chk2 activation in our POT1 KO cells
was a result of reduced telomere-associated TRF2 and
TIN2 upon POT1 deletion. These results further
highlight the complex mechanisms that are in place to
protect telomeres from DDR and the distinct signaling
events mediated by each subunit, and suggest that more
functional differences may exist between human and
mouse telomeric proteins in checkpoint response than
Telomere length maintenance and telomere overhang protection in inducible KO cells
Each of the six telomeric proteins participates in the
regulation of telomere length. Previous assessment of
their roles in telomere length control has mostly relied
on RNAi KD and overexpression of mutant proteins in
human cells, which has sometimes yielded conflicting
results. For example, overexpression and RNAi
experiments indicate that TPP1 and POT1 negatively
regulate telomere length [
21, 22, 24, 29, 66?68
disrupting the TEL patch (TPP1 glutamate (E) and
leucine (L)-rich patch) within TPP1 led to decreased
telomere length [
], the latter consistent with the
positive role TPP1 has in recruiting and promoting
telomerase activity [
25, 26, 70, 71
]. In this study, we
sought to better understand how inactivating
individual telomeric proteins may impact telomere length
control using the inducible KO cells.
Deleting the telomere duplex binding proteins TRF1
and TRF2 resulted in significantly elongated telomeres
within a few days following doxycycline addition
(Figure 4a and b, Supplementary Figure S5). In the
case of TRF2, increased telomere fusions following
induced KO (Figure 3c and d) likely led to the apparent
rapid increase in telomere length observed here. In
comparison, inducible RAP1, TIN2 and POT1 KO
cells showed a more gradual increase in telomere
length. The TPP1 KO cells exhibited moderate
acceleration of telomere shortening, which is more in line
with TPP1?s role in recruiting and promoting
Mammalian telomeres are thought to adopt the
t-loop structure, with the ss G overhang invading into
the duplex DNA [
]. The G overhangs are
maintained through telomere DNA synthesis and active
resection by exonucleases of the C-strand on 5? ends
]. Evidence suggests that the POT1?TPP1 complex
coats telomere G overhangs, inhibits nucleolytic
attacks and prevents the binding of the nonspecific
ssDNA binding protein RPA1 and subsequent
activation of ATR-mediated checkpoint responses [
]. Furthermore, TIN2 deletion in mice also led to
RPA1 accumulation and ATR activation, in line with
its role in tethering POT1?TPP1 to telomeres [
Consistent with these previous findings, inhibition of
TPP1, POT1 and TIN2 led to aberrant accumulation
of RPA1 at telomeres in 13.2%, 21.7% and 12.5% of the
cells, respectively (Figure 4c), indicating deprotected
G overhangs. In contrast, no upregulated telomeric
recruitment of RPA1 could be observed upon deletion
of TRF2, TRF1 or RAP1 (Figure 4c and
Supplementary Figure S6). It is possible that TRF1 and
TRF2 can each independently bring the TPP1?POT1
complex to telomeres to protect G overhangs.
In mice, ablation of Tin2, Tpp1 or Pot1a/b led to
extended overhang length [
48, 62, 63, 78?81
Surprisingly, of the six KO lines, only cells induced to KO
POT1 exhibited an increase in overhangs (Figure 4d
and e). We found extensive chromosomal fusions upon
TFR2 KO (Figure 3c and d), which likely
compromised overhang protection and caused the slight
decrease in G overhang length in TFR2 KO cells.
