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Department of Cell Biology, The University of Texas Southwestern Medical Center, Dallas, Texas .
Corresponding Author: Jerry W. Shay, The University of Texas Southwest-ern Medical Center, 5323 Harry Hines Boulevard, Dallas , Texas 75390-9039. Phone: 214-648-4201; Fax: 214-648-5814; E-mail: [email protected]
doi: 10.1158/2159-8290.CD-16-1050
©2016 American Association for Cancer Research.
Telomeres in mammals are chromosome ends that are
composed of thousands of the canonical repetitive sequence
TTAGGG ( 1 ). Telomeres shorten with each cell division due
to the end replication problem (incomplete lagging strand
DNA synthesis at the end) and postreplication end pro-
cessing (telo meric overhang generation processes). In pro-
liferating stem and germline cells, telomeres can be partially
maintained by the cellular reverse transcriptase, termed
telomerase. Telo merase adds new TTAGGG repeats onto
the telomeric overhangs, but due to lack of or insuffi cient
telomerase activity, human somatic cells can only divide
until at least some telo mere ends become critically shortened
(uncapped and recognized as a DNA double-strand break).
Most premalignant human lesions harbor very short telom-
eres, and it is generally thought that the repression of telom-
erase and progressive telomere shortening may have evolved
in large long-lived species as an initial antitumor protective
mechanism ( 1 ). To continue to divide, premalignant cancer
cells need to acquire a telomere maintenance mechanism dur-
ing neoplastic transformation. The vast majority of human
cancers (>85%) maintain their telomere length via telomerase
reactivation ( 1 ). All human tissues express the functional tel-
omerase templating RNA component ( TERC ), whereas most
human adult tissues do not express telomerase reverse tran-
scriptase ( TERT ). During early human fetal development,
telomerase is expressed and, in a tissue-specifi c manner, the
TERT gene becomes silenced. Although the regulation of
TERT is complex and involves transcriptional, posttranscrip-
tional, and epigenetic modifi cations, the precise mechanism
of activation of telomerase in cancer has not been resolved.
The TERT gene has a GC-rich core promoter containing
CpG islands that are mostly methylated in normal cells ( 2 ).
The hypermethylation of the TERT core promoter is believed
to be one of the major mechanisms for TERT repression, but
what changes the methylation status during fetal develop-
ment is not known. In addition, the TERT promoter does
not have typical transcriptional elements such as TATA boxes
or CCAAT boxes; instead it contains GC boxes, which are
the consensus binding sites for the transcription factor SP1.
Masking of the SP1 binding site by G-quadruplex formation
in GC boxes may be another mechanism for TERT promoter
repression. Thus, transcriptional upregulation or reactiva-
tion of the TERT gene is a critical step in tumorigenesis, and
multiple mechanisms have been proposed for reactivating
the TERT gene in cancer. These include mutation or deletion
in the TERT promoter, TERT gene amplifi cation, epigenetic
alterations, and TERT gene alternative splicing factors ( 1 ).
How cancer cells activate the silenced TERT gene still remains
largely unknown.
