Methods in
Molecular Biology 1587

Zhou Songyang Editor

Telomeres
and Telomerase
Methods and Protocols
Third Edition


Methods

in

Molecular Biology

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

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Telomeres and Telomerase
Methods and Protocols
Third Edition

Edited by

Zhou Songyang
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA


Editor
Zhou Songyang
Department of Biochemistry and Molecular Biology
Baylor College of Medicine
Houston, TX, USA

ISSN 1064-3745    ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6891-6    ISBN 978-1-4939-6892-3 (eBook)
DOI 10.1007/978-1-4939-6892-3
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Preface
In 2009, the Nobel Prize in Physiology or Medicine was awarded to Drs. Elizabeth
H. Blackburn, Carol W. Greider, and Jack W. Szostak for their pioneering work on telomeres
and telomerase, nearly 40 years after the first identification of telomeres. Our knowledge of
the telomerase and how telomeres are maintained has continued to grow, thanks in no small
part to the ever-expanding tools and platforms that are available to investigators. It is clear
that telomere maintenance is critically linked to cell growth, proliferation, aging, and diseases such as cancer. Active investigations are underway to untangle the complex signaling
events that lead from telomere dysfunction to premature aging and carcinogenesis.
In the second volume of Telomeres and Telomerase book (MiMB Vol. 735), a variety of
assays were presented that allowed investigators to query the activity of telomerase, function of telomere proteins, and the responses of the telomere DNA. Further advances in
technology have equipped us with new and improved assays that enable us to ask fundamental questions of telomere regulation in diverse model systems. This volume aims to
expand the scope further, incorporating some of the newest technologies in the field. This
combination of genetic, proteomic, genomic, biochemical, and molecular approaches will
afford us unprecedented insight into the complex protein interaction networks at work on
the telomere chromatin, and the detailed information regarding telomere dynamics in
response to stress or stimuli.
These protocols are detailed and easy to follow. It is our belief that this work will prove
useful and informative.
Houston, TX , USA

Zhou Songyang

v


Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
  1 Introduction to Telomeres and Telomerase . . . . . . . . . . . . . . . . . . . . . . . . . . .
Zhou Songyang
  2 Analysis of Average Telomere Length in Human Telomeric Protein
Knockout Cells Generated by CRISPR/Cas9 . . . . . . . . . . . . . . . . . . . . . . . . . .
Jun Xu, Zhou Songyang, Dan Liu, and Hyeung Kim
  3 Telomere Length Analysis by Quantitative Fluorescent in Situ
Hybridization (Q-FISH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isabelle Ourliac-Garnier and Arturo Londoño-Vallejo
  4 Telomere Strand-Specific Length Analysis by Fluorescent
In Situ Hybridization (Q-CO-FISH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isabelle Ourliac-Garnier and Arturo Londoño-Vallejo
  5 Telomere G-Rich Overhang Length Measurement: DSN Method . . . . . . . . . .
Yong Zhao, Jerry W. Shay, and Woodring E. Wright
  6 Telomere G-Overhang Length Measurement Method 2: G-Tail
Telomere HPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hidetoshi Tahara
  7 Telomere Terminal G/C Strand Synthesis: Measuring Telomerase
Action and C-Rich Fill-In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yong Zhao, Jerry W. Shay, and Woodring E. Wright
  8 Analysis of Yeast Telomerase by Primer Extension Assays . . . . . . . . . . . . . . . . .
Min Hsu and Neal F. Lue
  9 Assessing Telomerase Activities in Mammalian Cells Using the Quantitative
PCR-Based Telomeric Repeat Amplification Protocol (qTRAP) . . . . . . . . . . . .
Shuai Jiang, Mengfan Tang, Huawei Xin, and Junjiu Huang
10 Telomeres and NextGen CO-FISH: Directional Genomic Hybridization
(Telo-dGH™) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Miles J. McKenna, Erin Robinson, Edwin H. Goodwin,