Overlapping phenotypes in mouse cells knocked out of
Tin2, Tpp1 or Pot1a/b, such as TIF induction and
overhang elongation, underline the interdependence of
these proteins. The unexpected lack of overhang
elongation in our TIN2 and TPP1 KO cells suggests that
POT1 may have a protection function independent of
TIN2 and TPP1 in human cells, a major difference
between mouse and human cells in overhang
Human POT1 isoforms participate in telomere overhang regulation
The KO cell lines offer a unique opportunity for us
to investigate the possible functional significance of
splicing variants of telosome subunits. Although
human has one POT1 gene as opposed to two in mice, a
total of five alternatively spliced forms of hPOT1 have
been described to date [
]. hPOT1 V1 is the full-length
form that has been extensively studied (Supplementary
Figure S7A). Little is known about the functional
significance of the other isoforms, which appear to be
expressed in normal and cancer tissues as well as cancer
cell lines [
]. The V4 variant was not examined
here because POT1 coding sequences are interrupted
by an early stop codon. The remaining isoforms share
with V1 the N-terminal OB-fold domain but differ in
their C-termini. We have designed the dual sgRNAs to
inactivate all of the POT1 isoforms (Supplementary
Figure S7A), enabling us to determine the role of each
isoform individually. We expressed CRISPR-resistant
POT1 V1 isoform in the POT1 KO cells and examined
the cells for overhang status, TIF formation and RPA1
accumulation (Figure 5 and Supplementary Figure
S7B?D). As expected, POT1 V1 could localize to
telomeres (Figure 5b), and rescue the phenotypes of
increased TIFs, accumulated RPA1, and excessively
long overhangs (Figure 5c?f). These data support V1 as
the main POT1 isoform that caps telomere ends and
shields them from DDRs.
Studying telosome subunits using inducible knockout cell lines
Consistent with their lack of TPP1-interacting
domains, POT1 variants V2, V3 and V5 could not
interact with TPP1 (Figure 5a). All three isoforms do
contain intact OB-folds that can mediate telomere
ssDNA binding, and therefore may target to telomeres
independent of TPP1. In support of this notion, our
telomere ChIP experiments showed that POT1 V2, V3
and V5 could indeed associate with telomeres
(Figure 5b and Supplementary Figure S7B), albeit with
markedly reduced abilities compared with V1. Binding
of POT1 isoforms to telomeres appeared to increase in
the absence of endogenous POT1 (Figure 5b), probably
because of more sites becoming available upon POT1
deletion, or because of increased overhang length.
Individual expression of the short POT1 isoforms in
POT1 KO cells could not rescue the phenotypes of
RPA1 accumulation or increased telomere DNA
damage (Figure 5c and d and Supplementary Figure
S7C and D). However, they consistently reduced the
increase in telomere overhangs in these cells, albeit to
varying degrees (Figure 5e and f). These data indicate
that the shorter human POT1 isoforms can regulate
overhang length independent of TPP1. Furthermore,
this previously unknown function of the POT1
isoforms may help to explain some of the differential
phenotypes observed between mouse and human
Deletion of telosome subunits leads to metabolic perturbations in the inducible KO cells
Complete deletion of essential genes such as TRF2
and TIN2 causes cell cycle arrest and/or death, making
it difficult to isolate single KO clones or obtain large
numbers of cells for extensive biochemical studies. Our
inducible KO system bypasses the need of KO cell
cloning, and enables the expansion of cells to large
quantities before KO induction for biochemical
analysis such as metabolomic profiling.
Crosstalk between telomere maintenance and
metabolic pathways has been well documented, with
several key telomere regulators including TERT and
TIN2 implicated in more direct metabolic regulation
]. We therefore examined TIN2 KO cells and
determined how completely disrupting TIN2 affected
metabolic control, especially with respect to key
metabolites in glycolysis and the tricarboxylic acid
(TCA) cycle. Research has shown that cancer cells
often consume large quantities of glucose, which fuels
the TCA cycle, as well as pathways for macromolecule
synthesis (for example, nucleotides, amino acids and
lipids) (Figure 6a) [
]. Glucose and other
metabolites such as glutamine serve as substrates in various
bioenergetic pathways to support growth of cancer
cells in which upregulated glycolysis and
glutaminolysis pathways have often been found.
