Role of FANCM in Telomere Maintenance in Alternative Lengthening of Telomeres (ALT)

ROLE OF FANCM IN TELOMERE MAINTENANCE IN

ALTERNATIVE LENGTHENING OF TELOMERES (ALT) HUMAN

CELLS

Fathiya Al Murshedi

A thesis submitted in conformity with the requirements for the degree of

Master of Science

Graduate Institute of Medical Science

University of Toronto

Role of FANCM in Telomere Maintenance in Alternative Lengthening of Telomeres (ALT)

Human Cells

Master of Science 2010

Fathiya Al Murshedi

Institute of Medical Science, University of Toronto

Abstract

Most immortal human cells maintain their telomeres by up-regulating the enzyme telomerase. Approximately 10-15% of immortal cells maintain their telomere lengths by a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Human

ALT cells are characterized by ALT associated promyelocytic bodies (APBs) that contain proteins involved in DNA damage response and repair. Our lab has found significant colocalization of several components of the Fanconi Anemia (FA) pathway with telomeres and demonstrated that knockdown of FANCD2 leads to ALT-specific increase in the amount of telomeric DNA as well as increased aneuploidy and cell death. In this study, we examined the role of FANCM in telomere maintenance in ALT cells. We found a significant colocalization of FANCM with telomeres in two ALT cell lines. Knockdown of FANCM was associated with reduced growth, increases in the size of TRF2 foci and in the amount of telomeric DNA. These data suggest that FANCM plays a role in telomere length regulation and maintenance.

Acknowledgment

I would like to thank my supervisor, Dr Stephen Meyn, for his guidance, teaching, encouragement and support. I would like also to thank members of my committee Dr

Rosanna Weksberg and Dr David Malkin. Special thanks to Heather Root from Meyn‟s lab for her ongoing support and advice. I would like also to thank other members of Meyn lab

Paul Bradshaw, Magan Trottier and Martin Komosa for their help and advice.

Special thanks to my parents who inspired me to seek to learn and my husband, Ahmed, for his encouragement and support.

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Table of content

ABSTRACT ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS ix CHAPTER ONE: Introduction 1 1.1 Overview on telomere structure and maintenance 2

1.1.1 Telomere molecular structure 2 1.1.2 Shelterin Protein Complex 3 1.1.3 Non-shelterin proteins present at telomeres 5 1.1.4 Telomere induction of DNA damage signaling and replicative senescence 8 1.1.5 Telomere maintenance in immortal cells 10 1.1.6 Characteristics of ALT cells 12 1.1.6.1 Heterogeneous telomere length and ECTR in ALT 12 1.1.6.2 High instability at MS32 minisatellite 17 1.1.6.3 ALT associated promyelocytic bodies (APBs) 17 1.1.7Association of DNA recombination and repair proteins with telomeres in ALT 18 cells

1.2 Fanconi Anemia (FA) Pathway and telomere maintenance 21

1.2.1 Fanconi Anemia Pathway 21 1.2.2 Phenotype of FA mouse models 23 1.2.3 Role of FA Pathway at telomeres in ALT 24

1.3 Fanconi Anemia Complementation group M (FANCM) 25 1.3.1 FANCM protein structure 25 1.3.2 Functions and biochemical activity of FANCM 27 1.3.3 FANCM cellular phenotypes 28 1.3.3.1 Hypersensitivity to DNA cross-linking agents 28 1.3.3.2 Camptothecin and UV sensitivity 29 1.3.3.3 Increased sister chromatid exchanges 30 1.3.4 Role of FANCM in ATR/Chk1 checkpoint signaling pathway 31 1.3.5 FANCM human and mice disease phenotypes 32 1.3.6 FANCM and ALT telomere maintenance 33 1.4 Hypothesis 34 1.5 Objective 34 CHAPTER TWO: Analysis of the role of FANCM in telomere maintenance 35 iv

2.1 Experimental Approach 36

2.2 Methods 37

2.2.1 Cell lines and culture conditions 37 2.2.2 Antibodies 38 2.2.3 Immunofluorescence (IF) following detergent extraction in fibroblasts 38 2.2.4 Foci count and colocalization analysis 39 2.2.5 Transient knockdown of FANCM transcription using small interfering RNA 40 (siRNA) 2.2.6 Peptide nucleic acid- fluorescent In Situ Hybridization (PNA-FISH) 41

2.3 Results 43

2.3.1 FANCM forms discrete foci in ALT and telomerase positive cells 43 2.3.1.1 Antibodies against FANCM form discrete foci in GM00847 and 43 WI38-VA13/2RA ALT cells 2.3.1.2 Appearance of FANCM foci in GM00639 and HT1080 telomerase 45 positive fibroblasts 2.3.1.3 two different FANCM antibodies form highly colocalizing nuclear 47 foci

2.3.2 Colocalization of FANCM with TRF1/TRF2, FANCD2 and PML 48 2.3.2.1 Colocalization of FANCM with TRF1 and TRF2 48 2.3.2.2 Colocalization of FANCM with FANCD2 50 2.3.2.3 Colocalization of FANCM with PML 50

2.3.3 Effects transient depletion of FANCM using of siRNA 52 2.3.3.1 siRNA knockdown of FANCM resulted in poor growth and increased 52 cell death in ALT and telomerase-positive fibroblasts 2.3.3.2 FANCM knockdown resulted in a reduction of FANCM signal 53 2.3.3.3 FANCM knockdown resulted in larger TRF2 foci in GM00847 ALT 54 cells 2.3.3.4 FANCM knockdown resulted in distortion of TRF2 foci in 54 WI38VA13/2RA ALT cells 2.3.3.5 Preserved expression of FANCD2 in FANCM depleted 56 WI38VA13/2RA ALT 2.3.3.6 FANCM knockdown results in increased amount of telomeric DNA 57

CHAPTER 3: Discussion and Future Directions 60

3.1 Discussion 61

3.1.1 Validation of the IF staining of FANCM 61

3.1.2 FANCM is required for ALT telomere maintenance 62

3.2 Future Directions 65 3.2.1 Characterize the phenotype of FANCM depletion in ALT 65 3.2.2 Determine the mechanism for increased telomeric DNA synthesis in FANCM depletion 66

REFERENCES 67

List of Tables

Table 1-1 DNA damage response and repair proteins involved in telomere 7 maintenance Table 1-2 DNA damage response and repair protein colocalize with ALT 19 telomeres Table 2-1 Means of FANCM and TRF1 foci count and percentage of 50 colocalization

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List of Figures

Figure 1-1 How shelterin may shape telomeres 4 Figure 1-2 Telomere dysfunction and cell immortality 10 Figure 1-3 Homologous Recombination 14 Figure 1-4 Homologous recombination as the telomere maintenance mechanism 15 in ALT Figure 1-5 A model for rolling-circle amplification of telomeres 16 Figure 1-6 Signalling through the Fanconi anemia (FA)-breast cancer 22 susceptibility (BRCA) network Model Figure 1-7 Domain structure of human FANCM and FAAP24 26 Figure 2-1 Immunofluorescence appearance of FANCM foci under 20X and 44 63X magnification in randomly cycling ALT nuclei

Figure 2-2 Comparison between the immunofluorescence (IF) appearance of 46 FANCM foci in telomere positive and ALT cells Figure 2-3 Validation of FANCM foci by immunofluorescence (IF): high 47 colocalization of two different antibodies Figure 2-4 Graphic representation of mean percentage of colocalization of 49 FANCM foci with TRF1 foci in ALT and telomerase positive cells. Figure 2-5 Colocalization of FANCM foci with TRF1 foci in WI38-VA13/2RA 49 and GM00847 ALT cells Figure 2-6 Colocalization of FANCM with FANCD2, PML and TRF1 51

Figure 2-7 FANCM knockdown using siRNA resulted in depletion of FANCM 53 signal Figure 2-8 Effects of FANCM depletion on TRF2 foci in ALT 55

Figure 2-9 Preserved expression of FANCD2 in FANCM depleted 56 WI38VA13/2RA ALT cells Figure 2-10 FANCM depletion causes increased synthesis of telomeric DNA in 58 ALT but not in telomerase positive cells (20X magnification) Figure 2-11 FANCM depletion in ALT causes significant increase in telomeric 59 DNA synthesis (63Xmagnification)

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List of Abbreviations

53BP1 P53 binding protein 911 complex Rad9/Hus1/Rad1 complex ALT Alternative lengthening of telomeres APB ALT-associated promyelocytic bodies ATM Ataxia telangiectasia mutated ATR Ataxia telangiectasia and Rad3 related BIR Break Induced Repair BLM Bloom syndrome protein Chk1/2 Checkpoint kinase 1/2 DDR DNA damage response DEAD/DEAH motif Highly conserved Asp-Glu-Ala-Asp (DEAD) motif DNA Deoxyribonucleic acid DSBs DNA double strand breaks dsDNA double-stranded DNA ERCC1/ERCC3/ERCC4 Excision repair cross-complementing 1/3/4 FA Fanconi Anemia FAAP24 Fanconi Anemia Associated Polypeptide of 24 kD FANCA Fanconi Anemia Complementation Group A FANCB Fanconi Anemia Complementation Group B FANCC Fanconi Anemia Complementation Group C FANCD1 Fanconi Anemia Complementation Group D1 FANCD2 Fanconi Anemia Complementation Group D2 FANCE Fanconi Anemia Complementation Group E FANCG Fanconi Anemia Complementation Group G FANCI Fanconi Anemia Complementation Group I FANCJ Fanconi Anemia Complementation Group J FANCM Fanconi Anemia Complementation Group M FANCN Fanconi Anemia Complementation Group N HCLK2 Human CDC-like kinase 2

HU Hydroxyurea Ku70 and Ku80 proteins required for the NHEJ pathway MDC1 Mediator of DNA damage checkpoint 1 MEFs Murine Embryonic Fibroblasts MRE11 Meiotic recombination 11 NBS1 Nijmegen Breakage Syndrome 1 NHEJ non-homologous end joining ORC Origin recognition complex POT1 Protection of telomeres 1 RAD50 DNA repair protein RAD50 RAP1 Transcriptional repressor/activator 1 shRNA Small hairpin RNA siRNA Small interfering RNA SMC1 Structural Maintenance of Chromosomes 1 ssDNA Single-stranded DNA TIN2 TRF1-interacting protein 2 TMM Telomere maintenance mechanism TopoIIIa Topoisomerase III alpha TPP1 POT1- and TIN2-organizing protein 1 TRF1 Telomeric-repeat-binding factor 1 TRF2 Telomeric-repeat-binding factor 2 T-SCE Telomere-sister chromatid exchange UV Ultra-violet WRN Werner syndrome protein XPF Xeroderma Pigmentosum complementation group F

CHAPTER ONE

Introduction

1.1 Overview on telomere structure and maintenance

1.1.1 Telomere molecular structure

Telomeres are specialized nucleoprotein complexes at the end of linear chromosomes. They provide physical ends for chromosomes, prevent the triggering of the DNA-damage repair machinery and enzymatic attack (Verdun et al., 2007) and help to solve the end replication problem and so prevent the loss of genetic information. Telomeres are essential for genome stability and loss of telomere function has significant implications in human aging and cancer.

The telomere sequence is a repeat of the guanosine rich hexamer (5‟-TTAGGG-3‟)n with the complementary 5‟-end being rich in cytosine. Based on this uneven distribution of nucleotides, the two telomeric DNA strands are termed G-strand and C-strand. The number of TTAGGG repeats determines telomere length. Telomeres vary in length according to the age and the tissue of the cell of origin. The average length of human telomeres is 5-15 kb (de

Lange et al., 1990). The 5‟-end of human telomeres is accurately defined and predominantly ends on the sequence ATC-5‟ (Sfeir et al., 2005). The 3‟-end is longer and ends with a single strand, G-rich overhang (Figure 1-1A) (Hug and Lingner, 2006). This feature is conserved throughout eukaryocytes with the mammalian 3‟-end overhang being considerably longer than that of other eukaryotes and roughly ranging between 30-110 nucleotides (Chai et al.,

2006). The overhang can invade the complementary telomeric double strand-DNA (dsDNA) to form a displacement loop known as telomeric loop (t-loop) (Figure 1-1B). This strategy can keep it hidden within the telomere structure in order to protect it from enzymatic

degradation and prevent formation of complementary structures with other telomeres. T- loops differ in size according to the length of their telomere sequence, so that longer telomeres have larger t-loops (Matulic et al., 2007). This is because the 3‟-strand overhang invades the complementary strand near the centromeric end of the telomere border region.