Michael N. Cornforth, and Susan M. Bailey
11 Visualization of Human Telomerase Localization
by Fluorescence Microscopy Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Eladio Abreu, Rebecca M. Terns, and Michael P. Terns
12 Cytogenetic Analysis of Telomere Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . .
Rekha Rai, Asha S. Multani, and Sandy Chang
13 Probing the Telomere Damage Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rekha Rai and Sandy Chang

vii

1

15

29

41
55

63

71
83

95

103

113

127
133


viii

Contents

14 Induction of Site-Specific Oxidative Damage at Telomeres
by Killerred-Fused Shelretin Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rong Tan and Li Lan
15 Using Protein-Fragment Complementation Assays (PCA)
and Peptide Arrays to Study Telomeric Protein-Protein Interactions . . . . . . . . .
Wenbin Ma, Ok-hee Lee, Hyeung Kim, and Zhou Songyang
16 In Vitro Preparation and Crystallization of Vertebrate
Telomerase Subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jing Huang, Christopher J. Bley, Dustin P. Rand, Julian J.L. Chen,
and Ming Lei
17 Human Telomeric G-Quadruplex Structures and G-Quadruplex-Interactive
Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clement Lin and Danzhou Yang
18 Analysis of Telomere-Homologous DNA with Different Conformations
Using 2D Agarose Electrophoresis and In-Gel Hybridization . . . . . . . . . . . . . .
Zepeng Zhang, Qian Hu, and Yong Zhao
19 Analysis of Telomere Proteins by Chromatin Immunoprecipitation (ChIP) . . . .
Feng Liu, Xuyang Feng, and Wenbin Ma

139

147


161

171

197
205

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215


Contributors
Eladio Abreu  •  Department of Biochemistry and Molecular Biology, University of Georgia,
Athens, GA, USA; Department of Genetics, University of Georgia, Athens, GA, USA
Susan M. Bailey  •  Department of Environmental & Radiological Health Sciences,
Colorado State University, Fort Collins, CO, USA
Christopher J. Bley  •  Department of Chemistry and Biochemistry, Arizona State
University, Tempe, AZ, USA
Sandy Chang  •  Department of Laboratory Medicine, Yale University School of Medicine,
New Haven, CT, USA
Julian J.L. Chen  •  Department of Chemistry and Biochemistry, Arizona State University,
Tempe, AZ, USA
Michael N. Cornforth  •  Department of Radiation Oncology, University of Texas
Medical Branch, Galveston, TX, USA
Xuyang Feng  •  Key Laboratory of Gene Engineering of the Ministry of Education,
State Key Laboratory for Biocontrol, Department of Biochemistry, School of Life Sciences,
Sun Yat-sen University, Guangzhou, China
Edwin H. Goodwin  •  KromaTiD Inc., Fort Collins, CO, USA
Min Hsu  •  Department of Microbiology & Immunology, W.R. Hearst Microbiology
Research Center, Weill Medical College of Cornell University, New York, NY, USA

Qian Hu  •  Key Laboratory of Gene Engineering of the Ministry of Education, Higher
Education Mega Center, School of Life Sciences, Sun Yat-sen University,
Guangzhou, China
Jing Huang  •  State Key laboratory of Molecular Biology, National Center for Protein
Science Shanghai, CAS Center for Excellence in Molecular Cell Science,
Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences,
University of, Chinese Academy of Sciences, Shanghai Science Research Center,
Chinese Academy of Sciences, Shanghai, China
Junjiu Huang  •  Key Laboratory of Gene Engineering of the Ministry of Education,
SYSU-BCM Joint Center for Biomedical Sciences and Institute of Healthy Aging
Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, China
Shuai Jiang  •  Key Laboratory of Gene Engineering of the Ministry of Education,
SYSU-BCM Joint Center for Biomedical Sciences and Institute of Healthy Aging
Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, China
Hyeung Kim  •  Verna and Marrs McLean Department of Biochemistry and Molecular
Biology, Baylor College of Medicine, Houston, TX, USA
Li Lan  •  University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; Department
of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
Ok-Hee Lee  •  Department of Biomedical Science, CHA University, Seongnam-si,
Gyeonggi-do, Republic of Korea; Severance Integrative Research Institute for Cerebral
and Cardiovascular Diseases, Yonsei University Health System, Seoul, Republic of Korea
Ming Lei  •  State Key laboratory of Molecular Biology, National Center for Protein Science
Shanghai, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese

