Methods in molecular biology vol 1587 telomeres and telomerase methods and protocols

1 Zhou Songyang 2 Analysis of Average Telomere Length in Human Telomeric Protein Knockout Cells Generated by CRISPR/Cas9.. 1587, DOI 10.1007/978-1-4939-6892-3_1, © Springer Science+Busi

Telomeres

and Telomerase

Zhou Songyang Editor

Methods and Protocols

Third Edition

Methods in

Molecular Biology 1587

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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

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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Zhou Songyang

Department of Biochemistry and Molecular Biology

Baylor College of Medicine

Houston, TX, USA

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 dis-eases 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, tion 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 funda-mental 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

func-These protocols are detailed and easy to follow It is our belief that this work will prove useful and informative

Preface

Contents

Preface v Contributors ix

1 Introduction to Telomeres and Telomerase 1

Zhou Songyang

2 Analysis of Average Telomere Length in Human Telomeric Protein

Knockout Cells Generated by CRISPR/Cas9 15

Jun Xu, Zhou Songyang, Dan Liu, and Hyeung Kim

3 Telomere Length Analysis by Quantitative Fluorescent in Situ

Hybridization (Q-FISH) 29

Isabelle Ourliac-Garnier and Arturo Londoño-Vallejo

4 Telomere Strand-Specific Length Analysis by Fluorescent

In Situ Hybridization (Q-CO-FISH) 41

Isabelle Ourliac-Garnier and Arturo Londoño-Vallejo

5 Telomere G-Rich Overhang Length Measurement: DSN Method 55

Yong Zhao, Jerry W Shay, and Woodring E Wright

6 Telomere G-Overhang Length Measurement Method 2: G-Tail

Telomere HPA 63

Hidetoshi Tahara

7 Telomere Terminal G/C Strand Synthesis: Measuring Telomerase

Action and C-Rich Fill-In 71

Yong Zhao, Jerry W Shay, and Woodring E Wright

8 Analysis of Yeast Telomerase by Primer Extension Assays 83

Min Hsu and Neal F Lue

9 Assessing Telomerase Activities in Mammalian Cells Using the Quantitative

PCR-Based Telomeric Repeat Amplification Protocol (qTRAP) 95

Shuai Jiang, Mengfan Tang, Huawei Xin, and Junjiu Huang

10 Telomeres and NextGen CO-FISH: Directional Genomic Hybridization

(Telo-dGH™) 103

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 113

Eladio Abreu, Rebecca M Terns, and Michael P Terns

12 Cytogenetic Analysis of Telomere Dysfunction 127

Rekha Rai, Asha S Multani, and Sandy Chang

13 Probing the Telomere Damage Response 133

Rekha Rai and Sandy Chang

14 Induction of Site-Specific Oxidative Damage at Telomeres

by Killerred-Fused Shelretin Proteins 139

Rong Tan and Li Lan

15 Using Protein-Fragment Complementation Assays (PCA)

and Peptide Arrays to Study Telomeric Protein-Protein Interactions 147

Wenbin Ma, Ok-hee Lee, Hyeung Kim, and Zhou Songyang

16 In Vitro Preparation and Crystallization of Vertebrate

Telomerase Subunits 161

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 171

Clement Lin and Danzhou Yang

18 Analysis of Telomere-Homologous DNA with Different Conformations

Using 2D Agarose Electrophoresis and In-Gel Hybridization 197

Zepeng Zhang, Qian Hu, and Yong Zhao

19 Analysis of Telomere Proteins by Chromatin Immunoprecipitation (ChIP) 205

Feng Liu, Xuyang Feng, and Wenbin Ma

Index 215

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

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

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

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

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 chro-mosome 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 dis-rupting 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 mam-malian cells, the 5′-TTAGGG-3′ repeat sequences can reach hun-dreds 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 diversity

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 tures are dynamic and highly regulated throughout different cell cycle and developmental stages [21, 22]

struc-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 com-plex 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 tase (TERT) and a template RNA (TERC or TR) [27] Using the

transcrip-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 telomer-ase, 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 com-plexing 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 nucle-otide 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

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 struc-ture, 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–pro-tein interactions and cross talk between different pathways These proteins in turn recruit a multitude of factors to dynamically regu-late 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 somes 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 path-ways, cell cycle checkpoints, senescence, and apoptosis [25, 75, 76,

chromo-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 dyskera-tosis 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 sev-eral 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 telom-erase 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