1.1.2 Shelterin Protein Complex

The integrity of the t-loop is maintained by a specialized group of proteins known as the shelterin protein complex (de Lange, 2005; Hug and Lingner, 2006). Components of shelterin specifically localize to telomeres and are abundant at telomeres throughout the cell cycle (Palm and de Lange, 2008).

There are six proteins that make up the shelterin complex: telomeric-repeat-binding factor 1

(TRF1), TRF2, TRF1-interacting protein 2 (TIN2), the transcriptional repressor/activator protein (RAP1), protection of telomeres 1 (POT1) and the POT1- and TIN2-organizing protein (TPP1). Three members of this complex, TRF1, TRF2 and POT1, bind directly to telomeric DNA repeats and anchor the rest of the complex along the length of the telomeres.

TRF1 and TRF2 bind the double-stranded portion of telomeres while POT1 binds to 3- single-stranded G overhangs (Figure 1-1C). The amount of shelterin protein bound to telomeres is roughly proportional to their length (de Lange, 2005). All six shelterin subunits can be found in a single complex in fractionated nuclear extracts (Liu et al., 2004; Ye et al.,

2004). Further attempts using mass spectrometry on shelterin-associated factors failed to identify additional components (Liu et al., 2004; O‟Connor et al. 2004; Ye et al., 2004).

Figure 1-1: How shelterin may shape telomeres. (A) Generation of the telomere terminus. After replication, chromosome ends require processing in order to acquire a long 3 overhang. The nuclease involved is not known. The resulting 5 end always has the sequence ATC-5. (B) The t-loop structure. The 3 overhang is strand-invaded into the adjacent duplex telomeric repeat array, forming a D-loop. The size of the loop is variable. (C) Speculative model for t-loop formation by shelterin. TRF1 has the ability to bend, loop, and pair telomeric DNA in vitro and could potentially fold the telomere. The shelterin component TRF2 can mediate t-loop formation in vitro. Adapted from de Lange T. Genes and Development (2005).

Bradshaw et al., 2005 demonstrated that TRF2 moves from telomeres and migrates to sites of

DNA DSBs (Bradshaw et al., 2005). This suggested that TRF2 may have additional functions not directly related to telomere formation and maintenance. In fact, TRF2 is shown to be phosphorylated by ataxia telangiectasia mutated (ATM) in response to DNA damage, similarly to other proteins involved in DSB repair (Tanaka et al., 2005). In addition to widening the vision about the possible roles and interactions of shelterin proteins, these facts may also necessitate revisions to the criteria used to define members of the shelterin complex.

1.1.3 Non-Shelterin proteins present at telomeres

Mammalian telomeres also contain a large number of other proteins that make important contribution to their maintenance and protection. Many of these factors are involved in DNA damage response including proteins implicated in detection, signaling and repair of DNA damage, as well as factors associated with cell cycle checkpoint control and regulation of apoptosis. Examples are the non-homologous end joining (NHEJ) proteins, Ku70/80 (Hsu et al., 1999), xeroderma pigmentosum complementation group F (XPF/ERCC1) (Zhu et al.,

2003), mitotic recombination 11 (MRE11) complex (Zhu et al., 2000), DNA repair protein

RAD51D (Tarsounas et al., 2004), poly (ADP-ribose) polymerase 1(PARP1) (Palm and de

Lange, 2008), breast cancer susceptibility gene 1 (BRCA1), Rad9/Hus1/Rad1 (911) complex

(Francia et al., 2006), origin recognition complex (ORC) (Deng et al., 2007), WRN and BLM

RecQ helicases (Opresko et al., 2002) and heterochromatin protein 1 (HP1) (Garcia-Cao et al., 2004) proteins (Table 1-1).

The roles of several of the above proteins on telomere integrity have been studied.

Alterations in DNA damage response (DDR) proteins result generally in telomere dysfunction and subsequent chromosomal instability suggesting extensive functional interactions between telomere maintenance and DNA damage response mechanisms(Slijepcevic, 2006).

For example, McPherson and Hande et al., found that Brca1 knockout (Brca1-/-) in mice causes depletion of T cells, which can be rescued by deleting p53. Subsequently, telomere maintenance was investigated in Brca1−/− and p53−/− T-cells. These cells developed telomere erosions evidenced by reduced telomere length on quantitative fluorescent in situ

hybridization (qFISH) and flow-FISH (McPherson and Hande et al., 2006). These cells also had marked increased incidence of end-to-end fusions such as ring chromosomes, dicentric chromosomes and Robertsonian fusions. Some of these fusions had detectable telomeric signal suggesting a combination of telomere erosions and defective telomere capping as consequences of BRCA1 loss at telomeres (McPherson and Hande et al., 2006). Likewise, defects in ATM result in accelerated telomere loss, telomeric fusions and appearance of extra-chromosomal telomeric fragments in cells from ataxia telangiectasia patients or ATM defective mice (Hande et al., 2001). Another example of the role of DDR proteins at telomeres is the effect of WRN deficiency. WRN knockout mice had increased telomere sister chromatid exchange (T-SCE) suggesting that WRN servers as a repressor of homologous recombination (HR) at telomeres (Multani and Chang, 2007).

This impressive involvement of a variety of DNA damage response proteins in telomere maintenance, in addition to the role of shelterin proteins in coordinating the interactions of these factors as well as the identification of TRF2 as possible DNA repair protein, raised the possibility that telomere maintenance could be an integral part of a wider network of DNA damage responses, rather than an independent mechanism. This hypothetical model was introduced by Slijepcevic who introduced the term of an “integrative” model which refers to integration of telomere maintenance mechanisms into a complex set of mechanisms collectively known as DNA damage response (Slijepcevic, 2006).

Cell Telomere dysfunction Shelterin Protein Function Sensitivity origin interaction Length Fusions Other

Damage ATM Human IR Shorter Yes Yes Yes signaling

Ku Mouse NHEJ IR Shorter/longer Yes ND Yes

Mouse DNA-PKcs NHEJ IR Longer Yes ND Yes (scid) RAD54 Mouse HR IR Shorter Yes ND ND

RAD51D Mouse HR IR Shorter Yes ND Yes

Damage NBS1 Human IR Shorter No ND Yes sensing? Damage MRE11 Human IR ND ND ND Yes sensing? PARP-2 Human BER IR Normal No ND Yes ERCC1 Human NER UV ND No Yes Yes XPF Human NER UV ND No Yes Yes Topoisomerase WRN Human Helicase Shorter ND ND Yes inhibitors Topoisomerase BLM Human Helicase Shorter ND ND Yes inhibitors Damage FANCA Human MMC Shorter Yes Yes ND sensing? Damage RAD50 Human IR ND ND ND Yes sensing? BRCA1 Human HR IR Longer Yes ND Yes Damage Rad9 Human IR ND Yes ND Yes sensing? PARP-1 Human BER IR Normal Yes ND Yes Table 1-1: DNA damage response and repair proteins involved in telomere maintenance. IR, ionizing radiation; NHEJ, non-homologous end joining; HR homologous recombination; ND, not determined. Adapted from Slijepcevic, P. DNA Repair (2006).

1.1.4 Telomere induction of DNA damage signaling and replicative senescence

Most somatic human cells have limited replication potential (Gracia et al., 2007) due to an inability to fully replicate telomeric ends: The end replication problem. Primary human cells lose about 100–200 bases of TTAGGG repeats per cell division (Harley et al., 1999). As a result, their telomeres shorten with each cell division. The minimal functional telomere length to prevent cell cycle arrest has not been clearly defined. In mammalian cells, telomere tracts as short as 400 bases are sufficient to make a new telomere (Barnett et al., 1993). It had been shown that a single short telomere is sufficient to induce cell cycle arrest (Hemann,

2001) which explains the observation that senescent human cells can contain telomeric double-stranded repeats that are readily detectable (Verdun and Karlseder, 2007). The observation that primary human fibroblasts with TRF2 over-expression enter senescence with considerably shorter telomeres than control populations suggested that telomere structure, not telomere length, is the main determinant of functional telomeres (Karlseder et al., 2002).

Once telomeres reach a critical length, they can trigger the activation of DNA damage response. Cytological and chromatin immunoprecipitation (ChIp) data revealed the presence of p53 binding protein (53BP1), gamma- Histone 2AX (γ-H2AX), Mediator of DNA damage checkpoint 1 (MDC1), Nijmegen Breakage Syndrome 1 (NBS1) and Structural Maintenance of Chromosomes 1 (SMC1) at the shortened telomeres undergoing replicative senescence

(Palm and de Lange, 2008). Furthermore Chk1 and Chk2 become phosphorylated suggesting that both ATM and Ataxia telangiectasia and Rad3 related (ATR) are activated in this setting

(Palm and de Lange, 2008). Similar phenomena are observed when components of shelterin are disrupted. Deletion of TRF2 from mouse cells or expression of a dominant negative

TRF2 allele in human cells leads to ATM activation (Denchi et al., 2007; Celli and de Lange,

2005; Takai et al., 2003) and ATM-dependent formation of DNA damage foci at telomeres

(Denchi et al., 2006). The activation of the ATM pathway is triggered by the damage at the telomere itself rather than secondary to DNA damage accumulation (de Lange, 2005). In absence of ATM, the ATR kinase is thought to induce a cell cycle arrest. These telomere dysfunction-induced foci (TIF) contain many of the factors detected at DNA damage foci induced by DNA double strand breaks (DSBs) with similar cellular response to ATM activation including Chk2 and p53 induction.

The activation of DNA damage response leads the vast majority of cells at this stage to enter senescence and permanently lose their replication potential. Alternatively, cells may undergo apoptosis. The decision between replication senescence and apoptosis is dependent on several factors including the degree of genomic instability and the tissue of origin. This can be illustrated by the difference between fibroblasts and lymphoblasts in response to TRF2 inhibition (or treatment with DNA damaging agents) as most fibroblasts enter senescence whereas apoptosis is a more prominent outcome in lymphocytes and epithelial cells

(Karlseder et al., 1999).

Rare cells manage to bypass these protective mechanisms and continue to divide and accumulate further genomic instability leading to crisis (Figure 1-2). Although the majority of cells in crisis die, a few manage to activate a mechanism to maintain their telomeres and thus become immortal (Callé et al., 2004).

Critical telomere length

Activation of Genomic instability DNA damage response

Senescence Apoptosis Crisis

Immortal Die

Figure 1-2: Telomere dysfunction and cell immortality. Based on telomere length, human cells have limited replication potential. When telomeres become critically short most cells enter senescence. Cells that escape senescence become genetically unstable leading to “crisis”: (short telomeres + DNA damage + epigenetic factors), the majority die but some manage to restore and maintain their telomeres and so become immortal.

1.1.5 Telomere maintenance in immortal cells

Surveys across many tumor types have shown that about 85% of immortal cells and tumors maintain their telomeres by up-regulating the expression of telomerase enzyme (Kim et al.,

1994). Telomerase is a specialized reverse transcriptase complex that catalyzes the synthesis of telomeric sequence by adding telomeric repeats to the ends of chromosomes using its own internal RNA template (Verdun et al., 2007). The enzyme is a ribonucleoprotein complex

containing a reverse transcriptase subunit (hTERT), a template RNA (TER), and accessory components (Osterhage and Friedman, 2009). Telomerase activity is regulated through multiple levels. Transcription and posttranscriptional regulation of hTERT is tightly regulated in human cells where hTERT expression is low or absent in most somatic cells but is activated during embryonic development, in some stem cells, and in most tumors

(Osterhage and Friedman, 2009). Other regulatory mechanisms for telomerase include recruitment of telomerase to the chromosome terminus, and control of telomerase accessibility by telomeric binding factors of the shelterin complex.