Given that treatment of cells with drugs such as
doxycycline can drastically alter cellular metabolomes,
we decided to compare doxycycline-induced TIN2 KO
cells with doxycycline-treated Cas9-inducible parental
cells that did not express any sgRNA sequences. The
inducible TIN2 KO cells were expanded and then
treated with doxycycline for 6 days before being
collected for analysis by quantitative liquid
chromatography?mass spectrometry (LC-MS). As shown in
Figure 6b, TIN2 KO led to varying changes in a broad
range of metabolites in glycolysis, TCA cycle and
macromolecule synthesis. When we examined the other
telosome subunits, we found TRF1 KO to have the
least impact, only consistent increases in ribose (data
not shown). In comparison, TRF2 KO led to
reproducible increases in a number of metabolites
(Figure 6c). Similarly, knocking down the remaining
subunits resulted in reproducible and differential
changes in certain key metabolites in glycolysis and
macromolecule synthesis. These findings reaffirm the
distinct roles that each subunit has in ensuring the
growth and proliferation of the cell. None of the other
proteins examined had the same widespread effect on
metabolism as TIN2. For example, TIN2 was the only
telosome subunit whose KO affected multiple
metabolites in the TCA cycle, which occurs in the
mitochondria. This observation supports our previous
findings that TIN2 can localize to the mitochondria
and regulate the metabolic pathways in the
Work using mouse models, mutant proteins and
RNAi to probe the functional significance of the
telosome and its subunits has greatly advanced our
knowledge and understanding of telomere
homeostasis. Genetically modified mouse models have been
indispensable to loss-of-function studies, but
differences between mouse and human, as well as the cost
and efforts associated with mouse studies continue to
pose challenges. Major drawbacks of RNAi-mediated
KD include its off-target effects and the inability to
achieve complete inhibition. As a result, many
questions regarding human telomere maintenance remain
unanswered. The RNA-guided CRISPR/Cas9
genome-editing technology has enabled unprecedented
manipulations of the genome in a much more targeted
and efficient manner, particularly in somatic cells and
cell lines [
]. In this report, we describe the
systematic generation and profiling of inducible KO cell
lines for the six core telomere proteins. In all of the
experiments presented, the results came from multiple
independent doxycycline inductions of the inducible
KO cells. Such reproducibility and consistence
underscore the robustness of the inducible system and the
advantage of using polyclonal populations in cellular
assays. We can now more clearly define the function of
human telomeric proteins and identify differential
regulatory mechanisms in human vs mouse.
Organization of the human telosome/shelterin complex
With the inducible KO system, we now have a
clearer picture of how the human telosome complex
may be organized (Figure 6c). Of the six subunits,
RAP1 only interacts with TRF2. Except for telomere
length changes, induced RAP1 deletion had no major
impact in most of the telomere assays described here,
consistent with previous analyses using
RAP1inactivated mouse and cellular models [
addition, removing either TIN2, TPP1 or POT1
markedly impacted the telomere targeting of the other
two proteins, providing strong evidence that these three
proteins likely form a functional unit on telomeres.
Interestingly, TRF2 KO affected the telomeric binding
of all the other subunits except for TRF1, supporting
the existence of the five-protein TRF1-less complex
The role of POT1 in G overhang protection
In mice, both Pot1a and Pot1b can bind Tpp1 and
are tethered to telomeres through Tpp1-Tin2; however,
the two mouse POT1 proteins participate in distinct
signaling events for telomere regulation [
Human POT1, in comparison, appears to carry out the
functions attributed to both POT1a and POT1b. It is
therefore expected that depletion of TPP1 or TIN2 in
human cells would disrupt POT1 targeting to telomeres
and POT1-mediated protection and length regulation
of G overhangs. For example, Tpp1 KO MEFs showed
similar phenotypes as cells doubly knocked out for
]. Although POT1 KO in our inducible
cells led to expected increases in overhang length and
RPA1 staining, we were surprised to discover a lack of
accumulation of elongated telomere ssDNA in cells
knocked out of TIN2 or TPP1. Of the four splicing
variants of human POT1 examined in this study, only
V1 can interact with TPP1, likely because it is the only
variant that retains the C-terminal TPP1-interacting
domain (Figure 5a and Supplementary Figure S7A).