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 ane-mia, 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 character-ized by premature aging, increased cancer susceptibility, and criti-cally short telomeres For example, the gene defective in the

autosomal recessive Werner syndrome (WS) is WRN, a 3′-5′ case and exonuclease that participates in DNA replication, repair, recombination, and transcription [66, 100–107] WS cells display premature senescence, accelerated telomere attrition, and defec-tive telomere repair [108] Like WS, the gene mutated in Bloom syndrome (BS) also encodes a helicase (BLM) with diverse func-tions [109–111] Both WRN and BLM have been demonstrated

heli-to interact with the telomere binding protein TRF2 in ALT (alternative lengthening of telomeres) cells [58, 59] Ataxia tel-angiectasia (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 sible for NBS (NBS1) and AT (ATM) have been shown to inter-act with a number of telomere proteins [56, 73, 75, 76, 118–121] Fanconi anemia (FA) is a heterogeneous disorder characterized

respon-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 hematopoi-etic 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 develop-ment 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 prolifera-tive tissues [135, 136] Furthermore, inactivation of the gene encoding TERC in combination with the BLM hypomorphic muta-tion 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]

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 pos-sible 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 oped 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

devel-in telomeres and telomere protedevel-ins 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, bio-chemical 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-chromo-somal 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 sys-tems These assays, from TRF to STELLA to 3′ overhang mea-surements, 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 telo-meres 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

and temporal information regarding how proteins are targeted to telomeres The fluorescent and luciferase protein complementa-tion 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, chromo-some 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 telo-mere 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 meth-ods using nucleases, CRISPR/Cas9 promises much more expe-dient, 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 non-homologous 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

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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

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 ers 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).

research-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 telo-meres can be up to 150 kb, while somatic human cells have telo-meres 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 tory 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

regula-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 mainte-nance 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 hybrid-ization, 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 character-ized 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 restric-tion 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 estima-tion of both the telomeric repeats and sub-telomeric regions that

do not contain the particular restriction digest sites

2 Materials

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 fication 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)

2.1 For the

Generation of KO Cells

by CRISPR/Cas9

9 Two complementary oligonucleotides synthesized based on

gRNA design for the target gene locus (see Note 4.1.3).

10 T4 polynucleotide kinase used with T4 ligase buffer that tains ATP

11 T4 DNA ligase and buffer (quick reaction is preferable)

12 Bacterial competent cells for transformation

13 LB agar plates with appropriate antibiotics (ampicillin for px458) (100 mm/15 mm)

14 1× LB medium: dissolve 10 g of Bacto tryptone, 5 g of Bacto yeast extract, and 10 g of NaCl in ddH2O to 1 L Autoclave and store at room temperature

15 DNA miniprep and maxiprep kits

16 Tissue culture dishes (60 and 100 mm) and multi-well plates (6, 24, and 96-well (flat-bottom))

17 DNase/RNase-free ddH2O

18 A set of primers, which can amplify the region encompassing the target site, for Cas9 cleavage efficiency testing and clone

genotyping (see Note 4.1.4).

19 Filtered sterile pipette tips for PCR amplification

20 High-fidelity Taq polymerase for genomic DNA PCR.

21 QuickExtract™ DNA Extraction Solution (Epicentre)

22 T7 endonuclease I

23 A thermal cycler

24 Transfection reagents or an electroporation system (e.g., the Neon® Transfection System from Invitrogen) and the accom-

panying transfection kit (see Note 4.1.5).

25 A 37 °C incubator for bacteria growth

26 A 37 °C incubator with 5% CO2 for human cells

1 Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3

2 Restriction digestion enzymes RsaI and HinfI and DNase-free RNase

3 1× TAE as specified in item 7 of Subheading 2.1

4 DNA molecular weight markers: 1 kb DNA ladder and CHEF DNA size standard-5 kb ladder

5 Ethidium bromide stock solution at 10 mg/mL

6 Agarose gel electrophoresis apparatus

7 Depurination solution: 0.25 M HCl

8 Denaturation solution: 0.4 M NaOH

2.2 For TRF Analysis

9 Neutralization solution: 1.5 M NaCl, 0.5 M Tris–HCl, pH = 7.5.

10 20× SSC stock solution: 3 M NaCl, 0.3 M sodium citrate, pH

= 7 Dissolve 175.3 g NaCl and 88.2 g trisodium citrate (citric acid) in ddH2O to make 1 L

11 Hybond-N+ nylon membrane (GE Science) or equivalent

12 Prehybridization and Hybridization buffer: 0.5 M phosphate buffer (pH 7.2), 7% SDS, 1 mM EDTA (pH 8) 1 M phos-phate buffer (pH 7.2) stock solution can be made by mixing 17.1 mL of Na2HPO4 and 7.9 mL of NaH2PO4, Store the hybridization solution at −20 °C