ix


x


Contributors

Academy of Sciences, Shanghai, China; Shanghai Science Research Center, Chinese
Academy of Sciences, Shanghai, China
Clement Lin  •  Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy,
Purdue University, West Lafayette, IN, USA
Dan Liu  •  Cell-Based Assay Screening Service Core, Baylor College of Medicine, Houston,
TX, USA; Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, TX, USA
Feng Liu  •  Key Laboratory of Gene Engineering of the Ministry of Education, State Key
Laboratory for Biocontrol, Department of Biochemistry, School of Life Sciences, Sun Yat-sen
University, Guangzhou, China
Arturo Londoño-Vallejo  •  Telomeres & Cancer Laboratory, CNRS-UMR3244, Institut
Curie, Paris, France; UPMC University Paris 06, Paris, France
Neal F. Lue  •  Department of Microbiology & Immunology, W.R. Hearst Microbiology
Research Center, Weill Medical College of Cornell University, New York, NY, USA
Wenbin Ma  •  Key Laboratory of Gene Engineering of the Ministry of Education, State Key
Laboratory for Biocontrol, Department of Biochemistry, School of Life Sciences, Sun
Yat-sen University, Guangzhou, People’s Republic of China
Miles J. McKenna  •  Department of Environmental & Radiological Health Sciences,
Colorado State University, Fort Collins, CO, USA; KromaTiD Inc., Fort Collins, CO, USA
Asha S. Multani  •  Department of Laboratory Medicine, Yale University School of
Medicine, New Haven, CT, USA
Isabelle Ourliac-Garnier  •  Telomeres & Cancer Laboratory, CNRS-UMR3244, Institut
Curie, Paris, France; UPMC University Paris 06, Paris, France
Rekha Rai  •  Department of Laboratory Medicine, Yale University School of Medicine,
New Haven, CT, USA
Dustin P. Rand  •  Department of Chemistry and Biochemistry, Arizona State University,
Tempe, AZ, USA

Erin Robinson  •  KromaTiD Inc., Fort Collins, CO, USA
Jerry W. Shay  •  Department of Cell Biology, University of Texas Southwestern Medical
Center, Dallas, TX, USA
Zhou Songyang  •  Verna and Marrs McLean Department of Biochemistry and Molecular
Biology, Baylor College of Medicine, Houston, TX, USA
Hidetoshi Tahara  •  Department of Cellular and Molecular Biology, Graduate School
of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
Mengfan Tang  •  Department of Experimental Radiation Oncology, The University
of Texas MD Anderson Cancer Center, Houston, TX, USA
Michael P. Terns  •  Department of Biochemistry and Molecular Biology, University
of Georgia, Athens, GA, USA; Department of Genetics, University of Georgia,
Athens, GA, USA
Rebecca M. Terns  •  Department of Biochemistry and Molecular Biology, University
of Georgia, Athens, GA, USA; Department of Genetics, University of Georgia,
Athens, GA, USA
Rong Tan  •  University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; Xiangya
Hospital, Central South University, Changsha, Hunan, China; University of Pittsburgh
School of Medicine, Pittsburgh, PA, USA
Woodring E. Wright  •  Department of Cell Biology, University of Texas Southwestern
Medical Center, Dallas, TX, USA


Contributors

xi

Huawei Xin  •  Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX, USA
Jun Xu  •  Cell-Based Assay Screening Service Core, Baylor College of Medicine, Houston,
TX, USA

