FUNCTION OF PROTEIN RAP1 AND TELOMERIC

TRANSCRIPT IN ANTIGENIC VARIATIION IN

TRYPANOSOMA BRUCEI

VISHAL NANAVATY

Bachelor of Science in Biochemistry, Vocational Biotechnology

Gujarat University

June 2007

Master of Science in Biotechnology

Gujarat University

June 2009

Submitted in partial fulfillment of requirements for the degree

DOCTOR IN PHILOSOPHY IN REGULATORY BIOLOGY

At the

CLEVELAND STATE UNIVERSITY

November 2016

© COPYRIGHT BY VISHAL PARAGBHAI NANAVATY, 2016

We hereby approve this dissertation for

VISHAL P NANAVATY

Candidate for the Doctor of Philosophy in Regulatory Biology degree for the

Department of Biological, Geological and Environmental Sciences

and the CLEVELAND STATE UNIVERSITY

College of Graduate Studies

------Date ------Dr. Bibo Li, CSU- BGES, Major Advisor

------Date ------

Dr. Valentine Börner, CSU-BGES, Advisory Committee Member

------Date ------

Dr. Aaron Severson, CSU-BGES, Advisory Committee Member

------Date ------

Dr. Michelle Longworth, CCF-LRI, CSU-BGES, Advisory Committee Member

------Date ------

Dr. Sailen Barik, CSU-BGES, Internal Examiner

------Date ------Dr. Derek Taylor, CWRU-Pharmacology, External Examiner

Student’s date of Defense: 30th November 2016

DEDICATION

To my wife, Puja Nanavaty and daughter, Geet Nanavaty’s dreams To supreme power of Almighty and my parents

ACKNOWLEDGMENTS

I would like to give my deepest gratefulness to my major advisor and mentor Dr. Bibo Li. She has always supported me and was there as a constant pushing force behind me to push to thorough all the closed doors. I joined her lab in Feb 2010, as a non-English speaker from India. She had vision to see my love for the science and since than she has been tremendous guidance and force to cultivate me as a scientist, an individual thinker and pushed me to think thorough impossible. She has not only seen me growing as a graduate student but also as a husband in 2011 and father in 2013. She has been amazing help for my family and I am obliged to her for all the help she has given me. Thank You so much Dr. Li.

I would also like to acknowledge Dr. Valentine Boerner and Dr. Aaron Severson for tireless guidance throughout the graduate student life. Your suggestions and vision have helped me to push through rough time of the project in the graduate life. You have been toughest critiques at every stage of my PhD, be it in journal club presentations, committee meetings or candidacy examination. Your constant help has developed an essential skill, “of asking questions”, in me. Thank you Dr. Boerner and Dr. Severson.

I would like to thank Dr. Michelle Longworth, for her critiques in the committee meetings. Your questions and suggestions have brought detailed insight in my thesis. Thank you Dr. Longworth.

I would like to thank Dr. Sailen Barik and Dr. Derek Taylor for serving as an internal and external examiner on my thesis.

I would also like to thank, Dr. Crystal Weyman, Dr. Anton Komar, Dr. Girish Shukla, Dr. Barsanjit Mazumder and Dr. Roman Kondratov for their valuable questions during my presentations in departmental and GRHD seminars. Your questions and suggestions have pushed me to think out of the box and look for the answers in the dark. Kudos to BGES department! You have given such a brilliant training and making me a confident science thinker.

I miss Dr. Unnati Pandya, Dr. Sanaa Jehi and Dr. Ranjodh Sandhu for their suggestion in writing this thesis. More than that, thank you so much for lifelong friendship. I wish we all sustained competitive as we were here at Dr. Li’s lab. Besides them, I want to say thank you to Dr. Xiafong Yang, Dr. Imaan Benmerzouga, Nicole Kresak, Miao wang, Fan wu, and Grace wang to make working environment exceptional. Thank you all current Li lab members.

I certainly want to thank Dr. Sujata Jha and Dr. Arnab Gosh from Dr. Anton Komar’s Lab, for their help and motivation. I would also like to thank Jagjit Singh from Dr. Shukla’s lab and Rima Sandhu from Dr, Boerner’s lab for scientific and non-scientific discussions.

Survival in the Cleveland’s harsh weather would have been almost impossible without selfless love and friendship of Maulik Gajjar, Mona Patel, Ritesh Patel and Vatsal Patel. Whenever there is a need to find a family required, you guys were there as a family. Thank you all for all these wonderful years and many more to come.

Words cannot describe the happiness of my parents, Parag Nanavaty (Father) and Meena Nanavaty (mother), to see this thesis. I would like to thank for their unconditional love, support and trust in me. I cannot describe the proud feeling of my parent-in-law Dr. Sarvang Desai (an Orthopdeic surgeon, himself) and Toral desai (Mother). Their blessings and guidance have been always there to push me forward. I also thank my siblings Krishna and her husband Malav, Tosha and Haresh and their son Aarav and Raam, and Samarth for giving me patience and hope.

Lastly, I would like to thank my wife PUJA NANAVATY for understanding the most when it requires in the PhD life. Your love has always pushed me to break the boundaries. I started believing in myself more only after you enter in my life back in our master studies. I would never be able to this thesis without your divine support. Thank you for staying strong in ups and downs of this difficult journey. Thank you for blessing us with GEET, our daughter.

FUNCTION OF TELOMERE PROTEIN RAP1 AND TELOMERIC

TRANSCRIPT IN ANTIGENIC VARIATIION IN TRYPANOSOMA BRUCEI

VISHAL NANAVATY

ABSTRACT

Trypanosoma brucei is a parasite that causes fatal African sleeping sickness. Antigenic variation is an obligatory mechanism for long-term survival of

T. brucei inside its mammalian host. T. brucei expresses VSG as its major surface antigen and regularly switches its VSG coat to evade the host immune response.

Although T. brucei genome has more than 2,500 VSG and pseudogenes, it only expresses one VSG from one of 15 subtelomeric VSG expression sites (ESs) at any time. VSG switching can be transcriptional (in situ switching) or be mediated by DNA homologous recombination (such as conversion and reciprocal DNA crossover/telomere exchange). However, regulation of VSG switching is poorly understood. We previously found that T. brucei RAP1, a telomere protein, is essential for silencing subtelomeric VSG genes. Here we found that transient depletion of TbRAP1 increases the VSG switching frequency, and most switchers in TbRAP1-depleted cells arose thorough VSG-associated gene conversion events. Also, we detected increased amount of DSBs in the active and silent ESs upon TbRAP1 depletion. However, the underlying mechanisms of how TbRAP1 suppresses DSBs at /subtelomere remained unknown.

vii

T. brucei telomeres are transcribed, generating long, non-coding RNA called TERRA (Telomeric repeat-containing RNA). Now, we found that depletion of TbRAP1 not only leads to derepression of telomeric silent VSGs, but also results in increased TERRA levels. In addition, we observed a sixteen-fold increase in telomeric RNA:DNA hybrids (R-loops) in TbRAP1-depleted cells. R-loop has been shown to induce DSBs in yeast, mouse, and human cells and are sensitive to

RNaseH, a ribonuclease that cleaves the RNA strand of the RNA:DNA hybrid.

Ectopic expression of TbRNaseH1 in TbRAP1 RNAi cells resulted in reduction of the telomeric R-loop levels and reduced the DSB amount back to the WT level and suppressed the elevated VSG switching frequency phenotype. Therefore, we propose that increased TERRA and R-loop levels in TbRAP1-depleted cells mediate DSB-induced VSG gene conversion, resulting in increases VSG switching frequency. We have also identified that read-through into the telomere repeats from the adjacent active ES (but not from silent ESs) contributes to TERRA synthesis.

viii

TABLE OF CONTENTS

ABSTRACT ...... vii

LIST OF TABLES ...... xiv

LIST OF FIGURES ...... xv

CHAPTERS

I. INTRODUCTION ...... 1

1.1 African trypanosomiasis ...... 1

1.1.1 Epidemiology ...... 1

1.1.2 Symptoms ...... 4

1.1.3 Diagnosis ...... 5

1.1.4 Treatment ...... 6

1.2 Life cycle of T. brucei ...... 7

1.3 T. brucei genome ...... 9

1.4 Organization of T. brucei genes ...... 11

1.5 Surface coat protein and regulation of its expression in T. brucei ...... 13

1.6 Antigenic variation in T. brucei ...... 16

1.6.1 Monoallelic expression of VSG and regulators of the VSG expression

...... 17

1.6.2 VSG Switching mechanisms and regulators of VSG switching in T.

brucei ...... 20

1.7 Telomeres and telomere protein complexes in several eukaryotic organisms

...... 24

ix

1.8 Telomere and telomere protein complexes in T. brucei ...... 29

1.9 Telomere transcription and RNA:DNA hybrids ...... 33

1.10 Significance of this study ...... 36

II. MATERIALS AND METHODS ...... 38

2.1 Plasmids ...... 38

2.1.1 TbRAP1 RNAi construct – p2T7-TABlue-0760-RNAi ...... 38

2.1.2 Ectopic TbRAP1 expression construct – pLEW111-phleo-F2H-0760-

FL ...... 38

2.1.3 Ectopic TbRNaseH1 expression construct –pLew100V5-phleo-

TbRNaseH1-2HA ...... 39

2.2 Trypanosoma brucei strains ...... 39

2.2.1 The parent strain for VSG switching assays (S) ...... 39

2.2.2 The control strain with an empty RNAi vector for VSG switching

assays (S/EV) ...... 40

2.2.3 The TbRAP1 RNAi strain for VSG switching assays (S/RAP1i) ..... 40

2.2.4 TbRAP1 RNAi cells with ectopic expression of TbRAP1 (S/RAP1i +

F2H-TbRAP1) ...... 40

2.2.5 S/EV with ectopic expression of TbRNaseH1 (S/EV + TbRNaseH1-

2HA) ...... 40

2.2.6 TbRAP1 RNAi strain with ectopic expression of TbRNaseH1

(S/RAP1i + TbRNaseH1-2HA)...... 41

2.2.7 FLAG-HA-HA tagged TbRAP1 in WT427 procyclic form cells ...... 41

2.3 Growth curve analysis ...... 41

x

2.4 Western blotting ...... 42

2.5 VSG Switching Assay ...... 43

2.6 VSG Cloning ...... 44

2.7 Pulse field gel electrophoresis (PFGE) ...... 44

2.8 Southern blotting ...... 45

2.9 R-loop assay ...... 46

2.10 Isolation of RNA for TERRA analysis ...... 46

2.11 TERRA - Northern blots/ slot blots ...... 47

2.12 PCR of TERRA origin ...... 47

2.13 Chromatin immunoprecipitation (CHIP) ...... 49

2.14 Ligation mediated PCR assay (LMPCR) ...... 50

2.15 DNA isolation using DNAzol for PCR screening of switchers ...... 52

2.16 dot-blot western blot for false positive clone removal ...... 52

2.17 Isolation of genomic DNA (large quantity) ...... 53

2.18 Reverse transcription PCR for checking derepression of VSGs ...... 53

2.19 Preparation of probes for southern blot and northern blot ...... 55

2.20 Co-immunoprecipitation ...... 55

III. CHARACTERIZATION OF TbRAP1 FUNCTIONS IN VSG SWITCHING .... 57

3.1 Introduction ...... 57

3.2 Results ...... 65

3.2.1 Generation of strains to study the roles of TbRAP1 in VSG

switching ...... 65

xi

3.2.2 TbRAP1 suppresses VSG switching frequency ...... 71

3.2.3 TbRAP1 suppresses VSG gene conversion ...... 74

3.2.4 TbRAP1 is essential for maintaining subtelomere and telomere

integrity ...... 82

3.3 Discussion ...... 93

IV. THE TELOMERE TRANSCRIPT (TERRA) AND ANTIGENIC VARIATION . 99

4.1 Introduction ...... 99

4.2 Results ...... 106

4.2.1 Detection of telomeric transcript in wild type bloodstream form and

procyclic form T. brucei cells ...... 106

4.2.2 Identification of the origin of TERRA transcription in T. brucei ..... 111

4.2.3 Regulation of TERRA expression by T. brucei telomere proteins . 118

4.2.4 Increased TERRA levels results in more RNA:DNA hybrids (R-loops)

at the telomeres, in TbRAP1 deletion condition ...... 129

4.2.5 Identification of RNAseH1 gene and generation of ectopic expressing

TbRNaseH1 strain ...... 132

4.2.6 Ectopic expression of TbRNaseH1 reduces telomeric R-loop levels

and partially suppresses the elevated TERRA levels in TbRAP1

depleted cells ...... 137

4.2.7 Consequence of ectopic expression of TbRNaseH1 in suppression of

subtelomeric DNA DSBs...... 142

4.3 Discussion ...... 150

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V. TbRAP1 INTERACTING CANDIDATES ...... 159

5.1 Introduction ...... 159

5.2 Results and Discussion ...... 161

5.2.1 Large-scale immunoprecipitation and mass-spectrometry analysis of

TbRAP1 protein complex ...... 161

VI. FUTURE PERSEPECTIVES ...... 169

6.1 TbRAP1’s role in antigenic variation ...... 169

6.2 Role of TERRA in wild type cells ...... 172

BIBLIOGRAPHY ...... 177

APPENDICES ...... 212

APPENDIX – A ...... 214

APPENDIX – B ...... 215

APPENDIX – C ...... 216

APPENDIX – D ...... 218

APPENDIX – E ...... 219

APPENDIX – F ...... 222

APPENDIX – G ...... 229

APPENDIX – H ...... 230

xiii

LIST OF TABLES

1. Drugs used for the treatment of human African Trypanosomiasis ...... 6

2. List of primers used for TERRA’s origin experiment ...... 48

3. List of primers used for the ChIP-qPCR experiment ...... 50

4. List of primers used for LMPCR experiments ...... 51

5. List of primers used for the RT PCR experiment ...... 54

6. Phenotype and genotype characterization for switcher from S +Dox cells

...... 214

7. Phenotype and genotype characterization for switcher from S/ev + Dox

cells...... 215

8. Phenotype and genotype characterization for switcher from S/RAP1i -

Dox cells ...... 216

9. Phenotype and genotype characterization for switcher from

S/RAP1i+F2H-TbRAP1 + Dox cells ...... 218

10. Phenotype and genotype characterization for switcher from S/RAP1i

clone B1 + Dox cells ...... 219

11. Phenotype and genotype characterization for switcher from S/RAP1i

clone C1 + Dox cells ...... 222

12. Phenotype and genotype characterization for switcher from S/RAP1i +

TbRNaseH1-2HA cells ...... 229

13. Phenotype and genotype characterization for switcher from S/ev +

TbRNaseH1-2HA cells ...... 230

xiv

LIST OF FIGURES

1.1 Tse-Tse fly, an insect vector for T. brucei and blood smear of the patient

suffering with African trypanosomiasis...... 2

1.2 Life cycle of T. brucei...... 8

1.3 Genome organization in T. brucei ...... 10

1.4 T. brucei surface expression sites in different forms of T. brucei..... 14

1.5 Electron microscope picture of Variant surface glycoproteins (VSGs). .. 15

1.6 VSG switching pathways, principles ...... 21

1.7 Telomere protein complex in different organisms...... 25

3.1 Generation of the HSTB261/TbRAP1 RNAi strain ...... 67

3.2 Western blot analysis of TbRAP1 protein ...... 68

3.3 Establishing the TbRAP1 complementation strain ...... 70

3.4 A transient depletion of TbRAP1 increases VSG switching frequency .. 73

3.5 Different VSG switching mechanisms ...... 75

3.6 TbRAP1 suppresses VSG gene conversion ...... 78

3.7 Validating selected switchers by PFGE...... 81

3.8 Depletion of TbRAP1 increased the number of DSBs in and downstream

of subtelomeric VSG genes...... 84

3.9 Depletion of TbRAP1 led to increased number of DSBs in and downstream

of subtelomeric VSG genes ...... 86

3.10 Characterization of antibody specifically recognize the phosphorylated

and unphosphorylated H2A by western blotting...... 88

3.11 TbRAP1 depletion leads to more DSBs at telomeres ...... 89

xv

3.12 TbRAP1 depletion increases the amount of DSBs at the subtelomeric VSG

loci...... 92

4.1 Northern analysis to detect TERRA in wild type bloodstream form

cells ...... 108

4.2 Northern analysis to detect TERRA in wild type procyclic form cells ... 110

4.3 TERRA can be transcribed from the active ES-adjacent telomere ...... 113

4.4 Transcription of TERRA from the active VSG9-expressing

subtelomere ...... 115

4.5 Transcription of TERRA from the active VSG3-expressing

subtelomere ...... 117

4.6 TERRA expression in TbRAP1-depleted cells ...... 120

4.7 TERRA expression in TbTRF RNAi condition ...... 122

4.8 TERRA levels increase upon TbRAP1 depletion...... 124

4.9 Depletion of TbTRF rsults in an increase in TERRA levels ...... 125

4.10 Transcription of TERRA in the presence and absence of TbRAP1 .... 127

4.11 Depletion of TbRAP1 leads to increased amount of telomeric RNA:DNA

hybrid ...... 130

4.12 Protein sequence alignment using ClustalX for RNaseH1

homologues...... 133

4.13 Ectopic expression of TbRNaseH1 delays TbRAP1 RNAi growth

phenotypes...... 135

4.14 Ectopic expression of TbRNAseH1 does not affect the VSG derepression

phenotype upon TbRAP1 depletion ...... 136

xvi

4.15 Ectopic expression of TbRNaseH1 in TbRAP1 RNAi strain suppresses

the amount of telomeric RNA:DNA hybrid ...... 138

4.16 Changes in TERRA levels upon ectopic expression of TbRNAseH1 in

TbRAP1 depletion cells...... 140

4.17 TbRAP1 depletion leads to an increase in DSBs at

telomeres/subtelomeres, but the ectopic expression of TbRNaseH1 in the

TbRAP1 RNAi background suppresses the amount of DSBs at

telomeres/subtelomeres...... 143

4.18 TbRAP1 depletion increased DSBs at the subtelomeric VSG loci but

ectopic expression of TbRNAseH1 suppresses this phenotype ...... 145

4.19 A transient depletion of TbRAP1 increased VSG switching

frequency ...... 146

4.20 Ectopic expression of TbRNaseH1 in TbRAP1 RNAi strain reverted

TbRAP1 RNAi switching pattern to that in wild type cells ...... 148

4.21 A working model of the function of TbRAP1 linking TERRA to DSB-

associated VSG switching...... 158

5.1 Western analysis of immunoprecipitation of the TbRAP1 protein

complex ...... 162

5.2 Western analysis of protein samples collected from 20% - 60% sucrose

gradient...... 163

5.3 Coommassie-blue staining of TbRAP1 IPed protein complex ...... 164

5.4 Western analysis of CO-IP products done in cells expressing Ty1-

TbNOT1...... 166

xvii

CHAPTER I

INTRODUCTION

1.1 African trypanosomiasis

1.1.1 Epidemiology

Trypanosomes are obligate parasites belonging to class Kinetoplastida and genus Trypanosoma, among them Trypanosoma brucei (T. brucei) is a unicellular and flagellated parasite that lives and multiplies in the extracellular spaces such as in the bloodstream and tissue fluids of its mammalian hosts (Fig. 1-1B).

Recently, T. brucei have also been found in the adipose tissues and beneath the skin tissue (Trindade et al., 2016). Most of the trypanosomes are heteroxenous, and it need multiple hosts to complete their life cycle (Yao, 2010). T. brucei is highly pathogenic and infect humans as well as mammals. Infection of humans by T. brucei results in human African trypanosomiasis (HAT) or ‘sleeping sickness’ while infection in animals results in animal African trypanosomiasis (AAT) or ‘Nagana’.

T. brucei is transmitted by the bite of a blood sucking fly known as Tse-Tse

(Glossina spp.) that acts as the insect vector (Fig. 1-1A). The sub-Sahara region

1 of Africa that is affected by trypanosomiasis corresponds to the living range of Tse-

Tse (Simarro et al., 2012).

Figure 1-1: Tse-Tse fly, an insect vector for T. brucei and blood smear of the patient suffering with African trypanosomiasis. (A) Tse-Tse fly (Picture taken by Anthony Bannister, Encyclopedia Britanica) (B) Giemsa stain of blood-smear of the patient suffering from sleeping sickness. (Picture taken from www.cdc.gov)

T. congolense, T. evansi, T. vivax and T. brucei spp. are the major causative agents of nagana. In domestic animals, nagana is often fatal and is one of the most economically important livestock diseases in Africa. Nagana has a devastating impact on the rural areas. Approximately 50 million cattle are threatened by animal African trypanosomiasis (Holmes, 2013). It has been predicted that more than $4 billion are wasted by animal African trypanosomiasis

(Holmes, 2013). Severe outbreaks of this disease in cattle lead to low productivity and stunted the economic development of these regions. Moreover, domestic livestock acts as a reservoir of parasites for human infections (Shaw, 2004). HAT is caused by two subspecies of T. brucei, T. brucei gambiense and T. brucei rhodesiense. HAT is widespread and covers an area of approximately 1.55 million km2 in Africa (Simarro et al., 2012). If untreated, HAT is inevitably fatal. T. b.

2 gambiense is the causative agent of chronic and more prevalent form (97% of the cases) of HAT in Western and Central Africa, while T. b. rhodesiense causes a rare (3% of the cases), but acute, and more severe infection in Eastern and

Southern regions of Africa (Simarro et al., 2008). If left untreated, patients infected with T. b. rhodesiense die within months after infection while patients with T. b. gambiense infection die within few years of infection (Pepin, 2010). Trypanosome species causing AAT are unable to infect humans due to the presence of trypanosome lytic factors (TLFs) in human serum (Capewell et al., 2011). T. b. rhodesiense that causes severe form of the HAT expresses a serum resistance protein SRA (Van Xong et al., 1998). Instead, T. b. gambiense reduces the expression of a receptor that is involved in uptake of the lytic factor in trypanosome cells (Kieft et al., 2010). T. b. brucei is very closely related to T. b. rhodesiense and

T. b. gambiense, but it is sensitive to the human lytic factors. This makes T. b. brucei a very useful model organism for molecular studies in research labs.