13 (TTAGGG)3 telomeric probe Labeling may be carried out in

a 20 μL reaction with 2 μL of T4 polynucleotide kinase (NEB), 1× kinase reaction buffer (NEB), 7 μL γ-32P ATP (3000 Ci/mmol), and 10 pmol of the oligonucleotide probe for 1 h at

37 °C Remove unincorporated labels with QIAquick tide removal kit (QIAGEN)

14 Low stringency wash buffer: 4× SSC(from 10)/0.1%SDS

15 High stringency wash buffer: 2× SSC(from 10)/0.1%SDS

3 Methods

1 Design the gRNA oligos: Identify the genomic DNA region to

be targeted for KO (e.g., a 1–2 kb region within the first or second exon) (Fig 1) Use an online tool (e.g., http://tools

as instructed Select 2–3 potential gRNA sites from the output

(see Note 4.1.6).

2 Order both the sense and antisense oligos for each site, with the overhang sequence for cloning into the PX458 vector If the gRNA sequence does not start with a G, add a G to make the guide sequence 21 nt long

Myb

445 TRF2

*

73 Trf2_gRNA 5'-GCCTTTCGGGGTAGCCGGTA-3' Fig 1 A CRISPR gRNA is chosen for the telomeric protein TRF2 and predicted to result in truncation of TRF2 at

residue 73

incubate the mixture in a thermocycler, 37 °C for 30 min, 95 °C for

5 min, and then cool to 25 °C at 5 °C per min (see Note 4.1.7).

100 μM sense strand oligo (Oligo 1) 1

100 μM antisense strand oligo (Oligo 2) 1 10× T4 ligation buffer (NEB) 1

4 Digest 5 μg of PX458 with Bbs I for 1 hr at 37 °C Then add

1 μL of CIP and incubate the reaction at 37 °C for another

30 min quickly purify the digested vector through spin- columns to remove salt and enzymes

5 Set up a 10 μL ligation reaction with 50 ng of digested vector (from step 5), 1 μL diluted oligos (from step 4), and T4 DNA

ligase, and incubate the mixture at room temperature The length

of incubation time depends on the particular enzyme used

6 Transform bacterial competent cells with 1 μL of the ligation reaction, and plate the competent cells on an agar plate con-taining 100 μg/mL ampicillin

7 Pick a few single bacterial colonies for plasmid DNA extraction using the miniprep kit Sequence the plasmid DNA using the U6 sequencing primer

1 Transfect the target cell line with the CRISPR/gRNA vector Alternatively use an easy to transfect cell line (e.g., 293 T cells) Either a transfection reagent or electroporation system may be used Follow the directions of the manufacturer of the electro-poration system for the voltage and pulse for the specific cell type used

2 Collect and pellet the cells at 48 h after transfection and extract the genomic DNA using the QuickExtract™ DNA Extraction Solution (Epicentre) Follow the manufacturer’s directions

Please see Fig 2 for an example of testing the CRISPR gRNA that targets TRF2

3 Set up a PCR reaction using the genomic DNA from Step 2 above, and the primer set specified in item 18 of Subheading

2.1, to amplify the genomic region containing the gRNA

tar-get site (see Note 4.1.4) The exact conditions depend on the

Taq polymerases used and the length of the predicted ucts Follow the manufacturer’s directions

prod-3.1.2 Validate

CRISPR/Cas9 gRNA Vector

Cleavage Efficiency (See

Note 4.1.8)

4 Once the PCR reaction is done, anneal the reaction products

on a thermal cycler using the following conditions:

Lyse 10 5 cells in 200ml QuickExtract soluon

Vortex Incubate for 6 min

at 65⁰C Vortex Incubate for 2 min

at 98⁰C Use 4ml for PCR

Fig 2 An example of cleavage efficiency analysis Left, a flowchart for extracting

genomic DNA for cleavage analysis Right, the extracted DNA was used to PCR amplify the genomic region encompassing the TRF2 gRNA target site The PCR products were denatured and then annealed before being incubated with T7 endonuclease I (T7EN) The digested products were resolved on an agarose gel Parental cells were used as control (Con)

1 Prepare large quantities of the CRISPR gRNA vector using a DNA maxiprep kit.

2 Transfect your cells of interest as in Subheading 3.1.2 Scale up the amount of DNA for the transfection as more cells need to

be transfected

3 (Optional) At >24 h after transfection, sort the GFP+ cells on a FACS sorter directly into 96-well plates at no more than one cell per well Alternatively, the cells can be sorted as a pool and then

plated into 96-well plates by serial dilution (see Note 4.1.10).