However, about 10-15% of immortal and tumor cells lack telomerase (Kim et al., 1994).

These cells maintain their telomeric length through an Alternative Lengthening of Telomeres mechanism and thus are known as ALT cells. The preference to activate ALT versus telomerase varies between tumor types; where sarcomas in general have a higher tendency to utilize ALT. For example, ~100% of adenocarcinomas (Kammori et al., 2002) express telomerase where up to 47% of osteosarcomas (Henson et al., 2005) depend on ALT as a telomere maintenance mechanism (TMM). Tumors can also change their primary TMM as some ALT primary tumors can give rise to telomerase positive secondary tumors and vice versa (Henson et al., 2002). Furthermore there is evidence in liposarcomas (Johnson et al.,

2005) that both TMMs can be active in the same cell line as a proportion of these tumors were found to express telomerase and markers for the ALT mechanism (Johnson et al.,

2005).

1.1.6 Characteristics of ALT cells

There are several unique features that characterize ALT cell lines, including heterogeneous telomere length, presence of extra-chromosomal circular and linear telomeric DNA (ECTR),

ALT associated promyelocytic bodies (APBs), a high frequency of telomere-sister chromatid exchanges (T-SCE) and high instability at a specific GC-rich minisatellite, MS32 (D1S8)

(Royle et al., 2008).

1.1.6.1 Heterogeneous telomere length and ECTR in ALT

Telomere length in ALT cells is highly variable. Analyses using terminal restriction fragment

(TRF) analysis in pulse-field gels (Bryan et al., 1995), Q-FISH analysis of metaphase chromosomes from ALT cells (Henson et al., 2002) and single telomere length analysis

(Jeyapalan et al., 2008) revealed that telomere length in ALT cell lines ranges from a few hundred base pairs to over 50 kb. Telomeres of ALT cells can undergo both gradual and rapid changes in their length (Murnane et al., 1994). The gradual changes are due to the shortening of telomeres at a rate similar to that reported for telomeres of somatic cells without telomerase that is mainly due to incomplete replication. Rapid lengthening or rapid shortening has been observed as well (Murnane et al., 1994). These rapid changes can be explained by homologous recombination (HR) with unequal T-SCE. In yeast, these rapid changes in telomere length have been associated with telomere loss and chromosome instability.

Homologous Recombination (HR) and sister chromatid exchanges (SCE):

HR is one of the essential pathways in the repair of DSBs. HR is a highly accurate mechanism that relies on using the information on the undamaged sister chromatid or homologous chromosome. The process of HR starts by processing of DNA ends to produce molecules with 3‟ single-stranded tails (Figure 1-3). Following end resection, single- stranded DNA (ssDNA) strands are coated with the ssDNA binding protein, replication protein A (RPA). RPA is then replaced by RAD51 forming a nucleoprotein filament (Sung,

1997). Several proteins, known as HR mediators, are known to help the loading of RAD51 on the ssDNA including Rad52 and Brca2 (Brugman et al., 2007). RAD51-coated nucleoprotein filament can undergo homology-driven invasion of the intact sister chromatid, creating a joint molecule. This is followed by invasion of the second resected single- stranded DNA tail into the joint molecule creating a double Holliday junction. Template guided DNA synthesis then provides the damaged molecule with a copy of the undamaged strand. Resolution of the joint homologous recombination structures can result in exchange of sister chromatids, a process known as sister chromatid exchange (SCE).

Several lines of evidence support the role of genetic recombination in ALT TMM (Figure 1-

4). It was demonstrated that tagged telomere sequences can move from one telomere to another in mammalian ALT cells (Dunham et al, 2000). In addition, ALT telomere maintenance is dependent on RAD52 (Muntoni and Reddel, 2005) and Rad50-P recombination protein in yeast (Teng et al., 2000).

Figure 1-3. Homologous recombination. This is a highly accurate repair mechanism, relying on the presence of a homologous DNA fragment that can be used as template. Free DNA ends that are formed at the site of a DSB are first recognized and processed. After that a nucleoprotein filament is formed which searches for homologous DNA. Subsequently, a joint molecule is formed between the damaged and undamaged DNA molecules. Template guided DNA synthesis then provides the damaged molecule with a copy of the undamaged strand. Adapted from Brugmans et al., Mutation Research (2007).

In further support of the role of HR in ALT TMM is that chromosome orientation FISH (CO-

FISH) has shown a remarkable increase of telomeric post-replicative exchange events (range

28-280/100 metaphases) and rarely or never in non-ALT cells (Londono-Vallejo et al.,

2004). On the other hand, SCE was not found to be increased at interstitial sites in ALT cells

(Londono-Vallejo et al., 2004), and the overall frequency of homologous recombination was found to be similar in ALT and telomerase-positive cell lines (Bechter et al., 2003). Although simple HR-driven reciprocal exchanges between or within telomeres can explain the sudden changes in telomere size, they fail to explain the net gain in telomeric DNA observed in ALT cells. It also does not explain the presence of linear and circular extra- chromosomal telomere repeat (ECTR) DNA, which is another characteristic of ALT. Although short linear telomeric

DNA repeats were shown to be preferentially located within APBs (Fasching et al., 2007), these repeats are about a few kilobases long and cannot explain the rapid gains of telomeric

sequence seen in ALT cells (Muntoni and Reddel, 2005). The t-loops and circular ECTR can be large and so could be responsible for the increased amount of telomeric DNA either by end-joining or if used as template for telomeric replication.

Figure 1-4: Homologous recombination as the teloemere maintenance mechanism in ALT: Post-replicative chromosomes have telomeres of essentially equal length (A). If the sister chromatids align their telomeres asymmetrically (B) and exchange telomeric sequences (C), this will result in two sister chromatids (D) and ultimately two daughter cells (E) with different telomere lengths. The daughter cell with shorter telomeres will soon senesce and be lost from the population, whereas the one with longer telomeres will have an extended proliferative potential. Adapted from Muntoni and Reddel, Human Molecular Genetics (2005).

To explain the total gain of telomeric sequence observed in ALT, a rolling-circle amplification of telomeres model was suggested by Stavropoulos et al (Stavropoulos et al.,

2002). In this model, both the 3' and 5' ends of the telomere pair with their complementary strands at the base of the t-loop so as to form a four-stranded rolling circle (Figure 1-5A).

Both strands will be elongated using the base's strands as templates. Subsequent branch migration of the Holiday junction that forms behind the replication machinery allows for continuous replication of the rolling circle and results in extensive lengthening of the telomere (Figure 1-5B). This model can be used to explain the presence of both the intra- chromosomal gain in telomere sequence as well as the extra-chromosomal circles of

telomeric DNA (Stavropoulos et al., 2002). This mechanism is supported by the recent finding that telomere tag can be amplified without the involvement of other telomeres

(Muntoni et al., 2009). However, the previous well established observation that tagged telomeric sequences do move in between different chromosomes suggested that ALT mechanism involves more than one method of telomere elongation. Another model that can explain the inter-chromosomal exchange is called break-induced replication (BIR) in which the 3' end of the G-strand of a shortened deprotected telomere invades the telomeric DNA on another „donor‟ chromosome (McEachern and Haber, 2006). Further replication can continue using the roll-circle method described above.

Figure 1-5. A model for rolling- circle amplification of telomeres. In this model, BLM and TRF2 cooperate in forming a t-loop in which both the 3' and 5' ends of the telomere pair with their complementary strands at the base of the t-loop so as to form a four- stranded rolling circle (A). The replication machinery then adds to both strands of the telomere end using the base's strands as templates. Subsequent branch migration of the Holiday junction that forms behind the replication machinery allows for continuous replication of the rolling circle and results in extensive lengthening of the telomere (B). While an intra- telomeric loop is illustrated, this scheme also can apply to the use of extra-chromosomal circles of telomeric DNA as the template. Adapted from Stavropoulos et al., Human Molecular Genetics (2002).

1.1.6.2 High instability at MS32 minisatellite

MS32 Specific minisatellite (MS32) was found to be highly unstable in a subset of ALT cell lines, but not in telomerase positive or mortal cultures (Jeyapalan et al., 2005). High mutation rates at this particular locus were also seen in a subset of ALT-positive soft tissue sarcomas, indicating that minisatellite instability occurs in vivo and is not a culture artifact. In this study

(Jeyapalan et al., 2005); four other subterminal minisatellites that are highly recombinogenic in the germline were analyzed. None of these other minisatellites were unstable in ALT tumors indicating that minisatellite instability in ALT is not a generalized phenomenon and the instability at the MS32 is specific to ALT (Jeyapalan et al., 2005). The initiation of

MS32 instability seems to coincide with activation of the ALT mechanism and this suggests there is overlap between the underlying mechanism and at least some of the proteins involved

(Royle et al., 2008).

1.1.6.3 ALT associated promyelocytic bodies (APBs)

A morphologic feature of ALT is the presence of unique large specialized ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) (Yeager et al., 1999). PML bodies are

0.1–1 µm diameter nuclear matrix-associated structures of unknown function that contain

PML protein (Seeler and Dejean, 1999) and have been proposed to play a role in a wide range of cellular processes including oncogenesis, apoptosis and repair of DNA damage

(Quignon, 1998). PML in APBs is also accompanied by SP100 protein. In APBs, PML protein colocalizes with telomeric DNA; telomere binding proteins, TRF1 and TRF2 and

telomere-associated proteins (Yeager et al., 1999). Many of these telomere-associated proteins in APBs are known to be involved in DNA replication, recombination, and repair

(Henson et al., 2002) (see section 1.1.7).

Cell cycle analysis has shown that large APBs are most abundant during the G2 phase of the cell cycle, which seems to coincide with the timing of the telomere elongation in ALT cells

(Grobelny et al., 2000). Although the role of APBs in ALT telomere maintenance is still not clear, previous findings might suggest that APBs may be the site for telomere elongation in

ALT cells (Royle et al., 2008).

From a practical point of view, APBs are essential markers for the identification of cell lines that utilize the ALT mechanism (Henson et al., 2005; Costa et al., 2006). APBs appear as soon as ALT is activated (Yeager et al., 1999) and disappear when ALT is repressed in somatic cell hybrids (Perrem et al., 1999). Tumors are classified into four groups according to the presence of telomerase activity and/or APBs: telomerase positive, ALT positive, tumors with an unknown TMM (lack telomerase and APBs), and tumors that express both telomerase and markers for ALT (Costa et al., 2006).

1.1.7 Association of DNA recombination and repair proteins with telomeres in ALT cells

Similar to telomeres from mortal cell cultures and telomerase positive immortal and tumor cells, several studies have demonstrated the presence of proteins involved in DNA damage response, homologous recombination and non-homologous end joining (NHEJ) at the telomeres in ALT cells (Table 1-2). The presence of proteins involved in DNA replication

and genome maintenance is not surprising in ALT, since HR is thought to be one of the main

mechanisms in telomere maintenance.

The role of DDR proteins in telomeres of ALT might be different from their roles in telomere

maintenance in non-ALT. A number of studies have demonstrated preferential colocalization

of DNA replication, recombination and damage response proteins to telomeres of ALT

compared to non-ALT cells. Knockdown of others has resulted in telomere erosion and

genome instability that was either specific to or more marked in the ALT cell lines.

