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Blood First Edition Paper, prepublished online October 15, 2014; DOI 10.1182/blood-2014-06-582254
Article title
An anti-apoptotic role for telomerase RNA in human immune cells independent of
telomere integrity or telomerase enzymatic activity
Short title
An anti-apoptotic role for telomerase RNA in CD4 T cells
Authors and affiliations
Francesca S. Gazzaniga1-2 and Elizabeth H. Blackburn1*
1
Department of Biophysics and Biochemistry, University of California, San Francisco,
600 16th St Genentech Hall S-312F San Francisco, CA, 94158 USA;
2
currently at Department of Molecular Biology and Immunobiology, Harvard Medical
School, 77 Ave Louis Pasteur NRB 1058 Boston, MA 02115 USA
*Corresponding author; email: [email protected]
The online version of the article contains a data supplement.
Category
IMMUNOBIOLOGY
Text word count: 4000
Abstract word count: 197
Figures: 6
References: 48
1
Copyright © 2014 American Society of Hematology
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Key Points
Telomerase RNA component hTR, but not the core enzymatic protein component
hTERT, protects T cells from apoptosis.
hTR prevents dexamethasone-induced apoptosis specifically when in a telomerase
enzymatically-inactive state.
2
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Abstract
Telomerase is a ribonucleoprotein complex that adds telomeric DNA to the ends of
linear chromosomes. It contains two core canonical components: the essential RNA
component, hTR, which provides the template for DNA synthesis, and the reverse
transcriptase protein component, hTERT. Low telomerase activity in circulating
peripheral blood mononuclear cells has been associated with a variety of diseases.
However, whether telomerase, in addition to its long-term requirement for telomere
maintenance, is also necessary for short-term immune cell proliferation and survival has
been unknown. We report that overexpression of enzymatically inactive hTR mutants
protected from dexamethasone-induced apoptosis in stimulated CD4 T cells.
Furthermore, hTR knock-down reproducibly induced apoptosis in the absence of any
detectable telomere shortening or DNA damage response. In contrast, hTERT
knockdown did not induce apoptosis. Strikingly, overexpression of hTERT protein
caused apoptosis that was rescued by overexpression of enzymatically-inactive hTR
mutants. Hence we propose that hTR can function as a noncoding RNA that protects
from apoptosis independent of its function in telomerase enzymatic activity and longterm telomere maintenance in normal human immune cells. These results imply that
genetic or environmental factors that alter hTR levels can directly affect immune cell
function to influence health and disease.
3
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Introduction
Telomerase is a ribonucleoprotein complex with a well-established function of
adding telomeric DNA to the ends of linear chromosomes. In addition to associated
factors, the two core human telomerase enzyme components are the reverse
transcriptase protein hTERT, and the RNA templating component hTR, (also called
hTER or hTERC). Many studies have associated telomerase activity levels in resting
adult human peripheral blood mononuclear cells (PBMCs) to different health and
disease states. For example, inherited telomerase mutations resulting in
haploinsuffiency cause telomere syndromes, characterized by lung fibroses, cancer
predisposition, and bone marrow failure. In wild type individuals, low telomerase activity
in resting PBMCs is associated with risk factors for aging-related diseases and chronic
stress 1–3, suggesting that low telomerase might indicate, or promote, certain disease
states. While increases in resting PBMC telomerase activity have been reported
associated with meditation, healthy lifestyle changes, decreased measures of
psychological distress 4–6 and decreased LDL 7, the combination of short white blood
cell telomeres with high telomerase activity has also been associated with a variety of
disease risk factors including chronic psychological stress 8–12. Hence regulating
telomerase activity levels is important for maintaining health and proper immune
function, but the complicated relationships observed between telomerase levels in
PBMCs and health and disease states indicate the need for a fuller understanding of
roles of telomerase components.
The PBMC studies described above compared average telomerase activity from
heterogeneous populations containing several different cell types, and are potentially
confounded by changes in fractions of specific cell types. In vitro studies, including the
current study, have investigated associations between telomerase activity levels and
immune cell function in individual cell types. For example, CD4 T lymphocytes greatly
modulate telomerase activity levels 13,14, from very low in the resting state to large
increases, upon stimulation, of telomerase enzymatic activity, hTERT mRNA, and
hTR14–16; as cell proliferation slows, the levels of all three decrease 16–19. Furthermore, if
T cell proliferation in vitro is hindered by cortisol, actinomycin D, cycloheximide, or
Herbimycin A, telomerase activity is also reduced 16,20. While CD4 T cell proliferation
and telomerase activity correlate, it is unknown whether telomerase activity is necessary
for or even quantitatively coupled to this proliferative response. As proliferation upon
stimulation is an essential function for CD4 T cells, understanding the role of telomerase
in T-cell proliferation is important for understanding normal T-cell and immune function.
4
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We report here that unexpectedly, hTR specifically is important for short-term
CD4 T cell survival. While the level of telomerase activity is important for long-term CD4
T cell survival, this work identifies a new, telomere- and telomerase activity-independent
function of telomerase RNA in immune cells that we postulate acts in a cell-protective,
anti-apoptotic pathway that can be influenced by stress or other regulatory factors.