4 At >24 h after transfection, plate the cells into 96-well plates by serial dilution, with the final dilution at <60 cells/plate The total number of cells plated depends on the specific cell line and the cleavage efficiencies of the gRNAs tested in Subheading 3.1.2

We recommend at least 600 cells (i.e., ten plates) as a start

5 Culture the cells and replenish with fresh media as needed Clones of cells should form around 2–4 weeks after serial dilu-

tion plating, depending on the growth rate of the cells (see

Note 4.1.11).

6 Expand the clones as the cells grow back, by transferring the cells to larger dishes or plates as needed It is normal for differ-ent wells to exhibit differences in growth rates

7 Once there are enough clones that have grown back, mine if the gene of interest has been knocked out by several methods, including PCR, western blotting, and immunofluo-rescence Western blotting and immunofluorescence can be quick methods to identify candidate KO clones (Fig 3) All positive clones should be verified by genotyping The region encompassing the target site should be PCR amplified using

deter-the primer set for cleavage verification and sequenced (see Note 4.1.12) Because of potential clonal variation, it is also recom-

mended that multiple KO clones be selected and analyzed for TRF below

TRF2 KO clones

Fig 3 Western blotting analysis of clones of cells that grew back after single cell

cloning, using antibodies against TRF2 Three individual clones were tested here, with parental cells as control An antibody against actin served as a loading control

1 Harvest at least 300,000 cultured cells and wash with PBS Collect the cells in a microcentrifuge tube (if possible)

Spin at 1300 × g in a microcentrifuge at room temperature, wash in 1× PBS and collect the pellet (see Note 4.2.1) Cell

pellets can be assayed immediately, or directly frozen and stored at −80 °C

2 Extract genomic DNA using the QIAGEN DNeasy Tissue kit (QIAGEN) Standard genomic DNA extractions usually require several phenol–chloroform extraction steps, which makes processing multiple samples (routine for TRF analysis)

time consuming (see Note 4.2.2) Estimate the amount of

DNA based on the number of cells used (see Note 4.2.2).

3 Mix ~2–5 μg of extracted genomic DNA with 15 units each of

RsaI and HinfI, and 1 μg of RNase A Incubate at 37 °C for

≥12 h The digested DNA mixture may be stored at −20 °C

until further use (see Note 4.2.3).

1 Prepare a large agarose gel (0.7%, 20–25 cm long) (roughly

300 mL) in 1× TAE buffer containing ethidium bromide (2–3

μL) (see Note 4.2.4).

2 Load 1–2 μg of digested genomic DNA per lane Load DNA molecular weight markers (preferably mixed with 1–3 × 105cpm radiolabeled DNA marker) to aid visualization under UV

and to facilitate quantification steps (see Note 4.2.5) Run the

gel at 1.5 volts/cm until the 1 kb marker is at the bottom of

the gel (see Note 4.2.6).

3 Visualize and document the gel under UV Handle with care as the gel can be fragile and prone to breakage Use a ruler to note the positions of the DNA ladder relative to the wells

4 Soak the gel in depurination buffer for 15–20 min with gentle

agitation (see Note 4.2.7).

5 Discard the solution, briefly rinse the gel in ddH2O, and soak the gel in denaturation buffer for 30 min with gentle agitation

6 Discard the solution, rinse the gel in ddH2O, and neutralize the gel in neutralization solution for 30 min with gentle agitation

7 Equilibrate the gel in 2× SSC for 5–10 min, and wet the Hybond

N+ nylon membrane in 2× SSC Mark the gel and membrane for easy orientation during hybridization and analysis Set up capil-

lary transfer in 2× SSC for >12 h (see Note 4.2.8).

8 Disassemble the transfer assembly, and UV cross-link the DNA

to the membrane (120 mJ/cm2) with the DNA side facing up The membrane can be stored in a sealed plastic bag with sup-port at −20 °C until ready to use (see Note 4.2.9).

1 Pre-hybridize the membrane in Hybridization buffer in a sealed bag or roller bottle at 50 °C for ≥2 h Use 10–20 mL of buffer depending on the size of the blot.

2 Prepare purified radiolabeled telomeric probe as described in

item 13 of Subheading 2.2 Determine the specific activity of

the probe using a liquid scintillation counter (see Note 4.2.10).