Mechanism Protein DNA damage response ATM1, ATR1, MRE112,3, NBS13,4, BRCA14, RAD502,3

HR RAD11, RAD171, RAD514, RAD524, WRN5,6, BLM5,7

NHEJ Ku702,8,9, Ku 809, Ku861

Table1-2. DNA damage response and repair protein colocalize with ALT telomeres: Multiple factors involved in DNA recombination and repair have been found to associate with ALT telomeres by Immunoflouresence (IF) and by telomeric Chromatin Immunoprecipitation (ChIP). 1 Denchi et al, 2007; 2 Verdun and Karlseder, 2006; 3 Jiang et al, 2007; 4 Wu et al, 2003; 5 Opresko et al, 2005; 6 Opresko et al, 2004; 7 Stavropoulo et al, 2002; 8 Celli et al, 2006; 9 Zellinger et al, 2007

The MRE11/RAD50/NBS1 (MRN) protein complex has essential roles in the early stages of

DNA damage response and cell cycle checkpoint control. In addition, MRN has a direct role

in DNA repair by HR in higher vertebrates (Tauchi et al., 2002). MRN complex may be a

necessary component of APBs in ALT cells (Wu et al., 2003; Jiang et al., 2007). Small

hairpin RNA (shRNA)-mediated knockdown of NBS1, with or without depletion of other

members of the complex, resulted in decreased numbers of APBs and decreased telomere

length (Zhong et al., 2007). In contrast, depletion of NBS1 in telomerase-positive cells did

not result in telomere shortening (Zhong et al., 2007). Another study showed that NBS1 was

required for the formation of extra-chromosomal telomeric circles in ALT (Compton et al.,

2007). Together, these findings suggest that MRN complex and specifically NBS1 is required for ALT telomere maintenance.

Another example that highlights the role of DNA repair proteins in ALT maintenance is

Bloom syndrome protein (BLM). BLM is a RecQ helicase that is involved in resolution of

DNA structures that arise in the process of homologous recombination repair. It also catalyzes Holliday junctions (Karow et al., 2000) branch migration and help unwind G- quadriplex-DNA (Sun et al., 1998). Using immunofluorescence staining, BLM protein was shown to colocalize with telomeric foci in ALT human cells but not telomerase positive immortal cell lines or primary cells (Stavropoulos et al., 2002, Lillard-Wetherell et al., 2004).

Transient over-expression of BLM resulted in marked, ALT cell-specific increases in telomeric DNA (Stavropoulos et al., 2002).

The partner of BLM, Topoisomerase III alpha (TopoIIIa) colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells (Temime-Smaali et al., 2008).

TopoIIIa has double stranded DNA relaxation activity and it binds, cuts and re-ligates single stranded DNA (Royle et al., 2008). Depletion of TopoIIIa using small interfering RNA

(siRNA) resulted in a reduction of TRF2 and BLM levels. Moreover TopoIIIa depletion resulted in loss of G-strand overhangs, an increase in anaphase bridge formation and the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells (Temime-Smaali et al., 2008).

In summary, the studies above revealed the important role of MRN, BLM and TopoIIIa in telomere maintenance in ALT. Moreover, depletion of these DNA maintenance proteins selectively affected telomere maintenance in ALT rather than in telomerase positive cells.

This has significant impact on understanding the mechanism of telomere maintenance in

ALT that can used down the road for developing specific targeted anti-ALT tumor therapies.

1.2 Fanconi Anemia Pathway and Telomere Maintenance

1.2.1 Fanconi Anemia Pathway

Fanconi anemia (FA) is a chromosome instability syndrome characterized by congenital malformations in about 60-75% of patients, childhood-onset bone marrow failure and predisposition to hematological malignancies and solid tumors. On the cellular level, cells from FA patients are uniformly hypersensitive to crosslinking agents, a clinical diagnostic feature. The proteins that cause FA appear to be involved in a pathway that promotes genome stability. The number of known members of this pathway is increasing as knowledge about components and function of this group evolves. The FA pathway is activated during S phase and also in response to certain DNA damaging agents. Monoubiquitination of two of the FA proteins, FANCD2 and FANCI, is a key step in the activation of the FA pathway. Currently there are 13 known complementation groups that can be subdivided according to their role in

FANCD2/FANCI monoubiquitination. Eight FA proteins (FANCA, FANCB, FANCC,

FANCE, FANCF, FANCG, FANCL and FANCM) form a nuclear protein complex, the FA core complex, which is required for FANCD2 and FANCI monoubiquitination (Taniguchi and d‟Andrea, 2005). FANCD2 and FANCI then accumulate in nuclear foci and interact with the other FA proteins (FANCN, FANCJ and FANCD1/BRCA2) (Figure 1-6).

Figure 1-6: Signalling through the Fanconi anemia (FA)-breast cancer susceptibility (BRCA) network Model. a) A stalled replication fork activates ataxia telangiectasia and Rad3-related protein (ATR) and its downstream kinase checkpoint kinase 1 (CHK1). They activate the FA core complex and ID complex by phosphorylation. The core complex translocates and is recruited to sites of DNA damage by FA proteins FANCM and FAAP24, where it monoubiquitylates the FA–ID complex, and might also help to recruit the ID complex to the chromatin. FANCM and FAAP24 might also directly process the DNA as part of a DNA-repair reaction. b) A model for the downstream FA–BRCA network. ATR phosphorylates the histone H2A variant H2AX (making gammaH2AX), along with BRCA1 and BRCA2. The BRCT domains in BRCA1 recognize phosphorylated ATR or ataxia telangiectasia mutated (ATM) sites, and might be involved in the recruitment of the repair proteins. A complex of BRCA2 and partner and localizer of BRCA2 (PALB2) can promote a homologous recombination- dependent restart of the replication fork, whereas FANCJ (also called BRCA1-interacting protein 1 (BRIP1)) probably promotes translesion bypass. The BLM helicase complex could work together with the FA core complex to stabilize and re-start the stalled replication forks. P, phosphorylation; Ub ubiquitylation. Adapted from Weidong Wang. Nature Reviews Genetics (2007).

The hypersensitivity of FA cells to crosslinking agents implies a role for FA proteins in the repair of inter-strand DNA crosslinks. The pathways to repair inter-strand DNA cross-links can generate DSB-like intermediate structures that need further processing by HR or non- homologous end joining (NHEJ). There is evolving evidence that FA proteins facilitate recombination. First, several studies showed molecular interactions between FA proteins and

HR proteins (e.g. BLM) (Meetei et al., 2003). Monoubiquitinated FANCD2 and FANCI colocalize with Rad51 and BRCA1 in nuclear foci (Taniguchi et al., 2002, Smogorzewska et al.,

2007). Second, FANCD2 knockout mice have reduced rates of homologous chromosome pairing and increased mispairing of chromosomes during meiosis (Houghtaling et al., 2003).

Third, one of the FA proteins, BRCA2/FANCD1, interacts directly with RAD51 and has a well-defined role in homologous recombination. Fourth, a subset of FA patients possess mutations in breast cancer (BRCA) pathway genes that have an established role in HR such as BRCA2/FANCD1 and in the BRCA1 and BRCA2 interacting proteins BRIP1/FANCJ

(Levitus et al., 2005) and PALB2/FANCN (Reid et al., 2007).

1.2.2 Phenotype of FA mouse models

Several FA mouse models have been published. These included mice deficient for core complex complementation groups Fancc (Chen et al., 1996; Whitney et al., 1996), Fanca

(Cheng et al., 2000; Wong et al., 2003), Fancg (Koomen et al., 2002; Yang et al, 2001), and

Fancl (Agoulnik et al., 2002; Meetei et al., 2003), and one for Fancd2 (Houghtaling et al.,

2003). All FA mouse models displayed gonadal abnormalities and reduced fertility, which are common in FA patients. Unlike FA patients, mouse models lack the developmental

abnormalities observed in some FA human patients except for microphthalmia in Fanca mice. Also in contrast to human FA patients, none of the FA mice developed spontaneous bone marrow failure or acute myeloid leukemia. However, hematopoietic failure in FA mice could be induced by administration of sublethal doses of mitomycin C (MMC) (Carreau et al., 1998). Cells derived from FA mouse models are hypersensitive to DNA cross-linking agents and had no detectable FANCD2 monoubiquitination (Chen et al., 1996; Whitney et al., 1996; Cheng et al., 2000; Wong et al., 2003; Koomen et al., 2002; Yang et al., 2001).

1.2.3 Role of FA pathway in ALT

FA proteins have known interactions with several proteins that are known to localize to or shown to have an effect on telomere maintenance in ALT. Furthermore, chromosome instability that characterizes FA cells is the major phenomenon seen in cells that have dysfunctional telomeres. Being involved in HR and DNA repair, it is logical to presume that members of the FA pathway may have a role in ALT telomere maintenance. Data from our lab on the role of FA proteins in telomere maintenance in ALT cells showed significant colocalization of FANCA, FANCG, FANCD2 (Root, unpublished data) and FANCJ foci with telomeric TRF1/TRF2 foci in ALT cells (Komosa and Yeh, unpublished data).

Transient siRNA-mediated Knockdown of FANCD2 in ALT cells was shown to cause very large TRF1/TRF2 foci, increased number of DNA damage response foci (e.g. 53BP1) at telomeres, increased amounts of telomeric DNA, and subsequent increased frequencies of aneuploidy and cell death (Root, unpublished data). Fan et al., 2009, also demonstrated the colocalization of FANCD2 with telomeric foci. In this study, transient depletion of FANCD2 or FANCA using siRNA resulted in a dramatic loss of detectable telomeres in ALT cells but

not in telomerase-expressing cells (Fan et al., 2009). The authors also found reduced T-SCE rate following FANCD2 or FANCA depletion in ALT cells.

1.3 Fanconi Anemia Complementation group M (FANCM)

1.3.1 FANCM protein structure

FANCM or FAAP250 is a 250kD protein that was first described to be a FANCA-interacting protein in co-immunoprecipitation experiments in 2003 (Meetei et al., 2003). Two years later, in 2005, it was identified as FA complementation group M after a patient (EUFA867) with FA and bi-allelic mutations in FAAP250 (Meetei et al., 2005) was diagnosed. It is the most highly conserved component of the FA pathway with a sequence similarity to DNA repair proteins archaeal Hef, yeast MPH1 and human ERCC4 or XPF (Mosedale et al.,

2005). FANCM contains two conserved enzymatic domains, helicase and endonuclease

(Figure 1-7A). FANCM is assigned to the helicase superfamily 2 and contains seven helicase-specific motifs (I, Ia II, III, IV, V, and VI) (Wu et al., 2006). Motif II, the Walker B motif, consists of a DEAD or DEAH box and is necessary for the interaction with Mg2+ and thus for ATP hydrolysis and ATP-dependent DNA translocase activity. The endonuclease domain is homologous to the endonuclease domain of ERCC4 (XPF) but is degenerate and has not been shown to be functional.

Figure 1-7: Domain structure of human FANCM and FAAP24: (A) FANCM contains an active ATPase/helicase domain in its N-terminus and a degenerate ERCC4-like domain in its C- terminus. FANCM interacts with FAAP24 via its C-terminal endonuclease domain. FANCM contains two HhH domains in its c-terminus. (B) FAAP24 contains an ERCC4-like domain and interacts with FANCM. FAAP24 also contains two HhH domains in its c-terminus. Adapted from Ali et al, Mutation Research (2009).

FAAP24, Fanconi anemia A-associated polypeptide of 24 kDa mass, was discovered by a database search for proteins with homology to the C-terminus of FANCM (Ciccia et al.,

2007). It is a 253 amino acid protein and shares 20% identity (31% similarity) with the C- terminal 300 amino acid region of FANCM. Both FANCM and FAAP24 harbor two helix- hairpin-helix (HhH) motifs in tandem in their C-terminal regions (Figure 1-7A and B)

(Ciccia et al., 2007). The HhH is a domain of approximately 20 amino acids that is involved in sequence-nonspecific DNA binding and which is found in proteins that are essential for the repair of DNA structure. Similar to the ERCC4 domain of FANCM, the FAAP24 nuclease domain is degenerate at several residues essential for endonuclease function (Ciccia et al., 2007).