From genetic linkage analysis and whole-genome sequenc-
ing, it has been recently reported that the TERT gene can
be reactivated by proximal promoter mutation ( 3, 4 ). These
are single-nucleotide mutations in the proximal promoter
of the TERT gene: cytosine to thymidine transition at −124
bp and −146 bp upstream of the translation start site. These
TERT promoter mutations are very close to the transcription
start site of the TERT gene (−46 bp and −68 bp upstream of
transcription start site). Importantly, these are also located in
the GC boxes in the TERT core promoter (−90 bp to −22 bp
upstream of transcription start site). These two TERT pro-
moter mutations (−124C>T, −146C>T) are now considered
to be among the most common noncoding mutations in
cancer. For example, monoallelic TERT promoter mutations
are common in melanomas (∼85%), glioblastomas (∼84%),
hepatocellular carcinomas (∼44%), liposarcomas (∼79%), and
urothelial cancers (∼47%; refs. 3–5 ). However, TERT pro-
moter mutations are much less common (<10%) in lung,
colon, esophageal, pancreatic, breast, and prostate cancers
( 6 ). It is believed that these mutations generate a de novo
consensus binding motif (GGAA) for the E-twenty-six (ETS)
IN THE SPOTLIGHT
TERT Promoter Mutations Enhance Telomerase Activation by Long-Range Chromatin Interactions Jaewon Min and Jerry W. Shay
Summary: Although single-nucleotide somatic mutations in the proximal promoter of the human telomerase reverse
transcriptase ( TERT ) gene create novel consensus sequences for transcription factors that enhance TERT expression,
the precise mechanism of how telomerase is reactivated in cancer cells remains poorly understood. In this issue,
Ak ı nc ı lar and colleagues identify a potential mechanism of TERT reactivation that is mediated by a novel long-range
chromatin interaction between the TERT promoter on chromosome 5p and a 300-kb upstream region. This permits
recruitment of the transcription factor GABPA in mutant TERT promoters but not in wild-type promoters. Cancer
Discov; 6(11); 1212–4. ©2016 AACR.
See related article by Ak ı nc ı lar and colleagues, p. 1276 (2).
on October 4, 2020. © 2016 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
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NOVEMBER 2016�CANCER DISCOVERY | 1213
transcription factor family. The ETS family transcription
factors GABPA and ETS1 have been identifi ed as the binding
factor in most cancer cells with TERT promoter mutations ( 7,
8 ). A better understanding of what facilitates specifi c proxi-
mal promoter mutations in the TERT gene could provide
clues for developing therapeutic strategies to block telomer-
ase activation in most human cancers. In this issue of Cancer
Discovery , Ak ı nc ı lar and colleagues ( 2 ) demonstrate a novel
mechanism of mutant TERT promoter reactivation via long-
range chromatin interactions.
Taking advantage of CRISPR/Cas9 genome editing tech-
niques, Ak ı nc ı lar and colleagues ( 2 ) generated a series of
isogenic cancer cell lines with wild-type and mutant TERT pro-
moters. They showed that reversal of the mutant to wild-type
TERT promoter led to heterochromatin changes in the TERT
promoter region as well as reduction in telomerase activ-
ity. This observation is consistent with the previous report
that the mutant TERT promoter allele exhibits H3K4me2/3
(active chromatin histone mark) and recruits the GABPA
transcription factor, whereas the wild-type TERT allele retains
H3K27me3 (a repressed chromatin histone mark) and does
not recruit the GABPA transcription factor ( 9, 10 ). Because it
has been recently proposed that GABPA may have a potential
role in engaging long-range chromatin interactions, Ak ı nc ı lar
and colleagues investigated whether GABPA bound to the
mutant TERT promoter can engage long-range chromatin
interactions. They performed circular chromosome confor-
mation capture assays to test whether the three-dimensional
genome organization changed in the vicinity of the mutant
TERT promoter locus compared with wild-type TERT . Impor-
tantly, they found that mutant TERT promoters display
long-range chromatin interactions with a region 300 kb
upstream of the TERT promoter (chr5: 1,556,087–1,558,758),
and that these interactions are mediated by GABPA.
By performing additional genome-editing experiments,
they also showed that introducing a mutant sequence in the
wild-type TERT promoter could change long-range chroma-
tin interactions. Moreover, the acute depletion of GABPA by
using GABPA siRNA induced signifi cant reduction of long-
range chromatin interaction. These experiments indicate that
long-range chromatin interactions are reversible changes and
dependent on a single-nucleotide mutation sequence via
GABPA binding. The most striking part of this study is that
Ak ı nc ı lar and colleagues ( 2 ) generated knockout cell lines tar-
geting the interacting region (chr5: 1,556,087−1,558,758) far
from the TERT locus. The removal of the interacting region
reduced TERT expression and altered the epigenetic status
in the proximal TERT promoter region without any change
in GABPA expression. These experiments strongly support
the importance of the interaction between the proximal
TERT promoter and the chr5: 1,556,087−1,558,758 intergenic
region in driving mutant TERT promoter activity ( Fig. 1 ).