The number of HAT cases has declined in recent years. The latest epidemic of HAT was reported in 1970s and it lasted till the late 1990s (Berrang-Ford et al.,

2006). The rise of HAT has been finally decreased due to better treatment and more efficient control of the insect vector. In 2009, World Health Organization

(WHO) reported less than 10,000 cases of HAT. This decline in the HAT case number of cases has continued as of 2012, only 7,216 cases were reported by

WHO. However, this is just a fraction of the actual number of cases, as many are not recognized or reported due to the incomplete screening (Chappuis et al.,

3

2010). According to WHO, the actual number of cases is approximately 30,000 per year.

1.1.2 Symptoms:

Symptoms of HAT are divided into two stages: early stage and late stage.

These stages are mostly difficult to differentiate as these often merge with each other. The early stage is called hemolymphatic stage. The onset of this disease is variable and it usually takes 1 to 3 weeks post bite from an infected Tse-Tse fly for symptoms to appear. A painful swelling called “Chancres” appears at the site of the bite. In the early stage of HAT, trypanosomes are limited to the blood and lymphatic system. Main symptoms of the early hemolymphatic stage are episodes of fever that last for 1 to 7 days, lymphadenopathy, malaise, headache, weight loss, and painful joints, etc. Patients may also develop clinical features such as enlarged spleen and liver, cardiovascular, endocrine and ophthalmic abnormalities, etc. (Kennedy, 2004) (Kennedy, 2013). The late stage of the HAT is called the encephalitic stage, in which, trypanosomes are detectable in cerebrospinal fluid. In this stage, trypanosomes cross the blood brain barrier and enter the central nervous system. A wide variety of symptoms occur at this stage that include various psychiatric, reflex, and sleep abnormalities. Other common features of this stage are meningoencephalitis, tremors, and speech abnormality

(Blum et al., 2006) (MacLean et al., 2012). Almost all parts of the nervous system are affected during the late stage. Without proper treatment, patient’s condition worsens and symptoms such as seizures, cerebral edema, systemic organ failure,

4 and severe somnolence appear, which is followed by an inevitable death (Bouteille and Buguet, 2012).

1.1.3 Diagnosis:

Current scenario of the diagnosis is solely dependent on the early signs of fever and headaches in areas where endemic T. brucei infections persist.

However, it is also important to distinguish it from other diseases that show similar symptoms. Subsequently, it is essential to establish the strain responsible for the disease as it can be acute or chronic. Diagnosis of the species and stage decides the course of treatment. Detection of the parasitemia by simple blood smear based detection is the widely accepted and common method for diagnosis of T. brucei infection. However, if the infection is due to the presence of T. gambiense, it is hard to detect parasitemia thorough blood smear analysis as the number of parasitemia tends to be low. On the contrary, if the infection is due to T. rhodesiense it is fairly easy and quick to detect parasitemia. To overcome these difficulties in parasite detection, antibody based (against anti surface Tat1.3) filter associated method called Card Agglutination Trypanosomiasis Test (CATT) is developed (Truc et al, 2002.). Another diagnostic tool used to detect the early stage trypanosome infection is through PCR. PCR is a highly sensitive and specific technique to diagnose the strains responsible for the disease. In spite of high efficiency, advanced techniques such as PCR are not available in the field due to application limitations (Mugasa et al., 2012) (Kennedy, 2004).

5

Table 1: Drugs used for the treatment of human African Trypanosomiasis. (Table taken from Kennedy, 2013, IM = intramuscular and IV =intravenous)

1.1.4 Treatment:

Identification of the correct stage of the disease is essential because treating patients with early stage of sleeping sickness with drugs that are only effective at the later stage can increase the complication further. Patients at the late stage of the infection also require to be treated the drugs that are effective in the CNS system as well. Four drugs have been identified and approved so far (Table-1).

Suramin and Pentamidine are the first line treatment for the early stage infection.

For the late stage infection, Melarsorol and Eflornithine with Nifurtimox are available. They are given either intravenously or intramuscularly (Kennedy, 2013).

All these compounds lead to multiple side effects such as hypoglycemia/hyperglycemia, hypertension, gastrointestinal effects, renal failure, eye sight lost, bone marrow complications, white blood cell lost, peripheral neuropathy, and cardiac arrhythmia, just to name a few (Franco et al., 2012,

6

Kennedy, 2004, Barrett et al., 2007, Willert and Phillips, 2012). Even after treating with these drugs, clearance of the parasite is not 100%. Current research is focused on kinetoplast and glycosome associated metabolism. However, results from these new targets are far from market available.

1.2 Life cycle of T. brucei

Since T. brucei is a heteroxenous organism, it requires different hosts to complete its life cycle: a vector (Tse-Tse fly) and a mammalian host. Beginning of the life cycle of T. brucei is inside the midgut of the Tse-Tse fly (Fig.1-2). Here they are known as procyclic form. It is a proliferative stage. After the parasite migrates into the salivary gland of the Tse-Tse fly, it differentiates into the quiescent metacyclic form and they acquire infectivity. Upon biting a mammalian host, the parasite migrates into the bloodstream of the mammalian host and differentiates into bloodstream form. The long slender bloodstream form T. brucei is proliferative.

Once the number of slender bloodstream form is increased to a certain extent, T. brucei metamorphoses to the short stumpy bloodstream form (Matthews et al.,

2005), which is non-proliferative. This change is beneficial for the parasite for two reasons. First, stumpy form is non-proliferative, which helps prolong host survival time and is beneficial for T. brucei to build a reservoir. Second, stumpy form cells are arrested in the G1 phase of the cell cycle, which allows them to efficiently differentiate into procyclic form once they are picked up by a Tse-Tse fly again

(Vickerman, 1985, Matthews 2005).

7

Figure 1-2: Life cycle of T. brucei. A schematic representation of life cycle of T. brucei between two hosts. Life cycle begins in the midgut of the fly with Procyclic form and later goes to the salivary gland of the vector a stage is known as metacyclic form. In the mammals it is called bloodstream form and after reaching certain population it converts in to the stumpy form. (Dreesen et al., 2007).

There are several key differences between the procyclic, metacyclic, and bloodstream forms of the T. brucei. One major difference is the expression of their surface coat protein during the different forms of life cycle. In the midgut of the

Tse-Tse fly, procyclic form T. brucei expresses procyclins as a major surface protein. In the salivary gland of the Tse-Tse fly, metacyclic form T. brucei expresses metacyclic variant surface glycoprotein (mVSGs) on its cell surface.

8

While bloodstream form T. brucei expresses bloodstream form variant surface glycoprotein (VSG) as the major surface antigen (Vickerman, 1969, Cross, 1975

& 1977, Roditii et al., 1989 & 1998). The change of surface coat allows T. brucei to survive in various environments. Surface coat proteins’ expression and functions are discussed in separate section (1.5) below.

1.3 T. brucei genome

T. brucei has a genome that is mostly diploid, containing a mitochondrial

(kinetoplast DNA, kDNA) and a nuclear genome. The nuclear genome of T. brucei is linear and divided into three types of chromosomes based on their sizes (Fig. 1-

3). There are eleven pairs of the megabase chromosomes ranging 0.9-5.7 megabase (Mb) in size, 2-4 intermediate chromosomes ranging from 300-900 kb, and around 100 minichromosomes about 50-100 kb (Melville et al., 1998,

Wickstead et al., 2004 and Berriman et al., 2005). All these chromosomes carry telomeres at their ends, thus there are ~250 telomeres in the nucleus. The majority of the coding DNA is distributed in the megabase chromosomes and they are unidentical homologous pairs (Berriman et al., 2005). Heterogeneity in size of homologous chromosomes is largely due to differences in the subtelomeric regions. These differences are attributed to large gene conversions including subtelomeric regions consisting of VSG gene arrays (Callejas et al., 2006).

Sequencing of 11 megabase chromosomes suggests that there are 9,000 genes possibly expressing in the form of proteins and among them more than 2,500 genes and pseudogenes are VSGs (Cross et al., 2014).

9

Figure 1-3: Genome organization in T. brucei. There are three types of chromosomes present in the T. brucei genome. There are about 11 megabase chromosomes encodes for all the essential genes in the form of polycistronic transcription units. 2-4 intermediate chromosomes which potentially contains ES in subtelomeric regions. Hypothetical representation of the intermediate chromosome is drawn due to lack of complete sequence and exact repetition of 177 bp makes it harder to sequence. About 33% of the minichromosomes contains VSG genes and it is mostly made up of 177 bp repeats. (figure is adapted from Akiyoshi and Gull, 2013).

The intermediate chromosomes are still somewhat mysterious as their polyploidies are completely unclear. The intermediate chromosomes carry a core of non-repetitive sequences and 177 bp repeats at their subtelomeric regions (Fig.

1-3). Some of the intermediate chromosomes harbor VSG expression site (ES) at their subtelomeres but no housekeeping genes (Van der Ploeg et al., 1984,

Wickstead et al., 2004). Minichromosomes are hypothesized to be a reservoir of

VSG genes. Each minichromosomes is made up of 177 bp repeats of unknown function at its core and about one third of minichromosomes ends contain a VSG

10 gene or gene arrays (Borst, 1986, Gilbson and Borst, 1986, Weiden et al., 1991,

Alsford et al., 2001, Wickstead et al., 2004 & Cross et al., 2014).

1.4 Organization of T. brucei genes

Nearly all the genes in T. brucei are arranged in the polycistronic transcription units, which is often observed in prokaryotes. Large arrays of genes are transcribed from a single and are matured in an unusual way

(Tschudi and Ullu, 1988, Daniels et al., 2010). The same polycistronic transcription unit often does not include genes with the related functions. Recent data from

RNA-seq and ChIP-seq experiments combined with whole genome sequence data suggest that the genome of T. brucei (excluding subtelomeric regions) is arranged into approximately 150 polycistronic units transcribed by RNA II (Kolev et al., 2010, Siegel et al., 2009, Daniels et al., 2010). These polycistronic transcription units (PTUs) have an average size of 153 kb and contain about 55 genes each PTU (Daniels et al., 2010).

Genes within a polycistronic unit that are transcribed from a DNA strand and the region that separates two neighboring polycistronic units is called strand switch region (SSR). SSRs can be divergent or convergent. Transcription initiates at divergent SSRs, thus they are believed to contain the promoter and the transcription start sites. Although recent studies have provided some evidence that chromatin composition is involved in the transcription initiation and termination, much remains unclear (Siegel et al., 2009). As the promoter requirements for RNA pol II dependent transcription are unknown in T. brucei, it is unclear from where

11 transcription begins and terminates (Gunzl et al., 2007) (Martinez-Calvillo et al.,

2010).

After transcription, a polycistronic transcript is processed by the trans- splicing process (Huang and Van der Ploeg, 1991, LeBowitz et al., 1993). A 39-nt long spliced-leader is added to each individual mRNAs at the 5’ end (Parsons et al., 1984). T. brucei transcripts have an unusual cap structure that is called ‘cap 4’

(Perry et al., 1987). ‘Cap 4’ differs from the typical 5’cap of eukaryotic mRNA due to additional four methyl groups that are present at the first four nucleotides of SL

RNA (Bangs et al., 1992). Polyadenylations at the 3’ end of the RNA is coupled with the addition of the spliced leader. Two neighboring genes are also separated by intergenic sequences (LeBowitz et al., 1993).

Despite of having highly conserved copies of all the three RNA , T. brucei has evolved to associate them with parasite specific functions (Palenchar and Bellofatto, 2006). RNA pol II transcribes most of the protein coding genes in the polycistronic manner as discussed above. However, the spliced leader sequences are transcribed monocistronically (Gilinger and

Bellofatto, 2001). RNA pol II transcribes polycistronic clusters of the snoRNA from the megabase chromosomes. RNA pol III transcribes mostly the t-RNAs and the snRNAs with high uridine contents (Fantoni et al., 1994). RNA pol I transcribes pre-rRNAs (28S, 18S and 5.8S) as expected. However, RNA pol I also transcribes

VSG expression sites (ES), the last PTUs on the megabase chromosomes. The

ES contains genes encoding the major surface coat proteins (Gunzl et al.,2003,

Pays, 2005). Details of the transcription and regulation of ESs are discussed below

12

(in section 1.5). In procyclic form of T. brucei, RNA pol I transcribes the procyclin genes either from chromosome 6 or chromosome 10 (Gunzl et al., 2003). Lastly in the metacyclic form, RNA pol I transcribes the metacyclic VSGs in a monocistronically manner, although the metacyclic VSG expression sites are also located at the subtelomeric regions of the megabase chromosomes (Gunzl et al.,

2003, Ginger et al., 2002).

Genome sequence of three Trypanosomatid species, namely T. brucei, T. cruzi, and Leishmania major (also called as tritryps), not only confirmed the unusual organization of the genes in polycistronic transcription units but also revealed a high degree of the synteny among these three Trypanosomatid species

(El-Sayed et al., 2005). Genome organization maps based on this synteny have revealed that the chromosomes can be divided into two units based on the types of gene clusters (El-Sayed et al., 2005). First is the central core of the chromosomes that contains the housekeeping genes and is conserved across the tritryps. Second is the species specific subtelomeric proximal region, and the genes located at subtelomeres are mostly devoted to parasite-host interactions and the evasion of host’s immune system.

1.5 Surface coat protein and regulation of its expression in T. brucei

T. brucei expresses distinctly different cell surface coat proteins at all the different life cycle stages. In the midgut of the Tse-Tse fly it expresses the procyclins as the major surface proteins. In the salivary glands of the vector it expresses the metacyclic VSGs on its surface. Lastly, upon entering the mammalian hosts, it expresses the bloodstream form VSGs on the cell membrane.

13

Parasites survival depends on the expression regulation of these surface proteins as they are essential to mask other non-variable surface proteins (McCulloch,

2004).

Figure 1-4: T. brucei cell surface expression sites in different forms of T. brucei. (A) Subtelomeric bloodstream form VSG expression site is drawn. VSG gene is the last transcribing gene. It is masked by 70 bp repeats and telomere. All other gene in the transcription units are known as ES-associated genes (ESAGs). Transcription of the ES is polycistronic. The mature mRNAs are trans-spliced. Transcription is done by RNA PolI and promoter can be upstream of 40-60 kb. (B) Expression site in the metacyclic form of the VSG. mVSG transcription is done in monocistronically manner by RNA Pol I. Promoter can be 5 kb upstream. (C) Procyclic form expression site for the expression of procyclins. RNA pol I transcribes the internal locus present either at chromosome 6 or chromosome 10. Multiple procyclins can be present at the surface.

In the insect host’s mid-guts, the procyclins are expressed from the chromosomal internal loci and make a little less dense coat compare to the VSG

14 coat in bloodstream form (Fig. 1-4C) (Mowatt and Clayton, 1987). The procyclins are resistant to the proteases present in the insect mid-gut, which allows T. brucei to survive this hostile environment. Four types of the procyclin genes have been identified so far. Three of them expresses EP proteins (glutamic acid (E) – proline

(P) dipeptide repeats) (Roditi et al., 1998). The fourth gene encodes for a penta- called as GPEET proteins. GPEET does not contain any glycosylation site.

Two or more types of the procyclin proteins can be expressed simultaneously on the surface (Mowatt et al., 1989, Roditi et al., 1998).

In the salivary gland of the fly, metacyclic form of the T. brucei expresses the metacyclic VSGs (mVSGs) (Fig.1-4B). Metacyclic VSG expression sites (MESs) are located at the subtelomeric regions of the megabase chromosomes (Lenardo et al., 1984). The MESs are monocistronically transcribed by RNA pol I and the promoter is located approximately 5 kb upstream of the VSG genes (Alarcon et al.,1984, Ginger et al., 2002). Among a metacyclic T. brucei population 15-20 mVSGs can be expressed. However, only one type of mVSG is present on any T. brucei surface (Barry et al., 1998). Variation in the mVSGs expression during the metacyclic stage in a population, increases the infectivity during the first round of the infection.

15

Figure 1-5: Electron microscope picture of Variant surface glycoproteins (VSGs). (Vickerman et al., 1969). 15 nm thick VSG coat can be detected in the cross- section of T. brucei. VSG codes for 60 kDa protein.

In the bloodstream form T. brucei, cell surface is covered by a 15 nm thick coat of VSG consisting of ~10 million molecules (Fig. 1-5) (Cross, 1975). These

VSGs are highly immunogenic, and present on the extracellular surface of the parasite, and are attached to the membrane by a glycosylphosphtidylionositol anchor (GPI anchor) (Ferguson et al., 1988-1999, Schwede and Carrington, 2010,

Horn et al., 2001). Although the T. brucei genome contains more than 2,500 VSG genes and pseudogenes (Cross et al., 2014), only one is expressed at any time.

VSGs are expressed exclusively from the bloodstream form VSG expression

(BES) sites at subtelomeric loci (Fig.1-4A) (De lange and Borst, 1982, El-sayed et al., 200). The ESs are 40-60 kb PTUs, transcribed by RNA polymerase I (Gunzl et l., 2003, Hertz-Fowler et al., 2008). So far, 15 BESs have been identified in the

Lister 427 strain, majorly used in this work. In any T. brucei cell, only one ES is transcribed at any moment while the other ESs are kept silent (Bernards et al.,

1984). VSGs not only are present in subtelomeric ESs but also are found in the long tandem VSG gene arrays at the subtelomeric loci, which can be hemizygous.

The VSG gene arrays lack the promoters and are normally not expressed.

1.6 Antigenic variation in T. brucei

T. brucei can live in multiple different environments in two different hosts, and it is fully exposed to the immune surveillance of its mammalian host. In order to evade the host’s immune response, T. brucei regularly changes its surface coat,

16 which is known as the antigenic variation. The first medical written evidence of the antigenic variation is provided by Ronald Ross, a medical officer, and David

Thomson, a practicing physician, suggested that the regular increase in the parasitemia that overlaps with the variation in body temperature was due to the parasite’s ability to change its surface coat. This constant variation eventually exhausts the host immune defense system leading to the host death. The antigenic variation allows the parasite to continuously survive in the host. The underlying molecular mechanisms responsible for these events have been immense focus of the T. brucei research. However, despite of immense previous efforts, much is still unknown about the antigenic variation and its regulation in T. brucei. The antigenic variation in T. brucei has two important aspects: monoallelic expression of VSGs and VSG switching, which are discussed in details below.

1.6.1 Monoallelic expression of VSG and regulators of the VSG expression

Successful antigenic variation requires three conditions. First, a large pool of genes encoding antigenically distinct surface coats is necessary. Approximately

30% of the T. brucei genome is dedicated to the expression of VSGs (Cross et al.,

2014). There are more than 2,500 VSG genes and pseudogenes in the T. brucei genome and they can recombine to form a new mosaic VSG, making countless number of possible outcomes for new VSGs (Horn, D., 2014). Second, only one

VSG should be expressed at any given time. This is known as monoallelic expression. Third, the parasite should be able to switch from one VSG to another.

T. brucei exploits both transcription and recombination mechanisms to achieve this goal (Li, B. 2010).

17

Among 15 BESs identified in the T. brucei genome, only one ES is fully transcribed and others are not fully transcribed (Fig. 1-4A). Multiple mechanisms have been identified that play roles in regulating monoallelic VSG expression: (1) restricted transcription elongation, (2) a unique sub nuclear locus for VSG transcription, (3) chromatin remodeling, (4) replication machinery and (5) telomeric silencing.

Transcription initiates at multiple ES promoters, but transcription elongation quickly attenuates from other ES, resulting in only one ES being fully transcribed.

This is proposed to happen due to selective recruitment of the RNA elongation/processing machinery (Vanhamme et al., 2000). Another interesting feature observed in T. brucei is the presence of an extranucleolar polymerase I containing focus. It has been determined that only an active ES is associated with this extranucleolar focus called an ES body (ESB) (Navarro and Gull, 2001). An

ESB is required for stable inheritance of the active VSG, thus suggesting its role in the monoallelic expression (Landerira et al., 2009). Many proteins have been reported to suppress formation of multiple ESB inside the nucleus such as

(TbRAP1, TbPIP5 and TbVEX1) (Yang et al., 2009, Cestari, I., 2015, Glover et al.,

2016). However, regulation of the ESB formation is not fully understood.

The chromatin structure has been shown to play an important role in regulating the ES transcription. It was determined that the silent ESs have regularly spaced while the active ES is highly devoid of the nucleosomes

(Figueiredo and cross, 2010, Stanne and Rudenko, 2010). However, it still needs to be determined whether such occupancy in the active ES is a cause

18 or a consequence of the RNA polymerase I transcription. Many chromatin remodeling factors have been identified that affect the ES promoter functions. The depletion of histone H3 (Alsford and Horn, 2012), linker histone H1 (Povelones et al., 2012), and histone chaperones such as FACT subunits (Denninger et al.,

2010), NLP (Narayan et al., 2011) and ASF-1a and CAF-1b (Alsford and Horn,

2012) led to the derepression of the silent ES without affecting the VSG silencing.

Similar effects on the ES promoter were observed upon depletion of the chromatin remodeler ISWI (Stanne et al., 2011). Depletion of the SIR2rp1 (a homologue of yeast Sir2) (Alsford et al., 2007), MYST-family histone methyltransferase DOT1B led to an ~10 fold derepression of the silent VSG suggesting that it is required for monoallelic VSG transcription (Figueiredo et al., 2008).

The DNA replication machinery is suggested to have link with the antigenic variation in T. brucei. The depletion of ORC1 (origin recognition complex) led to 4-

12 fold derepression of the silent VSGs (Benmerzouga et al., 2013). The knockdown of MCM-BP (MCM binding protein, a component of replication complex) also led to 12-30 fold derepression of silent VSGs (Kim et al., 2013).