1.3.2 Functions and biochemical activity of FANCM

FANCM is constitutively associated with chromatin, either with or without exposure to

MMC or to blockage of replication (Kim et al., 2008). The localization of FAAP24, and other FA core complex proteins, to chromatin is defective in cells depleted of FANCM, suggesting that FANCM is required for chromatin localization of FA core complex proteins

(Kim et al., 2008). FANCM is hyperphosphorylated in response to DNA damage. The kinase responsible for the phosphorylation of FANCM has not been identified. Phosphorylation of

FANCM increases its binding affinity for chromatin (Kim et al., 2008). The phosphorylation of FANCM is dynamically regulated during the cell cycle, being moderately phosphorylated during S phase, extensively phosphorylated during mitosis, but is dephosphorylated after mitotic exit (Kim et al., 2008). Since FANCM activity is regulated by phosphorylation/dephosphorylation events, FANCM has been proposed as a signal transducer that regulates the activity of the FA core complex (Kim et al., 2008; Ali et al.,

2009).

Unlike core complex proteins, FANCM has a DNA binding activity. Full-length FANCM, or

FANCM- N1-754, shows strong specificity and affinity for branched DNA structures like

Holliday junctions and replication forks (Meetei et al., 2005; Gari et al., 2008; Xue et al.,

2008). FANCM- N1-754 has higher affinity for Holliday junctions than for fork-structured

DNA (fsDNA) (Xue et al., 2008).

In vitro, FANCM is able to dissociate DNA triplex (Meetei et al., 2005) and large recombination intermediates via branch migration of Holliday junctions (Gari et al., 2008;

Xue et al., 2008). The ability of FAAP24-FANCM to bind structures that arise during

27 A

replication or repair suggests that FANCM-FAAP24 may act as a sensor of the blockage of

DNA replication or of DNA damage (Ali et al., 2009). FANCM is able to dissociate D-loops that are free of, or bound with, RPA (Gari et al., 2008). These findings support FANCM direct contribution in FA-BRCA DNA repair pathway. The contribution of FANCM in FA-

BRCA pathway might be regulatory but not essential for homologous recombination (HR) as

EUFA867 lymphoblasts showed normal Rad51 foci (Singh et al., 2009).

1.3.3 FANCM cellular phenotypes

1.3.3.1 Hypersensitivity to DNA cross-linking agents

FANCM was first identified as an FA core complex protein (Mosedale et al., 2005). Studies on EUFA867, the reference cell line for Fa-M, suggested that FANCM is essential for

FANCD2 monoubiquitination (Meetei et al., 2005) as in this cell line monoubiquitinated

FANCD2 was absent and the levels of FANCA and FANCG were reduced (Meetei et al.,

2005). However, Transient knockdown of FANCM protein in HeLa cells using siRNA does not result in defects in the stability or assembly of the FA core complex (Kim et al., 2008;

Xue et al., 2008). Also data on a mouse model for FANCM showed residual FANCD2 monoubiquitination (Bakker et al., 2009). A recent publication by Singh et al, 2009 identified the presence of bi-allelic pathogenic mutations in the FANCA gene in addition to FANCM

(Singh et al., 2009) in the EUFA867 cell-line. Restoration of FANCA expression by either retroviral transduction or cDNA transfection partially rescued the FANCD2 monoubiquitination defect seen in EUFA867 cells (Singh et al., 2009). In chicken B cell line

DT40 exposed to high doses of cisplatin, FANCD2 ubiquitination in the FANCM null cell line was defective compared to wild-type, but importantly was clearly detectable (Rosado et al., 2009). These findings are consistent with the mouse model and together indicate that

FANCM is a core complex-associated protein that is not essential for FANCD2 monoubiquitination, but may increase the efficiency of this process.

In a knockout FANCM mouse model, murine embryonic fibroblasts (MEFs) depleted of

FANCM developed FA-like phenotype with pronounced hypersensitivity to cross-linking drugs such as MMC resulting in increased chromosomal breakage, reduced cell viability and a pronounced G2 arrest (Bakker et al., 2009). Likewise, FANCA expression in EUFA867 did not restore MMC hypersensitivity nor changed the G2 arrest observed in EUFA867 cells suggesting that FANCM deficiency is associated with crosslinker hypersensitivity that characterize all FA complementation groups.

1.3.3.2 Camptothecin and UV sensitivity

In addition to the crosslinker hypersensitivity, FANCM deficiency is associated with several other cellular phenotypes that are not seen with other FA core complex groups. Cells from

EUFA867 and FANCM-/- chicken B cell line DT40 showed sensitivity to other DNA damaging agents. Singh et al., 2009 found through growth inhibition assays on EUFA867 lymphoblasts that this cell line is hypersensitive to the topoisomerase I inhibitor, camptothecin. This hypersensitivity was not found in lymphoblasts with a defect in FA core complex members or in FANCD2, FANCI or FANCJ (Singh et al., 2009). In contrast, in 4 independent cell lines deficient in FA proteins that are not directly involved in FANCD2/I

monoubiquitination like FANCD1/BRCA2 and FANCN/PALB2 deficient lymphoblasts, camptothecin sensitivity was similar to FANCM deficient cells. Furthermore, Singh et al.

2009 found that EUFA867 was sensitive to ultraviolet light (UV). Sensitivity of FANCM null DT40 cell lines to both camptothecin and UV light was also reported by Rosado et al.,

2009. They constructed FANCM DT40 cell lines by disturbing the exons encoding either the helicase (FANCM-∆hel) or the nuclease domain (FANCM-∆nuc) and found that camptothecin sensitivity is partially complemented by the construct containing the nuclease domain whereas the UV sensitivity is complemented by the helicase domain (Rosado et al.,

2009).

1.3.3.3 Increased sister chromatid exchanges

Increased background level of sister chromatid exchanges (SCE) was observed in the

FANCM deficient chicken B cell line DT40 (Rosado et al., 2009) and in FANCM deficient mouse embryonic fibroblasts (Bakker et al., 2009). Suppression of crossover recombination required the Walker DEAD box motif as the DT40 strains carrying homozygous deletion of the nuclease domain (FANCM∆nuc) was dispensable for SCE suppression (Rosado et al.,

2009).

BLM is the main protector against SCEs in eukaryotes, but the yeast FANCM orthologues were shown to function in a distinct SCE suppression pathway to their respective Blm orthologues (Sun et al., 2008). In human cells, FANCM co-purifies with the Blm-Topo III complex (Meetei et al., 2003). Rosado et al., 2009 have demonstrated that the number of

SCEs in the double mutant ∆Blm FANCM-D203A strain (FANCM helicase point mutation

D203A in a DT40 BLM knockout cell line) was equivalent to the ∆Blm strain with WT

FANCM (mean=14 SCEs per metaphase). This led to the suggestion that FANCM functions in the same pathway with the Bloom helicase to suppress SCEs (Rosado et al., 2009).

1.3.4. Role of FANCM in ATR/Chk1 checkpoint signaling pathway

In addition to its well-identified role as FA pathway protein, FANCM was identified to be involved in ATR-mediated checkpoint signaling. FANCM and its partner protein FAAP24 were found to interact with HCLK2, which interacts with and regulates the stability of ATR

(Collis et al., 2008). The interaction between FANCM and HCLK2 was found to be independent of the FA core complex. This was supported by the observation that FANCM and FAAP24 depleted cells fail to efficiently block inappropriate mitotic entry in response to replication stress or the presence of DNA damage. This was evident by the inefficient

Cdc25A degradation in cells depleted for FANCM, FAAP24 or HCLK2 treated with hydroxyurea (HU) (Collis et al., 2008). The lack of mitotic arrest in these cells upon exposure to ionizing radiation (IR) was also evident by the 4-fold increase in phosphohistone

H3-positive cells, when compared to control cells (Collis et al., 2008). Similar to cells with defective ATR signaling, FANCM depletion is associated with supernumerary centrosomes

(Collis et al., 2008).

Several studies attempted to dissect the helicase and endonuclease domains to gain more functional insights. Expression of the FANCM ATPase point mutant (K117R) was able to restore FANCD2 monoubiquitination but did not correct the cross-linker resistance (Singh et al., 2009). The biochemical activities of FANCM, such as branch migration, translocase

activity, D-loop dissolution and fork regression, all require ATPase activity and are very likely to be significantly impacted by mutation of the DEAD box. The nuclease domain appears to have binding ability to FAAP24 and HCLK2, and to fork structured DNA and contributed to camptothecin resistance.

All of these observations provide evidence that FANCM participation in DNA damage signaling and repair is complex. FANCM may act through ATR-mediated checkpoint signaling, FANCD2/I monoubiquitination and/or by direct involvement in DNA metabolism.

1.3.5 FANCM human and mouse disease phenotypes

To date, EUFA867 is still the only FA-M patient described. The clinical phenotype of this patient has never been disclosed in a publication other than a mention that EUFA867 clinical features are different from classical FA (Singh et al., 2009). As mentioned above (see section 1.3.3.1), it recently became apparent that this patient also carries FANCA mutations

(Singh et al., 2009), which cast doubt on the actual contribution of FANCM deficiency to the

FA phenotype. Bakker et al., 2009 generated a Fancm mouse model to study the effect of

Fancm deficiency in vivo. Similar to other FA mouse models, FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in murine embryonic fibroblasts (MEFs) (Bakker et al., 2009). There were no congenital abnormalities noted in the

Fancm∆2/∆2 mice. Also the bone marrow of these mice showed no signs of hypoplasia and the number of hematopoietic stem cells appeared normal. Fewer female Fancm mice were born than expected indicating female embryonic lethality. Tumor formation was as high as

52% in FANCM homozygote mice compared to 39% of the herezygotes for one FANCM

mutation and 14% of the wild type. In other words, Fancm mice showed increased cancer incidence and associated reduced overall and tumor-free survival (Bakker et al., 2009).

1.3.6 FANCM and ALT telomere maintenance

To date, there are no published studies of the effect of FANCM depletion on telomere maintenance either in ALT or in telomerase positive cells. The observation that FANCD2 knockdown was associated with disturbed telomere maintenance (Root, unpublished data;

Fan et al., 2009) suggests that FANCM might be also a participant in ALT telomere maintenance through its role in FANCD2 monoubiquitination. The findings that FANCM co- purifies with the Blm-Topo IIIa complex in ALT, and the previously discussed effects of

BLM over-expression (Stavropoulos et al., 2002) and TopoIIIa knockdown (Temime-Smaali et al., 2008) in ALT (see section 1.1.7) all suggest that FANCM can affect ALT telomere maintenance in a similar way to BLM-TopoIIIa. Knockdown of FANCM thus may be expected to be associated with increased rate of T-SCE. Similar to BLM-TopoIIIa; this effect is expected to be specific to ALT rather than telomerase positive cells that did not seem to depend on HR as a TMM.

1.4 Objective

Understand the role of FANCM protein in telomere maintenance in Alternative Lengthening of Telomeres (ALT) human cells.

1.5 Hypothesis

FANCM plays a role in telomere integrity and maintenance in ALT cells.

CHAPTER TWO

Analysis of the role of FANCM in telomere maintenance in ALT human

cells

2.1 Experimental Approach

To study the role of FANCM in telomere maintenance in ALT cells, we first determined if

FANCM protein can form discrete foci that are detectable using immunofluorescence (IF) staining (section 2.2.3). We then assessed the availability of FANCM foci at ALT telomeres using two channel-IF staining for FANCM and telomere binding factors TRF1/TRF2

(section 2.2.4). Colocalization was computed on the images obtained and analyzed using

Volocity software (see sections 2.2.4 and 2.2.5). Comparison of the IF appearance of

FANCM foci and their colocalization with TRF1/TRF2 was made between different cell lines including two ALT cell lines, WI38VA13/2RA and GM00847; two telomerase positive fibroblasts, GM00639 and HT1080; a wild type lymphoblast cell line obtained from

SickKids tissue culture lab (WT36950) and a lymphoblast cell line obtained from the

EUFA867 patient.

The effect of FANCM depletion was studied using transient siRNA-mediated knockdown of

FANCM (see section 2.2.8). Effects of FANCM depletion on TRF2 foci were studied using

IF. Peptide nucleic acid- fluorescent In Situ Hybridization (PNA-FISH) (see section 2.2.9) was done to study the effect of FANCM depletion on the amount of telomeric DNA.