Materials and Methods
Cell culture
Human buffy coats from 9 healthy donors between 17 and 25 years old were purchased
from Stanford Blood Center. PBMCs were isolated from buffy coat by centrifugation with
Ficoll-Paque Plus (GE Healthcare), and CD4 T cells were isolated from PBMCs using
the Untouched CD4+ T cell Isolation Kit II Human (Miltenyi). Cells were stimulated 24
hours after isolation with 50 μl of Dynabeads Human T Cell Activator CD3/CD28 (Life
Technologies) per 1 million CD4+ T cells and cultured in RPMI with 10% FBS, 1%
Penicillin and Streptomycin, and 1% glutamine with 10 ng/ml IL-2. Cells were
transduced with lentivirus 24 hours after stimulation. 2 μg/ml of Puromycin was added
24 hours after transduction and kept in culture during the course of the experiment. Live
cells and percent live cells determined by Trypan blue exclusion were counted with the
TC-20 Automated Cell Counter (BioRad). Dexamethasone treatment was 1μM
concentrations for 72 hours.
Plasmids and lentivirus
The lentiviral vector system was provided by Dr. Trono (University of Geneva, Geneva
Switzerland 21). Lentivirus and shRNA expression vectors were prepared as described
previously 22. The following lentiviruses were generated from those previously
described in 22 but the CMV promoter was replaced with EF1alpha promoter driving
either Puromycin resistance or GFP: Empty vector, shScramble, shTR1. shTERT was
generated in the same lentiviruses based on the target sequence from 23. shTR2 was
generated based on a previously published target sequence 24. shBIM was generated
based on the previously published target sequence 25. CMV-GFP-IRES-puromycin and
CMV-TERT-IRES-puromycin lentiviruses were described in 23. The hTR overexpression
constructs were generated by PCR amplification of U3hTR500 from the following
plasmids from Kathleen Collins (University of California Berkeley, CA) and cloned into
the pHR vector with ef1alpha driving puromycin resistance, pBS’U3-hTR-500, pBS’U3Δ96-7-500, pBS’U3-C204G-500, pBS’U3-G305A-500, pBS’U3-hTR-U64-500 26,27.
Lentivirus was titered by qRT-PCR. See supplemental methods for protocol.
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Flow cytometry
Cell cycle state was measured by staining with Vibrant DyeCycle Green (Life
Technologies) according to the manufacturer’s instruction.
Apoptosis
Apoptosis was measured with the Caspase –Glo 3/7, Caspase-Glo 8, Caspase –Glo-9
kits (Promega) according to the manufacturer’s instructions. Luminescence was read
by the Veritas Microplate Luminometer (Turner Biosystems).
Telomere Repeat Amplification Protocol
Relative telomerase activity was measured by the real-time quantitative telomere repeat
amplification protocol RQ-TRAP and samples were determined to be in the linear range
of this assay 28,29.
Quantitative RT-PCR
hTR RNA levels and mRNA levels were measured as described in 29. See
Supplemental Table 2 for primers used.
Immunofluorescence (IF)-PNA-FISH
Cells were mounted on coated glass Cytoslides (Thermo Scientific) by centrifugation
with the CytoSpin (Thermo Scientific). Cells were fixed and permeabilized.
Immunostaining was performed with the primary antibodies anti-phospho histone H2A.X
(05-636, Millipore) or pAb anti 53BP1 (NB 100-304, Novus Biochemicals) and the
secondary antibody AlexaFluor 594 (Molecular Probes) Primary antibodies were diluted
1:500, secondary antibodies were diluted 1:750. DNA was visualized with 4’,6diamidino-2-phenylindole (DAPI, Life Technologies). IF was followed by telomere FISH
as described in 30 without pepsin treatment. The telomeric PNA probe used was FAMOO-ccctaaccctaaccctaa (Panagene) at 0.5μg/ml. All images were obtained using a
Deltavision RT deconvolutioon microscope (Applied Precision) with the 100x/1.4N
PlanApo objective (Olympus). Images were acquired in 0.5 μM increments,
deconvoluted, Z-projected in Softworx (Applied Precision), and adjusted for brightness
and contrast in FIJI 31. Telomeric and DNA damage foci and telomeric and DNA
damage integrated intensity were measured with CellProfiler image analysis software
(www.cellprofiler.org; pipelines available on request).
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Antibodies and Western Blot analysis
SDS-PAGE and Western blotting was conducted on whole cell lysates using the
Hybond-P PVDF membrane (GE Healthcare) with either the Western Lightning Plus
ECL kit (Perkin Elmer) or the SuperSignal West Femto Kit (Thermo Fisher) according to
the manufacturer’s instructions.
The following primary antibodies were used: Rabbit anti-Bid (human) at 1:1000
(Cell Signaling Technology 2002), Rabbit anti-Bim at 1:1000 (Cell Signaling Technology
2819), Rabbit anti-Puma at 1:1000 (Cell Signaling Technology 4976), Rabbit anti-Bad at
1:1000 (Santa Cruz Biotechnology sc-943), mouse anti-p53 (Abcam PAb 240), and
mouse anti-GAPDH at 1:20,000 (Millipore MAB374). The following secondary
antibodies were used: goat anti-rabbit at 1:2000 (Jackson Immunoresearch 111-035144) and sheep anti-mouse (Jackson Immunoresearch 515-035-003) at 1:2000 p53 and
1:10,000 for GAPDH.
Statistical analysis
All statistical analysis was performed using Prism (GraphPad Software). Significant
differences were assessed by either one-way ANOVA or by unpaired t-tests as
indicated. A cutoff of p<0.05 was used to determine significance.
Results
hTR overexpression protects from dexamethasone-induced apoptosis independent of
telomerase activity
Previous work has demonstrated that dexamethasone treatment reduces T cell
survival and telomerase activity in short term in vitro experiments 4–6,20. We tested
directly if increasing telomerase components mitigated the effects of dexamethasoneinduced apoptosis on T cell survival.
We overexpressed wild type (WT) hTR or different disease-causing
enzymatically-inactive mutants of hTR (Δ96-7, G305A, hTR-U64) from a lentiviral vector
in stimulated CD4 T cells incubated for 72 hours in the presence or absence of
dexamethasone, a corticosteroid that induces Bim-mediated apoptosis in T cells 32,33.