Danzhou Yang  •  Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy,
Purdue University, West Lafayette, IN, USA; Purdue Center for Cancer Research,
Purdue University, West Lafayette, IN, USA; Purdue Institute for Drug Discovery,
Purdue University, West Lafayette, IN, USA
Zepeng Zhang  •  Key Laboratory of Gene Engineering of the Ministry of Education,
Higher Education Mega Center, School of Life Sciences, Sun Yat-sen University,
Guangzhou, China
Yong Zhao  •  Key Laboratory of Gene Engineering of the Ministry of Education,
Higher Education Mega Center, School of Life Sciences, Sun Yat-sen University,
Guangzhou, China


Chapter 1
Introduction to Telomeres and Telomerase
Zhou Songyang
Abstract
Telomeres are ends of chromosomes that play an important part in the biology of eukaryotic cells.
Continued optimization and development of technologies have enabled researchers to probe the mechanisms of telomere maintenance in ever expanding areas. A combination of molecular, genetic, proteomic,
and biochemical approaches has revealed the complex and coordinated action of telomerase and telomere-­
associated proteins. The length and integrity of telomeres are maintained to prevent telomere dysfunction,
which has been linked to senescence, aging, disease, and cancer. The tools and assays used to study telomeres today have helped assemble a more detailed, high-resolution picture of the various players and
pathways at work at the telomeres.
Key words Telomere, Telomerase, Telomere interactome, Telomere dysfunction, Telomere length,
Telomere protection

1  Telomere Structure
In the years following the seminal work of Muller and McClintock
[1, 2], which led to the first recognition of the importance of chromosome ends—the telomeres, and the work from Blackburn and
Greider [3, 4] that demonstrated that telomerase was critical to
maintaining chromosomal end sequences, a tremendous amount

of information has been gained regarding the mechanisms of how
telomere maintenance is achieved, and the consequences of disrupting telomere homeostasis.
In most eukaryotes, telomeric DNA consists of tandem repeat
sequences, with the terminus ending in a single-stranded G-rich 3′
overhang [5, 6]. Both the length and exact sequence of the repeat
sequence and overhang may differ from species to species. In mammalian cells, the 5′-TTAGGG-3′ repeat sequences can reach hundreds of kilobases (such as certain mouse strains), with the 3′
overhangs ranging in length between 75–300 bases [7, 8]. In
between the repetitive telomeric ends and chromosome-specific
gene sequences are subtelomeric regions that exhibit great d
­ iversity
Zhou Songyang (ed.), Telomeres and Telomerase: Methods and Protocols, Methods in Molecular Biology, vol. 1587,
DOI 10.1007/978-1-4939-6892-3_1, © Springer Science+Business Media LLC 2017

1


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Zhou Songyang

and complexity between organisms and may contain genes [9] and
contribute to genetic diversity [10–14].
Telomeres appear to adopt a specialized structure with the
help of various telomere-binding proteins, as revealed by EM and
biochemical studies [15]. In this case, invasion by the 3′ overhang
into the double-stranded telomere DNA leads to formation of the
T-loop, while one strand is displaced to form the D-loop [15, 16].
Such configurations may protect telomere ends from nuclease
attack and prevent chromosome end-to-end fusion [17]. The long
G overhangs of mammalian telomeres may form the G-quadruplex,

which has been shown to inhibit telomerase access [18–20]. It
should be noted that such complex nucleoprotein structures need
to be resolved during DNA replication. As a result, telomere structures are dynamic and highly regulated throughout different cell
cycle and developmental stages [21, 22].