2.2 Methods

2.2.1 Cell lines and culture conditions

2.2.1.1 Lymphoblasts cell lines

Epstein-Barr virus-transformed EUFA867 lymphoblasts obtained from the only patient known with FA-M were donated by Hans Joenje (Amsterdam, Netherlands). WT35690 wild type lymphoblasts were obtained from the Hospital for Sick Children tissue culture services

(Toronto, ON). Lymphoblasts were cultured in RPMI1640 medium from Wisent (ST-Bruno,

Quebec) with L-glutamine and sodium bicarbonate supplemented with 15% fetal bovine serum (FBS) from Wisent and placed in a humidified 5% CO2 incubator at 37°C.

2.2.1.2 Fibroblasts cell lines

The ALT WI38-VA13/2RA cell line was obtained from the European Collection of Cell

Cultures (ECACC) (Salisbury, UK). Human ALT Simian Virus (SV) 40-transformed

GM00847 fibroblasts were purchased from Coriell Institute for Medical Research Cell

Repositories (CCR) (Camden, New Jersey). SV-40 transformed GM00639 telomerase positive fibroblasts were purchased from CCR and HT1080 (tumor-derived, telomerase positive) fibroblasts were obtained from American Type Culture Collection (ATCC)

(Manassas, VA). All human fibroblast cell lines were maintained in Eagle‟s Minimal

Essential Medium Alpha Modification (a-MEM), Invitrogen (Burlington, ON) supplemented

10% FBS from Wisent and cultured in a humidified 5% CO2 incubator at 37°C.

2.2.2 Antibodies

The main FANCM antibody was a polyclonal anti-rabbit antibody, ab26272, from Abcam

(Cambridge, MA). Four other anti-rabbit FANCM antibodies were obtained from the

Fanconi Anemia Research Foundation (FARF) (Eugene, OR): aM1-R4811, aM2-R3821, aM3-R6668 and aM4-R6667. Monoclonal anti-mouse FANCM antibody, sc-101389, was purchased from Santa Cruz (Santa Cruz, CA). Other antibodies used are anti-mouse TRF2

(IMG-124A) from IMGENEX (San Diego, CA); anti-goat TRF1 (sc-6165); anti-mouse

FANCD2 (sc-20022); anti-mouse PML and anti-mouse α-tubulin (sc-32293) all from Santa

Cruz.

2.2.3 Immunofluorescence (IF) following detergent extraction in fibroblasts

Fibroblasts were either seeded on 25mm round glass cover-slips in 2-ml culture dishes or in

4- well slides the day before fixation. Processing for IF was initiated by removing the culture media by suctioning, washing cells with phosphate buffered-saline (PBS) from Wisent for 5 minutes three times (standard wash) followed by permeabilization with Triton X-100 buffer

(0.5% Triton X-100, 20mM Hepes-KOH, pH7.9, 50mM NaCl, 3mM MgCl2, 300mM sucrose) for 5 minutes. Cells were then rinsed with PBS before adding ice-cold fixative (3% paraformaldehyde, 2% sucrose in H2O solution, pH 8.2) for 10 minutes. Cells were then washed and permeabilized again with Triton X-100 buffer for 10 minutes. Cells were washed again with PBS and blocked for 30 minutes at 37oC in filtered blocking solution, 0.3 mg/ml donkey serum from Jackson Immuno Research Laboratories (West Grove, PA) in 0.2% fish gelatin from Sigma (St. Louis, MO) and 0.5% bovine serum albumin (BSA), from Jackson

Immuno Research Laboratories, in PBS. Cells were then incubated with primary antibodies;

rabbit anti-FANCM antibody (1:800) and mouse anti-TRF2 (1:2000) diluted in a filtered

0.2% fish gelatin/0.5% BSA in PBS buffer overnight at 4oC. Cells were subsequently washed and then incubated with the secondary antibody solution; TRITC-conjugated donkey anti- rabbit antibody (1:1000), FITC-conjugated donkey anti-mouse antibody (1:1000) (both from

Jackson ImmunoResearch) in PBS containing 0.3 mg/ml donkey serum, 0.2% fish gelatin and 0.5% BSA for 1 hour at room temperature. Cells were washed and incubated for 1 minute in 0.1 µg/ml of 4, 6-diamino-2-phenylindole (DAPI) to stain DNA. Cells were then washed again, coverslips were allowed to air-dry and mounted with slowFade® anti-fade solution from Invitrogen and placed on 22X60mm slides. The other primary antibodies were used following the same protocol at the following dilutions: anti-mouse FANCD2 1:100, anti-goat TRF1 1:200, anti-mouse FANCM 1:100, and anti-mouse PML at 1:200.

2.2.4 Foci count and colocalization analysis

Images of IF stained slides were examined using 20X objective or 63X objective mounted onto a Zeiss Axioplan 2 microscope equipped with a Hammamatsu Orca ER camera. Images were captured using OpenLab 3.1.7 Software from Improvision (Coventry, UK). Stack images obtained at 0.2µm vertical intervals spanning each nucleus. On average 20 to 30 slice images were captured for each nucleus with two images being captured for each slice for the

TRITC and FITC channels. A file with series images was made in OpenLab 3.1.7 for each nucleus. These files were then analyzed by a 3-dimentional (3-D) imaging program, Volocity

3.7.0 from Improvision. In Volocity 3.7.0, all slices for each nucleus were de-convoluted and incorporated to provide 3-D images. Discrete signals of immunofluorescence were

considered foci. For standardization, a threshold of four standard deviations (SD) above mean of the total nuclear fluorescence intensity was used, so that only foci with greater than

4SD fluorescence intensity will be selected. Another threshold parameter was volume, so only signals above 0.1µm3 were selected as foci. All signals below either of these two thresholds were excluded. The program calculated the number of foci for each channel and provided spatial relationships between foci in the two channels represented in the number of

FANCM foci touching, overlapping or not touching TRF2 foci.

2.2.5 Transient knockdown of FANCM transcription using small interfering RNA

(siRNA)

Open reading frame (ORF) oligo-ribonucleotide FANCM siRNA sequence was adapted from

Xue et al., 2008 (26). The sense strand sequence is AGACAUCGCUGAAUUUAAA (Xue et al., 2008). The sequence was synthesized and purchased from Dharmacon (Lafayette, CO).

Random sequence siRNA oligo-ribonucleotide was designed by Heather Root from the Meyn

Lab and the sequence was synthesized by and purchased from Dharmacon.

One day before transfection, 5X104 cells/well were seeded into 6-well culture dish. Cells were cultured in antibiotic-free Dulbecco's Eagles Minimal Essential Medium Alpha

Modification (α-MEM) from Invitrogen supplemented with 10% fetal calf serum. For each 2 ml well, 10µl of 20µM siRNA was mixed with 240µl of Opti-mem® medium from

Invitrogen in a sterile tube. In a separate tube, 5µl of Lipofectamine® RNAiMAX (purchased from Invitrogen) was mixed with 245 µl of Opti-mem® and incubated for 5 minutes at room temperature. The 250 µl of siRNA-Optimem was then mixed with 250µl of Lipofectamine-

Optimem and the mix was incubated for 15 minutes at room temperature. For each well, the old culture medium was replaced with 1.5ml of Opti-mem. 500µl of siRNA-Lipofectamine mix was then added to each well drop-wise to ensure equal distribution. Cells are incubated for 3 to 4hours in a humidified 5% CO2 incubator at 37°C before adding 200µl of FBS

(Wisent) for each well. To minimize cytotoxicity, siRNA-Lipofectamine containing medium is replaced after 24 hours with α-MEM containing 10% FCS. A second round of siRNA transfection was performed 48 hours after the first siRNA treatment by repeating the above procedure. Cells were harvested for Western blotting, IF or PNA-FISH 96 hours after the first transfection.

2.2.6 Peptide nucleic acid- Fluorescence In Situ Hybridization (PNA-FISH)

A Q-FISH protocol was adapted from the Lansdrop laboratory (Terry Fox Laboratory,

Vancouver, B.C.): Cultured fibroblasts were treated with 100ng/ml Colcemid (KaryoMAX,

Gibco, Invitrogen) and incubated in humidified 5% CO2 incubator at 37°C for 3 hours.

Fibroblasts were detached using 0.05% Trypsin from Wisenet. Cells were then centrifuged for 1000 rpm for 8 min. To swell the cells, the pellet was resuspended gently in 10 ml of

75mM KCl buffer, pre-warmed at 37°C, for approximately 30 min in humidified 5% CO2 incubator. Next, one ml of ice-cold 3:1 methanol:acetic acid fixative was added slowly. Cells were centrifuged, supernatant removed, re-suspended and fixed with 5 ml of ice-cold 3:1 methanol:acetic acid. Fixed cells were stored in fixative at -20°C until hybridization.

Slide preparation for FISH: cells were centrifuged for 8 minutes at 1000rpm. Old fixative was then removed, 3-5ml of fresh, ice-cold fixative is added and cells were spun again. Most of the supernatant was then removed (amount left depended on the pellet size). In a

relatively humid room, several (usually 5 to 6) 10µl-drops were dropped on ethanol pre- cleaned slides. Slides were air-dried over night. For in situ hybridization cells were re- hydrated in PBS (pH=7.0-7.5) for 15 min. Cells were fixed in 4% formaldehyde in PBS

(pH=7.0-7.5) for 2 min and then washed in PBS for 5 minutes 3 times. Cells were treated with 1mg/ml Pepsin (Sigma) in 37oC for 10 min. (Pepsin was prepared freshly in acidified water (pH=2)). Cells were washed twice in PBS and fixed in 4% formaldehyde in PBS for 2 min. following fixation, cells were washed three times and dehydrated in 3 steps: 70% ethanol for 5 minutes, 90% ethanol for 5 min and 100% ethanol for 5 minutes. Slides were air-dried. After slides had dried, 2 drops (10 ul each) of pre-heated hybridization mix [70%

Formamide (ultra pure, pH=7.0-7.5), 0.1% blocking powder, 10 mM Tris, 5% MgCl2 Buffer

(82 mM Na2HPO4, 9 mM citric acid, 20 mM MgCl2), 0.5 µg/ml PNA Tel Cy-3 from

Applied Biosystems (Foster City, CA) diluted in H2O] were dropped on individual coverslips

(22x60 mm). Slides were then placed carefully (upside down) on the coverslips to avoid air bubbles and turned back up. Slides were then placed to denature in preheated oven (80oC) for

3 min. For hybridization to occur slides were placed in a plastic box and the box was positioned in a plastic beaker covered with parafilm for 2 hours at room temperature. Slides were placed in wash solution I (70% formamide, 10mM Tris, 0.1% BSA, pH=7.0-7.5) pre- warmed at 37C and coverslips were carefully removed. Slides were washed twice for 15 minutes in wash solution I and then for three times, 5 minutes each in wash solution II (0.1 M

Tris, 0.15 M NaCl, 0.08% Tween 20, pH=7.0-7.5). Cells were then stained with 0.1 g/ml of

DAPI, washed in PBS and dehydrated in 3 steps: 70% ethanol for 5 minutes, 90% ethanol for

5 min and 100% ethanol for 5 minutes. Slides were air-dried and mounted with slowFade antifade solution (Invitrogen).

2.3 Results

2.3.1 FANCM forms discrete foci in ALT and telomerase positive cells

2.3.1.1 Antibodies against FANCM form discrete foci in the nuclei of GM00847 and

WI38-VA13/2RA ALT cells

Immunofluorescence (IF) following detergent extraction (see section 2.2.3) was used to detect FANCM, TRF2 and TRF1 foci. Using the 20X objective, discrete focus formation was clearly noted in a proportion of the randomly cycling nuclei in both WI38-VA13/2RA and

GM00847 ALT cell lines. In WI38-VA13/2RA ALT cells, 9% of randomly cycling cells expressed FANCM foci, whereas in GM00847 cells FANCM foci were detected in 22% of the randomly cycling cells (Figure 2-1a and 2-1b). Of cells expressing FANCM foci, the average number of foci/nucleus obtained by deconvolution analysis was 26 and 27 in WI38-

VA13/2RA and GM00847 respectively. In WI38-VA13/2RA cells, the larger nuclei were more likely to express FANCM foci, whereas in GM00847, this tendency was less pronounced.

