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1212 | CANCER DISCOVERY�NOVEMBER 2016 www.aacrjournals.org

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: jerry.shay@utsouthwestern.edu

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).

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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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