3 Discard the prehybridization solution, add fresh Hybridization solution (10–20 mL) along with the labeled probe (~1–5 × 106cpm/mL), and incubate at 50 °C for at least 12 h

4 Properly dispose the hybridization solution Rinse the brane briefly in low stringency wash buffer to remove excess probes and hybridization solution Then wash the membrane

mem-in succession with low and high strmem-ingency wash buffers A minimum wash should have two low stringency and two high

stringency buffer washes Please see Note 4.2.11 for a guide to

the wash steps as the length and temperature of each wash step should be empirically determined

5 Blot-dry the membrane to get rid of excess wash buffer, wrap

it in plastic wrap Autoradiograph using KODAK X-OMAT film or equivalent for densitometric analysis, or expose the membrane in a PhosphoImager cassette for visualization and

quantification on a PhorsphoImager (see Note 4.2.12).

6 Use the Telorun spreadsheet to calculate average telomere length, which can be found at the homepage of the Shay and Wright laboratory (http://www.utsouthwestern.edu/labs/

4 Notes

1 The particular cell line of choice will be dictated by factors such as readout assays, biological questions asked, and the ease with which CRISPR reagents can be introduced The method described here requires transfection of CRISPR plasmids Cell lines that are less amenable to transfection may be more diffi-cult to use for KO purposes

2 There are many different CRISPR/Cas9 vectors, with ent designs, tags, and markers We use PX458 because of the GFP marker in the construct that enables FACS sorting of the transfected cells

3 The oligo sequences depend on the gRNA site chosen and the specific vector to be used

4 The gRNA vectors should be tested for cleavage efficiency And PCR-based genotyping is needed for clone analysis Therefore, a set of primers that robustly and specifically amplify

3.2.3 Hybridization

and Analysis

4.1 CRISPR/Cas9

KO Cells

the genomic region encompassing the target site is needed for each gRNA Use the NCBI/Primer-Blast to design genomic PCR primers The ideal PCR product size is from 300 bp to 1

kb Longer products are less efficient to amplify and smaller products may be difficult to detect on an agarose gel

5 The CRISPR plasmids can certainly be transfected into cells using transfection reagents, especially for cells that are easy to transfect We have found that electroporation routinely achieves higher efficiency without sacrificing viability Multiple rounds of transfection can be done to increase the efficiency Higher transfection efficiency should help improve KO frequencies

6 There are many free online tools for selecting gRNA sequences

To minimize off-target effects, the 20 nt gRNA sequence lowed by the NGG PAM) should have little homology to other genomic sites To achieve complete KO of a gene and avoid the expression of a truncated protein, the gRNA target site should locate within the first few exons of the coding sequence

(fol-In cases of the existence of alternative splicing products, the common region of all splicing isoforms should be targeted, whenever possible

7 The T4 ligation buffer should be fresh Aliquot and freeze if necessary Avoid frequent freeze–thaw cycles, which degrade the ATP in the buffer Alternatively, T4 polybucleotide kinase buffer and fresh ATP solution can be used instead of the T4 Ligation buffer

8 Cas9 cleaves at the position 3 nt 5′ of the PAM sequence, but the efficiency of this cleavage remains impossible to predict

We recommend making three different gRNA vectors and test them in cells If none works well, more gRNAs need to be screened

9 Use no more than 0.2U of T7 endonuclease I (NEB) per 20

μL of digestion reaction Cas9 cleavage results in indels in one

or both strands of genomic DNA Since not all of the cells will have been cleaved by Cas9, some PCR products will be from wildtype cells and some from mutant cells Annealed duplexes that are not precisely complementary to each other will create mismatches that can be recognized and cut by T7 endonucle-ase I Instead of T7 endonuclease I, the SURVEYOR nuclease can be used as well

10 If the CRISPR vector has a fluorescent marker (e.g., GFP for PX458), the transfected cells can be sorted by flow cytometry based on GFP expression Take care to avoid contaminating the cells during sorting Cells with high enough transfection efficiency (>80%) do not need to be sorted Sorting can also help separate cells into single-cell suspensions For some cell

types, sorting offers no increased advantage over serial dilution plating in terms of cost and excessive handling of the cells.