However, the detailed mechanism of the VSG silencing regulations is unknown.

Finally, and the most importantly, telomeres have been shown to play an important role in maintaining VSG silencing. TbRAP1, a telomeric protein, is known to be an important factor for maintaining the VSG silencing. The depletion of

TbRAP1 led to 10 to 100 fold derepression of all the silent VSGs tested (Yang et al., 2009). Proteins involved in the inositol phosphate pathways such as inositol phosphatase (TbPIP5), when depleted, result in VSG derepression by 100 to 1000

19 folds (Cestari et al., 2015). A genomic RNAi screen has identified TbVEX1 as an important VSG expression regulator - removal or overexpression of theTbVEX1 affects the steady state levels of the silent VSG mRNAs (Horn et al.,2014, Lucy et al., 2016).

1.6.2 VSG switching mechanisms and regulators of VSG switching in T. brucei

In the wild isolates of the bloodstream form T. brucei, the VSG switching rate is calculated to be one switching per 100 to 1000 cells per generation (Turner and Barry, 1989, Turner 1997). The VSG switching events can be broadly divided into two subtypes. The first is transcriptional mediated VSG switching and the second is recombination mediated VSG switching (McCulloch, 2014, Horn and

McCulloch, 2010).

20

Figure 1-6: VSG switching pathways, principles. VSG switching is divided in two major types. Transcription mediated and homologous recombination mediated. In transcription mediated switching, In-situ type of switchers arises. In homologous recombination, Crossover, gene conversion type of switchers arises. Mix of events can be observed as well.

In the transcription mediated VSG switching, in situ switch, an originally silent ES is fully expressed while the originally active ES is silenced without any

DNA rearrangements (Pays et al., 2014). Although there are 15 ES identified in

Lister 427 strain, this type of switching events is rare (Hertz-fowler et al., 2008).

The vast reservoir of VSG genes prompts the question why T. brucei needs to undergo antigenic variation via transcriptional switch. The answer to this came from the discovery of the organization of each ES. The ESs contain a number of

ES-associated genes (ESAG), among which ESAG6 and ESAG7 encodes for proteins involved in protein trafficking and has been hypothesized to play an

21 important function in active VSG expression. Different ESs often have different types of the ESAGs, and it has been proposed that different the ES expression allows the parasite to cope with the different types of host (Bitter et l., 1998, Liam et al., 2009).

Recombination mediated VSG switching involves two major pathways: gene conversion and reciprocal exchange of the genetic information (crossover).

In a gene conversion event the originally active VSG is lost and a silent VSG is duplicated into the active BES. The donor in gene conversion can be any functional

VSG from the genome. The duplicated sequence normally extends beyond the

VSG gene per se. Therefore, the terms of VSG gene conversion (VSG GC) and

ES gene conversion (ES GC) are used to differentiate different types of gene conversion events, depending on the region of the copied sequence relatively to the VSG gene (Kim, H. et al., 2010). In addition, more complicated switching events involving the loss of the active ES or VSG combined with an in situ switching event have been observed, such as ES loos + In-situ. (Kim, H., 2010). A reciprocal recombination between the active and a silent BES including their downstream telomere sequences has been observed as well, which is widely accepted as telomeric exchange or crossover.

Homologous recombination is a universally conserved DNA repair mechanism (San Filippo et al., 2008). DNA double strand breaks in the active ES has been proven to be the most important trigger of the recombination mediated

VSG switching events (Borst et al., 1996, Barry 1997). Recently, it has been shown that DSBs introduced in the active ES and immediately downstream of the 70 bp

22 repeats lead to a 250-fold increase in VSG switching (Boothroyd et al., 2009). Most

VSG genes have upstream 70 bp repeats, which allows these VSG genes to serve as a donor in gene conversion-mediated VSG switching (Hovel-Miner et al., 2016).

Furthermore, not only do DSBs in the active ES trigger high VSG switching frequency but also does the location of the DSB in the active the ES dictate the preferred type of switching events (Lucy et al., 2013).

An increasing amount of data has shown that the homologous recombination mediated gene conversion is the most dominant mechanism for

VSG switching. At the DSB site, RAD51 is the key factor that polymerizes (binds) on the single stranded DNA (ssDNA) after the 5’ end is resected. It catalyzes strand invasion in an ATP dependent manner (Holloman, 2011). Six RAD51 paralogues have been identified in T. brucei including RAD51, DMC1, RAD51-3, RAD51-4,

RAD51-4 and RAD51-6 (McCullcoch, 2005). Three of them, when deleted, impair

VSG switching in T. brucei. BRCA2 is a that helps load RAD51 onto the ssDNA. It has been shown that deletion of BRCA2 also affects VSG switching

(McCulloch, 1999, 2005 and 2008).

DNA breaks are almost evident during the DNA replication and it was expected that proteins involved in the DNA replication may also be involved in the regulation of VSG switching. Depletion of TbORC1 also increases the VSG switching frequency (Benmerzouga, et al., 2012). TbTOPO3α (a type IA ) and TbRMI1 (RTR complex associated partner), which play major role in dissolution of double Holliday junction intermediates and in the DNA replication have been shown to play a role in VSG switching (Kim et al., 2010 &

23

Kim et al., 2012). Increase observed in TOPO3α and RMI1 depletion is almost 100 fold to the control strains used in the assay. In addition, deletion of Histone H1 results in the increased VSG switching frequency (Povelone et al., 2013).

Telomeres are physical ends of chromosomes, and VSGs are located at the subtelomeric regions. It has been hypothesized that the telomeres may play important roles in the VSG expression regulation. Telomere length has been shown to play an important role in VSG switching, as when the active the ES adjacent telomere becomes short (~1.5 kb), VSG switching frequency is increased

6-36 fold (Hovel-Miner et al., 2012). Telomere proteins TbTRF and TbTIF2 have been shown to play an independent and overlapping roles in the VSG switching regulation (Jehi et al., 2014A, Jehi et al., 2014B, Jehi et al. 2016). The predominant

VSG switching pathway in cells depleted of TbTRF and/or TbTIF2 is gene conversion.

1.7 Telomeres and telomere protein complexes in several eukaryotic organisms

Telomeres are the complexes present at the ends of linear chromosomes. They are essential to maintain chromosome stability and genome integrity. The first observation indicating the importance of telomeres was made in

Drosophila by Hermann Muller in 1938, as the removal of chromosome ends led to the chromosome loss. Thus, it has been concluded that the linear chromosome ends are special and are required for maintaining the chromosome stability. The chromosome ends are also given the name “telomeres”, where “Telo” means

“ends” and “mere” means “part” (Muller, 1938). Sequencing of chromosome ends

24 in Tetrahymena revealed the actual structure of telomeres (Blackburn and Gall,

1978, Yao et al., 1981). It is now known that telomeres are tandem arrays of simple

DNA repeats of varying length but simple sequences. In most organisms, telomere

DNA consists of TG-rich repetitive sequences (Blackburn and Gall, 1978,

Blackburn and Challoner, 1984).

Figure 1-7: Telomere protein complex in different organisms. Schematic representation of the telomere proteins bound to telomere sequences in different organisms. G-overhang in T. brucei is 12 bp long (Sandu R. unpublished data), hence, keeping a question mark there (Sandhu, R, 2014).

25

The number of telomere repeats varies in different organisms. For example, in budding yeast lab strains, telomeres are on average 280-300 bp long, while human telomeres are 2-15 kb long (Shampay et al., 1984, Moyzis et al., 1988,

Lansdrop et al., 1996). Telomeres consist of mostly double stranded DNA, but at the very 3’ end of the telomere is usually single stranded and is known as telomere

G-overhang. There are many telomere specific proteins that associate with the telomere repeats. Binding of these proteins to telomeres maintains telomere structure intact.

Telomeres have two essential functions: 1. Protect the chromosome ends.

2. Solve the end replication problem. First, telomeres serve as a protective cap to prevent natural chromosome ends from being recognized as the DNA DSBs, thus preserving the chromosome stability (de Lange, 2009). Telomeres can form secondary structures such as the T-loop and G-quadruplex. Formation of the T- loop results in folding back and tucking in of the very end of the telomere and thus hiding the 3’ single stranded telomere G-overhang (Palm and de Lange, 2008).

Second, the ends of linear DNA molecules cannot be fully replicated by the conventional DNA polymerases. This problem stems from the use of small 5’ RNA primers by the DNA polymerases to initiate synthesis of the daughter DNA strand and DNA polymerases only synthesize DNA from 5’ to 3’. These short RNA primers are removed at the end of replication, creating a gap of 8-10 nucleotides at chromosome ends that cannot be filled by DNA polymerase. Most use , a ribonucleoprotein complex (RNP), to compensate for the loss of telomere sequence due to the end replication problem. Telomerase is a highly

26 specialized that uses a short stretch of its integral RNA component as the template to synthesize telomere DNA de novo.

Structural studies of telomere DNA binding proteins have shown that the

Myb motif is often used to recognize double stranded telomere DNA, while the OB

(oligonucleotide/oligosaccharide binding) fold often binds the single stranded telomere repeats (Horvath, 2008). Many non-telomeric proteins are recruited to the telomeres through their interaction with telomere DNA binding proteins. The resulting nucleoprotein complex maintains telomere function and lengths.

In S. cerevisiae, duplex telomere DNA is bound by ScRAP1 (repressor activator 1). ScRAP1 binds to DNA by its central myb and myb-like domains. It recruits many other proteins to the telomere by its c-terminal region (Longtine et al., 1989, Konig et al.,1996), including RAP1 interacting factor 1 (Rif1) and Rif2 that negatively regulate the telomere length (Wotton and Shore, 1997), Silent

Information Regulator 3 (Sir3) and Sir4 that are required for the formation of the telomere heterochromatin structure (Hardy et al., 1992, Moretti et al., 1994,

Moretin and shore, 2001, de Lange, 2012).

Cdc13 has been shown to bind single stranded telomere DNA and interacts with Stn1, Ten1 and EST1 (ever shorter telomeres) (Zakian, 1996, Bourns et al.,

1998, zakian, 2000). Cdc13 regulates the telomere length by recruiting telomerase to the short telomeres (Pennock et al., 2001, Chandra et al., 2001). S. cerevisiae

Ku proteins, a heterodimer composed of yKu70 and yKu80, bind to telomeres and are essential for non-homologous end joining (NHEJ) (Boulton and Jackson, 1996,

Gravel et al., 1998, Boulton and Jackson,1998). Genetic studies showed that yKu

27 interacts with TLC1 (the telomerase RNA component), which is important for the telomere maintenance (Peterson et al., 2001, Stellwagen et al., 2003). In addition, yKu also plays an important role in the chromosome end protection.

The telomere protein complex in mammalian cells is called “Shelterin” (de

Lange, 2005). Shelterin consists of six proteins: TTAGGG binding factor 1 and 2

(TRF1 and TRF2), TRF interacting factor 2 (TIN2), RAP1, TPP1, and protection of telomere (POT1) (de Lange, 2008). Among these proteins, TRF1 and TRF2 directly bind the duplex telomere DNA, while POT1 binds to the single stranded telomere DNA (Broccoli et al., 1997, Cech 2001). TRF1 and TRF2 form homodimers but do not interact with each other, although both interact with TIN2, while TRF2 interacts with RAP1. TPP1 interacts with TIN2 and POT1 (Li et al.,

2000, Kim et al., 1999, de Lange et al, 2012).

Recently, a trimeric CST complex containing CTC1, STN1, and TEN1 has been identified to bind the mammalian single stranded telomere DNA as well

(Miyake et al., 2009, Wan et al., 2009). It has been shown that the Shelterin complex is essential for the integrity of the mammalian telomeres. TRF1, TRF2,

RAP1, and TIN2 all play essential roles in the telomere length maintenance, while

POT1 controls the telomere G-overhang structure.

The shelterin complex of S. pombe is composed of 7 different proteins, many of which are structural and functional homologs of the mammalian Shelterin components. TAZ1 is the homologue of TRF1/2, and it binds to the duplex telomere DNA (Vassetzky et al., 1999, Spink et al., 2000). TAZ1 regulates telomere length and plays important roles in telomere replication and chromosome

28 end protection (Cooper et al., 1997, Miller et al., 2006, Verdun and Karlseder,

2006). RAP1 has more essential roles in telomere end protection and length regulation than its mammalian homolog (Fujita et al., 2012). POZ1 is an another novel protein associated to S. pombe’s telomeres. It does not contain any obvious sequence similarities with any of the telomere proteins and it has been shown to interact with TPZ1 (a POT1-interacting factor and a functional equivalent of mammalian TPP1) and RAP1 (Miyoshi et al., 2008). As POZ1 functionally correlated with TIN2, it may be a structural homologue of TIN2 (Miyoshi et al.,

2008).

1.8 Telomere and telomere protein complexes in T. brucei

T. brucei telomere sequence is the same as mammalian telomeres and consists of TTAGGG repeats (Blackburn and Challoner, 1984). T. brucei telomeres can be as long as 20 kb in length. The T. brucei genome has nearly 250 telomeres, and telomere sequence represents 10% of the entire genome. Telomeres in our lab strain are 3-20 kb long with an average size of 15 kb. (Munoz-Jordan et al.,

2001, Dreesen et al., 2005 & Sandhu et al., 2013). This is not the case in the wild isolates as they tend to have shorter telomeres (Dreesen et al., 2008). T. brucei telomere elongates on average 8 bp per population doubling. Deletion of TbTERT, the protein component of T. brucei telomerase, resulted in telomere shortening at a rate of 3-6 bp/ population doublings (Dreesen et al, 2005). Similar phenotype was observed when TbTR, the RNA component of the telomerase, is deleted

(Sandhu et al., 2013). T. brucei telomeres also have a 3’ single-stranded G-

29 overhang structure that is only ~ 12 nt long (Sandhu R., unpublished data). Three telomere proteins have been identified in T. brucei telomeres, TbTRF, TbTIF2 and

TbRAP1.

TbTRF is the first telomere protein to be identified in T. brucei (Li et al.,

2005). It is a functional homologue of mammalian TRF2. By fluorescent in situ hybridization/ immunofluorescent (FISH/IF) analysis TbTRF has been shown to colocalize with telomeres and chromatin immunoprecipitation (ChIP) indicates that

TbTRF is associated with the telomere chromatin. In vitro gel shift assays showed that TbTRF binds duplex telomere DNA directly (Li et l., 2005). Removal of TbTRF does not affect subtelomeric VSG silencing in BF cells (Yang et al., 2009).

However, depletion of TbTRF is detrimental to T. brucei cells. TbTRF has a similar structure as the vertebrate TRF1/2. At the c-terminus of the TbTRF protein there is a myb domain through which it binds the duplex telomere DNA (Li et al.,

2005). A homodimerization domain is present at the N-terminus of TbTRF, through which it forms homodimers. A number of have been generated in the

TbTRF myb domain and several exhibited weak telomere binding (Jehi et al.,

2014B). In addition, a transient depletion of TbTRF resulted in a 3-4 fold increase in VSG switching frequency (Jehi et al., 2014B). The majority of the switchers arose from TbTRF depleted cells were ES GC/ES loss+In-situ type switchers, which is similar to wild type switchers (Jehi et al., 2014B). Similarly, a significant increase in VSG switching frequencies was observed in TbTRF mutants with weaker telomere binding affinity, and switching mechanisms in these mutants were

30 similar to those in TbTRF depleted cells, indicating that the telomere binding activity of TbTRF is essential for VSG switching regulation (Jehi et al., 2014B).

TbTIF2 is an another telomere protein, which was identified in the TbTRF protein complex by immunoprecipitation. TbTIF2 is a functional homologue of mammalian TIN2. It has been shown to interact with TbTRF in vivo through Co- immunoprecipitation and immunofluorescence experiments (Jehi et al., 2014A).

TbTIF2 also plays an important role in VSG switching. A transient depletion of

TbTIF2 resulted in a 4-5-fold increase in VSG switching frequency and most of the switchers arose through ES GC/ES loss + In-situ, similar to wild type switchers

(Jehi et al., 2014A). Depletion of TbTIF2 led to increase in the DNA double strand breaks in the ES at 70 bpr, pseudogene, promoter and VSGs identified by the ligation mediated PCR assays (Jehi et al., 2014A). TbRAD51 was recruited to the telomere when TbTIF2 was depleted, suggesting that TbRAD51-mediated homologous recombination is important for the switching events in these cells (Jehi et al., 2014A).

TbTIF2 interacts with TbTRF and this interaction can be the one responsible reason that TbTIF2 stays at the T. brucei telomeres, however further detailed analysis is required to rule out other possibilities. Hence, it was expected that

TbTRF and TbTIF2 have similar functions at T. brucei telomeres. The switchers collected in TbTRF and TbTIF2 depleted cells both are enriched of ES GC/ES loss

+ in-situ events, which suggests that these proteins, indeed, function in the same pathway. Moreover, removal of both proteins showed reduction in G-overhang structure of T. brucei telomeres (Li et al., 2005, Jehi S., unpublished data).

31

However, TbTRF and TbTIF2 have independent roles, too. Although, TbTRF is involved for the subtelomeric VSG switching, removal of TbTRF for 24 hours did not depict an increase in DNA DSBs significantly in the LMPCR assay (Jehi, et al.,

2016). Moreover, ectopic overexpression of TbTRF in TbTIF2 RNAi cells, reduced the subtelomeric DNA DSBs compare to TbTIF2 RNAi strain (Jehi et al., 2016).

This observation suggests that although close but TbTRF and TbTIF2 function in the different pathways to protect telomeres. It is also being demonstrated that,

TbTIF2 is essential for the protein stability of TbTRF as removal of TbTIF2 reduced the TbTRF protein levels. However, addition of the protease inhibitor slowed down the TbTRF protein degradation in the TbTIF2 RNAi background suggesting that

TbTRF can be degraded by proteasome associated removal of protein mechanisms (Jehi, et al., 2016).

TbRAP1, another telomere protein, was identified in a yeast two hybrid screen using TbTRF as bait (Yang et al., 2009). It is an essential protein, and depletion of the TbRAP1 protein leads to robust growth arrest within 24 hours

(Yang et al., 2009). It has been shown to interact with TbTRF in vivo by co-IP. It has also been shown that TbRAP1 associates with the telomere chromatin by

ChIP. Depletion of TbRAP1 results in a striking phenotype: genes located close to telomeres are derepressed in a distance-dependent manner where derepression is stronger for the genes located closer to the telomere (Yang et al., 2009).

Depletion of TbRAP1 also derepresses the metacyclic VSGs located at subtelomeres (Pandya et al., 2013). In the procyclic form T. brucei cells, depletion of TbRAP1 also derepressed all the ES associated VSGs, which are normally

32 silent. Hence it is well-established that TbRAP1 controls VSG silencing (Pandya et al., 2013), a first essential aspect of the antigenic variation explained earlier.

Deletion of TbRAP1 also leads to more opened chromatin structure at the subtelomeres in the procyclic form T. brucei cells. However, in the bloodstream form T. brucei cells no significant change in chromatin structure was observed upon TbRAP1 depletion (Pandya et al., 2013).

1.9 Telomere transcription and RNA:DNA hybrids

Telomeres form a specialized heterochromatin structure that silences the expression of the genes located near the telomeres. Hence it was long standing belief that telomeres are not transcribed. However, contrary to popular belief, telomeres are transcribed into a long non-coding RNA called as telomere transcript or Telomere Repeat-containing RNA (TERRA) (Azzalin et al., 2007, Schoeftner and Blasco, 2008). TERRA ranges from 100 bp to 9 kb in mammals and is transcribed from the C-rich telomere strand giving rise to 5’-UUAGGG-3’ repeats

(Azzalin et al., 2007, Schoeftner and Blasco, 2008). TERRA forms a discrete focus in the cell and has been shown to be associated with the telomere in vivo (Azzalin,

2007). In budding yeast, TERRA can be transcribed from both telomere strands, giving rise to C-rich and G-rich telomere RNAs. In S. pombe, TERRA has two more variants known as ARRET (opposite of TERRA) and ARIA (TERRA molecule not associated with the subtelomeric region) (Bah et al., 2011). Telomere transcripts have been detected in T. brucei (Rudenko et al., 1989), and TERRA has the 5’-

UUAGGG-3’ repeats. It has been demonstrated that TERRA synthesis is not sensitive to α-amanitin treatment in T. brucei (an inhibits transcription of

33

RNA pol II) (Rudenko et al., 1989), suggesting that RNA Pol I transcribes telomere as a read through post transcription of VSG from an active ES.

TRF1 and TRF2 have been shown to interact with the TERRA molecule in the human cells. Overexpression of the TRF2 decreased the telomere length and reduced the TERRA expression (Benetti et al., 2007). These proteins regulate the

TERRA expression as deletion of the TRF2 elongates telomere length, while depletion of TRF1 reduces the telomere length and the TERRA expression is negatively regulated by the telomere length. As shrot telomeres shows an increase in TERRA expression, while elongated telomeres suppress TERRA expression

(Deng et al., 2009). In budding yeast, telomere binding protein ScRAP1 negatively regulates the TERRA expression (Luke et al., 2011). ScRAP1 interacts with Rif1 and Rif2. Together they regulate telomere length, and the differential telomere length affects TERRA expression (Iglesias et al., 2011). Forced transcription of yeast telomeres led to aberrant telomere shortening in a recombination dependent manner (Sandell et al., 1994, Maicher et al., 2012). TERRA is also proposed to negatively control telomerase-mediated telomere elongation in the mammals

(Blasco et al., 2008). Telomerase null cells showed reduction in the TERRA levels in mammals, suggesting ALT cells may differ in the TERRA expression regulation

(Schoeftner and Blasco, 2008). During in vitro assays, when TERRA is incubated with the telomerase complex, it competes with the RNA molecule of the telomerase complex (TR) and negatively regulates the telomerase association to short telomeres (Cusanelli et al., 2013). Chromatin structure is essential to control the

TERRA transcription. An open state chromatin is associated with the TERRA

34 expression (Azzalin, 2007). Mouse cell lines deficient in the histone methyltransferases Suv3-9h and Suv-20h displayed elevated levels of TERRA

(Schoeftner and Blasco, 2008). ScRAP1 also interacts with Sir3/Sir4 a chromatin modifier through it C-terminus domain and facilitates the telomere heterochromatin structure (Iglesias et al., 2011). in the THO complex, the RNA export machinery, increases the telomere transcription as well (Luke et al., 2014). In the human cells, TERRA transcription has been shown to initiate from promoters with the CpG islands (Deng et al., 2010). All these data suggest that the regulation of

TERRA in vivo is an essential aspect of the telomere structure and function regulation.