The hTR Δ96-7 mutation, located in the pseudoknot in the 5’ half of hTR, abolishes
telomerase enzymatic activity but allows normal hTERT binding 27. The G305A
substitution point mutation in the stem of the P6.1 stem-loop in the 3’ half of hTR also
completely abolishes catalytic activity and reduces binding to hTERT by 80%. As a
control for overexpression of a non-coding RNA, we used the hTR-U64 fusion chimeric
RNA, which contains the 5’ half of hTR (pseudoknot and template regions) but the 3’
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half (CR 4/5 and box H/ACA regions) is replaced with the H/ACA domain of the similarly
sized U64 snoRNA. This fusion RNA is stably expressed but cannot bind hTERT and
thus does not confer telomerase activity. 27
Without dexamethasone, overexpressing WT hTR increased telomerase activity
without increasing TERT mRNA (Fig. 1A,B), suggesting that either hTERT exists in the
cell without hTR or that hTR overexpression stabilizes hTERT protein (Fig. 1A,B). The
increased telomerase activity did not protect against dexamethasone-induced apoptosis
(Fig. 1C comparing red solid to checkered bars). As expected, overexpression of control
vectors or the catalytically inactive hTR mutants did not increase telomerase activity or
affect hTERT mRNA levels (Fig. 1A,B). Surprisingly however, overexpressing the
G305A point mutant hTR, but not WT hTR, or hTR-U64, protected from
dexamethasone-induced apoptosis (Figure 1C, solid to checkered bars). Thus,
protection from dexamethasone-induced apoptosis by catalytically inactive
overexpressed hTR occurs and requires a region(s) located in the 3’ half of hTR.
We determined whether fully WT-sequence hTR, in a catalytically inactive state,
can protect against dexamethasone-induced apoptosis. To increase the fraction of
catalytically inactive endogenous WT hTR, we knocked down hTERT using shRNA.
hTERT knock-down alone was sufficient to prevent dexamethasone-induced apoptosis
(Figure 1D, comparing solid to checkered pink bars). This dexamethasone effect also
extended to a natural corticosteroid; shRNA targeting hTERT also protected from
apoptosis induced by the clinically shorter-acting hydrocortisone, while the control
shScramble or hTR knockdown did not (Fig. S1). When hTERT knockdown was
combined with hTR overexpression, either WT hTR or G305A hTR protected from
dexamethasone-induced apoptosis. Thus when hTERT levels are reduced, both
endogenous and overexpressed WT hTR, as well as overexpressed G305A hTR, can
protect from dexamethasone-induced apoptosis (Fig. 1D). Again, hTR-U64 fusion RNA
overexpression, even when combined with hTERT knockdown, failed to protect from
dexamethasone-induced apoptosis. Because knocking down hTERT alone protected
from apoptosis, this suggests that the overexpressed hTR-U64 fusion interfered with the
ability of endogenous WT hTR to protect from apoptosis
In summary, WT hTR, when catalytically inactive, protects CD4 T cells from
dexamethasone-induced apoptosis and hTR-U64 fusion RNA interferes with this
protection.
hTR knockdown induces the intrinsic apoptotic pathway
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Because increasing catalytically inactive hTR protected from dexamethasoneinduced apoptosis we predicted that, conversely, reducing endogenous hTR would
increase apoptosis. We knocked down telomerase in human CD4 T cells, using
lentiviral vectors targeting hTERT (shTERT), and two different anti-hTR shRNAs
targeting the hTR templating sequence (shTR1) or the hTR pseudoknot structure
(shTR2), previously reported to specifically knock down telomerase RNA in human
cancer cell lines and primary fibroblasts 22,24. After 14 days, each of these shRNAs (see
Fig. S2 for optimization procedures) equally reduced telomerase activity levels
(approximately 80%) compared to two different controls: empty lentivector or
scrambled-sequence shRNA (shScramble) (Fig. 2A). Knock-down of either core
component of telomerase did not affect the transcriptional steady state level of the other
(Figure 2B). Notably, only hTR knockdown, and not hTERT knockdown, resulted in
fewer live cells compared to empty vector, shScramble, or shTERT, as measured by
Trypan blue staining (Fig. 2C; see day 15). This result was confirmed using CD4 T cells
isolated from 6 additional donors in 8 independent experiments (data not shown), and
by using GFP vectors instead of puromycin resistant vectors (Fig. S3). Furthermore,
hTR knockdown did not affect the percentages of naïve, central memory, effector
memory, or Th1 CD4 T cells (Fig S4). Additionally, the reduction of live cells with hTR
knockdown was observed when starting with only naïve CD4 T cells (Fig S5). The
reduction in live cells was not due to disruption of progression through the cell cycle (Fig.
2D). Instead, hTR knockdown, but not hTERT knockdown, induced apoptosis,
measured by increases in Caspase 3/7 and Caspase 9 activities, with no change
detected in Caspase 8 activity (Fig. 2E). Together these results indicated that hTR
knockdown activated the intrinsic apoptotic pathway in stimulated CD4 T cells, whereas
hTERT knockdown did not.
Western blotting measures of protein levels of the apoptotic proteins Bim, Bad,
Bid, Puma, and p53 showed upregulation of both Puma, which triggers Bim
upregulation, and of Bim, which triggers Caspase 9 activation, with hTR knockdown
compared to shScramble and shTERT (Fig. 2F). No changes were observed in p53
levels, suggesting that PUMA is triggered through a p53- and DNA damageindependent pathway 34,35. Combining Bim depletion with hTR knockdown increased
cell viability (Fig. 2G), confirming that Bim may be at least partially responsible for the
apoptosis induced by hTR knockdown.