11 Fast growing cells such as 293 T and Hela form colonies in 2 weeks, which are clearly visible under the microscope Slow dividing cells may take much longer to form visible colonies

In fact, some cells may not be easily cloned from low- density plating or single cells It is highly recommended to test whether the cell line of interest is clonable from single cells, if not already known

12 If a good antibody is not available for easy western blotting or immunostaining, we recommend designing two CRISPR gRNAs so as to delete an entire exon The two CRISPR gRNA target regions should be in the intron regions on each side of the exon Deletion of the exon should result in frameshift mutations and premature stop codons Deletion of the exon can then be more easily screened by genomic DNA PCR and sequencing

1 In general, 106 mammalian cells yield roughly 6 μg of genomic DNA For genomic Southern blotting analysis, at least 2 μg of DNA is needed Typically 5 μg of DNA is ideal for the analysis This may serve as a guide for calculating the number of cells needed per assay

2 The genomic DNA may also be extracted using standard genomic DNA extraction protocols An example using pro-teinase K is given below Please note that while spectrophoto-metric measurements are usually used to assess the quality and quantity of genomic DNA, many genomic DNA preparations often contain significant amount of RNA, which can skew the results

• Harvest cells in a DNase-free clean microcentrifuge tube

• Resuspend cells in 100 μL 1× PBS

• Add 200 μL of Lysis buffer (0.3 M Tris (pH 8), 0.15 M EDTA (pH 8), 1.5% SDS), plus 15 μL of freshly added proteinase K (10 mg/mL)

• Mix and incubate at 55 °C for 2–12 h Heat the sample to

70 °C for 30 min to inactive proteinase K

• Briefly centrifuge the tube to collect all liquid Add 200 μL

of lysis buffer and mix

• Add 500 μL of phenol–chloroform–isopropanol (1:1:1)

• Mix thoroughly by vortexing and spin in microcentrifuge

at top speed for 5 min

• Transfer the top aqueous phase to a new tube containing

500 μL chloroform–isopropanol (1:1) And repeat ning and transfer step

spin-4.2 TRF Analysis

• Add 200 μL 7.5 M ammonium acetate and 800 μL of 100% ethanol, mix by inverting the tube multiple times.

• Spin in microcentrifuge at top speed for 5 min

• Wash the pelleted DNA with cold 70% ethanol and repeat spinning step

• Resuspend the DNA pellet in 100 μL TE or appropriate buffers

3 The reaction volume will depend on the amount of DNA and enzymes used If TE is used as the final elution buffer for DNA extraction, the EDTA concentration will need to be diluted (at least tenfold) to ensure complete digestion Likewise, the amount of glycerol in the enzymes will dictate that their com-bined volume not exceed 10% of total reaction volume

4 Handle the gel with care as it contains ethidium bromide The gel should not be overly thick or thin A thin large gel may be too fragile to handle and can break easily during subsequent steps, whereas a thick gel can hinder DNA transfer Generally, a thickness of 0.5 cm is good Take care to select combs with the right thickness and width, which should permit sufficient load-ing of samples A small gel (less than half the size) may also be prepared to verify complete digestion of the genomic DNA samples Since 4 bp cutters are used here, they are expected to cut every 44 bps As a result, a completely digested sample should show a smear below the 1 kb DNA marker band For incompletely digested samples, more enzymes may be added for additional incubation at 37 °C Some samples may appear resistant to digestion (they float out the well when being loaded) Repeat purification steps to get rid of salt and other contaminants (such as phenol if using the protocol in Note 3)

may help Electrophoresis may also be carried out in 0.5–1× TBE buffer (1× TBE buffer: 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA) While TBE buffer has better buffering capacity, it is best for resolving smaller sized DNA fragments

5 A radiolabeled DNA marker will be visible by autoradiography

or phosphorImager exposure, which facilitates the calculations

in TRF analysis Radiolabeled DNA markers may be obtained through random priming labeling reactions using Klenow, or end-labeled using T4 polynucleotide kinase (as described below) The latter can be performed along with the telomere probe as described in 2.14 The 1 kb DNA ladder should first

be dephosphorylated using calf intestinal phosphatase (CIP, NEB) (1 μg of DNA ladder, 0.5 unit CIP, 1× NEB buffer,

60 min at 37 °C) The dephosphorylated DNA should be fied through gel purification, spin column, or phenol extrac-tion End labeling is then carried out in a 20 μL reaction with

puri-1 μL of T4 polynucleotide kinase (NEB), 2 μL 10× kinase

reaction buffer (NEB), 3 μL γ-32P ATP (3000 Ci/mmol), and

°C Unincorporated labels are removed with QIAquick otide removal kit (QIAGEN) Determine the specific activity

nucle-of the labeled marker using a liquid scintillation counter

6 Depending on the size of the gel, type of running buffer, power supply and gel apparatus, the electrophoresis process can take 24 h or longer TAE buffer generally requires lower voltage and longer running time In addition, slow low- voltage electrophoresis leads to better resolution