2  The Telomere Interactome: An Integrated Telomere Signaling Network
One inherent problem during mammalian DNA synthesis is the
replication of linear chromosomal ends [23–25]. Together with
telomerase (TERT and TERC), the telomeric nucleoprotein complex inhibits loss of genetic information due to incomplete end
replication [26–30]. Tremendous progress has been made in the
last decade in our understanding of the vast protein networks at
the telomeres—the Telomere Interactome [31]. The interactome
incorporates diverse telomere signaling pathways and represents
the molecular machinery that regulates mammalian telomeres. Key
protein–protein interaction hubs within the interactome include
telomerase, TRF1, and TRF2.
The telomerase contains a highly conserved reverse transcriptase (TERT) and a template RNA (TERC or TR) [27]. Using the
3′ telomere overhang as a primer to align with TERC sequences,
TERT adds telomeric repeats to chromosome ends. A number of
proteins and factors have been shown to interact with the telomerase, including dyskerin and TCAB1 [32–34], and the list continues
to grow. Cells that lack TERT extend their telomeres through the
alternative lengthening of telomeres (ALT) pathway [35–38].
TRF1 and TRF2 specifically bind to telomeric DNA through
their Myb domains [39–42]. It has been shown that TRF1 counts
and controls the length of telomere repeats, likely through complexing with TIN2, Tankyrase, PINX1, TPP1, and POT1 [43–54].
In comparison, TRF2 has an essential role in end protection,
recruiting the BRCT domain-containing protein RAP1, the nucleotide excision repair protein ERCC1/XPF, BLM helicase, and
DNA repair proteins PARP-1, Ku70/80, MCPH1, and the
Mre11/Rad50/NBS1 (MRN) complex [52, 55–63]. TRF2 is
thought to be critical for maintaining the T-loop that may be



Introduction to Telomeres and Telomerase

3

important for shielding the ends from being recognized as DNA
breaks [42, 64, 65]. Furthermore, TRF2 associates with the RecQ-­
like helicase WRN that regulates end protection, the D-loop structure, and replication of the G-rich telomere strand [62, 66–68]. In
fact, the six telomere-targeted proteins, TRF1, TRF2, TPP1,
TIN2, RAP1, and POT1, can form a core protein complex within
the telomere interactome [17, 31, 69], coordinating protein–protein interactions and cross talk between different pathways. These
proteins in turn recruit a multitude of factors to dynamically regulate telomeres.
Protein–protein interaction studies have also offered clues
regarding the posttranslational modifications that are important to
telomere maintenance. For example, kinases (such as ATM and
DNA-PK) have been shown to be recruited to telomeres [70–76],
while poly-ADP ribosylation plays an important role in regulating
TRF1 and TRF2 [47, 77, 78]. Furthermore, ubiquitination may
add another level of control for telomere proteins, as is the case for
the TRF1-specific E3-ligase FBX4 [79]. Further studies should
shed light on the signals that activate these modifying enzymes and
whether additional modifications are involved.

3  Telomere Dysfunction, Genome Stability, and Diseases
Without telomere protection, exposed or critically short chromosomes may result in chromosome end-to-end fusion, formation of
dicentric chromosomes, and ultimately aneuploidy [80]. The
unprotected ends can also activate DNA damage response pathways, cell cycle checkpoints, senescence, and apoptosis [25, 75, 76,
81–83]. Cancer development and aging are often associated with
changes in telomeres. Telomere erosion, found in many human cell

types, is thought to limit the proliferative capacity of transformed
cells. In humans, telomerase expression appears tightly regulated;
in >90% of human cancers normal telomere maintenance is often
bypassed and TERT expression upregulated [30]. The strongest
evidence to date for the importance and function of telomerase
and telomere maintenance comes from studies in human diseases
and mouse models. Patients with the rare human disease dyskeratosis congenita syndrome (DKC) have abnormally short telomeres
and lower telomerase activity [84], with clinical manifestions of
premature aging phenotypes and increased incidence of cancer
[84–87]. Depending on the underlying mutations, DKC has several modes of inheritance. X-linked DKC is due to a mutation in
dyskerin that can bind to the telomerase RNA template (TERC)
[88], and mutations in the TERC and TERT genes of the telomerase itself lead mostly to autosomal-dominant DKC [89, 90].
Mutations in the other subunits of the telomerase holoenzymes,
such as NOP10 and NHP2, have also been identified in