Besides, TERRA’s role in open chromatin formation and telomere length maintenance, it is also an essential component for the chromosome end protection. Excessive amount of the TERRA allows formation of RNA:DNA hybrids

(R-loop) at the telomere. R-loops have been detected in highly transcribed regions and It is easier to form R-loop at the G-rich non template strand of the DNA, making telomere DNA an excellent target for R-loop formation (Hamperl et al., 2014). R- loops have been shown to induce the DNA double strand breaks (DSBs) and the homologous recombination (Hamperl et al., 2014). Accumulation of R-loops is detrimental to the cells as there is an increased amount of the DNA damage in the region close to R-loops, which in turn leads to the increased recombination events

(Balk et al., 2013). R-loops have been shown to generate more DSBs in the telomere and impair the ability to repair broken telomeres, leading to telomere shortening (Balk et al., 2013, Pfeiffer et al., 2012). Chromatin remodeling FACT

35 subunits controls the R-loop mediated telomere shortening (Herrera-Moyano et al.,

2014). In yeast and human cells proteins involved in the higher TERRA expressions have been also shown to be involved in the high R-loop formation at the telomeres.

Ribonuclease H (RNaseH1), is an enzyme which cleaves the RNA strand of the RNA:DNA hybrids. Overexpression of RNaseH1 in yeast and human cells can remove R-loop at the telomeres (Balk et al., 2013, Pfeiffer et al., 2013, Arora et al., 2014). Besides its role at the telomere, R-loop formation is observed in the neurodegenerative diseases, , heart diseases and renal diseases (Groh M.

& Gromak N., 2014). Many proteins have been demonstrated to play important roles in the regulation of R-loop levels, including RNA processing and export proteins, ribonucleases, , and proteins responsible for genome integrity

(Groh M. & Gromak N., 2014). It has been suggested that the role of R-loops and regulation of R-loop is important for cell survival. All these data support the idea that, regulation of the telomere transcription and telomere transcript is very essential for the genomic stability of the chromosome ends. As increased TERRA and R-loop at the telomeres leaves the chromosome ends for higher recombination.

1.10 Significance of this study

T. brucei causes a devastating disease in humans and animals in the sub-

Saharan countries. African trypanosomiasis is detrimental for the development of those countries. Unrealistic targets of the treatment of the disease causes more damage than the immediate cure. Pharmaceutical companies are less involved in

36 the development of the drugs because of the small market and poor profits. Hence it is extremely important to study the disease which has been kept under the neglected tropical disease category.

T. brucei has been evolved to undergo antigenic variation to escape host immune defense. Surface coat protein coding genes are located close to telomeres, and telomeres have been shown to play important roles in the regulation of the antigenic variation. Although telomere proteins are functionally conserved to their mammalian counterparts, they have many unique feature and are only weakly homologous at the sequence level. Therefore, telomere proteins can serve as potentially good targets for anti-trypanosome drugs. Therefore, detailed knowledge about their function will allow us to design targets against these proteins better.

Understanding the detailed mechanisms for VSG switching will bring insights to identify potential targets of anti-parasite reagents. Although conserved telomere transcript (TERRA) is unique from T. brucei to humans. Understanding the roles of TERRA in wild type cells will increase our knowledge about previously unknown functions of telomeres. Hence studying TERRA’s role in T. brucei will not only expand our knowledge about non-coding RNA in T. brucei but also in other organisms as well. Altogether, identifying the strategies to fight against antigenic variation in T. brucei can serve as a platform to develop drugs against other potential antigenic variation associated pathogens (Plasmodium, Borellia and

Candida to name some).

37

CHAPTER II

MATERIALS AND METHODS

2.1 Plasmids

2.1.1. TbRAP1 RNAi construct – p2T7-TABlue-0760-RNAi

Full length TbRAP1 ORF (Tb927.11.370) was amplified and inserted in p2T7-TABlue plasmid using XhoI (NEB) and BamHI (NEB) restriction .

The final product is called p2T7-TAblue-0760-RNAi. There are two T7 promoters upstream and downstream of this ORF, which can be induced with doxycycline

(100 ng/ml) inside T. brucei cells. This plasmid was linearized using NOTI (NEB) restriction enzyme before transfected in T. brucei cells and integrated into the rDNA spacer region. Hygromycin (5 ug/ml) was used for selecting transfected candidates.

2.1.2. Ectopic TbRAP1 expression construct – pLEW111-phleo-F2H-0760-FL

pLEW111-BSD-F2H-Tb0760-FL was generated previously in the lab. I replaced the BSD resistance gene from this plasmid (by BamHI (NEB) and SalI

(NEB) restriction digestion) with Phleomycin resistance gene to generate

38 pLew111-phleo-F2H-Tb0760-FL. TbRAP1 is tagged with the FLAG-HA-HA epitope at its N terminus. This plasmid is linearized with NOT1 (NEB) before transfected in T. brucei.

2.1.3. Ectopic TbRNaseH1 expression construct – pLew100V5-phleo-

TbRNAseH1-2HA

pLew100V5-phleo was first linearized with HindIII and BamHI. The full length TbRNAseH1 ORF (Tb927.7.4930) was PCR amplified (900 bps) using a forward primer with the HindIII site and a backward primer with two HA and the

BamHI site. The PCR product was digested with HindIII and BamHI before ligated with pLew100V5-phleo, digested with the same restriction enzymes. The final clones were sequenced to confirm its identity.

2.2 Trypanosoma brucei strains

2.2.1. The parent strain for VSG switching assays (S)

The parent strain for VSG switching assays (S) is obtained from Dr. Hee- sook Kim and Dr. George Cross (Kim, H. & Cross, G., 2010, referred as HSTB221).

The strain is doubly marked with Blasticidin resistance (BSD) and the Puromycin resistance-thymidine (PURO-TK) cassette. BSD is integrated immediately downstream of the BES1 (VSG2 containing ES) promoter. PURO-TK is inserted upstream of VSG2 and immediately downstream of the 70 bp repeat region. These cells grow the same as Lister 427 antigenic type MITat1.2 clone 221a strain frequently known as SM221+ (Doyle et al., 1980; Hirumi et al., 1989), with

Blasticidin (5 ug/ml) and Puromycin (0.1 µg/ml). Addition of 4 µg/ml of Ganciclover

39

(GCV) in the medium kills TK positive cells, which is used to select switchers that do not express TK anymore.

2.2.2. The control strain with an empty RNAi vector for VSG switching assays

(S/EV)

p2T7-TABlue-Phleo was transfected into S cells to generate the S/EV strain. After transfection, cells are selected with 2.5 µg/ml of phleomycin. The C1 clone was used for VSG switching analysis.

2.2.3. The TbRAP1 RNAi strain for VSG switching assays (S/RAP1i)

Linearized p2T7-TABlue-0760-RNAi plasmid is transfected into S cells in the presence of 5 µg/ml of hygromycin to generate the S/RAP1i strain. Clone B1 and C1 were used for the detailed characterizations.

2.2.4. TbRAP1 RNAi cells with ectopic expression of TbRAP1 (S/RAP1i + F2H-

TbRAP1)

pLew111-phleomycin-F2H-Tb0760 was transfected into clone B1 of

S/RAP1i and selected with 2.5 µg/ml of phleomycin. Several clones were obtained.

The expression of F2H-TbRAP1 was checked by western blotting using anti-HA antibody F7 before the cells were used in the study.

2.2.5. S/EV with ectopic expression of TbRNaseH1 (S/EV + TbRNaseH1-2HA)

pLew100V5-phleo-TbRNaseH1-2HA was transfected into the S/EV strain and selected with 2.5 µg/ml phleomycin. Two clones were finalized to perform further experiments.

40

2.2.6. TbRAP1 RNAi strain with ectopic expression of TbRNaseH1 (S/RAP1i +

TbRNaseH1-2HA)

pLew100V5-phleo-TbRNaseH1-2HA was transfected into S/RAP1i cells to generate S/RAP1i+TbRNaseH1-2HA. 2.5 µg/ml of phleomycin were used to select tranfectants. Clones that showed knockdown of TbRAP1 and overexpression of

TbRNaseH1 upon induction were used for the studies.

2.2.7. FLAG-HA-HA tagged TbRAP1 in WT427 procyclic form cells

WT427 procyclic form T. brucei cells were transfected by XhoI and NOTI linearized psk-5UTR-puro-F2H-TbRAP1NT.tar plasmid (Yang et al, 2009) to tag one endogenous TbRAP1 allele with an N-terminal F2H epitope. Pool of transfectants was selected with 1 µg/µl of puromycin. Clones were obtained by limiting dilution and expression of F2H-TbRAP1 was verified by western blotting.

The other endogenous allele of TbRAP1 was replaced with the hygromycin resistance gene by transfecting psk-5UTRTbRAP1-hygro-3UTRTbRAP1 plasmid

(Yang et al, 2009).

2.3 Growth curve analysis

All strains described above were grown with or without doxycycline (100 ng/ml) in parallel to study their growth rates. Cells were counted at 24-hour intervals and cell population doublings (PDs) were calculated. When necessary, cells were washed with media without doxycycline twice to remove doxycycline.

41

2.4 Western blotting

Cells from different strains were grown with or without doxycycline and washed with 1x TDB buffer (0.005M KCl, 0.080M NaCl, 0.001M MgSO4.7H2O,

0.02M Na2HPO4, 0.002M NaH2PO4, 0.02M glucose). Cells were harvested by centrifugation at 0, 24, 48 & 72 hours after induction and cell numbers were counted. Protein samples were prepared by re-suspending cell pellet with 2X

Laemmli buffer (0.1 M Tris-Cl, pH 6.8, 6% SDS, 20% glycerol, 0.004% bromophenol blue) at a concentration of 1 million cells/2 µl of buffer followed by boiling for 10 min. Samples were separated on 4-10% SDS poly-acrylamide gel at

90 volts in 1X running buffer (25 mM Tris-base, 190 mM glycine, 0.1% SDS) till the all blue protein marker (Bio-rad) reaches the bottom of the gel. Proteins were transferred onto 0.45 um nitrocellulose membranes using 1X transfer buffer (50 mM Tris-base, 95 mM glycine, 0.1% SDS, 20% methanol) at 4ºC. Proteins on blots were stained with Ponceau S (0.2% (w/v) Ponceasu S, 0.5% (v/v) acetic acid) S) for 5 min and de-stained with de-staining buffer (0.002 % HCl) for 5 min. Stained blots were scanned to keep a record of protein loading. Blots were then blocked with 10% milk, 1X PBS, 0.1% tween-20 for at least 30 min. After being washed once with 1x PBS, blots were incubated with 5% milk, 1X PBS, 0.1% tween-20 with desired primary antibody at 4ºC overnight. For detection of TbRAP1, we used rabbit anti-TbRAP1 antibody 598. As a loading control, we used either TAT-1 (anti- tubulin) antibody or EF-2 antibody (Santacruz). For detection of F2H-TbRAP1, we used anti-HA (F7) antibody (Santacruz). After incubating with primary antibody, blots were washed thourice with 1% milk, 1X PBS, 0.1% tween-20 for 5 min each

42 time. Blots were then incubated with the secondary antibody (For 598, anti-rabbit; for Tubulin, anti-mouse; and for EF-2, anti-goat) for 1 hour in 5% milk, 1X PBS,

0.1% tween-20. Blots were washed with 1% milk, 1X PBS, 0.1% tween-20 for four times with 5 min each. Amersham ECL (1 ml) was added on each blot and incubated for 2-5 min exposure to autoradiography.

2.5 VSG Switching Assay

HSTB261 (Kim and Cross., 2010) and its derivatives were normally maintained in the presence of blasticidin and puromycin to keep the population uniformly expressing the active VSG2. Cells were washed to remove puromycin and blasticidin at the start of switching assays. 1,100 cells were incubated with or without 100 ng/ml of doxycycline for 24 hours followed by recovery in the absence of doxycycline until cell density reaches ~1.5-1.7 million cells/ml. Cells were then evenly plated (at 1 million cells/6 x 96-well plates) in the presence of 5

µg/ml GCV to select for switchers or distributed (at 1 cell/well) in the absence of GCV to determine plating efficiencies. To reduce the possibility of missing slow- growing switchers, the GCV selection period was 10-11 days long. GCV resistant cells were confirmed by their sensitivity to 2 µg/ml puromycin and dot blots using

VSG2 antibody to verify that they lost the expression of VSG2. Plating efficiency was calculated by dividing the number of growing clones by the total number of wells plated with cells. The switching frequency was calculated by dividing the number of GCV-resistant, VSG2 negative clones by the total number of plated cells and normalized with the plating efficiency. Subsequently, all switchers were tested for their sensitivity to 5 µg/ml and 100 µg/ml blasticidin. Silent BES

43 promoters are weakly active. Therefore, in situ switchers weakly express BSD and is resistant to 5 µg/ml blasticidin but sensitive to 100 µg/ml blasticidin.

Genomic isolated from the switchers were analyzed by PCR using BSD and VSG2 specific primers. For selected switchers, the newly expressed VSG was determined by reverse transcribing total RNA using a random primer, PCR using a spliced leader primer and a primer common to all VSG genes, and sequencing. Genotypes of these switchers were then confirmed by PFGE of DNA plugs from the switcher, followed by Southern blotting using VSG2, the newly expressed VSG, and BSD probes.

2.6 VSG Cloning

Before running samples on the pulse-field gel, we need to identify the current active VSG. To clone active VSG, we have isolated RNA from the individual switchers and by using random hexamer primers, we have performed reverse transcriptase step to generate cDNA. We have done PCR using forward primer specific for the splice leader region in the 5’UTR of the VSG gene (5´-

GACTAGTTTCTGTACTATAT-3´) and common C-terminus region (5´-

GACTAGTGTTAAAATATATCA-3´). PCR product was purified from the gel and sent for the sequencing. At the end, results were blast to the T. brucei lister 427 genome.

2.7 Pulse field gel electrophoresis (PFGE)

DNA agarose plugs were prepared as described by Horn and Cross, 1997.

Briefly, 2 x 108 cells were harvested, washed in 1xTDB and resuspended in L-

44 buffer (0.1 M EDTA pH 8.0, 10 mM Tris-HCl pH 7.6, 20 mM NaCl) and was left it at 50C. Equal amount of low melting agarose (Sigma) in L-buffer was added to the cells, mixed and poured into Plug Molds (Bio-Rad Laboratories). Plugs were treated in 5 ml of L-buffer with 1% N-L-sarcosyl for 2 days with 1 mg/ml of proteinase K at 50C. Upon washing twice for 15 minutes with L-buffer, proteinase

K treatment and washing is repeated. Intact DNA were electrophoresed in 1.2% agarose in 0.5x Tris-borate-EDTA gel.

Running condition

Loaded gels were inserted into gel chamber carefully and set up running parameters. Initial pulse: 1500 s, Ending pulse: 700s, Voltage: 2.5 V/cm, Length of time: 120 hours, Temperature: 12ºC.

2.8 Southern blotting:

Genomic DNA was isolated using DNAzol method. 2-5 µg of genomic DNA was digested overnight with desired endonuclease in not more than 40 µl of reaction volume. DNA was separated on 0.7% agarose gel prepared in 0.5xTAE at 100 V until the orange G dye reaches the bottom of the gel. The gel was scanned on Typhoon FLA 9140 by placing a ruler next to it. DNA was depurinated (0.25 M

HCL) for 30 minutes followed by denaturation (1.5 M NaCl; 0.5 M NaOH) for one hour (buffer was changed after 30 minutes). Finally, the DNA was neutralized (1

M Tris 7.4; 1.5 M NaCl) for one hour and blotted onto hybond nylon membrane

(GE healthcare life sciences) in 20 x SSC (3 M NaCl, 0.3 M sodium citrate). After overnight blotting, the membrane was cross-linked (Stratalinker UV crosslinker)

45 and pre-hybridized for ~1 hour in CHURCH mix (0.5M NaPi pH 7.2, 4 mM EDTA pH 8.0, 7% SDS, 1% BSA) at 65ºC. Hybridization was done at 65ºC overnight using the CHURCH mix and appropriate radiolabeled probe. The blot was washed three times using CHURCH wash (40 mM NaPi pH 7.2, 1 mM EDTA pH 8.0, 1%

SDS) for ~15 minutes each at 65ºC, wrapped, and exposed to a phosphorimager screen (GE Healthcare Life Sciences).

2.9 R-loop assay

Genomic DNA was isolated. Samples were sonicated at the medium voltage for 8 cycles. Samples were collected accordingly. Samples were treated with RNaseH (10 U) or kept on ice for without treatment. Samples were subjected for the IP overnight (either with mouse normal IgG and s9.6 mAb), and purified later using protein G beads. The IPed material were eluted. Samples were loaded on nitrocellulose plus (N+) membrane, (do not crosslink). Hybridize it with telomere probe. (TTAGGG)133 from psty-TTAGGG plasmid DNA product from EcoRI digestion.

2.10 Isolation of RNA for TERRA analysis

Cells were grown for 1.5-1.7 million/ml and centrifuged cells at 1500 rpm for

10 min at R9T. (NO TDB wash). Pellete were resuspended in 500 uls of the media.

RNA-STAT-60 (TEL TEST) based RNA purification was performed. RNA-STAT-

60 and Chloroform are used to isolate RNA. Equal volume of isopropanol was added to the aqueous phase. Precipitated RNA was washed twice with 70% EtOH.

Pellete was resuspended in 200 uls of the RNAse free water (or DEPC treated water). Repeat the above mention procedure but now dissolve pellete only in 30

46 uls of RNAse free water. Total RNA was digested with 10U DNAseI (Thermo fischer), make sure to use RNasein to protect RNA. After DNAse treatment isolate

RNA again by repeating above mention procedure. After washing the cells, pellete was resuspended in 30 uls of RNase free water for Northern blot or ~15-20 ul for

TERRA PCR (amount of volume is based on the pellete size.

2.11 TERRA - Northern blots/ slot blots

After isolation of the RNA, the RNA samples were digested for a positive control with RNase. Samples after RNase treatment were subjected either on the gel or on the blot for analysis. For running the samples on the gel 6X RNA loading dye (Bromophenol blue -0.005 gms, Xylene cyanol-0.005 gms, 50% Glycerol) was used, with Denaturing buffer (10x MOPS, 37% formaldehyde, Formamide).

Samples were loaded on the denaturing gel for northern analysis or subjected to nylon blot for slot blot analysis. Hybridize the blots with the respective probes to perform hybridization at 500C. Wash the blots with church wash for 20 mins three times. Signals are recorded in phosphoimager.

2.12 PCR of TERRA origin

Reverse transcription of the 1 ug isolated RNA was done, using TELC and

TELG primers. TELC 5’-CCCTAACCCTAACCCTAACC-3’ & TELG – 5’-

GGGTAAGGGTTAGGGTTAGG-3’.

Conditions: (RNA - 1 ug, Primer - 0.5 ul (20mM stock), dNTP (10mM) - 2 ul, RNase free water to Match volume to 13.5 uls). Mix well, kept at 65C for 5 min. (Small volume use thermocycler). Then use reagents from Thermoscript RNaseH-reverse

47 transcriptase kit (Thermo), 5x buffer - 4 uls, 0.1M DTT - 1 ul, rRNasein - 1 ul,

Reverse transcriptase - 0.5 uls, Total - 20 uls were used). Keep at 650C for 1 hour, reaction was stopped by keeping mixture at 950C for 15 mins. Reverse transcription was performed using Random Hexamer and No hexamers. 2 ugs of

RNA was reverse transcribed using standard lab protocol. M-MLV reverse transcription kit (Promega) was utilized. RNA - 2 ug, Random Hexamer (0.5 ug/ul)

- 2 uls, RNAse free water to bring volume to 28 uIs, Incubated at 650C for 5 mins.

Then add rRNAsein 1 ul, dNTPs (10mM) - 2 uls, 5X M-MLV buffer - 12 uls, M-MLV reverse transcriptase - 0.5 ul, Final volume of the mix should be 60 uls and incubated at 420C for 2 hours.

Once all the cDNAs were made, PCR was performed using 1 ul of cDNA for regular locus and 1 ul of 1:10 diluted cDNA for active locis (i.e. active VSG, tubulin and rDNA). All products ran on 0.7% agarose gel and results were scan by fluorescence.

Table 2: List of primers used for TERRA’s origin experiment.

Primer name Primer sequence

OBL-btubulin-RT-FW 5’-ATCTTTGGACAGTCTGGCGC-3’

OBL-btubulin-RT-BW 5’-TTCCACAAGTTGGTGCACGG-3’

OBL-rRNA-RT-FW 5’-ACGGAATGGCACCACAAGAC-3’

OBL-rRNA-RT-BW 5’-GTCCGTTGACGGAATCAACC-3’

OBL-VSG224-RT-FW 5’-CAGTCTTGTGCGCACTAGCT-3’

OBL-VSG224-RT-BW 5’-ATGCTGCTGCTGCTGTTACC-3’

48

OBL-VSGVO2-RT-FW 5’-AGCTTATCTAGCAGACGCCG-3’

OBL-VSGVO2-RT-BW 5’-TCCTCCCATTTCCTCCGATC-3’

OBL-VSG221-FW-3 5’-TGGATCGAGCCTGGAA-3’

OBL-VSG221-BW-3 5’-AAAGCAAAACTGCAAGCCAAAG-3’

OBL-70BP-RT-FW1 5’-TATCAGGAGAGTGTTGTGAGTGTG-3’

OBL-70BP-RT-BW1 5’-GGTAAGCACACACTCACAACAC-3’

2.13 Chromatin immunoprecipitation (CHIP)

Cells were fixed with 1.1% of formaldehyde for 20 mins at room temperature and cell lysate was sonicated in a Biorupor (Diagenode Corp.) at medium voltage for 4 cycles. IP was carried out using protein G Dynabeads (Life technologies) couples with appropriate antibodies followed by wash, elution, reverse crosslinking and DNA isolation. The ChIP products were subsequently analyzed by slot blot or q-PCR as explained below.