7 We find it easier to carry out steps 3–6 with the gel still in the

casting tray There is no need to slide the gel on and off during these steps, which can lead to gel breakage

8 There is no need to flip the gel upside down for the transfer Carefully slide it off onto the transfer surface Make sure to place several layers of 2× SSC soaked Whatman paper (cut to the correct size) followed by several dry layers on top of the membrane to ensure even transfer and minimize bubbles

9 The membrane may be further incubated in NaOH solution for 5–10 min to denature any remaining DNA, neutralized again, and rinsed in 2× SSC before cross-linking

10 While both the G and C probes can be used, the G probe erally yields better and stronger signals The specific activity of the probe should be ~0.5–1× 106 cpm/μL

11 The membrane is first washed in low stringency buffer once at room temperature and once at 37 °C, and then in high strin-gency buffer at least twice at room temperature The strin-gency of the wash may be further raised by increasing the number of washes, or the temperature for high stringency wash (to 37 °C or 50 °C if needed) The membrane should be checked with a Geiger counter periodically A good signal ratio between DNA-bound vs unbound portions of the membrane coupled with minimum signals from DNA-free portions of the membrane would indicate readiness Prolonged and overly stringent washes may result in weak signals that require extended exposure time

12 For samples with exceptionally long telomeres such as those from inbred laboratory mice, agarose plugs (available from commercial sources) with embedded cells should be prepared

to aid the digestion with protease and restriction enzymes

1 de Lange T (2002) Protection of mammalian

telomeres Oncogene 21:532–540

2 Xin H, Liu D, Songyang Z (2008) The

telo-some/shelterin complex and its functions

Genome Biol 9:232

3 Wang SS, Pluta AF, Zakian VA (1989) DNA

sequence analysis of newly formed telomeres in

yeast Prog Clin Biol Res 318:81–89

4 Moyzis RK, Buckingham JM, Cram LS, Dani

M, Deaven LL, Jones MD, Meyne J, Ratliff RL,

Wu JR (1988) A highly conserved repetitive

DNA sequence, (TTAGGG)n, present at the

telomeres of human chromosomes Proc Natl

Acad Sci U S A 85:6622–6626

5 Deng Y, Chan SS, Chang S (2008) Telomere dysfunction and tumour suppression: the senes- cence connection Nat Rev Cancer 8:450–458

6 Raynaud CM, Sabatier L, Philipot O, Olaussen

KA, Soria JC (2008) Telomere length, meric proteins and genomic instability during the multistep carcinogenic process Crit Rev Oncol Hematol 66:99–117

7 Artandi SE, DePinho RA (2010) Telomeres

and telomerase in cancer Carcinogenesis

31:9–18

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a biological marker in malignancy Biochim Biophys Acta 1792:317–323

Zhou Songyang (ed.), Telomeres and Telomerase: Methods and Protocols, Methods in Molecular Biology, vol 1587,

DOI 10.1007/978-1-4939-6892-3_3, © Springer Science+Business Media LLC 2017

Chapter 3

Telomere Length Analysis by Quantitative Fluorescent

in Situ Hybridization (Q-FISH)

Isabelle Ourliac-Garnier and Arturo Londoño-Vallejo

Abstract

Length is a functional parameter of telomeres, the nucleoprotein structures that protect chromosome ends The availability of highly specific, high affinity probes for telomeric repeat sequences allowed the development of quantitative approaches aimed at measuring telomere length directly on chromosomes or

in interphase nuclei Here, we describe a general method for telomere quantitative FISH on metaphase chromosomes and discuss its most common applications in research.

Key words Telomere, FISH, Q-FISH, Length, PNA, LNA

1 Introduction

The number of telomeric repeats at chromosome ends is important for telomere function [1–3] These repeats serve as a platform for the assembly of a protective protein complex, called shelterin [4]

or telosome [5] that will protect the extremity against degradation and fusion [6] The telomeric protein complex is also required for replication and repair and to prevent recombination [7] Maintaining telomere length is thus crucial to preserve function However, telomere repeats are lost at every cycle of DNA replica-tion both due to the “end replication problem” and due to the necessary processing of newly replicated ends to recreate a 3′ over-hang [8] In cells expressing telomerase, the dedicated enzyme able to add de novo telomere repeats, an equilibrium is reached between shortening and lengthening kinetics, thus defining a dynamic point of length homeostasis [9] However, most human somatic cells do not express, or else at low levels, telomerase activ-ity and telomeres inexorably shorten with cell division [10] As soon as telomere length is insufficient to ensure protection, a signal

is sent to command cell arrest and entry in mitotic senescence [11] Telomere length, therefore, directly influences cell prolifera-tion potential.