A detailed examination of FANCM foci shows that the larger FANCM foci can have a distinct structure (Figure 2-1c).

2-1a 2-1b

Figure 2-1: Immunofluorescence appearance of FANCM foci under 20X and 63X magnification in randomly cycling ALT nuclei. a) In WI38-VA13/2RA large discrete foci seen in 9% of nuclei which tend to be seen more often in larger nuclei. b) In GM00847, more nuclei have detectable FANCM foci (22%) where foci were detected in nuclei of different sizes. c) Enlarged image for a large FANCM focus showing a flower-like structure in GM00847 ALT nucleus

2.3.1.2 Appearance of FANCM foci in GM00639 and HT1080 telomerase positive fibroblasts

Using detergent extraction IF in HT1080 and GM00639 telomerase positive human fibroblasts, discrete FANCM foci were detected in both cell lines. 11 out of 198 (5%)

HT1080 nuclei expressed FANCM and 45 out of 552 (8%) GM00639 nuclei expressed

FANCM. In HT1080, detected foci were hardly visible under the 20X magnification and more visible under the oil –mounted 63X objective (Figure 2-2a to 2-2d) whereas GM00639 telomerase positive cells had larger FANCM foci. The size of FANCM foci in both of these telomerase positive cell lines was smaller than their counterparts in ALT cells. To provide an objective comparison between the size of FANCM foci in HT1080 telomerase positive cells compared to WI38-VA13/2RA ALT cells, binned volume for each focus was computed using Volocity software (see section 2.2.5). Mean volume of 174 WI38-VA13/2RA foci was

192 voxels whereas 146 HT1080 foci had a mean volume of 81 voxels. The distribution of foci volume was different between these two cell lines as well, with WI38-VA13/2RA having more foci that are considerably larger than the mean (Figure 2-2e and 2-2f). The difference of the distribution of voxel count between WI38-VA13/2RA and HT1080 is statistically significant (p 3.8X10-11 using Mann Whitney u test). GM00639 telomerase positive nuclei had larger FANCM foci than HT1080 but smaller than ALT cells with an average voxel count of 112 in 345 foci.

a) HT1080 b) GM00639

c) WI38-VA13/2RA d) GM00847

e) f)

Figure 2-2: Comparison between the immunofluorescence (IF) appearance of FANCM foci in telomere positive and ALT cells. Appearance of foci detected by ab26272 FANCM antibody using 63X objective in a) HT1080 and b) GM00639 telomerase positive cell lines. Foci formed are smaller with large number of background speckles when compared to the foci formed in c) WI38-VA13/2RA and d) GM00847 ALT cells. e) distribution of the mean volume of 174 FANCM foci in WI38-VA13/2RA ALT cells is skewed to the right compared to f) the distribution of the mean volume of 146 FANCM foci in HT1080 which indicates the presence of more FANCM foci that are larger than the mean in the ALT cell line.

2.3.1.3 Two different FANCM antibodies form nuclear foci that highly colocalize

To further validate the specificity of IF detection of FANCM by the anti-rabbit FANCM antibody (ab26272), IF was performed using the anti-mouse FANCM antibody (sc-101389).

There was excellent colocalization between the foci formed by each antibody (Figure 2-3).

Figure 2-3: Validation of FANCM foci by Immunofluorescence (IF): high colocalization of two different antibodies. Using IF following detergent extraction, anti-rabbit and anti- mouse FANCM antibodies form nuclear foci that colocalize >90%.

2.3.2 Colocalization of FANCM with TRF1/TRF2, FANCD2 and PML

2.3.2.1 FANCM colocalizes with TRF1 and TRF2 in ALT more than telomerase positive cells

IF staining of the two ALT cell lines, WI38-VA13/2Ra and GM00847 using antibodies against TRF1 or TRF2 showed that FANCM is commonly present at telomeric foci in ALT.

In a sample of 75 WI38-VA13/2RA ALT cells that expressed FANCM foci, 72% of their

FANCM foci were present at telomeres. Also the majority of telomeric TRF1 foci (59%) localized with FANCM foci. In 50 randomly cycling GM00847 ALT cells, 57% of their

FANCM foci colocalized with telomeric foci and 42% of telomeric TRF1 foci colocalized with FANCM (Figure 2-4). In HT1080 and GM00639 telomerase positive cells, 10 nuclei each, mean percentage of colocalization of FANCM with TRF1 foci of 28% and 31% respectively. Likewise, the colocalization of TRF1 foci with FANCM foci was lower in telomerase positive cells with mean percentage of colocalization of 25% and 20% in HT1080 and GM00639 respectively (Figure 2-5 and Table 2-1). The colocalization of FANCM foci with TRF1 and the colocalization of TRF1with FANCM were both more pronounced in ALT compared to telomerase positive cells (p0.0057 and p0.0004 respectively using Fisher exact test).

FANCM merge TRF1

2-4 WI38- VA13/2RA

GM00847

Figure 2-4: Colocalization of FANCM foci with TRF1 foci in WI38-VA13/2RA and GM00847 ALT cells. From left to right, IF appearance of FANCM and TRF1 foci and the merge image that demonstrates colocalization in randomly cycling WI38-VA13/2RA (top lane) and GM00847 nuclei (bottom lane).

2-5 Percentage of FANCM and TRF1 colocalization in various cell types

80 70 60 percentage of 50 FANCM co-localize with TRF1 40 percentage TRF1 30 colocalize with

percentage FANCM 20 10 0

HT1080 GM00847 GM00639 WI38-VA13

Figure 2-5: Colocalization of FANCM foci with TRF1 foci in ALT and telomerase positive cells. Graphic representation of the average percentage of FANCM and TRF1 colocalization in WI38-VA13/2RA and GM00847 ALT and HT1080 and GM00639 telomerase positive cells. FANCM localization to telomeric TRF1 (blue bars) is higher in ALT WI38-VA13/2RA and GM00847 (72% and 57%) than telomerase positive HT1080 and GM00639 (28% and 31%). Similarly TRF1 colocalization with FANCM is lower in telomerase positive cells (red bars)

FANCM/nucleus TRF1/ FANCM colocalizes TRF1 colocalizes with nucleus with TRF1 FANCM

ALT WI38- 25 30 72% 59% VA13/2RA

GM00847 23 31 57% 42%

Telomerase HT1080 18 21 28% 25% positive GM00639 32 49 31% 20%

Table 2-1. Means of FANCM and TRF1 foci count and colocalization in ALT and telomerase-positive cells.

2.3.2.2 Colocalization of FANCM with FANCD2 primarily occurs at telomeres

Three-channel IF in WI38-VA13/2RA ALT cells using antibodies against FANCD2,

FANCM and TRF1 antibodies showed that the majority of FANCM foci that colocalized with telomeric foci did colocalize with FANCD2 foci as well (figure 2-6a and 2-6b). On average, 65% of FANCM foci colocalized with FANCD2 foci whereas 55% of FANCD2 foci colocalized with FANCM foci. Out of 928 TRF1 foci from asynchronously cycling

WI38VA13/2RA ALT cells, 418 (50%) contained FANCM and FANCD2. This indicates that in this cell line, when FANCD2 and FANCM are located together, most of the time they were at telomeres.

2.3.2.3 FANCM colocalizes with PML bodies

In WI38-VA13/2RA ALT cells, the majority of FANCM foci colocalized with PML bodies.

The average percentage of FANCM foci that colocalized with PML bodies out of total

FANCM was 75% (figure 2-6c). Most of FANCM foci that colocalized to PML, colocalized with TRF1 at the same time.

FANCM FANCD2 Merge

2-6a

2-6b

47 192 416

418

107

TRF1 FANCM FANCD2 Total Total Total 928 764 660 2-6c

DAPI FANCM PML FANCD2 Merge

Figure 2-6: Colocalization of FANCM with FANCD2, PML and TRF1 in WI38-VA13/2RA ALT. a) IF appearance of FANCM and FANCD2 foci demonstrates the high colocalization in randomly cycling WI38- VA13/2RA nucleus. b) A graph representation to the overlap between FANCM, FANCD2 and TRF1 in WI38-VA13/2RA. Over 50% of telomeric TRF2 foci colocalized with FANCM and FANCD2 at the same time. c) IF appearance of FANCM, PML and FANCD2 in a WI38-VA13/2RA nucleus demonstrates FANCM and FANCD2 colocalization with PML.

2.3.3 Effect of transient depletion of FANCM using siRNA

2.3.3.1 siRNA knockdown of FANCM results in poor growth and increased cell death in

ALT and telomerase-positive fibroblasts siRNA knockdown of FANCM was conducted in WI38-VA13/2RA and GM00847 ALT and

GM00639 and HT1080 telomerase-positive cell lines. Within two days after the first siRNA transfection, it was observed that all cell-culture wells from all cell lines received the

FANCM siRNA had decreased growth compared to cells treated with the control GL2 siRNA and growth reduction was more apparent by the fourth day (96 hours) after the first siRNA knockdown. Reduced growth was more pronounced in the ALT cell lines where

FANCM knockdown WI38-VA13/2RA and GM00847 culture wells were about 50% less confluent compared to GL2 siRNA groups. Increased cell death in the FANCM knockdown groups was also noticed; as there were more floating cells compared to the cells treated with the GL2 siRNA.

FANCM knockdown resulted in increase in the percentage of cells with larger nuclei (~20% of total population) compared to the random siRNA control (5-10% of total population) (data not shown). This phenomenon was also observed in FANCM-depleted WI38-VA13/2RA and

GM00847 ALT cells but not seen in telomerase-positive HT1080 upon FANCM depletion.

2.3.3.2 FANCM knockdown resulted in a reduction of FANCM signal

Using detergent extraction IF in WI38-VA13/2RA and GM00847 ALT, cells that received the FANCM siRNA had significantly lower FANCM signal intensity compared to the cells treated with the random siRNA (Figure 2-7). This finding was important for the validation for the specificity of the FANCM antibody used in the IF experiments. It was also an important indicator to the successful knockdown of FANCM transcription.

GL2 siRNA FANCM siRNA

WI38- VA13/2RA

GM00847

Figure 2-7: FANCM knockdown using siRNA resulted in depletion of FANCM signal. Appearance of FANCM under the 20X magnification using GL2 siRNA (left) and FANCM siRNA (right) in WI38-VA13/2RA (top) and GM00847 (bottom): FANCM siRNA resulted in depletion of the FANCM signal.

2.3.3.3 FANCM knockdown resulted in larger TRF2 foci in GM00847 ALT cells

In GM00847 ALT cells, FANCM knockdown resulted in significant increase in the size of

TRF2 foci. Nuclei that expressed abnormally large TRF2 foci had absent or faint FANCM foci (Figure 2-8a).

2.3.3.4 FANCM knockdown resulted in distortion of TRF2 foci in WI38VA13/2RA ALT cells

A minority of randomly cycling WI38VA13/2RA ALT cells, had nuclei with predominantly large TRF2 foci. FANCM depletion was associated with increased frequency of cells that expressed large TRF2 foci (Figure 2-8b). Of 234 nuclei with decreased FANCM staining from the FANCM knockdown group, 45 nuclei (19%) expressed large TRF2 nuclei compared to 44 out of 405 total nuclei (11%) from the random knockdown group (Chi square

10.104, p<0.001). A phenotype that was never seen before in WI38-VA13/2RA ALT cells, where TRF2 foci looked irregular and disorganized (Figure 2-8c), was seen only in FANCM knockdown (13/234 nuclei compared to zero/405 nuclei in the random knockdown group).

All nuclei expressed the disorganized TRF2 foci had absent or faint signal for FANCM.