Although isoforms V2, V3 and V5 do not bind TPP1,
they can still associate with telomeres (Figure 5a and
b). Given that our POT1 KO strategy disrupts isoforms
V2, V3 and V5 as well, we speculate that these POT1
isoforms may participate in overhang protection
independent of TPP1 and that the unexpected results seen
in TIN2 and TPP1 KO cells help to highlight this
shared function between different human POT1
isoforms (Figure 6e).
Based on this model, telomere targeting of the
fulllength variant POT1 V1 is disrupted in TPP1 and TIN2
KO cells; however, telomere ssDNA overhangs can
still be protected by other variants, which can localize
to telomeres independent of TPP1-TIN2. These
OBfold only POT1 proteins may associate with telomeres
directly or through interaction with other OB-fold
containing proteins. Indeed, when we ectopically
expressed V2, V3 or V5 in the POT1 KO cells, they
could rescue the overhang length phenotype to varying
degrees. Similar findings were previously reported for
POT1 V5 in POT1 KD cells [
]. Interestingly, POT1
V2, V3 and V5 could not rescue the TIF or RPA1
phenotypes of POT1 KO cells, suggesting distinct
pathways for different POT1 isoforms in regulating
overhang length vs DDR. It is possible that once V2,
V3 and V5 are recruited to telomeres, they can block
exonucleases such as Exo1 from further recessing the 3?
end of telomeres. Collectively, our study supports a
new model of both TPP1-dependent and -independent
regulation of telomere overhangs by human POT1, in
contrast to the TPP1-dependent model for mouse
The role of telomeric proteins in metabolic control
Although previous studies have implicated telomeric
proteins in metabolic regulation, this is the first time
that metabolic changes were systematically
investigated upon deletion of individual subunits of the
shelterin/telosome complex. It is possible that the
metabolic alterations observed in our KO cells were
indirect results of activation of DDR pathways and
changes in telomere length. However, the differences in
the metabolomes in these cells suggest distinct and
significant impact on cellular metabolism as a result of
inhibition of different human telomeric proteins. For
instance, TIN2 KO appeared to impact many of the
metabolites in different metabolic pathways, including
the TCA cycle. The TCA cycle, which occurs in the
mitochondria where it metabolizes the end products of
glycolysis and feeds into oxidative phosphorylation, is
central to energy production and biosynthesis. The
finding that only TIN2 KO appeared to impact TCA
cycle metabolites supports our previous findings of
TIN2 targeting to the mitochondria, and is consistent
with idea that TIN2 can directly regulate metabolism.
Interestingly, although TRF2 KO appeared to also
affect multiple glycolytic, glutaminolytic and nucleic
acid synthesis intermediates, knocking out the
remaining subunits was more restricted in terms of
changes in the metabolome. Whether such differences
are linked to the predominant function of TRF2 in
ATM-mediated DDR response warrants further
investigation. Taken together, these data underline the
complex crosstalk between telomere maintenance and
Application of the inducible CRISPR/Cas9 KO system
For genes essential for growth and survival, the
inducible KO cell lines afford the time window needed
to carry out biochemical studies before the cells
undergo growth arrest. For example, we could not
obtain straight KO clones of cells deleted for TIN2,
TRF2 or POT1, but the inducible KO cells have
allowed us to explore the functions of these proteins in
a variety of assays. In theory, a single sgRNA targeting
a specific site within a locus should effectively generate
cells with frame-shift indels that inactivate the target
gene. In our induced single sgRNA-targeted KO cells,
we often found low expression levels of the target genes
after Cas9 induction. This residual expression is likely a
result of the induced KO cells containing a mixture of
alleles with different indels (some of which cannot
completely disrupt target gene function), and differs
from that observed in RNAi KD cells. In the latter,
every cell likely still expresses the target gene at a
certain level following incomplete suppression by small
interfering RNAs/short hairpin RNAs. The polyclonal
induced KO cells, on the other hand, comprise mostly
of cells completely knocked out for the target gene,
with a small fraction of cells that may contain in-frame
indels or can ?restore? expression following extended
culturing. This is an important distinction because
residual expression in nearly every RNAi KD cell may
be sufficient for the entire population to ?behave?
normally in an assay. However, a very small fraction of
cells with heterozygous KO or wild-type alleles are
unlikely to dilute or mask the response of the whole
population, if the overwhelming majority of cells have
no expression of the gene.