For slot blot of CHIP samples (Telomere, 50 bpr probes)

For input 1:10 dilution of sample was used & final volume was brought to

50 uls. NaOH and EDTA were added before boiling the samples. Samples are eventually subjected on the nylon membrane with the help of vacuumed manifold.

Each samples are divided in two slots for two different probes.

For Q-PCR

Each well was prepared for total 25 uls (2 uls of CHIP eluted sample (for input samples are 1:10 diluted), with 12.5 uls of SsoD sybr green mix, 0.5 uls of

49 forward primer, 0.5 uls of backward primer and 9.5 uls of water). PCR was done at 60C for 35 cycles and signals are quantified by Cq values from Bio-rad CFX software.

Table 3: List of primers used for the ChIP-qPCR experiment

Primer name Primer sequence

OBL-221ESPr-FW 5’-GTTACCGGAGTGCCCGTATT-3’

OBL-221ESPr-BW 5’-CAAAACAACACACCAGCCCTC-3’

OBL-VSG221-FW1 5’-GTCCTAGCCCAAGTTCTTC-3’

OBL-VSG221-BW1 5’-GCTGTTGCAGTAGCTGTTAC-3’

OBL-VSG21-RT-FW 5’-ACATGCGTCACATAACGCGG-3’

OBL-VSG21-RT-BW 5’-TTGTTTGCTGGCCTGCTAGC-3’

OBL-btubulin-RT-FW 5’-ATCTTTGGACAGTCTGGCGC-3’

OBL-btubulin-RT-BW 5’-TTCCACAAGTTGGTGCACGG-3’

OBL-rRNA-RT-FW 5’-ACGGAATGGCACCACAAGAC-3’

OBL-rRNA-RT-BW 5’-GTCCGTTGACGGAATCAACC-3’

2.14 Ligation mediated PCR assay (LMPCR)

LMPCR were performed according to Boothouroyd et al., 2009. Genomic

DNA was isolated from BF cells according to de Lange et al., 1990. The adaptor was prepared by annealing 5 nmole of LMPCR long and LMPCR short primers in

300 µl of 1X buffer (Life Technologies). In each ligation reaction, 2 µg of genomic DNA was either treated or not treated with 2 µl of T4 DNA Polymerase

50

(3000 U/ml, New England BioLabs) in the presence of 200 µM dNTP and then ligated with 10 µl annealed adaptor. 3 rounds of 1:3 serial dilutions of the ligated products were prepared and used in subsequent PCR reaction using hotstart

Platinum® Taq DNA Polymerase (Life Technologies) and a touchdown PCR program that transits from initial annealing temperature of 65ºC to final annealing temperature of 56ºC in 10 cycles followed by amplification for additional 25 cycles.

All LMPCR reactions were done using the same conditions. PCR products were then separated by agarose gel electrophoresis. The agarose gels were then dried and hybridized with end-labeled oligo probes.

Table 4: List of primers used for LMPCR experiments.

Primer Name Primer sequences

Linker short 5'- GTGAATTCAGATC -3' Linker long 5'- GCGGTGACCCGGGAGATCTGAATTCAC -3' OBL-BeforeESPr-LMPCR- 5'- AGAAATCTCGGATATCAGACTCAC -3' FW OBL-SNAP50-unique - 5’- TGAACATTTCAAAGA LMPCR-FW ATGCATTTTTCTTTATTGGA -3’ OBL-VSG pseudogene - 5'- ACCGGAACTGCAGGAACAAATG -3' FW OBL-LMPCRTEL122VSG 5'- GAAAAGGCTAAGA CGCTAAGAATC -3'

pseudo-unique-FW 70 bp oligo probe 5'- ATACGAATATTATAATAAGAGCAGTA -3' BES oligo probe 5'-GACCGTTGTCGGTTCATG

CATGACTTTAATCTCATC -3' BES11-VSGpseudogen 5'- GACGCAGAAGAAGAC oligo probe ACCAATAACACAAAC -3' SNAP50 oligo probe 5’- GGATGGTGCAACCATT ACTTTTACCTTAGTTCAG-3’

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2.15 DNA isolation using DNAzol for PCR screening of switchers

Cells were grown upto 1.5 to 2.0 million cells/ml. Only 2 ml of cells are used.

On the cell pellete 0.5ml of DNAzol was added and lyse the cells by repeating pipetting, and incubate the mix for 10 mins at RT. 100% EtOH (ethanol) was added and mixed till the white cloud is homogeneous. After centrifugation pellet was washed twice with 70% ethanol at RT. After discarding all ethanol, add 15 uls of

TE buffer was added and the g.DNA was stored in -200C DNA. For switchers screenings, 2-3 uls were used as template.

2.16 dot-blot western blot for false positive clone removal

Cell count: 1.5 – 2.0 million/ml, take 1-1.5 ml of this material to perform dot slot blot experiment. Preparation of membrane: pre-wet hybond-ECL, membrane in a clean container with 20 ml of distilled water for 5 mins. (An essential step for the proper signals at the end, signals spread a lot if you forget or reduce the time for this step). Discard the water and soak membrane in 1x PBS buffer for 5 mins.

Remove the solution and let the membrane dry on a clean c-fold towel. Do not let it over dry, co-ordinate your procedure with the cell preparations. Spot the parasites on the membrane: Centrifuge cells at 2000 RPM for 10 mins in an

Eppendorf tube, discard all the media, leaving 5 uls behind. (Little tricky step, I usually discard all and add fresh 5 uls of media). Resuspend the pellet in 5 uls of media and put a spot on the pre-equilibrated membrane with entire intact cells. Let the spot dry for 5 mins. Do not overlap spots. Block the membrane with 3% milk solution in 1x PBS, with 0.1% tween-20, for 45 mins at RT. Hybridize with primary antibody in the same solution for 1 hour. Antibody dilutions depends on the

52 antibody. For VSG221 its 1:10K, hence I add 1 ul of antibody in the container.

Wash membrane 4 times with 1x PBST for 5 mins each round. Hybridize with secondary antibody for 1 hour. Secondary antibody dilution varies between antibodies. Repeat step 6 exactly same. Develop the blot by adding ECL solution and look for the positive control spot.

2.17 Isolation of genomic DNA (large quantity)

Total genomic DNA was isolated as previously described. 200 million BF or PF cells were washed and resuspended in 1 ml of TNE (10 mM Tris pH 7.4,

10 mM EDTA, 100 mM NaCl). 1 ml of TNES/proteinase K (10 mM Tris pH 7.4,

100 mM NaCl, 10 mM EDTA, 1% SDS + 100 µg/ml proteinase K (Roche)) was added and samples were incubated for 16 hours at 37°C. After overnight incubation DNA was extracted using phenol/chloroform. DNA was precipitated with sodium acetate and isopropanol and gently spooled out and was resuspended in

300 ul TNE+100 mg/ml RNase. After 2-hour incubation with RNase at 37°C, 300 ul of TNES/proteinase K was added and reaction samples were incubated for 1 hour at 37°C. Following incubation DNA was extracted and precipitated as mentioned above and resuspended in 100 ul of TE (10 mM Tris pH 7.5/1 mM

EDTA).

2.18 Reverse transcription PCR for checking derepression of VSGs

After isolation of RNA, clean RNA using RNAeasy clean up protocol

(quiagen). 2.4 ug of RNA is converted into cDNA using, random hexamer and M-

MLV reverse transcriptase. For active VSG, tubulin and rrna samples are diluted

53 to 1:10. Each well is prepared for total 25 uls (2 uls of CHIP eluted sample (for input samples are 1:10 diluted), with 12.5 uls of SsoD sybr green mix, 0.5 uls of forward primer, 0.5 uls of backward primer and 9.5 uls of water). PCR was done at 60C for 35 cycles and signals are quantified by Cq values from Bio-rad CFX software.

Table 5: List of primers used for the RT PCR experiment.

Primer name Primer sequence

OBL-VSG221-RT-FW 5’-GTCCTAGCCCAAGTTCTTC-3’

OBL-VSG221-RT-BW 5’-GCTGTTGCAGTAGCTGTTAC-3’

OBL-VSG224-RT-FW 5’-CAGTCTTGTGCGCACTAGCT-3’

OBL-VSG224-RT-BW 5’-ATGCTGCTGCTGCTGTTACC-3’

OBL-VSG1.8-RT-FW 5’-CTACCAATTGCAGGGCTCAC-3’

OBL-VSG1.8-RT-BW TTACGGCCAGCAGCTTGGAT-3’

OBL-VSG1.13-RT-FW 5’-ATAACGCATGGCCATCTTGAC-3’

OBL-VSG1.13-RT-BW 5’-GTCGTTGCTGTGGATTGCTC-3’

OBL-VSG18-RT-FW 5’-ACTGCTAAAGAAGGTCTGGG-3’

OBL-VSG18-RT-BW 5’-TCGCCCTTTGAGATAGGTGG-3’

OBL-mVSG397-RT-FW 5’-GATACCAAGCAGTACTGGCC-3’

OBL-mVSg397-RT-BW 5’-CGTGAATTGTTCACCCGGG-3’

OBL-btubulin-RT-FW 5’-ATCTTTGGACAGTCTGGCGC-3’

OBL-btubulin-RT-BW 5’-TTCCACAAGTTGGTGCACGG-3’

OBL-rRNA-RT-FW 5’-ACGGAATGGCACCACAAGAC-3’

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OBL-rRNA-RT-BW 5’-GTCCGTTGACGGAATCAACC-3’

2.19 Preparation of probes for southern blot and northern blot

Regular probe preparation: Take 100-150 ng of complementary DNA needs to be labelled. Add, Random Hexamer or poly dT as a primer bring the final volume to

39 uls. Boil the samples at 95 C, either on heat block or sand bath for 5 mins. After

5 mins, keep the probe for 5 mins on ICE. Add 5 uls of 10x OLB-dCTP, 1 ul of klenow polymerase (NEB) and lastly add 5 uls of dCTP32, incubate the mix for 90 mins at RT. After 90 mins stop the reaction by adding equal volume of TNES (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM EDTA, 0.1% SDS) buffer. Purify the mix with G-50 sepharose beads column. Heat the mixture 5 mins at 95 C. and mix it with church-mix to filter sterilize to remove contaminants.

For C-rich and G-rich probe preparation: All the steps are similar as above.

Replace the chemicals as follow. Use 10x OLB without dCTP & dGTP. Telomere

DNA (250 ng) is hybridized with dCTP32 for C-rich probe and dGTP32 for G-rich probes. Increase incubation for 2:30 hours instead of 90 mins.

Preparation of 10x OLB: 0.5 M Tris-HCl pH 6.8, 0.1 M MgOAc, 1mM DTT, 0.5 mg/ml BSA. Add 0.6 ul of desired dNTP (100 mM stock) to 100 uls of OLB. Store it in -80C.

2. 20 Co-immunoprecipitation

Cell lysate was prepared from 200 million cells from WT427-Ty1-

TbNOT1/hygro-KO cells using acid-washed glass beads (Sigma) by alternating

55 vortexing (30 S) and sitting on ice for (10 s) for 6 times. Lysis buffer (50 mM TrisCl pH7.5, 0.02% NP-40, 2mM EDTA, 20% glycerol, 0.15M NaCl, 1X protease inhibitor

(Sigma), 1mM PMSF, 1mM DTT, 4ug/ml pepstatin A, and 0.5 mg/ml TLCK). IP was performed with 2 ug of anti-TY1 BB2 monoclonal antibody (MSKCC antibody core) and anti-RAP1 (598, Yang et al., 2009).

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CHAPTER III

CHARACTERIZATION OF TbRAP1 FUNCTIONS IN VSG SWITCHING

3.1 Introduction

RAP1 (Repressor/Activator Protein 1) is one of the intrinsic and well characterized factor of the telomere complex in mammals, budding yeast, fission yeast, and T. brucei (Conrad et al.,1990, Li et al., 2000, Kanoh and Ishikawa, 2001,

Yang et al., 2009). It plays various important roles in the telomere end protection, the telomere length regulation, and the telomeric gene silencing. In addition, RAP1 also acts as a and controls the expression of various genes in

S. cerevisiae, vertebrates, and T. brucei (Shore and Nasmyth, 1987, Lieb et al.,

2001, Martinez et al., 2010, Lickwar et al., 2012).

ScRAP1 regulates the telomere length by recruiting Rif1 and Rif2 to telomeres (Wotton and Shore, 1997). C-terminal truncation of ScRAP1 or deletion of Rif1/Rif2 proteins results in the telomere elongation, suggesting that ScRAP1 is a negative regulator of telomere length (Hardy et al., 1992, Wotton and Shore,

1997). Consistent with its role as a negative regulator, overexpression of ScRAP1 led to the telomere shortening. Based on the ScRAP1 tethering experiments a

57

‘protein counting’ model has been proposed (Marcand et al., 1997). According to this model, the number of ScRAP1 molecules bound to telomeres serves as a gauge for the telomere length sensing mechanism. Although it is not clear how

ScRAP1 exerts its role in the telomere length regulation, recent studies have suggested two possible mechanisms. First, ScRAP1, together with Rif1 and Rif2, restricts telomerase activity in the G1 phase (Gallardo et al., 2011) thereby preventing the telomere elongation. Second, ScRAP1 forms complex with Rif2 and, to a less extent, with Rif1 that regulates the telomere end processing by the

MRX complex (Bonetti et al., 2010) (Vodenicharov et al., 2010). A study in cdc13 temperature sensitive mutant background revealed that ScRAP1, along with Rif1 is important for telomere capping and is thus required for the telomere protection function (Anbalagan et al., 2011a, Xue et al., 2011). Also, ScRAP1 inhibits NHEJ at the telomeres by multiple pathways (Pardo and Marcand, 2005). Along with Rif2,

ScRAP1 prevents NHEJ at the telomeres most likely by preventing the activation of MRX complex (Marcand et al., 2008). Moreover, studies targeting ScRAP1 and

Rif2 to non-telomeric DSB sites revealed that the ScRAP1-Rif2 complex acts as

NHEJ inhibitor independent of telomere sequence. At telomeres, ScRAP1 inhibits

NHEJ also by recruiting Sir4 (Marcand et al., 2008). However, the mechanism by which Sir4 blocks NHEJ at telomeres is not clear. Lastly the DNA binding domain of ScRAP1 inhibits NHEJ independently of Rif2 and Sir4 (Marcand et al., 2008).

In S. pombe, SpRAP1 along with Taz1 maintains the telomere length homeostasis by inhibiting the telomere elongation and controlling the telomere 3’ overhang generation (Kanoh and Ishikawa, 2001, Miller et al., 2005). SpRap1

58 prevents the NHEJ at the telomeres but is shown to promote the homologous recombination in the absence of Taz1 (Subramanian et al., 2008). Also SpRAP1 is important for the telomere clustering towards the spindle body at the premeiotic horsetail stage, as depletion of SpRAP1 leads to multiple (>4) SpRAP1 foci, suggesting reduction in telomeric gene silencing (Chikashige and Hiraoka,

2001). In mammals, RAP1’s function at telomeres is much more complicated and diverse. In humans, RAP1 is an essential protein, while in mice it is non-essential

(Li et al., 2000, Martinez et al., 2010). In humans, RAP1 negatively regulates the telomere length and is required for the inhibition of NHEJ at the telomere, similar to yeast counterparts (Li et al., 2000, Bae and Baumann, 2007, Sarthy et al., 2009).

In contrast, mice have mildly shortened telomeres when RAP1 is deleted (Sfeir et al., 2010). Intriguingly, mouse RAP1 is not required to inhibit NHEJ but is important for suppressing the directed repair (Sfeir et al., 2010). Regardless of the species-specific differences in the functions of RAP1, it is evident that RAP1 is important for the telomere length maintenance and protection. In T. brucei, preliminary efforts have been made to understand the role of TbRAP1 in telomere length regulation (Pandya & Li, unpublished data). However, depletion of TbRAP1 have very strong growth phenotypes and in relatively short period of time it is difficult to catch changes in the telomere length unless they are very obvious.

Hence, functions of TbRAP1 are not studied in terms of telomere protection, yet.

Since, no homolog of ligase IV has been identified in T, brucei, it is proposed that

NHEJ is either absent or unique in T. brucei (Burton et al., 2007). In-vitro study confirmed that T. brucei cell extract has DNA end ligating activity that is

59 independent of the Ku heterodimes, suggest that NHEJ might be absent in T. brucei (Burton et al., 2007). Importantly, T. brucei has a very efficient homologous repair pathway (Conway et al., 2002). Conversely, micro-homology mediated end joining (MMEJ) repair pathway has been shown to perform repair in T. brucei

(Glover et al., 2008). Therefore, it is important to study the role of TbRAP1 in T. brucei telomere end protection and repair mechanism to better understand this parasite’s telomeres. T. brucei undergoes antigenic variation and homologous recombination has been proposed to be the central player for the antigenic variation (Mcculloch et al., 1999). That also suggest us to study the role of TbRAP1 in antigenic variation and homologous recombination.

Genes present on the subtelomeric region are often silenced due to the heterochromatic nature of telomeres. This phenomenon is known as telomere position effect (TPE) or telomeric silencing. So far, TPE has been identified in

Drpsophila melanogaster, yeast, human, mouse, T. brucei, and Plasmodium falciparum (Hazelrigg et al. 1984, Gottschling et al. 1990, Horn and Cross 1995,

Baur et al. 2001, Frietas-Junior et al. 2005, Duraisingh et al. 2005, Pedram et al.

2006). RAP1 is known to play an important role in TPE and in the silencing of subtelomeric genes.

In S. pombe, the deletion of SpRAP1 led to loss of telomere silencing as well as a loss in telomere clustering towards spindle pole body (Kanoh and

Ishikawa, 2001). In human cells a telomere position effect has been identified.

However, the proteins essential for TPE are currently unknown (Baur et al., 2001).

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ScRAP1 plays an important role in TPE. It recruits Sir3/Sir4 to the telomere to regulate the telomere silencing and the heterochromatin formation/maintenance

(Moretti et al., 1994, Cockell et al., 1995), and the mechanism is well characterized.

Sir3 and Sir4 together recruit Sir2, an NAD+-dependent histone deacetylase that can remove the acetyl group from histone H3 at K9 and K14 residues, and from histone H4 at K16 (Moazed et al., 1997, Bourn et al., 1998, Imai et al., 2000, Moretti et al., 2001). Sir3 in turn recognizes the unacetylated H4K16 (Hect et al., 1995).

Therefore, binding of Sir3 and Sir4 to ScRAP1 recruits Sir2, which deacetylates neighboring histone tails and allows the subsequent binding of Sir3 and Sir4 to neighboring nucleosomes, allowing propagation of the heterochromatin structure from the telomere to subtelomeric region (Rusche et al., 2002). The loss of RCT domain of ScRAP1 is required for the proper telomere silencing (Kyrion et al.,

1993). In T. brucei homologs of Sir3 and Sir4 are not identified yet. However, homolog of Sir2, TbSir2rp1, is identified and required for the silencing of a reporter gene targeted to a subtelomeric locus, however it not essential for VSG silencing

(Alsford et al., 2007). TbRAP1 has been shown to play an important role in the

VSG silencing, the first essential aspect of antigenic variation (Yang et al., 2009,

Pandya et al., 2013). The effects of the VSG silencing were much stronger close to the telomeres than farther in the subtelomeres. Also, depletion of TbRAP1 did not affect the chromatin much at the telomeres as no significant difference was observed between the active and the silent ES through the Formaldehyde associated isolation of regulatory elements (FAIRE) analysis and the micrococcal nuclease digestion analysis (Pandya et al., 2013). Hence, it has been speculated

61 that the TbRAP1 protein associates differently with the active and the silent ES to control transcription elongation at different ESs.

The second most essential factor of the antigenic variation, after VSG silencing, is VSG switching. VSG switching events occur at a rate of 0.001% in laboratory cultured strains than in wild isolates. (Horn, D. 2014). Several studies have been done to identify molecular players involved in the regulation of VSG switching. As expected, TbRAD51, an essential factor for the homologous recombination, and its paralogues TbRAD51-3, TbRAD51-5, and BRCA2 (a mediator for RAD52) play an important role in VSG switching (McCulloch, 1999;

McCulloch, 2005). Depletion of TbRAD51 or its paralogues hinders the parasites recovery efficiency, hence, suppresses the VSG switching frequency. These observations have established the idea that the DNA recombination is a central mechanism of VSG switching. In addition, TbORC1 (origin recognition complex 1),

TOPO3α, and TOPO3α interacting protein RMI1, have been shown to suppress the VSG switching frequency (Benmerzouga et al., 2012, Kim, H. & Cross, G.,

2012, Kim, H. & Cross, G., 2013). Metabolic enzymes of the inositol phosphate pathway intermediates PIP5, PLC and PKC have also been shown to play an important role in VSG switching (Cestari, I. & Stuart, K., 2015). Besides these proteins, histone protein H1 also influences VSG switching (Povelone et al., 2013).

Every switcher can switch their new VSG from two different mechanisms.