Additional experiments eliminated extrinsic factors as contributing to hTR
knockdown-induced apoptosis. Media from hTR knockdown cells did not induce
apoptosis in control cells (Fig. S6). Similarly, when hTR was knocked down with
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lentiviral vectors expressing GFP, survival of surrounding GFP- cells was not affected
(Fig. S3). Furthermore, genome-wide microarray analysis of hTR or hTERT knockdown
compared to shScramble did not reveal any transcriptional changes of genes involved in
apoptosis, suggesting that hTR knockdown triggers apoptosis at a post-transcriptional
level. After validation by qPCR, only three immune-function genes were statistically
significantly upregulated by hTR knockdown compared to shScramble: CXCL10,
CXCL11, and IL6. As they are not directly involved in apoptosis, this may instead result
from apoptosis. Interestingly several snoRNAs significantly changed with hTR
knockdown compared to hTERT knockdown, suggesting potential involvement of
snoRNA pathways (Fig. S7). The combined results indicate that in stimulated CD4+ T
cells, hTR knockdown was sufficient to induce cell-intrinsic Bim-mediated apoptosis.
Apoptosis induced by hTR knockdown is telomere length- and damage-independent
Short or deprotected telomeres can trigger a p53-mediated DNA damage
response to induce apoptosis or senescence in normal cells (Karlseder et al., 1999; de
Lange, 2009a). Although the short duration of our experiments and the lack of p53
upregulation (Fig. 2F) argued against short or damaged telomeres as the trigger for the
observed hTR knockdown-induced apoptosis, we directly analyzed telomere shortening
and telomere damage. Telomere length distributions, determined by measuring the
integrated intensity of individual telomeric PNA foci, showed no significant shortening or
change in length distribution in shTERT, shTR1, or shTR2 cells compared to each other
and to no virus, empty vector, and shScramble (Fig. 3A; Fig. S8A). We also found no
significant difference in the numbers or length frequency distributions of telomeres
detected/area in controls, hTERT knockdown or hTR knockdown cells (Fig. 3B; Fig.
S8B). This ruled out the possibility that the shortest (and therefore most likely
uncapped) telomeres were not detected by this method. As a positive control, shRNAmediated knockdown of TIN2, a known telomere-protective shelterin protein, resulted in
significant telomere PNA-FISH signal reductions and concomitantly decreased
telomeres detected/area (Fig. S9 A,B). Thus neither hTERT nor hTR knockdown
induced significant telomere shortening in the timeframe of our experiments.
To assess if DNA damage at the telomeres occurred independent of telomere
length, we measured co-localization between telomeres and the DNA damage pathway
proteins γH2AX or 53BP1; such co-localizations are termed TIFs (Telomere DNA
damage-Induced Foci). hTR knockdown did not induce an increase in γH2ax or 53BP1
TIFs/telomere or TIFs/DNA damage foci (Fig. 3C, D; Fig. S8C,D). or increase the total
amount γH2ax of 53BP1 foci in nuclei regardless of localization (Fig. 3E; Fig. S8E), in
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contrast to the shTIN2 positive control (Fig. S9C, D, E). These results together
provided additional evidence that hTR knockdown does not induce apoptosis via
telomere shortening or a telomeric DNA damage response, in the timeframe of our
experiments.
Overexpression of hTERT but not of the catalytically inactive β- hTERT isoform is
sufficient to induce apoptosis
We also manipulated hTERT, without manipulating the endogenous hTR, to
further test the hypothesis that hTR in a state incapable of supporting telomerase
enzymatic activity is the form of hTR that protects from apoptosis. Overexpression of full
length, enzymatically competent hTERT increased telomerase activity, indicating that
some hTR normally exists in CD4 cells unbound to hTERT. We predicted that the
overexpressed hTERT would increase the fraction of hTR assembled into catalytically
active telomerase, thus reducing the level of catalytically inactive hTR. Consistent with
this prediction, full-length hTERT overexpression decreased the live cell number and
increased Caspase 3/7 activity (Fig. 4A-D). We overexpressed the β-splice variant of
hTERT protein. This natural major isoform of hTERT lacks a portion of the telomerase
enzyme active site but binds hTR efficiently, hence assembling it into a catalytically
inactive protein-hTR complex 29. Overexpression of either full-length or β- hTERT did
not affect total hTR levels. Overexpression of the β-isoform did not increase telomerase
activity, as expected 29, nor apoptosis (Fig. 4A-D). Because hTR complexed with βhTERT did not impede hTR anti-apoptotic function, these data further support the
hypothesis that hTR in catalytically inactive form protects from apoptosis and binding of
hTR per se does not affect its anti-apoptotic role.
Catalytically inactive hTR mutants protect from hTERT-induced apoptosis
Because hTERT overexpression alone was sufficient to induce apoptosis under
endogenous hTR conditions, we predicted that apoptosis would be rescued by cooverexpression of catalytically inactive hTR mutants but not WT hTR. Cooverexpressing WT hTR and hTERT, while increasing telomerase activity (Fig. 5A,B)
comparing solid to checkered red bars), decreased live cell counts (Fig. 5C) and
increased apoptosis compared to co-overexpression of hTR with GFP (Fig. 5D). These
results suggest that hTERT overexpression was high enough to bind up the
overexpressed hTR molecules to increase telomerase activity, preventing accumulation
of catalytically inactive hTR. In marked contrast, co-overexpressing Δ96-7 hTR or
G305A hTR and hTERT protected from the hTERT-induced apoptosis (Fig. 5D). Hence,
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a catalytically inactive hTR, bound either efficiently (Δ96-7) or poorly (G305A) to fulllength hTERT, can protect from hTERT overexpression-induced apoptosis. Again,
overexpression of the hTR-U64 fusion failed to protect from hTERT-induced apoptosis
(Fig. 5D), further supporting the results showing that the hTR 3’ portion is necessary for
the anti-apoptotic role of hTR.