Based on our data, more complete deletion and
inactivation can be achieved with two sgRNAs. A
second sgRNA significantly reduced the possibility of
in-frame ligations, as well as the ability of cells to
overcome inactivating mutations, although with the
caveat of possibly increasing off-targets. As the
induced cells are not single clones, possible
complications in data interpretation from off-target effects may
be less likely. Moreover, expression of
CRISPRresistant constructs could rescue the observed
phenotypes, which helped to rule out potential off-target
It appeared to take similar amount of time for our
inducible cells to achieve efficient KO as for cells
transfected with plasmids encoding Cas9 and sgRNA
to generate straight KO. The isolation of straight KO
cells, however, requires significantly longer time (for
non-essential genes), whereas large numbers of induced
KO cells can be more quickly obtained. The inherently
mixed nature of the induced KO population does have
the potential to introduce variations. The distribution
of various populations (+/+ vs +/ ? vs ? / ? ) should in
theory be similar each time the cells are induced. Deep
sequencing of the induced KO cells may help to shed
light on the exact dynamics of the KO populations, but
the short reads of such methods will fail to capture any
large deletions, especially with our dual sgRNA cells.
Any variability in population dynamics is more likely
to be caused by variable Cas9 induction than the
constitutively expressed sgRNAs. Hence, using the same
inducible Cas9 clone to generate all six cell lines for
comparative studies should help minimize variations.
Furthermore, each of our cell lines had been induced
multiple times independently for each assay and the
results were found to be similar, attesting to the
reproducibility of the system.
The six inducible cell lines should prove particularly
useful to investigators who may be interested in
studying different aspects of telomere maintenance.
For instance, in addition to the ability to assay cells in
which essential genes are deleted, this system also
enables real-time comparisons of specific protein
complexes before and after the removal of a key
subunit. Furthermore, our method makes possible both
the production of large numbers of genetically edited
cells for studies of essential genes and the generation of
more well-defined snapshots of cells in response to
Materials and Methods
Sequences encoding the humanized Cas9 gene under the
control of the tetracycline-responsive promoter were cloned into
a lentiviral vector that also encodes rtTA [
sgRNA sequences (Supplementary Table S1) were cloned into
modified vectors encoding different antibiotic resistance genes
(puromycin, blasticidin or hygromycin). These vectors are based
on the LentiCRISPR vector (GeCKO) but no longer contain
Cas9 sequences [
]. Complementary DNAs encoding wild-type
and rescue mutants for sgRNA-resistant hTIN2 and hPOT1
isoforms were cloned into a pHAGE-based lentiviral vector for
C-terminal tagging with HA and FLAG epitopes [
constructs were generated by introducing either
singlenucleotide mutations in the PAM sequence (TIN2 sgRNA1:
GGG ? GAG, TIN2 sgRNA2: TGG ? TGA, POT1 sgRNA1:
AGG ? AGA) or double-nucleotide mutations in the sgRNA
target region (POT1 sgRNA2: GGAGGTACCAGTTAC
GGTCG ? GGAGGTACCAGTTACGGAAG).
Generation of inducible CRISPR KO cell lines
Hela cells stably expressing doxycycline-inducible Cas9 were
first generated by lentiviral transduction. A single clone with
robust and efficient Cas9 induction was selected for further
experiments. Vectors expressing single or dual sgRNAs were
stably introduced into the Cas9-inducible cells by lentiviral
transduction followed by selection with appropriate antibiotics.
The appropriate concentrations for Cas9 induction and efficient
cleavage at the intended locus were determined for each cell line.
We have found that 6 days of incubation in 1 ?g ml?1 of
doxycycline is optimal for our cells. Successful inactivation of each
gene was confirmed by western blotting with the appropriate
antibodies. Further validation was conducted by extracting
genomic DNA from the cells either for direct sequencing or for
TOPO cloning before Sanger sequencing.