4

Zhou Songyang

DKC patients [91, 92]. In addition, some patients with leukemia
have mutations in the TERC and TERT genes [84, 85]. More
recently, mutations in core telomere proteins TIN2 [93–96], TPP1
[97, 98], and POT1 [99] have been found in patients suffering
from cancer and telomere-related diseases such as DC, aplastic anemia, and Revesz syndrome.
Several DNA repair proteins can localize to telomeres and
interact with telomere-binding proteins [85], a number of which
are mutated in human genome instability syndromes characterized by premature aging, increased cancer susceptibility, and critically short telomeres. For example, the gene defective in the
autosomal recessive Werner syndrome (WS) is WRN, a 3′-5′ helicase and exonuclease that participates in DNA replication, repair,
recombination, and transcription [66, 100–107]. WS cells display

premature senescence, accelerated telomere attrition, and defective telomere repair [108]. Like WS, the gene mutated in Bloom
syndrome (BS) also encodes a helicase (BLM) with diverse functions [109–111]. Both WRN and BLM have been demonstrated
to interact with the telomere binding protein TRF2 in ALT
(alternative lengthening of telomeres) cells [58, 59]. Ataxia telangiectasia (AT) and the Nijmegen breakage syndrome (NBS)
share many characteristics including developmental retardation
and predisposition to lymphoid malignancy [112–117]. Cells
from these patients exhibit pronounced genome instabilities such
as chromosome end-to-end fusions. The gene products responsible for NBS (NBS1) and AT (ATM) have been shown to interact with a number of telomere proteins [56, 73, 75, 76, 118–121].
Fanconi anemia (FA) is a heterogeneous disorder characterized
by bone marrow failure and high incidence of developmental
abnormalities and cancer [122–124]. In FA patients, there is a
strong correlation between telomere dysfunction and hematopoietic defects [125–127].
Several mouse models of the diseases mentioned above have
been generated. Mutations in a number of telomere regulators
(e.g., Rte1, RAD51D, ATM family kinases, and Ku) have provided
important evidence linking telomere dysfunction to the development of diseases such as cancer [128–132]. Homozygous knockout
for BLM is lethal [133], whereas TERC−/− and WRN−/− mice are
initially normal [134]. However, successive breeding of TERC−/−
mice leads to progressive telomere loss, chromosome end-to-end
fusions, and various age-related diseases affecting highly proliferative tissues [135, 136]. Furthermore, inactivation of the gene
encoding TERC in combination with the BLM hypomorphic mutation and/or WRN null mutation results in accelerated pathology
compared to TERC−/− [137, 138]. Similarly, mice homozygous
knockout for TERT also exhibit accelerated telomere shortening
and genomic instability [139]. Homozygous inactivation of the
core telomeric proteins TIN2 or TRF1 is lethal [140–142].


Introduction to Telomeres and Telomerase

5


In conditional POT1 knockout mice, mouse POT1a and POT1b
are shown to function distinctly, and both are required for normal
telomere maintenance [143, 144]. In adrenocortical dysplasia (acd)
mice (a spontaneous autosomal recessive mutant), a mutation in the
TPP1 gene results in aberrant splicing of TPP1, leading to adrenal
dysplasia, skin abnormalities, and defects in embryologic and germ
cells [145]. Interestingly, deletion of RAP1 in mice revealed a possible role of RAP1 in bridging telomere function, transcriptional
regulation, and metabolism [146, 147].

4  Tools for Studying Telomere Biology
Over the years, numerous tools and platforms have been developed for telomere studies, affording us unprecedented abilities to
probe the many aspects of telomere biology. At the same time,
techniques commonly used in other fields are being incorporated
and adapted for telomere studies. We are now able to assay changes
in telomeres and telomere proteins on a larger scale, at higher
resolution (e.g., within a single cell or chromosome), and with
better sensitivity and accuracy. One set of commonly used tools
for telomere studies focuses on the telomere DNA itself, biochemical approaches such as EM and NMR seek to probe the
structure and property of telomere DNA. For example,
G-quadruplexes may play an important part in telomere structure
and are being actively investigated as drug targets. In addition to
duplex repetitive sequences at chromosomal ends, extra-chromosomal telomere homologous sequences are also found in cells and
may be products of normal or dysfunctional telomere metabolism.
In the current series, we have added methods for detecting such
telomere DNA species. Assays to measure telomere length are the
workhorses in telomere biology, since telomere length is a major
indicator of telomerase function. A number of methods have been
developed to determine the length of telomere repeat DNA (and
its overhangs) under different conditions and in a variety of systems. These assays, from TRF to STELLA to 3′ overhang measurements, allow us to look at telomere length changes in a cell