DISCUSSION
Various studies have suggested non-telomeric roles for hTERT that implicate it in
proliferation, apoptosis, and mitochondrial function (for references see 23), in addition to
maintaining telomeres. However, only two studies have previously proposed
telomerase-independent roles for hTR: regulating p53 dependent cell growth in cancer
cell lines and fibroblasts 24 and regulating differentiation of myeloid cells in zebrafish 38.
Here, we have presented evidence indicating that hTR, specifically, has a new role in
protecting stimulated human CD4 T cells from apoptosis only when hTR is in an
enzymatically inactive state. In this context, enzymatic activity is defined in terms of the
canonical telomeric DNA polymerization reaction performed by the core active hTERThTR telomerase complex. We further report that the 3’ portion of hTR is necessary for
protecting from hTERT overexpression-induced or corticosteroid-induced apoptosis.
Our unexpected results from multiple shRNA and overexpression experiments,
summarized in Table 1, provide data consistent with hTR existing in more than one
functional form: the well-known enzymatically active form that complexes with
catalytically competent hTERT and other factors to synthesize telomeric DNA and
elongate telomeres, and an enzymatically inactive form(s) that protects from apoptosis.
In this model (Fig. 6), full-length hTERT overexpression, which reduces endogenous
catalytically inactive hTR by converting it into catalytically active hTR, induces apoptosis.
In contrast, overexpressing hTR mutants, or catalytically-dead β-hTERT, does not
induce apoptosis. This model (Fig. 6) is plausible given structural studies indicative of
multiple conformations for hTR: a predicted active pseudoknot conformation with a triple
helix that is stabilized by hTERT and required for telomerase activity, and an inactive
“open” pseudoknot conformation 39–41. We propose that hTR, possibly in an open
inactive conformation, can interact with other factors to protect from apoptosis.
The disease-linked hTR mutants utilized here disrupt distinct hTR structures:
Δ96-7 disrupts the pseudoknot triple helix and the G305A mutation disrupts the P6.1
stem loop. Both create catalytically inactive hTR that can bind hTERT either well (Δ967) or poorly (G305A). Our finding that overexpression of catalytically inactive G305A
hTR - but not of WT hTR or of full- length hTERT - protects from dexamethasone-
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induced apoptosis indicates that in the time frame of our experiments, increasing
telomerase enzymatic activity does not protect from apoptosis. The portion of hTR
present in G305A but missing from hTR-U64 is necessary for this protection.
Furthermore, hTR protection from apoptosis is not specific to the P6.1 disruption of
G305A, because in the setting of hTERT knock-down, endogenous or overexpressed
WT hTR protected from dexamethasone-induced apoptosis. In addition, binding to fulllength hTERT per se does not prevent hTR from protecting from apoptosis, because
overexpressed catalytically inactive Δ96-7 mutant, which binds hTERT, protects from
hTERT-induced apoptosis. In summary, our data support a model in which catalytically
inactive hTR protects from apoptosis and the 3’ half of hTR is necessary for this
protection.
Our data that hTERT overexpression induces apoptosis might superficially
appear to contradict previous studies reporting that hTERT overexpression extends
replicative lifespan in lymphocytes 42–44. However, differences in experimental time
lines (and potentially culture conditions) are likely to account for these discrepancies.
Previous studies used cells cultured for up to 300 days whereas our studies lasted 1-2
weeks. It is possible that hTERT overexpression might initially induce apoptosis by
reducing the amount of catalytically inactive hTR but, in long-term culture, extend
lifespan through better telomere maintenance. Hence, the present study suggests that
while increasing telomerase activity might increase the replicative lifespan of immune
cells, increasing the available catalytically inactive hTR can additionally increase T cell
survival by protecting from apoptosis. However, it is also possible that the hTR and
hTERT exert the effects on apoptosis described here via different mechanisms.
Recently, lncRNAs were shown to regulate gene expression and proliferation in
lymphocytes 45. Possible mechanisms for the anti-apoptotic effect of catalytically
inactive hTR could also involve binding to factors or being processed into microRNA(s).
The hTR 3’ portion could be important for these, and/or for localization, because the
hTR-U64 fusion RNA would be localized to the nucleolus whereas the other hTR RNA
constructs we investigated are not strictly nucleolar. Both hTR and the fusion hTR-U64
RNA have a 3’ H/ACA dyskerin-binding domain. The observed interference by hTR-U64
with the anti-apoptotic role of endogenous hTR in the setting of hTERT knockdown
suggests that hTR-U64 can compete with endogenous hTR for dyskerin, which might be
important for hTR to protect from apoptosis, potentially via hTR targeting for
pseudouridinylation RNA(s), as do other snoRNAs 46.
Our findings predict that human disease states of telomere syndromes 47 which
include immunodeficiency, might be more severe with mutations reducing hTR versus
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catalytically inactive hTR mutants or hTERT insufficiency mutants. Mutations in dyskerin,
which reduce hTR levels, have more severe disease phenotypes than other telomere
syndrome mutations 47. More specifically, a familial mutation in which the 74 nucleotides
of the 3’ end of hTR were deleted (and presumably decreased hTR levels) caused
lymphocytes from the patients to show increased baseline levels of apoptosis 48. Hence
we propose that mutations that reduce hTR levels can cause more severe telomere
syndromes through two mechanisms: telomere shortening due to low telomerase
(previous studies) as well as increased lymphocyte apoptosis due to reduced hTR
levels (this report). Furthermore, we propose that bone marrow failure, the most
common cause of death in individuals with telomere syndromes, may not be explained
solely by stem cell depletion through shortening telomeres, but also by continued drains
on the hematopoietic stem cell reserves to replace decreasing levels of immune cells,
caused by low hTR level-induced apoptosis as well as shortening telomeres.