Immunoprecipitation, immunoblotting and telomere ChIP assays
Co-immunoprecipitation studies were performed as
described previously [
]. Cells were lysed in 1 ? NETN buffer (1 M
Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl and 0.5%
Nonidet P-40) containing 1 mM DTT and a proteinase inhibitor
mixture (Roche Applied Science, Mannheim, Germany). The
lysates were then immunoprecipitated with appropriate
antibodies for sodium dodecyl sulfate?polyacrylamide gel
electrophoresis and western blotting.
Telomere ChIP assays were performed as described
previously with slight modifications [
]. Briefly, cells were
chemically crosslinked in1% formaldehyde in phosphate-buffered
saline, and sonicated to shear chromatin. Sonicated lysates were
pre-cleared before being incubated with 3 ?g of antibodies for
immunoprecipitation. The co-precipitated DNA was eluted and
analyzed by dot-blot and southern hybridization using the
32Plabeled telomere (TTAGGG)3 and Alu repeat probes.
Antibodies used for immunoprecipitation and western blot
analyses in this study are: horseradish peroxidase-conjugated
anti-glutathione S-transferase (GST) polyclonal antibody (GE
Healthcare Life Science, Pittsburgh, PA, USA), horseradish
peroxidase-conjugated anti-FLAG M2 antibody and
M2conjugated agarose beads (Sigma, St Louis, MO, USA), rabbit
anti-FLAG polyclonal antibody (Sigma), goat anti-actin
polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA)
and rabbit anti-SMC1 antibody (Bethyl Laboratories,
Montgometry, TX, USA), mouse anti-TRF2 monoclonal antibody
(Calbiochem, San Diego, CA, USA), rabbit anti-RAP1
polyclonal antibody (Bethyl Laboratories), rabbit anti-POT1
polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit
anti-TPP1 and anti-TIN2 polyclonal antibodies [
], and goat
anti-TRF1 antibody [
], rabbit anti-p-Chk1(Ser317) and
antiChk1 antibodies (Cell Signaling Technology, Danvers, MA,
USA), and rabbit anti-p-Chk2(Thr68) and anti-Chk2 antibodies
(Cell Signaling), mouse anti-p-ATM (Ser1981) and rabbit
antiATM antibodies (Cell Signaling), rabbit anti-p-ATR(Ser428)
and anti-ATR antibodies (Cell Signaling) and rabbit anti-HA
antibody (Santa Cruz Biotechnology).
Cell proliferation assay and cell cycle analysis
Cells were plated in 12-well plates at 1 ? 104 cells per well and
maintained for 10 days with or without 1 ?g ml?1 doxycycline.
The number of viable cells at various time points was determined
by Trypan blue exclusion. To determine DNA content, 1 ? 106
cells maintained with or without doxycycline (1 ?g ml?1) for
6 days were collected, washed with 1 ? phosphate-buffered
saline and then fixed in 70% ethanol at room temperature for
30 min. The fixed cells were then incubated in 0.5 ml 1 ?
phosphate-buffered saline containing 50 ?g ml?1 propidium iodide
and 0.2 mg ml?1 DNase-free RNase A (pH 7.4) at 37 ?C for
30 min. The cells were subsequently analyzed using an LSRII
flow cytometry analyzer (BD Biosciences, San Jose, CA, USA).
IF and telomere fluorescence in situ hybridization (FISH)
IF was performed as previously described [
]. Briefly, cells
grown on glass coverslips were permeabilized for 30 s with 0.2%
Triton X-100, fixed in 4% paraformaldehyde and then
permeabilized again with 0.5% Triton X-100, before being blocked in
5% bovine serum albumin. Cells were subsequently incubated
with appropriate antibodies and/or a telomere peptide nucleic
acid (PNA) probe (Bio-PNA). 4,6-Diamidino-2-phenylindole
was used to visualize the nuclei. For TIF assays, 4100 cells were
examined for each experiment, and cells with 45 co-stained foci
were counted as being TIF positive. At least three independent
experiments were performed for each cell line.