population or a single cell and provide more accurate information
regarding changes in telomere protection.
Another set of tools focuses on the proteins that are important
for telomere function. The most important enzyme at the telomeres is telomerase, for which a number of methods are available
to assess its activity and function. In addition, other telomerase and
telomere-associated proteins have also become the subjects of
intense investigation, and biochemical and molecular approaches
have been utilized to analyze these telomere factors. Examples
include proteomic tools to study the network of proteins at
telomeres and how they interact with each other, as well as spatial


6

Zhou Songyang

and temporal information regarding how proteins are targeted to
telomeres. The fluorescent and luciferase protein complementation assays in this series should greatly facilitate our investigation
into the protein–protein interaction network.
The last set of tools seeks to understand the consequences of
telomere dysfunction. While telomere length changes may reflect
such dysfunction, it usually takes longer for the effect to become
apparent in a population of cells. On the other hand, immediate
consequences as a result of decapping or deprotection at the
telomeres may be assayed by a variety of tools, many of which
rely on microscopy to visualize the damage and changes at the
telomeres. For example, one important indication of telomere
dysfunction is the telomeric recruitment of proteins involved in
DNA damage responses. This response can be examined by the
Telomere dysfunction induced foci (TIF) assay. The number of

telomere-specific DNA-damage foci can then be quantified and
compared. When used with a telomere-specific probe, chromosome orientation fluorescence in situ hybridization (CO-FISH)
can determine the absolute 5′ to 3′ pter-qter direction of a DNA
sequence. The technique has proven especially powerful for
assaying abnormalities associated with telomere dysfunction,
including telomere sister chromatid exchange (T-SCE) and telomere fusion.
One of the most exciting developments in the last couple of
years has been the CRISPR/Cas9 technology. Compared to
conventional approaches and other genome modification methods using nucleases, CRISPR/Cas9 promises much more expedient, convenient, and versatile means to precisely manipulate
the genome [148–157]. Cas9, guided to the target site by
sequence-specific guide RNAs, cleavages genomic DNA and
generates double-strand breaks, which in turn trigger the nonhomologous end joining (NHEJ) DNA repair mechanism in the
absence of a donor template [158–160]. NHEJ-mediated DNA
repair may generate small insertions and/or deletions (indels) at
the target site, potentially leading to loss of gene function if
cleavage occurs within protein coding sequences. CRIPSR/
Cas9 enables investigators to generate cells and cell lines
knocked out for their genes of interest. This edition includes
the generation of a CRISPR/Cas9 knockout human cell line for
telomere length analysis.
In conclusion, we have in our possession an ever-expanding
arsenal that continues to aid us in dissecting the function of
telomerase and telomere-binding proteins, probing the changes
in telomeres, and elucidating the consequences of telomere
dysfunction.


Introduction to Telomeres and Telomerase

7


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Chapter 2
Analysis of Average Telomere Length in Human Telomeric
Protein Knockout Cells Generated by CRISPR/Cas9
Jun Xu, Zhou Songyang, Dan Liu, and Hyeung Kim
Abstract
Telomeres play an important role in ensuring the integrity of the genome. Telomere shortening can lead
to loss of genetic information and trigger DNA damage responses. Cultured mammalian cells have served
as critical model systems for studying the function of telomere binding proteins and telomerase. Tremendous
heterogeneity can be observed both between species and within a single cell population. Recent advances
in genome editing (such as the development of the CRISPR/Cas9 platform) have further enabled researchers to carry out loss-of-function analysis of how disrupting key players in telomere maintenance affects
telomere length regulation. Here we describe the steps to be carried out in order to analyze the average
length of telomeres in CRISPR-engineered human knockout (KO) cells (TRF analysis).
Key words Telomere length, TRF, Telomere maintenance, CRISPR, Cas9, Knockout