Many studies have shown associations between telomerase levels in vivo and
human health and disease. Studies have associated healthy lifestyle and behaviors with
increased telomerase activity in PBMCs, and, conversely, shown that corticosteroid
treatment reduces T cell survival and telomerase activity 4–6,20. Our findings suggest a
new way, besides via telomere maintenance, in which altering hTR levels regulates
immune function. As new techniques become available for primary human immune cells,
future studies focusing on hTR genome editing, hTR localization, and investigating
potential hTR targets and interacting partners will be integral to identifying mechanisms
by which hTR influences apoptosis.
Acknowledgements
We thank Beth Cimini for designing the Cell Profiler programs used to analyze telomere
length and TIF data, Eric Verdin and Emmanuelle Passegue for insightful comments on
experimental design, Kathleen Collins and Michael McManus for plasmids, Richard
Novak for microarray analysis, Jue Lin, Bradley Stohr, Morgan Diolaiti, and Richard
Novak for comments on the manuscript.
This work was supported in part by NIH/NCI grants CA096840 and AG030424.
FSG was supported by the Graduate Education in Medical Sciences training program at
UCSF (http://physio.ucsf.edu/GEMS) and the David & Annette Jorgensen Foundation.
The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript. FSG and EHB conceived the study, designed
experiments and wrote the manuscript. FSG performed the experiments. The authors
declare no competing financial interests.
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References
1.
Epel ES, Blackburn EH, Lin J, et al. Accelerated telomere shortening in response to life
stress.
2.
Proc. Natl. Acad. Sci. U. S. A. 2004;101(49):17312–17315.
Epel ES, Lin J, Wilhelm FH, et al. Cell aging in relation to stress arousal and
cardiovascular disease risk factors.
3.
Epel ES, Lin J, Dhabhar FS, et al. Dynamics of telomerase activity in response to acute
psychological stress.
4.
Psychoneuroendocrinology. 2006;31(3):277–287.
Brain. Behav. Immun. 2010;24(4):531–539.
Daubenmier J, Lin J, Blackburn E, et al. Changes in stress, eating, and metabolic factors
are related to changes in telomerase activity in a randomized mindfulness intervention
pilot study.
5.
Psychoneuroendocrinology. 2012;37(7):917–928.
Jacobs TL, Epel ES, Lin J, et al. Intensive meditation training, immune cell telomerase
activity, and psychological mediators.
6.
Psychoneuroendocrinology. 2011;36(5):664–681.
Lavretsky H, Epel ES, Siddarth P, et al. A pilot study of yogic meditation for family
dementia caregivers with depressive symptoms: effects on mental health, cognition,
and telomerase activity.
7.
Int. J. Geriatr. Psychiatry. 2013;28(1):57–65.
Ornish D, Lin J, Daubenmier J, et al. Increased telomerase activity and comprehensive
lifestyle changes: a pilot study.
8.
Brydon L, Lin J, Butcher L, et al. Hostility and Cellular Aging in Men from the Whitehall
II Cohort.
9.
Lancet Oncol. 2008;9(11):1048–1057.
Biol. Psychiatry. 2012;71(9):767–773.
Damjanovic AK, Yang Y, Glaser R, et al. Accelerated Telomere Erosion Is Associated with
a Declining Immune Function of Caregivers of Alzheimer’s Disease Patients.
J. Immunol.
2007;179(6):4249–4254.
10. Steptoe A, Hamer M, Butcher L, et al. Educational attainment but not measures of
current socioeconomic circumstances are associated with leukocyte telomere length in
healthy older men and women.
Brain. Behav. Immun. 2011;25(7):1292–1298.
11. Wolkowitz OM, Mellon SH, Epel ES, et al. Resting leukocyte telomerase activity is
elevated in major depression and predicts treatment response.
Mol. Psychiatry.
2012;17(2):164–172.
12. Zalli A, Carvalho LA, Lin J, et al. Shorter telomeres with high telomerase activity are
associated with raised allostatic load and impoverished psychosocial resources.
Proc.
Natl. Acad. Sci. 2014;111(12):4519–4524.
13. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase
activity with immortal cells and cancer.
Science. 1994;266(5193):2011–2015.
14. Morrison SJ, Prowse KR, Ho P, Weissman IL. Telomerase activity in hematopoietic cells
is associated with self-renewal potential.
Immunity. 1996;5(3):207–216.
15
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
15. Broccoli D, Godley LA, Donehower LA, Varmus HE, de Lange T. Telomerase activation in
mouse mammary tumors: lack of detectable telomere shortening and evidence for
regulation of telomerase RNA with cell proliferation.
Mol. Cell. Biol. 1996;16(7):3765–
3772.
16. Weng NP, Levine BL, June CH, Hodes RJ. Regulated expression of telomerase activity in
human T lymphocyte development and activation.
J. Exp. Med. 1996;183(6):2471–2479.
17. Kosciolek BA, Rowley PT. Telomere-related components are coordinately synthesized
during human T-lymphocyte activation.
Leuk. Res. 1999;23(12):1097–1103.