In summary, the report by Ak ı nc ı lar and colleagues ( 2 )
identifies a novel mechanism of TERT reactivation by
cancer-specifi c TERT promoter mutations that are mediated
by a long-range chromatin interaction between the TERT
promoter and a region 300 kb from the TERT promoter.
Targeting this long-range chromatin interaction exclusively
harbored in cancers with TERT promoter mutation may be
a potential therapeutic strategy for inhibiting telomerase
activity and the indefi nite cell proliferation that is one of
the hallmarks of cancer. However, there are additional
Figure 1. TERT promoter mutation reactivates the silenced TERT promoter via generating long-range chromatin interactions . Ak ı nc ı lar and colleagues ( 2 ) show that TERT reactivation by the proximal promoter mutation is directly mediated by a novel long-range chromatin interaction between the TERT promoter and a 300 kb upstream region (chr5: 1,556,087–1,558,758). GABPA proteins might generate long-range chromatin interactions by linking the mutant TERT promoter and the specifi c interacting region.
GABPA
“Repressed” “Reactivated”
WT TERT promoter Mutant TERT promoter
Telomere
300 kb
Telomere
Chr 5p
Chr 5p
TERT
BR
D4
TERT
Pol2
GABPAH3K27
me3
H3K4
me2/3
-124C>T
/-146C>T
Long-range chromatin interaction
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1214 | CANCER DISCOVERY�NOVEMBER 2016 www.aacrjournals.org
fundamental questions that remain unsolved regarding the
potential roles of TERT promoter mutations in tumorigene-
sis. For example, we still do not know whether TERT promoter
mutations are suffi cient for reactivating completely silenced
TERT genes in normal human cells (e.g., normal fi broblasts).
In addition, we do not know why many common cancer types
do not demonstrate TERT promoter mutations. Perhaps in
these tumor types, genomic amplifi cations, rearrangements,
and TERT pre-mRNA alternative splicing factors may pre-
dominate. Finally, a potential mechanism to connect TERT
promoter mutations to the epigenetic changes observed by
Ak ı nc ı lar and colleagues ( 2 ) to the well-established fact that
most cancers occur in the older segment of the population
may be the location of the TERT gene in humans and other
large long-lived mammals. The human TERT gene is located
within a megabase to the telomere on chromosome 5p, and
one possibility is that TERT expression autoregulates itself.
Although it is not known why telomerase is silenced at
very specifi c times during human fetal development or why
human telomeres are maintained within a small range of
sizes, there is mounting evidence that when telomeres reach
a specifi c initial length, three-dimensional chromatin struc-
tures involving telomere position effects over long distances
(TPE-OLD) may silence many genes, including the TERT gene
( 1 ). This would serve as a mechanism to silence telomerase to
prevent the early onset of cancer. Thus, as we age and telo-
meres progressively shorten, it is reasonable to suggest that
in a monoallelic-specifi c manner, the chromatin silencing
effects at one TERT promoter may change, facilitating TERT
promoter mutations. This is an example of antagonist plei-
otropy when one gene controls more than one trait and one
of these traits is benefi cial to the organism fi tness early in life
(silencing telomerase, reducing the early onset of cancer) and
one is detrimental to the organism fi tness later in life (telom-
erase reactivation in cancer).
Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed .
Grant Support We acknowledge support from the National Cancer Institute
(Lung SPORE P50CA70907 ), R01 AG001228 , and a distinguished
chair from the Southland Financial Foundation in Geriatrics
Research . This work was performed in laboratories constructed with
support from NIH grant C06 RR30414 .
Published online November 2, 2016.
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2016;6:1212-1214. Cancer Discov Jaewon Min and Jerry W. Shay Long-Range Chromatin Interactions
Promoter Mutations Enhance Telomerase Activation byTERT
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