One it can switch using transcription mediated switch and this type of switchers are known as in-sit type of switcher, where they activate transcription form previously silent ES and turn off the transcription at the previously active ES,

62 without any gene rearrangement. Second types of switchers use, homologous recombination and can be sub-divided into crossover and gene conversion. During the crossover event, reciprocal recombination occurs between active and silent

VSGs with its surrounding regions and no genomic information is lost. During gene conversion event, previously active VSG is lost and new VSG from the genome can be duplicated in to the active ES. However, depending on the size and location of the duplicating regions. If duplication involves only VSGs and its downstream regions than its called VSG gene conversion (VSG GC). But, if the duplication is of an entire silent ES than its called as ES gene conversion (ES GC). Complex type of switching events also occurs where entire ES is lost but instead of duplicating the silent ES, transcription mediated switching is turned off and this type of switching event is known as ES loss + in-situ. All of the studies mentioned above, have shown that mutants of all those proteins either increase or decrease the VSG switching frequency and switchers isolated from these switching assays usually arose from the gene conversion events (McCulloch & Horn, 2014).

However, the regulation of VSG switching is still poorly understood and causes behind each type of switching mechanism are mere speculations.

Efforts have been made to understand the trigger of VSG switching.

Bloodstream form expression sites are fragile and the location of DNA double strand breaks in the active ES can suggest the pathway of VSG switching (Glover et al., 2013). Artificially generated DNA breaks in the 70 bp repeat region almost always lead to the gene conversion mediated switching events. If the break is at the promoter region, fewer switchers have been observed (Glover et al., 2013).

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Highly repetitive in nature, the 70 bp repeats have been shown to serve as a major recombining site in the homologous recombination associated switching events

(McCulloch and Barry, 1999). The presence of the 70 bp repeats does not restrict the recombination partners (the VSG donors) to be in the silent ESs. However, the removal of 70 bp repeats from the active ES restricts VSG donors to the ES associated VSGs (Hovel-miner et al, 2016). Breaks generated in the 70 bp repeat regions are mostly lethal with survival rate less than 22% (Glover et al., 2013).

Most of the survivors after having a break in 70 bp repeat regions are switchers that arose through the gene conversion (Glover et al., 2013). Hence, it is clear that the maintenance of 70 bp repeat region in the active ES is essential for the switching mechanisms. RecQ, a important for DNA repair and DNA replication, when depleted, increased VSG switching frequency. In RecQ depleted cells, breaks generated between the 70 bp repeat region and the active VSG generates more crossover and the VSG gene conversion associated switchers

(Devlin et al., 2016). The DSB location is one of the major factors that determines the pathway of the VSG switching. The breaks at the 70 bp repeat region have shown to increase more than 250-fold in the VSG switching frequency (Boothroyd et al., 2009). These observations confirm that expression sites are fragile and breaks in the active ES are a potent trigger for VSG switching.

Telomeres are nucleoprotein complexes at linear chromosome ends.

Telomere proteins are essential for the telomere functions (Welinger et al., 2000, de Lange, 1999). In T. brucei TbTRF and TbTIF2 have been identified to play important roles in the VSG switching regulation (Jehi et al., 2014A, Jehi et al.,

64

2014B). Most switchers isolated from either TbTIF2 or TbTRF depleted cells were the ES GC/ES loss + In-situ switchers. Depletion of TbTIF2 also increased DSBs in the active and the silent ESs, and an increase in the TbRAD51 foci was observed by the immunofluorescence and an increase in the TbRAD51 loading at the ES was observed (Jehi et al., 2014A). Depletion of TbTRF does not affect the amount of the DSBs at the ESs (Jehi et al., 2016). Hence, the telomere proteins achieve similar switching outcomes via potentially different mechanisms.

TbRAP1 is a component of the T. brucei telomere complex and its involvement in the VSG silencing is remarkable. Yet, it was unknown whether

TbRAP1 has any role in the VSG switching regulation. Since depletion of TbRAP1 affects the VSG silencing, we speculate that depletion of TbRAP1 may also affect the VSG switching and the majority of the switchers may arise thorough the in situ switching mechanism as, all ES are derepressed in TbRAP1 RNAi condition. If that is true then, we do not expect an increase in the DSBs in the ESs upon the TbRAP1 depletion. Here in this chapter, we set to characterize the functions of TbRAP1 in the VSG switching.

3.2 Results

3.2.1 Generation of strains to study the roles of TbRAP1 in VSG switching

It has been shown earlier that the TbRAP1 double knockout strains are not viable, and within 24 hours of the knockdown of TbRAP1 by RNAi cells experience the arrest (Yang et al., 2009). In order to study TbRAP1’s function in the VSG switching we needed to introduce the conditional TbRAP1 RNAi construct

65 into the VSG switching assay strain developed and used earlier in the Cross lab

(Kim, H. and Cross, G., 2010). The VSG switching assay strain is known as

HSTB261 (Kim, H. and Cross, G., 2010). In HSTB261, the blasticidin resistance gene (BSD) is inserted immediately downstream of the active BES promoter. The puromycin resistance gene (PUR) fused with the Herpes simplex gene (TK) is inserted immediately upstream of the active VSG2 gene and downstream of the 70 bp repeats (Figure 3-1A). The HSTB261 strain is a derivative of the bloodstream form single marker (SM) strain, which expresses T7 polymerase and Tet repressor and allows inducible expression of the double stranded TbRAP1 RNAi for conditional knockdown (Wirtz et al., 1999). We will refer the parent HSTB261 strain as “S” (for switching) and the TbRAP1 RNAi strain as “S/RAP1i”. We also generated strains carrying the empty RNAi vector as a control, which we will refer as “S/ev”. After collecting 5 individual S/RAP1i clones, we first checked the growth phenotype of all the clones upon induction of the

TbRAP1 RNAi. Two clones, S/RAP1i-B1 and S/RAP1i-C1, showed strong response to the induction and were used for the following investigation (Fig. 3-1B-

D).

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Figure 3-1: Generation of the HSTB261/TbRAP1 RNAi strain. (A) Cartoon representing the HSTB261 strain. The active ES has been marked with a BSD resistance gene downstream of the promoter and a PUR-TK cassette between the 70 bp repeats and VSG2. Red arrow indicates the active ES. (B) Growth curves for different strains are plotted. S, S/ev, and S/RAP1i strains were induced with doxycycline for 24 hours. (C) Growth curves for S/RAP1i-B1 with 24 hour induction and subsequent recovery. (D) Growth curves for S/RAP1i-C1 with 24 hour induction and subsequent recovery.

Previously it has been shown that cells experience the growth arrest upon

depletion of TbRAP1 for 24 hours, and continued lack of TbRAP1 results in

eventual cell death. Clones S/RAP1i-B1 and C1 both showed the similar growth

arrest phenotype (Fig. 3-1B-D), while S and S/EV strains showed no difference in

67 the growth upon addition of doxycycline (figure 3-1B-D). In order to carry out successful switching assay, it is important to recover switchers. Hence, it is essential to identify the best time-point, where maximum TbRAP1 protein is depleted and cells can still recover after removing from doxycyclin. Therefore, we only induced TbRAP1 RNAi with doxycycline transiently. We attempted 18, 24, 36 and 48 hours of TbRAP1 RNAi induction followed by removal of doxycycline by extensive cell washing. Cells induced for 36 and 48 hours did not fully recover even after 5 days of culturing, and cells induced for only 18 hours did not show a strong growth arrest (Data not shown). Cells induced for 24 hours, however, showed growth arrest after 24 hours of induction and were able to recover completely within

48 hours post removal of doxycyclin (Figure 3-1B). Therefore, we decided to perform the transient induction for 24 hours.

Figure 3-2: Western blot analysis of TbRAP1 protein in S, S/ev, S/RAP1i strains after inducing cells with doxycyclin at various time points. Cells were induced for 24 hours followed by recovery for 48 hours. Whole cell extracts were collected at

68 each 24 hour interval. The rabbit anti-TbRAP1 antibody, 598, was used for western blotting. Anti-tubulin and anti-EF-2 (Santa cruz biotechnologies) antibodies were used to detect tubulin and EF-2 protein levels as loading controls. We next examined the TbRAP1 protein level in S/RAP1i, S and S/ev strains with or without induction of RNAi. No change in the TbRAP1 protein level was observed when we added and washed away doxycyclin in S and S/ev cells (Fig.

3-2, left two). We also observed no change in the TbRAP1 protein levels when we did not induced cells in S/RAP1i cells (Fig. 3-2, bottom right). However, ~70% reduction in the TbRAP1 protein levels was observed after 24 hours of induction in the S/RAP1i cells. The TbRAP1 protein levels recovered to the wild type level within the 48 hours after removal of doxycyclin (Fig. 3-2, top right). Tubulin and

EF-2 protein levels are examined to serve as a loading controls in western blotting analyses (Fig. 3-2).

To check whether the S/RAP1i cells recovered after removal of doxycyclin because they were responsive to RNAi induction, we re-induced the recovered cells the second time after 5th and 4th day of recovering in S/RAP1i B1 and C1 clones, respectively (Fig. 3-1C-D). After re-induction, the recovered cells experienced growth arrest again. As a control we also induced S and S/ev strains twice and these cells did not show any growth changes (Fig. 3-1C-D).

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Figure 3-3: Establishing the TbRAP1 complementation strain. (A) Growth curves of S, S/RAP1i, and S/RAP1i+F2H-TbRAP1 strains in the presence and absence of doxycyclin. Ectopic expression of the F2H-TbRAP1 allele complements the growth arrest phenotype in TbRAP1 RNAi cells. (B) Western analysis showed F2H-TbRAP1 expression upon induction. Anti-HA antibody (HA-probe, F7, Santa Cruz Biotechnologies) was used to detect F2H-TbRAP1 and Anti-EF2 antibody (Santa Cruz Biotechnologies) to detect EF-2 as a loading control. To rule out off-target effects of the TbRAP1 RNAi we introduced an ectopic

WT TbRAP1 allele in the S/RAP1i strain. The FLAG-HA-HA (F2H) epitope is fused with the N-terminus of the full-length TbRAP1 protein and the expression of F2H-

TbRAP1 is driven by the T7 promoter and under the control of Tet repressor.

Hence addition of doxycycline will allow expression of both the TbRAP1 RNAi construct and the F2H-TbRAP1 at the same time. We will refer to this strain as

“S/RAP1i + F2H-TbRAP1”. Out of multiple clones we obtained, we used the clone

70 that complements best for the defective growth phenotype. (Fig. 3-3). Induced expression of F2H-TbRAP1 was observed upon adding doxycyclin.

3.2.2 TbRAP1 suppresses VSG switching frequency

After the basic characterization of all the established strains, we performed the VSG switching assays (see the details in the Chapter-2). The parent strain “S” is resistant to the BSD (100 µg/ml) and puromycin (1 ug/ml) and addition of both the drugs helps to maintain a pure population of cells that expresses VSG2.

Immediately before starting the switching assay, we removed the BSD and PURO from the medium, which allows switchers to arise. The S strain carries a TK gene fused with PURO that is inserted in the active ES immediately upstream of the active VSG gene and highly expressed. Expression of the TK renders ganciclovir

(GCV) toxic to the cell, because GCV, a nucleoside analogue, can be incorporated into DNA instead of thymidine by TK, causing replication problems (Kim, H &

Cross, G, 2010). Switchers will switch off the expression of VSG2 and at the same time, the expression of TK and Puro. Therefore, switchers and resistant to GCV and can be selected by GCV.

S, S/EV, S/RAP1i, and S/RAP1i + F2H-TbRAP1 cells were either induced or uninduced with doxycycline for 24 hours followed by 2 to 3 days of recovery, and all strains were grown for the same number of population doublings. S/RAP1i cells without any induction was used as a control. Switchers were selected among the recovered cells by GCV, as all the switchers are expected to either lost or not express the TK gene. We also tested plating efficiency of all strains by plating 1

71 cell / well in the medium without GCV. Plating efficiency was calculated by dividing the number of wells showing growth by the total number of wells plated. Raw switching frequency was calculated by dividing the number of GCV resistant clones by the total number of cells plated. Final switching frequency was obtained by normalizing the raw switching frequency with the plating efficiency (Figure 3-4).

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Figure 3-4: A transient depletion of TbRAP1 increases VSG switching frequency. (A) A transient depletion of TbRAP1 increased VSG switching frequency. VSG switching frequencies in S, S/ev, S/RAP1i, and S/RAP1i+F2H-TbRAP1 cells with (+) or without (-) doxycycline for 24 hours are shown in a bar graph. B1 and C1 are

73 independent clones of S/RAP1i. Average switching frequencies were calculated from at least four independent switching assays. Standard deviations are shown as error bars. P values (unpaired t tests) between the switching frequencies in S/ev and S/RAP1i cells are indicated. The difference is considered significant when the P value is less than 0.05. (B) Platting efficiency for S, S/ev, S/RAP1i, and S/RAP1i+F2H-TbRAP1 cells with (+) or without (-) doxycycline for 24 hours are shown in a bar graph. We observed a 6-7 fold increase in the VSG switching frequency when

TbRAP1 was transiently depleted in the S/RAP1i cells, compared to S/ev. Both

TbRAP1 RNAi clones showed similar levels of increase in the VSG switching frequency. S and uninduced S/RAP1i cells did not show any significant change in the VSG switching frequency when compared to S/ev cells (Fig. 3-4A). The small increase in switching frequency in the uninduced S/RAP1i cells is due to the leaky expression of the RNAi vector as we discussed above. The VSG switching frequencies observed in all control strains are similar to previously published results (Kim, H. & Cross, G., 2010, Jehi et al., 2014A, Jehi et al., 2014B, Povelone et al., 2012, Devlin et al., 2016, Jehi et al., 2016). Also, the S/RAP1i + F2H-

TbRAP1 cells showed no significant increase in the VSG switching frequency upon induction, indicating that the increase in the VSG switching frequency is due to the depletion of TbRAP1. The Plating efficiencies of all strains were similar (38% -

54%) (Fig. 3-4B).

3.2.3 TbRAP1 suppresses VSG gene conversion

One advantage using the S strain (HSTB261, Kim & Cross, 2010) to examine the VSG switching is that it allows us to determine the switching mechanism of each switcher we obtain. VSG switching can occur through an in- situ, a crossover, a VSG and/or ES gene conversion, and/or an ES-loss + in-situ

74 switch (details of VSG switching events are explained in chaper-1 & Fig. 3-5). All switchers were recovered and cultured followed by the characterization of their resistance to the BSD and puromycin. We also isolated their genomic DNA and performed PCR using primers specific for the BSD and the VSG2 gene to determine whether the switchers have lost these genes. Based on the results we can categorize each switcher for its switching mechanism (Fig. 3-6A).

Figure 3-5: Different VSG switching mechanisms. The S strain (Parent, HSTB261) is shown on top left and as described in figure 3.1. The parent strain will be resistant to BSD (100 µg/ul) and Puromycin (1 µg/ul). Red arrow indicates the active ES. In an in-situ switcher a previously silenced ES is fully transcribed, which is denoted by a long red arrow and the previously active ES is silenced. In a crossover switcher, reciprocal recombination exchanges the PUR-TK cassette and the active VSG (together with its downstream telomere sequence) with a silent ES- linked VSG. In a gene conversion, the originally active VSG (VSG2) is lost and a previously silent VSG is duplicated into the active ES. If the neighboring sequence encompasses only VSG neighboring sequences and the downstream telomere, it is known as the VSG gene conversion switch. If the entire ES is duplicated it is known as the ES gene conversion switch. In an ES loss + in-situ switching, the originally active ES is lost, and a previously silent ES becomes fully transcribed. The expected phenotypes and genotypes in S (parent) cells and different kinds of

75 the switchers are indicated. “S” means sensitive; “R” means resistant; “-“means the gene is lost; “+” means the gene is still present.

Although it is known that only one ES is fully active, genes that are immediately downstream of the silent promoters are not completely silenced

(Vanhamme et al., 2000). In fact, silent promoters are weakly active and the transcription goes down a few kb on the silent ESs. Hence, in in-situ switchers

(where an originally silent ES is fully transcribed while the originally active ES is silenced), the BSD marker is now transcribed at a basal level, and the switcher is resistant to 5 µg/ml BSD but not 100 µg/ml BSD (Fig. 3-6A) (Kim, H. & Cross, G.,

2010). Therefore, we tested all switchers to see whether they are resistant to the two concentrations of BSD. No genetic information should be lost in in-situ switchers. Therefore, PCR using BSD and VSG2 primers should yield positive results.

In crossover switchers, reciprocal recombination takes place between the active VSG (with its neighboring sequences) and a silent ES-linked VSG.

Therefore, crossover switchers should be resistant to 100 µg/ml BSD, and PCR using BSD and VSG2 primers should yield positive results (Fig. 3-5, Fig. 3-6A).

The VSG gene conversion switchers (Fig. 3-5) should be resistant to 100 µg/ml

BSD but sensitive to puromycin. PCR using VSG2 primers will yield a negative result and PCR using the BSD primers will yield a positive result. ES gene conversion switchers will be sensitive to both concentrations of BSD and

Puromycin. PCR using BSD and VSG2 primers will both yield a negative result.

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Finally, switchers can also arise thorough more than one simple pathway.

For example, in ES loss + in-situ switchers, the originally active ES is lost and a previously silent ES becomes fully transcribed. By examining phenotypes

(sensitivity to BSD and puromycin) and genotypes (test the presence of BSD and

VSG2 genes by PCR) of the switcher, it is impossible to separate ES gene conversion and ES loss + in-situ switchers. Therefore, we classify these two in one category. These can only be differentiated by examining switchers’ karyotype on pulsed-field gel electrophoresis (PFGE) and Southern blotting. ES gene conversion switchers will have the newly active VSG gene duplicated into the originally active ES, while the newly active VSG is not duplicated in ES loss + In- situ switchers. We also recovered few switchers that have lost the originally active

VSG2 while a previously silent ES became fully activated. This type of switchers was resistant to 5 µg/ml BSD, and VSG2 PCR was negative. This is called VSG loss+ In-situ. We also isolated few switchers that duplicated the newly active VSG to the originally active ES, but original donor gene was lost from its original locus.

We refer to these switchers with such complicated recombination mechanisms as

“others” switchers.

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Figure 3-6: TbRAP1 suppresses VSG gene conversion. (A) Principle of determining switching mechanisms by characterizing switcher phenotypes and genotypes. Cells are incubated with indicated amount of antibiotic and cell growth is scored after 3 days. Small scale genomic DNA was isolated from switchers and PCR analysis is done using primers specific to BSD and VSG2. (B) Percentage of different switching mechanisms among all obtained switchers in S, S/ev, S/RAP1i, and S/RAP1i+F2H-TbRAP1 cells is shown both in numbers and as different

78 colored bars (where the length of the bars represents the percentage). Total number of switchers is written on top of each bar. After collecting switchers from all the different strains, we characterized all switchers for their genotypes and phenotypes as described above to determine their switching mechanisms. All control strains, including S, S/ev, uninduced

S/RAP1i, and S/RAP1i + F2H-TbRAP1, showed similar distribution of different switching mechanisms, and the ES gene conversion/ ES loss + In-situ switchers are the predominant type (68% to 78%), while VSG GC switchers are less predominant (16% to 32%). However, in the TbRAP1 depleted cells, most switchers arose from VSG GC (62-74%), while a smaller portion of the cells switched through an ES GC or ES loss + in situ (19-26%). The portion of cells that switched through an in situ or a crossover/telomere exchange did not seem to change significantly upon TbRAP1 depletion. When the ectopic F2H-TbRAP1 was expressed in the S/RAP1i cells, ES GC or ES loss + in situ switch were again the most frequent events (50%) and a smaller portion of cells switched through the

VSG GC (43%) (Fig. 3-6B). The list of all characterized switchers and their phenotypes and genotypes are included in appendices (Table 6 – Table 13).

To confirm that we classified the switchers in the correct categories by examination of their genotypes and marker expression status, we randomly selected few switchers from different switching types, derived from the induced

S/RAP1i cells and further analyzed their karyotype. We first identified which VSGs were expressed in the recovered switchers by RT-PCR and sequencing analysis

(Chapter-2, cloning of the active VSG). These were confirmed to be all different from the originally active VSG2. Subsequently, intact genomic DNA of these

79 switchers were separated by PFGE and analyzed by Southern blotting using probes for BSD, the originally active VSG2, and the newly active VSG (Fig 3-7).

Interestingly, karyotype analysis showed that all analyzed switchers that could have arisen from an ES GC event or an ES loss + in situ switch turned out to be the latter type of switch. Although we cannot perform the extremely laborious karyotype study for all VSG switchers, these observations are based on a random set of samples, suggests that the ES loss + in situ switch is a frequent event among switchers from S/RAP1i cells (Fig. 3-7A & 3-7E). We also performed PFGE on the other types of switchers and all were correctly categorized, indicating robust methodologies of identifying switching events through characterization of switcher phenotypes and genotypes.

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Figure 3-7: Validating selected switchers by PFGE. DNA plugs were prepared from VSG9, VSG18 and VSG3 expressing switchers as explained in chapter 2. Samples were run on a CHEF DRII or a CHEF DRIII (Bio-rad) apparatus for 120 hours using the following conditions: 1500s-700s pulse, 2.5 Volt/cm voltage, at 140C. After electrophoresis, gels were stained with EtBr and the picture of the stained gel was presented on the left of each figure with molecular marker labeled. Switchers numbers are labeled on top of each well and details about each switcher have been categorized in table-11. (A & E) VSG 9 expressers. (B) VSG 3 expressers. (C & D) VSG 18 expressers. Switchers 1, 3, 4, 12, 26, 48, 93, 100, and 117 have lost the BSD and VSG2 genes. None of them have duplicated their newly active VSG into the active ES. Hence, they are ES loss + in-situ switchers. Switcher 82 has lost the BSD and VSG2 genes. It duplicated the newly active VSG9 gene to the active ES. However, VSG9 was lost from its original locus and hence we call this “other” switcher. Switchers 18, 34, 35, 63 are In-situ switchers, as they have not lost the BSD and VSG2 genes. VSG2 is still at its original locus. They are all VSG18 expressers and express it only from its original place. Switcher 15 has not lost the BSD gene but has lost VSG2. It has duplicated the active VSG18 into the active ES. This clone has undergone VSG gene conversion. Switcher 87 has lost VSG2 and kept BSD. It has translocated VSG3 to the active ES from its original locus. Hence this is an “other” switcher.