18. Liu K, Schoonmaker MM, Levine BL, et al. Constitutive and regulated expression of
telomerase reverse transcriptase (hTERT) in human lymphocytes.
Proc. Natl. Acad. Sci.
U. S. A. 1999;96(9):5147–5152.
19. Weng NP, Palmer LD, Levine BL, et al. Tales of tails: regulation of telomere length and
telomerase activity during lymphocyte development, differentiation, activation, and
aging.
Immunol. Rev. 1997;160:43–54.
20. Choi J, Fauce SR, Effros RB. Reduced telomerase activity in human T lymphocytes
exposed to cortisol.
Brain. Behav. Immun. 2008;22(4):600–605.
21. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector
achieves efficient gene delivery in vivo.
Nat. Biotechnol. 1997;15(9):871–875.
22. Li S, Rosenberg JE, Donjacour AA, et al. Rapid inhibition of cancer cell growth induced
by lentiviral delivery and expression of mutant-template telomerase RNA and antitelomerase short-interfering RNA.
Cancer Res. 2004;64(14):4833–4840.
23. Gazzaniga FS, Listerman I, Blackburn EH. An investigation of the effects of the
telomerase core protein TERT on Wnt signaling in human breast cancer cells.
Mol. Cell.
Biol. 2013;
24. Kedde M, le Sage C, Duursma A, et al. Telomerase-independent regulation of ATR by
human telomerase RNA.
J. Biol. Chem. 2006;281(52):40503–40514.
25. Del Gaizo Moore V, Brown JR, Certo M, et al. Chronic lymphocytic leukemia requires
BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737.
J.
Clin. Invest. 2007;117(1):112–121.
26. Fu D, Collins K. Distinct biogenesis pathways for human telomerase RNA and H/ACA
small nucleolar RNAs.
Mol. Cell. 2003;11(5):1361–1372.
27. Robart AR, Collins K. Investigation of human telomerase holoenzyme assembly, activity,
and processivity using disease-linked subunit variants.
J. Biol. Chem.
2010;285(7):4375–4386.
16
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
28. Wege H, Chui MS, Le HT, Tran JM, Zern MA. SYBR Green real-time telomeric repeat
amplification protocol for the rapid quantification of telomerase activity.
Nucleic Acids
Res. 2003;31(2):E3–3.
29. Listerman I, Sun J, Gazzaniga FS, Lukas JL, Blackburn EH. The major reverse
transcriptase-incompetent splice variant of the human telomerase protein inhibits
telomerase activity but protects from apoptosis.
Cancer Res. 2013;73(9):2817–2828.
30. Diolaiti ME, Cimini BA, Kageyama R, Charles FA, Stohr BA. In situ visualization of
telomere elongation patterns in human cells.
Nucleic Acids Res. 2013;41(18):e176.
31. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for
biological-image analysis.
Nat. Methods. 2012;9(7):676–682.
32. Erlacher M, Labi V, Manzl C, et al. Puma cooperates with Bim, the rate-limiting BH3-only
protein in cell death during lymphocyte development, in apoptosis induction.
J. Exp.
Med. 2006;203(13):2939–2951.
33. Heidari N, Miller AV, Hicks MA, Marking CB, Harada H. Glucocorticoid-mediated BIM
induction and apoptosis are regulated by Runx2 and c-Jun in leukemia cells.
Cell Death
Dis. 2012;3(7):e349.
34. Erlacher M, Labi V, Manzl C, et al. Puma cooperates with Bim, the rate-limiting BH3-only
protein in cell death during lymphocyte development, in apoptosis induction.
J. Exp.
Med. 2006;203(13):2939–2951.
35. Yu J, Zhang L. PUMA, a potent killer with or without p53.
Oncogene. 2008;27(Suppl
1):S71–S83.
36. Karlseder J, Broccoli D, Dai Y, Hardy S, Lange T de. p53- and ATM-Dependent Apoptosis
Science. 1999;283(5406):1321–1325.
37. Lange T de. How Telomeres Solve the End-Protection Problem. Science.
Induced by Telomeres Lacking TRF2.
2009;326(5955):948–952.
38. Alcaraz-Pérez F, García-Castillo J, García-Moreno D, et al. A non-canonical function of
telomerase RNA in the regulation of developmental myelopoiesis in zebrafish.
Nat.
Commun. 2014;5:
39. Hengesbach M, Kim N-K, Feigon J, Stone MD. Single-Molecule FRET Reveals the Folding
Dynamics of the Human Telomerase RNA Pseudoknot Domain.
Angew. Chem. Int. Ed.
2012;51(24):5876–5879.
40. Qiao F, Cech TR. Triple-helix structure in telomerase RNA contributes to catalysis.
Nat.
Struct. Mol. Biol. 2008;15(6):634–640.
41. Yeoman JA, Orte A, Ashbridge B, Klenerman D, Balasubramanian S. RNA Conformation
in Catalytically Active Human Telomerase.
J. Am. Chem. Soc. 2010;132(9):2852–2853.
17
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
42. Menzel O, Migliaccio M, Goldstein DR, et al. Mechanisms Regulating the Proliferative
Potential of Human CD8+ T Lymphocytes Overexpressing Telomerase.
J. Immunol.
2006;177(6):3657–3668.
43. Röth A, Yssel H, Pène J, et al. Telomerase levels control the lifespan of human T
lymphocytes.
Blood. 2003;102(3):849–857.
44. Röth A, Baerlocher GM, Schertzer M, et al. Telomere loss, senescence, and genetic
instability in CD4+ T lymphocytes overexpressing hTERT.
Blood. 2005;106(1):43–50.
45. Carpenter S, Aiello D, Atianand MK, et al. A Long Noncoding RNA Mediates Both
Activation and Repression of Immune Response Genes.