In vivo, telomere lengths decrease with aging and premature aging syndromes are associated with accelerated telomere shorten-ing [12] Both observations have lent support to the hypothesis that cell senescence triggered by short telomeres is responsible of most, if not all, manifestations connected to aging [12] The dis-covery that mutations in components of the telomere enzyme are implicated in aging syndromes and aging-related manifestations, has established a definitive link between short telomeres and organ-ismal aging [13] As a result, telomere length has become an obliged biomarker in all studies related to aging [14]

Classically, telomere length is measured by telomere restriction fragment (TRF) analysis in Southern blots, which, in spite of its technical problems and relative imprecision, remains the gold stan-dard [15, 16] On the other hand, PCR-based quantitative analy-ses, which require little material, have been developed and used with success by some laboratories [17, 18] Both methods, though, have the inconvenience of providing a rough estimate of either mean telomere length or total telomere repeat content of a cell population, thus underestimating the relative proportion of short, potentially dysfunctional, telomeres which may be sufficient to trigger cell senescence in any single cell [19, 20] In fact, telomere length heterogeneity is the rule in normal somatic cells [21–23] and single telomere lengths are allele-specific, transmitted to the offspring and stable throughout life, thus representing a constitu-tive characteristic of any given individual [24, 25] It has been also shown that chromosome arms carrying the shortest telomeres are the first to become unstable upon unrestrained cell proliferation [2] Moreover, the distribution of single telomere lengths among chromosome arms determines the way a particular cell accumulates chromosome abnormalities during tumor transformation and has a major impact on the karyotypic characteristics of spontaneously immortalized cells in vitro [2 26]

Therefore, telomere length analysis on chromosome meta phase provides not only an estimation of telomere length of the cell population but allows the evaluation of other parameters such as heterogeneity of telomere lengths and the presence of critically short telomeres Telomere length heterogeneity tends to be reduced and almost disappears when cells express high levels of telomerase (tumor cells or cells expressing hTERT transgene) [21] On the other hand, telomere length heterogeneity is characteristically exag-gerated in cells using recombination to maintain telomeres (ALT)

so that very long telomeres coexist with chromosome extremities on which no telomere signals are detectable (signal free end, SFE) [27] Interestingly, the re-expression of telomerase

activity in these cells leads to a significant reduction of SFEs thus constituting a readout for telomerase activity, a test which can be used to evaluate the performance of modified telomerase complexes [28, 29] Finally, allele specific analyses can be carried out to study length differences between homologous chromosomes [21].

The method described hereafter is based on the original method described by the group of P Lansdorp [30] Very little modifications need be introduced if PNA probes are used However, it is also possible to use telomeric LNA probes, which also have high specificity and high affinity for telomere repeats [31] Contrary to PNA, LNA probes tolerate a few mismatches [32] and therefore signals may no longer exclusively originate from canonical sequences but may instead comprise telomere repeats variants, whose composition and length presumably vary from an extremity to another and therefore between individuals [33, 34] However, the number of juxtatelomeric variants is limited, relative

to the number of canonical repeats and therefore the contribution

to the total fluorescence intensity from probes hybridizing to ant repeats is most likely negligible

vari-2 Materials and Reagents

1 0.1 μg/mL colcemid (KaryoMax, Invitrogen)

2 Trypsin–EDTA solution: Trypsin 0.05%, EDTA 0.53 mM

3 Hypotonic solutions (see Note 1): 8 g/L Na citrate, 0.075 M

KCl

4 Fixative solution for metaphase preparation: ethanol–acetic

acid (3:1, v/v) (see Note 2).

1 Clean superfrost slides (Menzel-Gläser from D Dutscher S.A.) with pure ethanol

2 Coverslip 24 × 60 (VWR)

PNA probes can be ordered from Panagene (www.panagene.

com) The most common telomeric probe is (CCCTAA)3 which can be labeled with red (Cy3) or green (Fam/FITC) fluoro-chromes (Note 3).

1 Pepsin solution: 1 mg/mL, pH 2 For 100 mL: 100 mg pepsin (Sigma-P7000)–10 mL 0.5 M citric acid pH 2–water

(see Note 4).

2 Fixative solution: 3.7% formaldehyde–PBS 1×

For 100 mL: 10 mL PBS 10×–10 mL formaldehyde 37% (SIAL from Sigma-F1635)–water