Antibodies used for IF are: mouse anti-FLAG M2 and rabbit
anti-FLAG polyclonal antibodies (Sigma), rabbit anti-53BP1
(NB100-304; Novus Biologicals) and mouse anti-53BP1 (BD
Biosciences) antibodies, rat anti-RPA1 polyclonal antibody
(Cell Signaling), goat anti-TRF1 polyclonal antibody, mouse
anti-TRF2 monoclonal antibody (Calbiochem), rabbit
antiRAP1 polyclonal antibody (Bethyl Laboratories), rabbit
antiTPP1 and anti-TIN2 polyclonal antibodies [
], rabbit anti-POT1
polyclonal antibody (Bethyl Laboratories) and rabbit anti-HA
antibody (Santa Cruz Biotechnology).
Metaphase spread and telomere fluorescence in situ
hybridization analysis was performed as previously described [
Briefly, cells were incubated with 0.1 ?g ml?1 colcemid
(KaryoMax, Invitrogen, Carlsbad, CA, USA) for 3 h and harvested.
The cells were then incubated in hypotonic solution (0.075 M
KCl) for 25 min at room temperature, fixed in methanol/glacial
acetic acid (3:1) solution for 5 min and spread onto clean slides.
The slides were treated with pepsin, fixed in 4% formaldehyde,
dehydrated in successive ethanol baths (70, 90, and 100%) for
5 min each and air dried. The slides were subsequently
denatured at 80 ?C for 3 min and then hybridized with telomere PNA
probes (Bio-PNA) at 25 ?C for 2 h in the dark. The slides were
then washed, dehydrated and mounted with VectaShield
mounting medium containing 4,6-diamidino-2-phenylindole
(Vector Laboratories, Burlingame, CA, USA). At least 50
metaphase spreads were captured using a Zeiss Imager Z1
microscope (G?ttingen, Germany) and analyzed using
AxioVision 4.8(G?ttingen, Germany).
TRF assay and telomere overhang analysis
Cells were first induced with doxycycline for 6 days, and then
collected for TRF analysis at various time points to estimate the
average length of telomeres using TeloRun [
]. The in-gel
detection of telomere ssDNA overhangs was performed as
previously described with slight modifications . Genomic
DNA was digested with HinfI and RsaI (New England Biolabs,
Ipswich, MA, USA) for 16 h. In all, 5 ?g each of the digested
DNA was then incubated with and without 20 U of Exonuclease
I (New England Biolabs) for 6 h. The reaction mixtures were
fractionated on a 1.2% agarose gel in 1 ? Tris/borate/EDATA
(TBE) buffer for 1.5 h at 60 V and dried on a gel dryer at 50 ?C
for 2 h. The dried gel was pre-hybridized in hybridization buffer
(0.5 M Na2HPO4 pH 7.2, 1 mM EDTA, 7% sodium dodecyl
sulfate) and hybridized in fresh buffer with a 32P-labeled
C-strand probe (CCCTAA)3. The gel was washed 3 ? with
2 ? SSC containing 0.1% sodium dodecyl sulfate for 30 min at
room temperature and analyzed on a PhosphorImager (GE
Healthcare Life Science). The gel was subsequently denatured
(0.5 M NaOH, 1.5 M NaCl for 30 min), neutralized (0.5
MTrisHCl for 30 min), and then hybridized with the C-strand or Alu
repeat probes as control.