3.2.4 TbRAP1 is essential for maintaining subtelomere and telomere integrity

Although there is a difference in the VSG switching pattern between control and S/RAP1i cells, the majority of the switchers arose through the gene conversion events in both cells. Gene conversion requires the duplication of a silent VSG and its neighboring sequences into the active ES. As DNA recombination frequently initiates with the DSBs, we anticipated that depletion of TbRAP1 might increase the DSB amount at the telomeric and the subtelomeric regions, especially at the active VSG locus, as suggested previously (Glover et al, 2013). We therefore performed the Ligation-Mediated PCR as previously published (Boothroyd et al.,

2009, Glover et al., 2013, Jehi et al., 2014A) and examined the ES regions for the

DSBs before and after the depletion of TbRAP1 in S/RAP1i cells.

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In a LMPCR assay, isolated genomic DNA was treated with or without T4

DNA polymerase. The T4 DNA polymerase will polish any sticky broken DNA ends into the blunt ends. Subsequently, a double stranded adaptor is prepared so that it has a blunt end on one side and an overhang on the other. This adaptor is then used to ligate with the genomic DNA. Only genomic fragments with the blunt ends

(due to DSBs) can be ligated to the blunt end of the adaptor (Chapter - 2). After ligation we performed PCR using a forward primer that is specific to a genomic locus and the bottom strand of the adaptor as the backward primer. The PCR products were subsequently separated by the gel electrophoresis and detected by the locus specific probes in Southern blotting. This protocol amplifies all the DSBs at the locus of interest, as single stranded breaks cannot be ligated to the adaptor.

Because the DSBs can occur at different places, the PCR products often have heterogeneous sizes (Fig. 3-9F).

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Figure 3-8: Depletion of TbRAP1 increased the number of DSBs in and downstream of the subtelomeric VSG genes. (A) The principle of LMPCR. After the DSBs occur, the adapter is ligated with the genomic DNA at the break sites if they have blunt ends. Treating genomic DNA with T4 DNA polymerase will convert staggered broken ends into the blunt ends. The ligated products are then amplified by the PCR using the locus-specific forward primer and the adapter specific backward primer. The PCR amplified products are subsequently detected by locus-specific probes in Southern analysis. LMPCR analyses were performed in S/RAP1i clone B1 (B-D) and S/ev (E) cells. The LMPCR products were hybridized with VSG2 (B, E), VSG21 (C), and the ES promoter (D) probes. In each panel, the Ethidium bromide (EtBr)-stained LMPCR products are shown at the top, the Southern blot result is shown in the middle, and the PCR products using primers specific to the TbRAP1 gene (as a loading control) are shown at the bottom. The amounts of input genomic DNA, either treated (+) or untreated (−) with T4 DNA polymerase, were marked on top of each lane. In (C), the black bar on the right of

84 the Southern blot marks the position of the LMPCR products more than 1.6 kb long. Interestingly, we detected more DSBs in TbRAP1-depleted cells compared to the uninduced S/RAP1i cells only when subtelomeric ES-linked VSG probes were used, including the active VSG2 (Fig. 3-8B) derepressed VSG21 (Fig. 3-8C), and derepressed VSG15 (Fig. 3-9A), but not when ES promoter (Fig. 3-8D) or the

70 bp repeat (Fig. 3-9B) probes were used. After quantifying the signals, it is observed that the number of DSBs at VSG loci increased mildly but significantly

(Fig. 3-9E). In addition, depletion of TbRAP1 does not increase the number of

DSBs at a random chromosome internal SNAP50 locus (Fig. 3-9C), indicating that

TbRAP1 depletion affects the chromosome integrity specifically at the subtelomeric/telomeric regions. We did not detect any change in the DSB levels in S/ev control cells at the VSG2 or the ES promoter regions (Fig. 3-8E, Fig. 3-9D).

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Figure 3-9: Depletion of TbRAP1 led to increased number of the DSBs in and downstream of the subtelomeric VSG genes. LMPCR analyses were performed in S/RAP1i clone B1 (A-C) and S/ev (D) cells. The LMPCR products were hybridized with VSG15 (A), 70 bp repeat (B), SNAP50 (A random chromosome internal gene) (C), and ES promoter (D) probes, respectively. In each panel, the Ethidium bromide (EtBr)- stained LMPCR products are shown at the top, the southern blot result shown in the middle, and the PCR products using primers specific to the TbRAP1 gene (as a loading control) are shown at the bottom. The amounts of input genomic DNA, either treated (+) or untreated (-) with the T4 DNA polymerase, were marked on top of each lane. (E) Quantification of fold changes in DSB levels at ES promoter (EsPr), 70 bp repeats, VSG2, VSG21, VSG15 and SNAP50 loci before and after depletion of TbRAP1. Averages were calculated from at least 3 independent experiments and standard deviations were shown as error bars. (F) Different sized LMPCR products are generated when DSBs are located at different places downstream of the forward primer using in PCR, which is represented by a blue arrow in (a), (b) and (c). (a) DSBs are within VSG gene. (b) DSBs are near

86 the telomere/subtelomere junction. (C) DSBs are in telomeric repeats. The adaptor is represented in blue lines. Red bolts, represent DSBs. The differences between VSG gene and the telomeric repeats and the distance between the forward primer and most DSBs detected in the assay are marked. Theoretically, in the LMPCR analysis, a locus specific probe can detect all the DSBs located downstream of the probe, and the sizes of the PCR products vary with the positions of the DSBs. However, DSBs located close to the forward primer used in the PCR amplification would be more easily detected because shorter ligation products are amplified at a higher efficiency by PCR (Boothroyd et al., 2009). All subtelomeric VSG probes detected major LMPCR products of ~1 kb, while the forward primers lie at the N-terminus of each VSG gene, suggesting that

DSBs are concentrated inside the VSG genes. Using the VSG21 probe, we also observed low amount of longer LMPCR products when more input DNA (54 ng) was used (Fig. 3-8C, marked by a black bar on the right), suggesting that DSBs also occurred near the telomere/subtelomere junction (Fig. 3-9F) and in the telomeric repeats downstream of the VSG21 gene (Fig. 3-8C). However, DSBs located more downstream and those in the telomeric repeats would produce longer ligation products with more TTAGGG repeats, which are often poorly amplified by

PCR (Boothroyd et al., 2009). Therefore, it is likely that loss of TbRAP1 also results in more telomeric DSBs but these are rarely detected by the LMPCR using VSG probes. Still, since ES-linked VSGs are located within 2 kb from the telomeric repeats, processing DSBs in the telomeric DNA can easily allow breaks to migrate into the subtelomeric VSG loci. We detected increased number of DSBs inside the

VSG genes, which would explain why most VSG switching events in TbRAP1- depleted cells are VSG GC events.

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Although LMPCR can detect breaks at subtelomeric VSG locus, it is really difficult to identify breaks at the telomeres due to repetitive sequences. It is also dependent on the size to accurately measure the amount of breaks present at the repetitive regions. Due to these reasons we examined the association of TbRAD51 and phosphorylated histone 2A (γH2A, a DNA damage marker) (Lucy, G. & Horn,

D., 2012) with the active and silent BES chromatin and telomere chromatin, which are indicative of DSBs at these regions. This way we can detect the DSBs at the telomere, which is not possible using the LMPCR approach.

Figure 3-10: Characterization of antibody that specifically recognize the phosphorylated and unphosphorylated H2A by western blotting. WT cells were grown for 16 hours with or without 1.5 µg/ml phleomycin (Glover et al., 2013). (Phleomycin causes DNA DSBs). Increase in γH2A level was observed in cells treated with phleomycin. No change in the total H2A protein level was observed with anti-H2A antibody.

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Figure 3-11: TbRAP1 depletion leads to more DSBs at the telomeres. Chromatin immunoprecipitation was done using anti-TbRAD51 and anti-γH2A antibodies. (A) Uninduced (0 hour samples) and induced (24 hour samples) S/ev and S/RAP1i cells were used for ChIP and isolated samples were blotted on the Nylon membrane for subsequent Southern hybridization using either the telomere probe or the 50 bp repeat probe. Blots were exposed to phosphor imagers.

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Quantifications of the signals from hybridization with the TTAGGG probe (B) or the 50 bp repeat probe (C) are shown. At least three experiment results are used for calculating the average and standard deviation (shown as error bars). Unpaired t- test was performed to calculate the P values (a value of <0.05 is considered as significantly different). To examine whether the DSBs occur at the telomere after TbRAP1 is depleted, we performed CHIP assays using anti-TbRAD51 (a kind gift from Dr.

Richard McCulloch, University of Glasgow) and anti-γH2A antibodies. The rabbit anti-γH2A antibody was custom-raised against the phosphorylated H2A peptide and affinity purified. To confirm the purity of anti-γH2A antibody, we artificially generated DNA damage in WT cells, by incubating them with 1.5 ug/ul of phleomycin and expression of γH2A was confirmed by western blot analysis

(Figure 3-10). After confirming the antibody, CHIP was performed in the S/ev and

S/RAP1i cells at 0 and 24 hours after adding doxycycline. The ChIP products were blotted on the nylon membranes and hybridized with a telomere probe containing

800 bp of (TTAGGG)n or a 50 bp repeat probe (Fig. 3-11A). The 50 bp repeats are located immediately upstream of the ES promoters. The blots were exposed to phosphorimagers and hybridization signals were quantified using image-quant software (GE) (fig. 3-11B and Fig. 3-11C). Upon depletion of TbRAP1, we observed that significantly more TbRAD51 and γH2A were located at the telomeres. No significant change in the association of both proteins with the telomere and 50 bp repeats was observed in S/ev and S/RAP1i cells (Fig. 3-11C).

All the ESs including the active one and all the silent ones are flanked by the same downstream telomere repeats and upstream 50 bp repeat. Therefore, the above described ChIP-slot blot cannot deduce whether the DSBs are at the

90 active ES or at the silent ones. However, we did not detect more DSBs at the ES promoter regions upon depletion of TbRAP1 using the LMPCR analysis (Figure 3-

8D). Conversely, in ChIP-slot blot analysis, we detect an increase in the association of γH2A with the ES promoter chromatin.

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Figure 3-12: TbRAP1 depletion increases the amount of DSBs at the subtelomeric

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VSG loci. ChIP was performed using anti-TbRAD51 antibody (Top), anti-γH2A antibody (Bottom), and IgG (as a control) in S/ev and S/RAP1i strains at 0 and 24 hours after induction with doxycycline. ChIP products were quantified by quantitative PCR using primers specific to VSG2 (the active VSG), VSG21 (a Silent VSG), BES Promoters, rRNA and tubulin as indicated. The enrichment of ChIP product over the input was calculated. Average calculated from at least three experiments are shown in the bar graph, with the Standard deviations shown as the error bars. P-values are calculated using unpaired t-test.

To obtain results with a higher resolution and determine whether DSBs are found along the subtelomere ESs, we further performed the ChIP-qPCR using the same anti-RAD51 and anti-γH2A antibodies. The ChIP products were analyzed by quantitative PCR using primers specific to VSG2 (the active VSG), VSG21 (a Silent

VSG), ES Promoters, rRNA and tubulin. We detected significant increases in the association of TbRAD51 and γH2A at the active VSG2, a silent VSG21, and ES promoter regions (Fig. 3-12) upon depletion of TbRAP1 for 24 hours. No significant changes were seen in S/ev cells. No significant increase of the DSB amounts at the rRNA and tubulin loci. We detected more DSBs at the active and silent VSGs by the ChIP-qPCR (Fig. 3-12), which is consistent with the LMPCR data (figure 3-

8 and 3-9). Therefore, upon depletion of TbRAP1, we detect significant more DSBs at the telomeres and VSGs.

3. 3. Discussion

Ever since the discovery that the VSGs are expressed from the subtelomeric regions (de lange, 1982) it has been proposed that the telomeres play important roles in the VSG silencing and the VSG switching regulation

(Dreesen et al., 2007). Indeed, when the telomere next to the active VSG gene

93 becomes extremely short, the VSG switching frequency is increased approximately ten fold (Hovel-Miner et al., 2012). In addition, we have shown recently that the duplex telomere binding factor TbTRF and its interacting factor

TbTIF2 both suppress the VSG switching (Jehi et al., 2014A, Jehi et al., 2014B).

Furthermore, we have shown that TbRAP1, an intrinsic component of the T. brucei telomere complex, is essential for the silencing subtelomeric VSGs (Yang et al.,

2009, Pandya et al., 2013). TbRAP1 interacts with TbTRF (Yang et al., 2009), and we now show that TbRAP1 also suppresses the VSG switching (Fig. 3-4).

However, functions of TbRAP1 in VSG regulation are quite different from that of

TbTRF and TbTIF2. First, TbRAP1 is essential for subtelomeric VSG silencing

(Yang et al., 2009, Pandya et al., 2013), while TbTRF and TbTIF2 do not affect

VSG silencing (Pandya et al., 2013, Jehi, et al., 2014A). Second, the most frequent

VSG switching events in TbRAP1-depleted cells and those in TbTRF and TbTIF2- depleted cells are different (Jehi et al., 2014A, Jehi et al., 2014B).

We transiently depleted TbRAP1 in the switching strain, S/RAP1i. As expected and reported previously (Yang et al., 2009), knockdown of TbRAP1 arrests T. brucei cells within 24 hours. They could be rescued by extensively washing them from the doxycycline, suggesting that cells can recover post induction. Also, they did not lose their RNAi construct from the cells as they could be reinduced at later time points. Moreover, the ectopic expression of F2H-

TbRAP1 could revert the growth arrest phenotypes completely. After performing the switching assay we observed 6-7 fold increase in the VSG switching rate in

TbRAP1 RNAi strain. Thus we prove that, indeed, TbRAP1 is involved in the VSG

94 switching regulation.

In cells transiently depleted of TbRAP1, most VSG switchers arose from the

VSG gene conversion events (Fig. 3-6B). This was completely opposite to all the control strain used in the assay, as their most VSG switchers arose through the

ES GC/ ES loss + in-situ. This is the only telomeric protein which switches through a different switching mechanism. Previously TbTIF2 and TbTRF, telomeric proteins, were reported to be involved in the VSG switching and their major switching mechanism stays similar to the wild type of strain used in each respective experiment (Jehi et al., 2014A, Jehi et al., 2014B). Hence, we wondered what could be the reason behind this shift. However, to prove this is true we may need to perform more experiments in different type of backgrounds and in-vivo in the mouse models, to remove the possibility of strain specific mechanisms. However, other groups have performed switching assays in moderately different type of switching background and reported the ES GC or the in-situ to be a predominant switching mechanism (McCulloch, 1999, Povelones et al., 2013). Depletion of cohesin subunit TbSCC1 led to the premature dissociation of the sister chromatids that contain the active VSG ES, which leads to more frequent VSG switching with many in situ VSG switchers (Landeira et al., 2009), suggesting that inheritance of the ES expression status helps suppress the VSG switching. Depletion of TbRAP1 leads to disruption of the telomeric silencing, and the silent ESs become expressed at a significantly higher level than those in the WT cells (Yang et al., 2009).

Expression of the multiple ESs simultaneously presumably disrupts the inheritance of the ES expression status and may result in the higher VSG switching frequency.

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However, should this be the case, we expect more VSG switchers arose from in situ switching in TbRAP1-depleted cells. Since the VSG GC is the most frequent event in TbRAP1-depleted cells, it is unlikely that more frequent VSG switching is directly due to the derepression of silent ESs.

Upon depletion of TbRAP1, we observed slightly more DSBs at the VSG loci (both the active and the derepressed) that are immediately upstream of the telomeric repeats (within 1.5 kb), but not around the 70 bp repeats that are several tens of kb and at least 2.5 kb upstream of the telomeric DNA, respectively (Hertz-

Fowler et al., 2008). On the contrary, we observe increased association of

TbRAD51 and γH2A, at the VSG loci (both the active and the silent), the ES promoter (near the 50 bp repeat region) and the telomeres. Although there is a clear increase in both TbRAD51 and γH2A loading at the subtelomeres and the

ES promoters (Fig. 3-12, Fig. 3-13), only γH2A seems to have significant increase in loading at the 50 bp repeat region. This difference may be due to different mechanisms of the loading of two proteins. γH2A is known to be loaded for nearly

2 Mb away from the site of break on the chromosomes in mammals (Kinner et al.,

2008). Hence it is possible that the majority of the DSBs are present only at the telomeres and telomere proximal VSG loci in TbRAP1 depleted T. brucei cells, but

γH2A loading is spread far upstream. These observations provide us the first view of TbRAP1’s involvement in the telomere end protection in T. brucei. In contrast, in cells transiently depleted of TbTRF and TbTIF2, most VSG switchers arose from the ES GC or ES loss coupled with an in situ switch (Jehi, S. et al, 2014 A & B).

Particularly, loss of TbTIF2 dramatically increased the subtelomeric DSB numbers

96 at the active and the silent VSG loci, 70 bp repeats, VSG pseudogenes in ESs, and ES promoters (Jehi, S., 2014 B). These observations clearly indicate that

TbRAP1 and TbTRF/TbTIF2 have distinct functions in regulation of the VSG silencing and switching.

TbRAP1 depletion did not increase the DSB numbers when the ES promoter and the 70 bp repeat probes were used in the LMPCR (Fig. 3-8 and Fig.

3-9). However, significantly more TbRAD51 and γH2A are associated with the ES promoters upon depletion of TbRAP1 when analyzed by ChIP-qPCR (Fig. 3-12).

The ES-linked subtelomeric VSGs are located adjacent to the telomeric repeats.

In the Lister 427 strain used in this study, the distance from the end of each VSG gene to the first TTAGGG repeat is 0.7 kb (VSG2), 0.8 kb (VSG15), and 1.5 kb

(VSG21), respectively, while the distance between the last 70 bp repeat and the first telomeric repeat is 2.4 to 8.9 kb. ES promoters are 40-60 kb upstream of the telomeric repeats (Hertz-Fowler et al., 2008). DSBs at the subtelomeric VSGs are significantly higher when detected either by the LMPCR or via the ChIP-qPCR, suggesting deletion of TbRAP1 leaves the junction of telomeres and subtelomeres unprotected. We observed a similar gradient effect for TbRAP1-mediated telomeric silencing, where depletion of TbRAP1 leads to strongest derepression of

VSG genes located within the 2 kb from the telomere, intermediate level of derepression of the VSG pseudogenes located 10-20 kb upstream of the telomere, and weakest derepression of selectable markers next to the ES promoter that are

~50 kb upstream of the telomere (Yang et al., 2009). Both TbRAP1’s effects on the ES silencing and the genome integrity are strongest at the telomere, which is

97 consistent with the idea that the TbRAP1 protein is concentrated at the telomere and spreads its effects from telomere towards chromosome internal regions.

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CHAPTER IV

THE TELOMERE TRANSCRIPT (TERRA) AND ANTIGENIC VARIATION

4.1 Introduction

Telomeric position effect has been identified in Drosophila melanogaster, yeast, human, mouse, T. brucei, and Plasmodium falciparum (Hazelrigg et al.

1984, Gottschling et al. 1990, Horn and Cross 1995, Baur et al. 2001, Frietas-

Junior et al. 2005, Duraisingh et al. 2005, Pedram et al. 2006), which is widely accepted to play an important role in strong heterochromatic structure formation at the telomeres. Strong heterochromatic structures suppress the expression of subtelomere genes. Hence, telomeres have long been considered transcriptionally silent. However, recent studies showed that telomeres are transcribed into a long non-coding RNA called TElomeric Repeat containing RNA (TERRA) in birds, trypanosomes, diptera, humans, rodents, budding yeast, fission yeast, and zebra fish (Morcillo et al. 1988, Rudenko & Ploeg 1989, Solovei et al. 1994, Azzalin et al.

2007, Luke et al. 2008, Schoeftner and Blasco 2008, Greenwood and Cooper

2011). In mammals, TERRA is transcribed from the C-rich telomere strand, giving rise to UUAGGG repeats with sizes ranging from 100 bp - 9 kb (Azzalin et al. 2007).

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In budding yeast TERRA is usually <500 bp long. Along with TERRA, budding yeast also expresses molecules called ARRET that are antisense to the subtelomere and do not contain any detectable telomeric repeats (Luke et al.

2008). On the other hand, the telomere transcriptome of fission yeast is much more complicated and contains multiple transcripts including TERRA, ARRET, ARIA, and αARRET (Bah et al. 2012), suggesting both the strands of telomeres are transcribed. In the bloodstream form T. brucei, telomeres are also transcribed from the C-rich strand ranging from 0.5 - 4 kb (Rudenko and ploeg 1989). Therefore, it is clear that telomere transcription and the generation of heterogeneous RNA molecule is conserved from yeast to mammals.

TERRA is transcribed by RNA polymerase II in humans, mice, and budding yeast (Schoeftner and Blasco, 2008, Luke et al. 2008). In trypanosomes, however, it is recognized that TERRA is not sensitive to α-amanitin (Rudenko and Ploeg

1989). One simple hypothesis proposed was that in T. brucei TERRA is transcribed from the active ES-adjacent telomere as a result of read through pass the last VSG gene in the PTU. However, in T. brucei TERRA promoters have not been identified and further investigation is required. In humans, a CpG island consisting of “61-

29-37” repeats located in the subtelomeric region functions as the promoter for the

TERRA transcription, and in yeast TERRA promoters are well defined (Azzalin et al. 2007, Schoeftner and Blasco 2008, Nergadze et al. 2009). TERRA is found mostly in the nucleus co-localizing with telomeres (Azzalin et al. 2007, Schoeftner and Blasco 2008). Not only does TERRA co-localizes physically with telomeres, but also it interacts with telomere proteins, such as TRF1 and TRF2. In humans

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TERRA interacts with TRF2 and ORC (origin recognition complex) to form ternary structure close to telomeres (Deng et l., 2009). The interaction between TERRA and several heterochromatin-associated factors including heterochromatin protein

1 (HP1) and H3K9 methyltransferases has been established (Deng et al. 2009).