These abnormal TRF2 foci were mainly seen in the large nuclei whereas FANCM depletion did not seem to alter the appearance of TRF2 in small nuclei with small TRF2 foci.

2-8a DAPI FANCM TRF2 Merge GM00847

GL2 siRNA

FANCM siRNA

2-8b

Merge WI38-VA13/2RA DAPI FANCM TRF2

GL2 siRNA

FANCM siRNA

2-8c

WI38-VA13/2RA

Figure 2-8: Effects of FANCM depletion on TRF2 foci in ALT. a) In GM00847, FANCM depletion resulted in the appearance of large TRF2 foci. b) In WI38-VA13/2RA, no significant change in the size of TRF2 foci but FANCM depletion was associated with increasing frequency of nuclei with large nuclei that contained large TRF2 foci. c) FANCM depletion was associated with appearance of disorganized TRF2 foci in 4% of the randomly cycling WI38-VA13/2RA. These foci were not detected in FANCM depleted GM00847.

2.3.3.5 Preserved expression of FANCD2 in FANCM depleted WI38VA13/2RA ALT

Knockdown of FANCM did not appear to have a major impact on FANCD2 focus formation

(Figure 2-9). This is opposite to knockdown of other core complex proteins which abolishes

the appearance of FANCD2 foci (Root, unpublished data). Bakker et al, 2009 noted a

reduction in FANCD2 foci formation in their murine models, but FANCM in these mice was

completely knocked-out.

GL2 siRNA FANCM siRNA

FANCD2 FANCD2

Figure 2-9: Preserved expression of FANCD2 in FANCM depleted WI38VA13/2RA ALT. No significant reduction in FANCD2 signal was apparent in WI38-VA13/2RA ALT cells after FANCM knockdown.

2.3.3.6 FANCM knockdown result in increased amount of telomeric repeat DNA

Transient siRNA knockdown of FANCM in WI38VA13/2RA and GM00847 ALT cells resulted in a significant increase in the nuclear telomeric repeat DNA. This difference can be readily appreciated under the 20X magnification (Figure 2-10). In telomerase –positive

HT1080, FANCM knockdown was not associated with noticeable changes in amount of telomeric repeat DNA.

The increase in the telomeric repeat DNA is more prominent at 63X magnification (Figure

2-11). About 50% of randomly cycling ALT cells depleted of FANCM had significantly increased amount of telomeric repeat DNA when compared to the cells with the control GL2 knockdown. There were rare nuclei from the FANCM knockdown group in which no telomeric signal was detected.

GL2 siRNA FANCM siRNA

GM00847 ALT

WI38VA13/2RA ALT

HT1080 telomerase- positive

Figure 2-10: FANCM depletion causes increased synthesis of telomeric DNA in ALT but not in telomerase positive cells. Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) in interphase nuclei of GM00847 and WI38VA13/2RA ALT cells and HT1080 telomerase-positive cells at 20X magnification. There is significant increase in telomeric DNA in ALT cells at 96 hours after first FANCM knockdown compared to cells transfected with GL2 control siRNA. FANCM knockdown in telomerase-positive HT1080 was not associated with increase in telomeric DNA signal intensity. Note also the increase in the size of ALT nuclei after FANCM knockdown.

GL2 siRNA FANCM siRNA

GM00847

WI38VA13/2RA

Figure 2-11: FANCM depletion in ALT causes significant increase in telomeric DNA synthesis. Interphase nuclei from GM00847 and WI38VA13/2RA 96 hours after GL2 and FANCM siRNA knockdown at 63X magnification showing that FANCM knockdown causes significant increase in the PNA signal. All images were taken at same exposure time. Vertical streaking in FANCM siRNA images is due to the extreme intensity of the PNA telomeric signal exceeding the capacity of the CCD camera.

CHAPTER THREE

Discussion and Future Direction

3.1 Discussion

3.1.1 Validation of the immunofluorescence staining of FANCM

Previous characterizations of FANCM expression in the literature were all done through

Western blotting (WB) and immunoprecipitation (IP) and no published study reported the use of IF to study and characterize FANCM protein. My results show that FANCM protein does form visible nuclear foci in two ALT cell lines, WI38-VA13/2RA and GM00847. FANCM foci were also detectable by detergent extraction IF in telomerase-positive fibroblasts,

GM00639 and HT1080. However, FANCM foci seen in telomerase-positive cells were smaller than those in ALT (see section 2.3.1.2).

The finding that two different antibodies against FANCM formed similar, highly colocalizing foci supports that the foci formed by these antibodies are staining the FANCM protein (see section 2.3.4.1). The other finding that supports the specificity of the FANCM antibody is the fact that FANCM depletion using a published siRNA for FANCM resulted in a clear reduction in nuclear foci detected by the ab26272 FANCM antibody (see section 2.3.3.2).

The findings that FANCM foci in ALT were easily detectable, had significant colocalization with FANCD2, similar FANCM foci are formed by two different antibodies targeting different domains of FANCM protein and the reduction of FANCM signal upon FANCM depletion are all suggestive that the FANCM foci formed in ALT cells are a real representation of FANCM protein expression.

3.1.2 FANCM is required for ALT telomere maintenance

Many FANCM foci in ALT cells colocalize with TRF1 and TRF2 telomeric foci. There is some difference in the extent of colocalization to telomeric foci in the two ALT cell lines, with a greater tendency in WI38-VA13/2RA; mean 72%, compared to GM00847, mean 57%.

In about a quarter of WI38VA13/2RA nuclei that expressed FANCM, colocalization with

TRF1/TRF2 foci was 100% meaning that FANCM foci were only visible at telomeres.

FANCM foci in WI38VA13/2RA were mainly seen in large nuclei that have large telomeric

TRF1/TRF2 foci. Larger nuclei had greater telomeric localization of FANCM than smaller nuclei. In GM00847, there were more small nuclei expressing FANCM (see section 2.3.1.1 and figures 2-1a and 2-1b). When these were excluded, FANCM expressed in the larger nuclei has a strong tendency to be colocalized to telomeres similar to WI38VA13/2RA.

The finding that FANCM foci were localized to telomeres in the telomerase positive HT1080 and GM00639 was novel. This indicates that, unlike other FA proteins studied (Root, unpublished data), FANCM associates with telomeres in telomere positive cells as well as

ALT cells. The difference in the amount of colocalization (72% and 57% in WI38-

VA13/2RA and GM00847 to 28% and 31% in HT1080 and GM00639) may be related to any additional roles FANCM may have at the telomeres of ALT cells.

The colocalization of FANCM and FANCD2 foci in ALT nuclei is not surprising, as this was seen with several members of the FA core complex proteins (Root, unpublished data).

FANCM colocalization with PML bodies is in keeping with the previously reported association of other DNA repair and recombination proteins with ALT telomeres that make

up the APBs (see section 1.1.7). This high level of colocalization may be an indication that

FANCM is directly involved in maintaining the integrity of ALT telomeres.

The fact that FANCM siRNA knockdown affected cell growth and cell death in both ALT and telomerase positive cells may be a reflection of an important role(s) FANCM plays in

DNA metabolism. Absence or depletion of FANCM can be speculated to affect cell growth and survival through failure to resolve stalled replication intermediates, achieve or regulate appropriate homologous recombination (HR) or through effects on disturbance of cell cycle regulation through inhibition of ATR/Chk1 and related accumulation of DNA damage (see sections 1.3.2-1.3.4). However, the concurrent observation that FANCM depleted cells had larger and -sometimes- irregular telomeric foci suggests that disruption of telomere maintenance may be, in part, responsible for the effect of FANCM depletion on cell growth and viability.

I expected to see a reduction in signal intensity of FANCD2 foci in ALT after FANCM knockdown. This is based on the previous observation by Bakker et al, 2009 of partial preservation of FANCD2 foci formation in their FANCM knockout murine model. FANCM knockdown in WI38-VA13/2RA did not result in visible reduction in FANCD2 signal (see section 2. 3.3.5). It might be possible that total knockout of FANCM is needed to have a visible reduction of FANCD2 foci formation. Alternatively, FANCM is not required for

FANCD2 foci formation.

The increase in the amount of telomeric DNA is consistent with data from our lab on

FANCD2 knockdown in ALT. Root, in the Meyn Lab, had shown that FANCD2 knockdown resulted in increase in the amount of telomeric DNA of ALT cells. This is contradictory to

the findings published by Fan et al 2009 who reported loss of detectable telomeres in ALT cells but not in telomerase-expressing cells after FANCD2 or FANCA knockdown. Absence of detectable telomeric signal was observed in a rare population (about 1:500) of FANCM depleted ALT cells, but the major phenotype was the large increase in telomeric signal intensity in the two ALT cell lines, WI38-VA13/2RA and GM00847. Both findings could be explained on the basis of failure to regulate HR, the mechanism thought to be responsible for telomere length maintenance and hence immortality of ALT cells.

Although FANCM foci in HT1080 and GM00639 telomerase positive cells colocalized with telomeric proteins, knockdown of FANCM did not seem to affect the amount of telomeric repeat DNA. This may suggest that the role that FANCM plays in telomere maintenance in telomerase positive cells is different from its role in ALT cells where HR is thought to play an essential role as a TMM.

The finding of disorganized TRF2 foci resulting from FANCM knockdown is important for two reasons. First, it indicates that FANCM might be important for the normal binding of

TRF2 with the telomeric DNA or associated with the protein component of APBs. Second, disturbance of TRF2 might add to the phenotype of FANCM knockdown including the increase in cell death and perhaps the increased amount of telomeric DNA and ECTD. In vitro TRF2 promotes Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities (Poulet et al., 2009). Synergistic effect of FANCM depletion and secondary TRF2 disruption, might lead to the substantial increase in the amount of telomeric repeats and amplification of telomeric DNA. Furthermore, knockdown of TRF2 is known to be associated with telomeric fusions.

In summary my data suggest that FANCM is required for normal telomere maintenance in

ALT cells by the involvement of FANCM in regulation of HR-mediated replication/amplification of telomeric repeat DNA and possibly by regulating TRF2 binding with the telomeres.

3.2 Future Directions

3.2.1 Characterize the phenotype of FANCM depletion in ALT

For validation purposes I would like to duplicate my findings using a different siRNA to knockdown FANCM expression. To determine if the increased amount of telomeric DNA is intra-chromosomal or part of the ECTR seen in ALT, PNA FISH on a metaphase spread should be done. Metaphase FISH will also provide additional information on the effect of

FANCM depletion on chromosomal and nuclear integrity.

The finding of disorganized TRF2 foci appearance upon FANCM depletion in ALT needs to be further characterized. IF to look for similar changes in other proteins present in ALT telomeres like TRF1 and PML in FANCM-depleted ALT should be done. This will help to delineate if the disorganized structure of TRF2 is a specific effect to a possible FANCM interaction with TRF2 or a secondary result of a general effect on the integrity of the APB.

3.2.2 Determine the mechanism for increase telomeric DNA synthesis in FANCM depletion

The next step at this point is to find out the mechanism of the increase in telomeric DNA in

FANCM knockdown. As regulation of HR and thus on the rate of T-SCE is a likely mechanism, assessment of changes in T-SCE associated with FANCM depletion by chromosome orientation-FISH (CO-FISH) should be the next step. Assessment of FANCM colocalization with other proteins that have an established role in HR like RAD51 and BLM is another step to take. In addition, studying the effect of FANCM/BLM double knockdown on ALT telomeres should be performed. Illustration that FANCM/BLM double knockdown abolishes the increase in ECTR will suggest that loss of FANCM regulation on HR and SCE is the likely mechanism.

FANCD2 knockdown in ALT resulted in an increase in telomeric repeat DNA similar to the effect seen in FANCM knockdown (Root, unpublished data). FANCM however was shown to have other functions besides its role in the FA pathway (see sections 1.3.2, 1.3.3 and

1.3.4). Studying the effect of a FANCD2/FANCM double knockdown in ALT will help to know if the effect of FANCM knockdown on telomeric repeat DNA is related to its role in

FA pathway or due to another function that is unique to FANCM.

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