Metabolomic analysis by liquid chromatography-mass spectrometry
Each cell line was induced with 1 ?g ml?1 doxycycline,
cultured and harvested in multiple (3?4) replicates (~5 ? 106 cells
each), and frozen in aliquots before metabolome extraction as
described previously [
]. Briefly, cells were three times frozen
and thawed and resuspended in ice-cold methanol:water (750 ?l,
4:1) containing 20 ?l of internal standards (Tryptophan-15N2,
Glutamic acid-d5, Thymine-d4, Gibberellic acid, Trans-Zeatin,
Jasmonic acid, Anthranilic acid and Testosterone-d3, all from
Sigma-Aldrich). Homogenization entailed 2 ? 30-s pulses,
10min vortex mixing with ice-cold chloroform (450 ?l), and 2-min
vortex mixing with ice-cold water (150 ?l). The homogenate was
incubated at ? 20 ?C for 20 min and centrifuged at 4 ?C for
10 min to partition aqueous and organic layers for drying at
37 ?C for 45 min. The aqueous extract was reconstituted in
500 ?l of ice-cold methanol:water (50:50) and filtered at 4 ?C for
90 min through 3 kDa molecular filters (AmiconUltracel ? 3 K
Membrane, Millipore Corporation, Billerica, MA, USA). The
filtrate was dried for 45 min at 37 ?C before resuspension in
100 ?l of methanol:water (50:50) containing 0.1% formic acid
(Sigma-Aldrich). High-performance liquid chromatography
(HPLC) analysis was performed using an Agilent 1290 series
HPLC system equipped with a degasser, binary pump,
thermostatted autosampler and column oven (Agilent Technologies,
Santa Clara, CA, USA). The multiple reaction
monitoringbased measurement of relative metabolite levels used reverse or
normal phase chromatographic separation. All samples were
kept at 4 ?C and 5 ?l was used for analysis.
TCA metabolites were separated through normal phase
chromatography. The binary pump flow rate was 0.2 ml min?1
with 80% B to 2% B gradient over 20 min, 2% B to 80% B for
5 min and 80% B for 13 min. The flow rate was gradually
increased as follows: 0.2 ml min?1 (0?20 min), 0.3 ml min?1
(20.1?25 min), 0.35 ml min?1 (25?30 min), 0.4 ml min?1
(30?37.99 min) and 0.2 ml min?1 (5 min). Metabolites were
separated on a Luna Amino (NH2) column (4 ?m, 100A
2.1 ? 150 mm, Phenominex, Torrance, CA, USA) in a
temperature-controlled chamber (37 ?C). All columns used were
washed and reconditioned after every 50 injections.
HPLCgrade acetonitrile, methanol and water were from Burdick &
Jackson (Morristown, NJ, USA). The calibration solution
containing multiple calibrants in acetonitrile/trifluroacetic acid/
water was from Agilent Technologies. Data were curated
through quality control assessment (using data from sample
pools) and normalization using internal standards.
All experiments were independently repeated at least three
times and presented as mean ? s.d. or mean ? s.e. Statistical
analyses were performed using either Student?s t-test or one-way
analysis of variance. Significant differences were defined as
P o 0.05 or lower.
Conflict of Interest
The authors declare no conflict of interest.
This work was supported by the National Key Research and
Development Program of China (2017YFA0102800 and
2017YFA0102801), National Natural Science Foundation of
China (NSFC 91640119 and 81330055), Science and
Technology Planning Project of Guangdong Province
(2015B020228002) and Guangzhou Science and Technology
Project (201605030012). We would also like to acknowledge the
support of NIGMS GM095599, NCI CA211653, CPRIT
RP160462, the Welch Foundation Q-1673, HL131744 and the
C-BASS Shared Resource (with special thanks to Dr JX) at the
Dan L Duncan Cancer Center (DLDCC) of Baylor College of
Medicine (P30CA125123). This research was also made possible
through the support by the CPRIT Core Facility Support
Award RP120092, Proteomic and Metabolomic Core Facility,
NCI/2P30CA125123-09 Shared Resources Metabolomics core,
and funds from the Dan L Duncan Cancer Center (DLDCC).
HK, FL, QH, TD and FJ performed experiments; HK, FL,
QH and NP performed data analysis; HK, DL and ZS designed
experiments; HK, DL and ZS wrote the manuscript.
Studying telosome subunits using inducible knockout cell lines
(Supplementary Information is linked to the online version of the
paper on the Discovery website.)
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credit line; if the material is not included under the Creative
Commons license, users will need to obtain permission from the
license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
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