1  Introduction
In eukaryotic cells with linear chromosomes, the chromosomal
ends—telomeres—are maintained and protected through the
coordinated action of telomerase and telomere binding proteins
[1, 2]. Different organisms display remarkable variability in the
makeup and exact length of the repetitive telomeric elements in

their telomere DNA sequences. For example, in yeast, the sequence
is 350 ± 75 bps of C1–3A/TG1–3 [3], whereas mammalian telomeres
contain (TTAGGG)n. Among mammalian species, mouse telomeres can be up to 150 kb, while somatic human cells have telomeres of 5–15 kb in length [4]. Even in a relatively homogenous
population such as cultured mammalian cell lines, telomeres exhibit
great heterogeneity in length.
Perturbations in the intricate telomere interacting and regulatory network can lead to changes in telomere structure and exposed
chromosomal ends [5, 6]. In telomerase-active cells such as cancer
cells and during development, such changes in turn impact the
length of telomeres and the status of the cell such as its replicative
Zhou Songyang (ed.), Telomeres and Telomerase: Methods and Protocols, Methods in Molecular Biology, vol. 1587,
DOI 10.1007/978-1-4939-6892-3_2, © Springer Science+Business Media LLC 2017

15


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Jun Xu et al.

potential [7, 8]. The advent of new genome-editing tools such as
CRISPR/Cas9 has enabled investigators to better understand how
inactivation of individual telomere regulators affects the maintenance and status of telomere length.
The CRISPR/Cas9 system, adapted from the acquired
immune systems of bacteria and archaea, consists of the Cas9
nuclease and a guide RNA (gRNA). Through RNA-DNA hybridization, the 20 nucleotide sequence at the 5′ or 3′ end of the gRNA
determines the target site, a feature that has simplified the process
of targeting endogenous loci for disruption due to the ease with
which gRNA sequences can be manipulated. Streptococcus pyogenes
Cas9 (SpCas9), which has a specific protospacer adjacent motif
(PAM) preference of 5′-NGG-3′, is the most extensively characterized and widely used in genome editing.

In this chapter, we describe the steps for generating telomeric
protein KO cells using the CRISPR/Cas9 platform. These cells are
then used to study how inactivation of a telomere regulator impacts
telomere length control using terminal restriction fragments (TRF)
analysis. Genomic Southern blotting has been adapted to assess the
average length of telomeres in populations of cultured mammalian
cells. Here, genomic DNA is digested with frequent cutting restriction enzymes, to which repetitive telomeric sequences are resistant,
thereby allowing for the analysis of the length of chromosomal
terminal restriction fragments. The final results reflect the estimation of both the telomeric repeats and sub-telomeric regions that
do not contain the particular restriction digest sites.

2  Materials
2.1  For the
Generation of KO Cells
by CRISPR/Cas9

1.A human cell line of interest and the appropriate medium and
supplements for culturing the cell line (see Note 4.1.1).
2.The CRISPR/Cas9 vector pSpCas9(BB)-2A–GFP (PX458)
from Addgene (Plasmid# 48138) (see Note 4.1.2).
3.The restriction enzyme BbsI and its digestion buffer, and calf
intestinal alkaline phosphatase (CIP) and its reaction buffer.
4. DNA spin-columns for purification (e.g., QIAquick PCR purification kit from Qiagen).
5.Agarose.
6. Agarose gel electrophoresis apparatus.
7. 50× TAE buffer: Mix 242 g Tris base, 57.1 mL acetic acid, and
18.6 g EDTA in ddH2O to final volume of 1 L. Make 1× TAE
buffer from this stock solution.
8. Gel extraction DNA purification kit (such as the QIAquick Gel
Extraction Kit).