Science. 2013;341(6147):789–
792.
Nat. Rev. Genet. 2011;12(12):861–874.
47. Armanios M, Blackburn EH. The telomere syndromes. Nat. Rev. Genet.
46. Esteller M. Non-coding RNAs in human disease.
2012;13(10):693–704.
48. Knudson M, Kulkarni S, Ballas ZK, Bessler M, Goldman F. Association of immune
abnormalities with telomere shortening in autosomal-dominant dyskeratosis congenita.
Blood. 2005;105(2):682–688.
18
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Table 1. Summary of experimental results: Treatments of CD4 T cells with RNA
expression constructs
RNA name
shScramble
shTERT
shTR1,2
WT hTR
Δ96-97
G305A
hTR-U64
hTERT
β-hTERT
Telomerase
activity
level
normal
decreased
decreased
increased
catalytically
inactive
catalytically
inactive
catalytically
inactive
increased
catalytically
inactive
Dexamethasoneinduced
apoptosis
susceptible
resistant
Stimulationassociated
apoptosis
normal
normal
increased
normal
normal
susceptible
resistant
resistant
normal
resistant
susceptible
normal
susceptible
susceptible
hTERTinduced
apoptosis
increased
normal
Figure Legends
Figure 1. hTR overexpression protects from dexamethasone induced apoptosis
independent of telomerase activity. (A) Telomerase activity with hTR variant
overexpression. Error bars show standard deviation of biological triplicates. (B) RNA
levels of hTR overexpression represented as fold change over empty vector. Error bars
show standard deviation of PCR triplicates. (C) Caspase 3/7 activity measured by
luminescence. Luminescence normalized to background levels. Error bars represent
biological triplicates (D) Caspase 3/7 activity measured by luminescence when hTERT
is knocked down. Error bars represent biological triplicates. Solid bars represent no
dexamethasone treatment. Checkered bars represent treatment with 1 μM
dexamethasone.
Figure 2. hTR knockdown induces Bim-mediated apoptosis. (A) Telomerase activity
in CD4 T in culture with different lentiviral vectors. Error bars show standard deviation of
biological triplicates. Representative example of 10 different experiments using cells
from 8 different donors; each experiment was performed in triplicate. (B) hTERT mRNA
and hTR RNA levels in CD4 T cells. Error bars show standard deviation of the average
of two experiments. (C) Live cell counts measured by trypan blue exclusion. Error bars
19
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show standard deviation of biological triplicates. Representative example from 10
different experiments using cells from 8 different donors. (D) Stages in cell cycle
measured by DyeCycle green. Error bars show standard deviation of biological
triplicates. (E) Caspase 8, 9, 3/7 activity. Caspase activity was measured by
luminescence. Error bars represent standard deviation of biological triplicates. One-way
ANOVA was performed for each caspase. * represents p<0.05. (F) Western blot
measuring Bim, Puma, Bad, Bid, p53 in shScramble, shTERT, shTR1, shTR2 cells. (G)
Cell survival measured by trypan blue exclusion with Bim knockdown followed by hTR
knockdown compared to empty vector knockdown followed by hTR knockdown.
Figure 3. Telomerase knockdown does not induce significant telomere shortening
or TIFs in the timeframe of this experiment. (A) Cumulative frequency of telomere
lengths measured by PNA intensity. (B) Telomeres/area. (C) TIFs/telomere measured
by co-localization of γH2ax foci and PNA foci. (D) TIFs/γH2ax foci. (E) γH2ax foci/area.
Statistical significance was assessed using one way ANOVA and Dunn’s multiple
comparison test with a significance cutoff of p <0.05 in Prism (GraphPad). No virus (NV)
Empty vector (EV).
Figure 4. hTERT overexpression increases telomerase activity and induces
apoptosis. (A) Telomerase activity with overexpression of hTERT variants. Error bars
show standard deviation of biological triplicates. (B) RNA levels with overexpression of
hTERT variants. Error bars show standard deviation of PCR triplicates. (C) Live cell
counts with overexpression of hTERT variants. (D) Apoptosis measured by Caspase3/7.
Error bars show standard deviation of biological triplicates. * represents statistical
significance of p<0.05 assessed by one-way ANOVA .
Figure 5. Overexpression of catalytically inactive hTR mutants protects from
hTERT-induced apoptosis. (A) Telomerase activity with hTR and hTERT cooverexpression. (B) RNA levels with hTR and hTERT co-overexpression. (C) Live cell
counts with hTR and hTERT co-overexpression. (D) Caspase 3/7 activity measured by
luminescence. Significance assessed by unpaired t-test. ** represents p<0.01, ***
represents p<0.001, **** P<0.0001.
Figure 6. Two functions for hTR. hTR and hTERT complex to form catalytically active
telomerase to maintain telomeres. In this catalytically active conformation, hTR
complexes with hTERT and other factors to elongate telomeres. hTR also functions in a
20
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catalytically inactive state (shown here as unbound to hTERT with a disprupted
pseudoknot) to prevent apoptosis. In a catalytically inactive state, hTR may be able to
bind other factors to protect from apoptosis. Dyskerin is depicted as it is necessary for
hTR accumulation, but some other binding partner might be involved with hTR to
prevent apoptosis. Red: template, green line: Δ96-7 hTR mutant. Orange: P6.1 stem
disrupted by G305A.
21
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Prepublished online October 15, 2014;
doi:10.1182/blood-2014-06-582254
An anti-apoptotic role for telomerase RNA in human immune cells
independent of telomere integrity or telomerase enzymatic activity
Francesca S. Gazzaniga and Elizabeth H. Blackburn
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