These RNA-protein interactions are essential for several telomere maintenance mechanisms. A portion of TERRA is modified post-transcriptionally. Although, majority of the TERRA is found to be inside the nucleus, a portion of TERRA is poly-adenylated in humans, yeast, and trypanosomes (Rudenko and Ploeg 1989,

Azzalin and Lingner 2008, Luke et al. 2008). Importance of this post-transcriptional modifications are not explored yet.

Telomere proteins are known to influence TERRA expression levels. In budding yeast, ScRap1/Sir and ScRap1/Rif complexes regulate the TERRA transcription, and the TERRA levels are upregulated upon introducing the ScRap1 mutant that fails to recruit Sir proteins (Iglesias et al. 2011). In fission yeast, deletions of Taz1 or SpRap1 lead to upregulation of TERRA transcription

(Greenwood and Cooper 2011). Thus, it is clear that telomere proteins are important in regulation of TERRA expression. However, importance of T. brucei telomere proteins in TERRA expression regulation is not studied. Depletion of

TbTRF and TbTIF2 did not show derepression of VSGs, but TbRAP1 depletion did

(Pandya et al.,2013, Jehi et al., 2014A, Yang et al., 2009). If above stated hypothesis that TERRA in T. brucei is a product of RNA pol I read-through post

VSGs, then we expect to see TbRAP1 affect the TERRA expression regulation.

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Depletion of TERRA by siRNA led to loss of cell viability and the formation of telomere dysfunction induced foci (TIFs), a hallmark of telomere DNA DSBs, in mammalian cells (Deng et al. 2009). Interestingly, depletion of TERRA also affected telomere heterochromatin with reduced recruitment of ORC complex and decreased amount of H3K9 trimethylation (Deng et al. 2009). This observation suggests that TERRA might have a role in regulation of the telomere chromatin structure. Conversely, increase in telomere length increases H3K9 trimethylation deposition to the telomeres, suppressing TERRA expression levels (Arnoult et al.,

2012). Hence telomere lengths may play a significant role in the TERRA expression regulation. In T. brucei, depletion of all the three telomere proteins leads to severe growth arrest in comparatively short period of time, hence, detecting differences in telomere length is technically limited. Therefore, to study different effects of TERRA expression levels in T. brucei due to telomere length variation requires us to develop better methodologies. We have not pursued to analyze this particular role of TERRA here in this thesis.

It has also been shown recently that, majorly higher amount of TERRA can form a RNA:DNA hybrid (R-loop) with the telomere, which prompts DSB formation at nearby regions (Balk et al., 2013, Arora et al., 2014). R-loops (RNA:DNA hybrids) are the three stranded nucleic acid structures where a complementary

RNA strand hybridizes with a DNA strand, leaving the other strand as a single strand DNA. R-loops have paradoxical effects on cell fate (Groh and Gromark,

2014). In some cases, it is beneficial to the cells. For example, during class switch recombination (CSR) in human cells, formation of R-loops provides generous

102 diversity for the development of multiple antibodies in the activated B-cells

(Chaudhari et al., 2004). In bacteria E. coli, replication of COlE1 type plasmid requires the R-loops to form and overlap with the region, which later can be cleaved by RNaseH and leaves the 3’-OH end of the RNA strand open for further replication of the plasmid (Hamperl et al, 2014). On the other hand, the

R-loop has one single strand DNA that is exposed, which can be easily nicked and cause DNA damage at the near vicinity (Hamperl et al, 2014). In yeast and human cells R-loops have been shown to facilitate telomere recombination in a telomerase independent manner, which happens in ALT cells that are present mostly in somatic cancer cells (Arora et al., 2014, Balk et al., 2013). Regulation of the R- loop is necessary for the proper cell segregation (Groh and Gromark, 2014).

Removal of the R-loop is also essential for the embryo development, as R-loop formation hinders mitochondrial DNA replication (Ceritelli et al., 2009).

Ribonuclease H is an enzyme that cleaves the RNA strand of the RNA:DNA hybrid. It can control the presence of R-loops in the cell and can also resolve the effects exerted by R-loop. In prokaryotes there are two types of RNaseH enzymes, which are known as RNaseHI and RNaseHII, and both are monomeric (Ceritelli et al., 2009). Similarly, in eukaryotes two RNaseH complexes are present and known as RNaseH1 and RNaseH2. RNaseH1 is a monomeric protein. However,

RNaseH2 is a heterotrimeric complex (Ceritelli et al., 2009). Both proteins can cleave the RNA strand from the substrate RNA:DNA hybrids efficiently, although with different mechanisms. Deletion of individual RNaseH enzyme complexes has shown increased recombination in YACs (Yeast artificial chromosomes)

103 significantly, which suggests that suppression of R-loop clearly minimizes the illegitimate recombination in the cells (Hamperl et al., 2014). In yeast cells, deletion of THO/TREX complex, the proteins involved in RNA processing, results in R-loop formation at telomeres, which in turn leads to telomere elongation through recombination (Pfeiffer et al., 2013). These recombination events are mostly utilizing RAD52 to repair the breaks inside the telomeres independent of telomerase and the survivors can be of type I and type II survivors (Pfeiffer et al.,

2013, Balk et al., 2013). Deletion of the functional RNaseH genes rnh and rnh1 overturns phenotypes of RAD52 deletion, suggesting the role of R- loop in cell growth (Balk et al., 2013). Mutations of nse3 and mms21 proteins, components of structural maintenance proteins (SMC) 5/6 complex has been shown to increase the TERRA expression from subtelomeres containing the X elements. In S. pombe, telomeres are clustered together and IFA of RAP1 shows

3-5 foci, suggesting subtelomeric silencing. SMC5/6 complex mutants show multiple RAP1 foci, which suggests the loss of subtelomeric silencing, and overexpression of TERRA and formation of telomeric R-loop is responsible for this

(Moradi-fard et al., 2016). Hence, it is essential to maintain telomere chromatin structure and telomere replication to control the illegitimate telomere recombination.

Knowledge of telomere transcription can potentially provide significant insight about the telomere functions. T. brucei telomeres are transcribed

(Rudenko, 1989), however, we still do not know exactly from where TERRA expression originates. We previous found that depletion of TbRAP1 resulted in

104 derepression of all ES-linked silent VSGs (Yang et al., 2009). However, depletion of TbRAP1 did not affect ES chromatin structure in bloodstream for T. brucei cells when analyzed by Formaldehyde-Assisted Isolation of Regulatory Elements

(FAIRE) and micrococcal nuclease (Mnase) digestion (Pandya et al., 2013), suggesting that additional factors are also involved in telomere chromatin structure determination. It is possible that TERRA may play an important role in telomere chromatin structure formation/maintenance. Previously, it was hypothesized that read-through of RNA Pol I from the active ES into the downstream telomere repeats results in TERRA transcription (Rudenko, 1989). However, this has not been confirmed. In addition, TERRA is also transcribed at the insect stage of T. brucei, where all subtelomeric ESs are silenced, suggesting that this hypothesis is not true at least in procyclic form T. brucei cells. Hence, it is extremely important to identify origins of TERRA in bloodstream form cells to investigate the outcome of the conundrum.

Depletion of TbRAP1, increased the VSG switching frequency and the majority of the switchers arose through VSG gene conversion (chapter – 3). This suggests that T. brucei telomeres have undergone recombination event. However, the initiation of these recombination is unknown. We have also detected higher amount of breaks at the VSGs (active and silent), telomeres and ES promoter, upon depletion of TbRAP1 (Chapter - 3). Increase in the DNA DSBs were observed at subtelomeres/telomeres, with the increased amount of TERRA due to formation of R-loop at the telomeres, ultimately leading to telomere recombination (Bah et al., 2011, Balk et al., 2013, Arora et al., 2011). Therefore, we hypothesize that

105 removal of TbRAP1 in T. brucei cells may increase TERRA levels. Also, we hypothesize that, increase in the TERRA may lead to formation of R-loop at the telomeres. We further hypothesize that removal of the R-loop from telomeres with overexpressed RNaseH in TbRAP1 depleted cells should suppress VSG switching frequency. Since RNaseH can cleave the RNA strand of the DNA:RNA hybrid, we expect to see a reduction in R-loop levels similar to what have been reported in yeast and human cells (Balk et al., 2013, Arora et al., 2014). However, no RNaseH has been characterized in T. brucei, so far. Hence, identifying the role of TERRA thus helps us to understand the cause of DNA double strand break at the subtelomeres in TbRAP1 RNAi cells.

4.2 Results

4.2.1 Detection of telomeric transcript in wild type bloodstream form and procyclic form T. brucei cells

Previously telomeric transcript has been identified as a heterogeneous long non-coding RNA ranging from 0.4 kb to 4 kb (Rudenko & Ploeg, 1989). It can be detected only with the CCCTAA probe, suggesting that a G-rich telomere transcript is synthesized according to the C-rich strand of telomere DNA (Rudenko & Ploeg,

1989). This telomere transcript is not sensitive to α-amanitin, a compound that inhibits RNA pol II effectively. We decided to first confirm these findings. To confirm that WT bloodstream form SM cells, a derivative of Lister 427 strain that expresses T7 polymerase and Tet repressor (Wirtz et al., 1999) and WT procyclic cells, WT427, were used. We first decided to detect TERRA by northern blotting.

Total RNA was isolated with treatment with 10U DNAseI according to the protocol

106 described in Chapter -2 (T. brucei genome has ~250 telomeres, which can affect the hybridization result if residue DNA is left behind). 10 ug of the total RNA was loaded on each lane with or without treatment with RNase (including both RNaseA and RNase one). The G-rich telomere probe was prepared by random primer extension reaction using telomere DNA as template and only dATP, dTTP, and radioactive dGTP were added as substrates. Similarly, the C-rich telomere probe was prepared by random primer extension reaction using telomere DNA as template and only dATP, dTTP, and radioactive dCTP as substrates. The G-rich and C-rich telomere DNA probes were used to specifically identify the C-rich and

G-rich telomere transcript, respectively. After hybridization, northern blots were exposed to phosphorimager and results are shown in Fig. 4-1 (Bloodstream form) and Fig. 4-2 (Procyclic form).

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Figure 4-1: Northern analysis to detect TERRA in wild type bloodstream form cells. Total RNA was isolated followed by DNAseI treatment, 10 ug of total RNA is loaded in each lane. (A) Detection of TERRA molecules using the C-rich telomere probe. The EtBr stained agarose gel is shown on the left. 0.5 – 10 kb RNA ladder is run in the first lane. Samples without and with RNase treatment are loaded in lane two and lane three, respectively. Three distinct rRNA precursor bands are visible in the center of the gel. Northern blotting hybridized with the C-rich telomeric repeats (CCCTAA)n detects 0.3 – 5.0 kb long UUAGGG telomere transcript, which is

108 shown in the middle. The same northern blot is stripped and hybridized with a TbTR probe, and the result is shown on the right (TbTR is the RNA component of the telomerase, Sandhu et al., 2012). A distinct 0.9 kb band is identified. (B) Detection of TERRA molecule using the G-rich telomere probe. The EtBr stained gel and hybridization results are arranged the same way as in (A) No TERRA signal was detected when the G-rich telomere probe is used, indicating no transcription from the G-rich strand of the telomere.

Northern blot analysis revealed that in the bloodstream form of the T. brucei cells TERRA is only transcribed using the C-rich strand as a template, detected by the C-rich telomere DNA probe. (Fig. 4-1A, center). TERRA molecules consist of

5’-UUAGGG-3’ repeats, suggesting that transcription runs in a direction towards telomere ends (Fig. 4-1A). The lengths of the TERRA molecules are heterogeneous (0.3 kb - 5 kb). We did not detect any signals when we probed the same RNA sample with the G-rich telomere DNA probe (Fig. 4-1B). TbTR is the

RNA subunit of the telomerase enzyme. We have used it as a loading control

(Sandhu et al. 2013). All the preparation of RNA was also subjected to 10U RNase

A and 20U RNase one combine as a control.

.

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Figure 4-2: Northern analysis to detect TERRA in the wild type procyclic form cells. RNA Isolation, RNA electrophoresis, and northern hybridization was performed the same way as in figure 4-1. (A) The C-rich telomere probe was used in the hybridization. (B) The G-rich telomere probe was used in the hybridization. EtBr- stained gel and TbTR hybridized blots are shown as controls.

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When similar experiments were performed on the procyclic form T. brucei cells, we detected the TERRA species using both C-rich and G-rich telomere DNA probes (Fig. 4-2). Similar to previously published report we detect heterogeneous length of both the TERRA molecules but of lesser intensity. Despite of detecting heterogenous smear for the TERRA signals, we also detected one sharp band at around ~ 800 bp when either the C-rich or G-rich telomere probes was used.

In the previous work (Rudenko & Ploeg, 1989), telomeric transcripts were detected in both bloodstream and procyclic form T. brucei cells. In procyclic form cells, Rudenko & van de Ploeg have detected telomeric transcript from both telomere strand. While in bloodstream form they have identified the transcript only transcribed from the C-rich telomere strand, although data not shown. The telomeric transcripts detected by these authors were heterogeneous in length, ranging from 0.5 to 4.0 kb. Similarly, we observed telomeric transcripts of heterogeneous sizes, too, although their sizes ranged from 0.3-5.0 kb. This difference can stem from different strains used in the earlier and current studies.

The TRUE927 strain was used in the previous study, while we used Lister 427 strain (Doyle, 1980).

4.2.2 Identification of the origin of TERRA transcription in T. brucei

As explained in the introduction (Chapter - 1), T. brucei has only one active subtelomere transcription at a time due to monoallelic VSG expression.

Transcription of the active ES is mediated by RNA pol I in a polycistronic manner.

T. brucei transcripts are trans-spliced later to generate mature RNAs. During trans- splicing, a 39 nt long RNA molecule, the spliced leader, is ligated to the 5’ end of

111 each pre-mRNA (Gunzl, 2005, Huang and Van der Ploeg, 1991, LeBowitz et al.,

1993). The spliced leader sequence is present the 5’ end of each mature transcript of T. brucei. We hypothesize that TERRA can be transcribed from the active ES- adjacent telomere as RNA Pol I reads thorough the ES and continues into the telomere repeats immediately downstream of the last VSG gene in the ES. If this is true, a small amount of unspliced RNA containing both the telomeric sequence and its adjacent VSG sequence should be present in the cell (Fig. 4-3B).

To test this, we isolated total RNA from WT BF cells and reverse transcribed the RNA by using TELC20 (5’-CCCTAACCCTAACCCTAACC-3’), TELG20 (5’-

GGGTTAGGGTTAGGGTAAGG-3’), random hexamer, or no hexamer as primers

(Fig. 4-3A). The resulting cDNA product was then amplified by PCR using primers specific for the active VSG, silent VSGs, rRNA, and tubulin (the latter two were used as controls). The final RT-PCR products were separated on a 0.7% agarose gel (Fig. 4-3).

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Figure 4-3: TERRA can be transcribed from the active ES-adjacent telomere. (A) A cartoon explaining transcription of both strands of telomeres. Transcription of the C-rich telomere strand will generate 5’-UUAGGG-3’ repeat (indicated as transcript I in the figure), and transcription of the G-rich strand will generate 5’-CCCTAA-3’

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(indicated as transcript II in the figure). TELC20 and TELG20 primers can be used to anneal the G-rich and C-rich telomere transcripts, respectively. (B) The active and the silent VSGs in the T. brucei strain used in the RT-PCR analysis. VSG2 is the active, while VSG9 and VSG3 are the silent VSG loci. (C) and (D) RT-PCR results. EtBr-stained gel with the RT-PCR products. TELC20 and TELG20 are the primers used in the RT reaction. Locus-specific primers used in PCR reactions are labeled on the top of each lane.

We can only detect PCR products with VSG2 primers when the RT reaction is done using the TELC20 primer, suggesting that the TERRA is transcribed from the active ES-adjacent telomere (Fig. 4-3B, Fig. 4-3C). We did not detect any PCR products when rRNA, tubulin, and VSG9 primers were used, indicating that telomeres adjacent to silent ESs are not transcribed (Fig. 4-3C).

We did not detect any PCR products if TELG20 was used as the RT primer, indicating that TERRA is generated only from the C-rich telomere strand (Fig. 4-

3C). As expected, when a random hexamer is used as the RT primer, we can obtain the PCR products using primers specific to all transcribed genes including rRNA and tubulin (Fig. 4-3D). When no primer is added to the RT reaction, no PCR products were detected for any primers used (Fig. 4-3D).

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Figure 4-4: Transcription of TERRA from the active VSG9-expressing subtelomere. (A) The subtelomere VSG expression sites. VSG9 is the active, while VSG2 and VSG3 are the silent VSG loci. (B) and (C) show RT-PCR results separated on EtBr-stained agarose gels. TELC20 and TELG20 are primers used in the RT reaction. Locus-specific primers used in different PCR reactions are labeled on top of each lane.

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To confirm that TERRA always expresses from the active ES-adjacent telomere, regardless of which ES is active, we have repeated the same experiment in the PVS 3.2/OD1-1 strain (Yang et al., 2009) that expresses VSG9 and the

SM/plew111-Ty1-TbTIF2 strain (Pandya et al., 2013) that expresses VSG3.

Similar results were observed in these strains as well. PVS3.2/OD1-1 cells (VSG9- expressor) yielded a RT-PCR product only with VSG9 primers (Fig. 4-4B) and the

SM/plew111-Ty1-TbTIF2 strain (VSG3 expresser) yielded a RT-PCR product only with VSG3 primers (Fig. 4-5B) only when cDNA is prepared with the TELC20 primer. Primers specific to the silent VSG2 and VSG3 in the PVS3.2/OD1-1 strain

(Fig. 4-4A) and primers specific to the silent VSG9 and VSG8 in SM/plew111-Ty1-

TbTIF2 strain (Fig. 4-5A) did not yield any PCR products. In both these strains, we did not detect any signals when cDNA was prepared using TELG20 as RT primer or when no primer is used for the RT reaction (Fig. 4-4B, Fig.4-4C, Fig. 4-

5B, Fig. 4-5C). However, as a control, we detected PCR products when primers specific to the active VSG, rRNA and tubulin were used for the PCR after the cDNA was synthesized using the random hexamer as the RT primer (Fig. 4-4C and Fig.

4-5C). This allowed us to conclude that TERRA originates from the active ES, suggesting RNA Pol I reads through the last gene of the ES and continues to transcribe the downstream telomere. However, the T. brucei genome very likely contains telomeres that do not host any subtelomeric ESs, and whether TERRA is also transcribed from these telomeres cannot be determine by our current assay.

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Figure 4-5: Transcription of TERRA from the active VSG3-expressing subtelomere. (A) The subtelomere VSG expression sites in VSG3-expressors. VSG3 is active, while VSG8 and VSG9 are silent. (B) and (C) showed RT-PCR products separated on EtBr-stained agarose gels. TELC20 and TELG20 are primers used in the RT reaction. Locus-specific primers used in different PCR reactions are labeled on top of each lane.

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4.2.3 Regulation of TERRA expression by T. brucei telomere proteins

TERRA expression depends on multiple factors such as chromatin structure, length of telomeres, telomere proteins, and RNA processing factors

(Azzalin, 2013). Among all of these factors, telomere proteins have been shown to play an important role in the regulation of TERRA expression in yeast and mammals (Bah et al., 2013, Azzalin et al., 2007, Schoeftner and Blasco, 2008). In budding yeast, RAP1 binds to telomeric DNA and also binds to Sir3/4 and Rif1/Rif2

(Iglesius et al., 2011). Sir3/4 recruits Sir2, which is a histone deacetylase and have been shown to negatively regulate the TERRA expression (Iglesius et al., 2011).

Similarly, Rif1/Rif2 negatively regulate the TERRA expression, suggesting RAP1’s role in TERRA expression regulation (Iglesius et al., 2011). In fission yeast, TAZ1, the telomere DNA binding protein, and its interactor RAP1 negatively regulate

TERRA expression as well (Bah et al., 2012). In human cells, telomere DNA binding protein TRF2 interacts with TERRA and ORC protein to form a stable ternary structure, hence it is clear that telomeric proteins play important role in

TERRA regulation (Chawla & Azzalin, 2008, Deng et al., 2009).

We have identified three T. brucei telomere proteins: TbTRF, TbTIF2, and

TbRAP1. TbTRF and TbTIF2 both have been shown to play an important role in

VSG switching (Jehi et al., 2014A, Jehi et al., 2014B). TbTIF2 is essential for subtelomere stability (Jehi et al., 2014A). TbTRF protects G-overhang structure (Li et al., 2005). Depletion of TbTIF2 also affects the G-overhang structure of the telomere, but detailed characterization is required (Jehi, S. & Li, B., unpublished data). On the other hand, TbRAP1 is required to silence subtelomeric VSG

118 expression (Yang et al., 2009, Pandya et al., 2013). Whether any of these telomeric proteins plays any role in the regulation of TERRA expression is unknown. We hypothesized that depletion of TbRAP1 may upregulate the TERRA levels since increased transcription of silent ESs in TbRAP1-depleted cells may allow read-though from all the silent ES associated VSGs and ultimately allowing an increase in the TERRA level. We will also examine the role of TbTRF as a comparison, as depletion of TbTRF did not derepressed silent ES-associated

VSGs transcription (Pandya et al., 2013).

To examine whether TERRA levels are affected by TbTRF and TbRAP1, we knocked-down the expression of TbTRF and TbRAP1 by RNAi for 24 and 48 hours. We isolated total RNA at respective time points and performed northern blotting analysis using the C-rich telomere probe to detect TERRA. The TbRAP1

RNAi and TbTRF RNAi strains used for VSG switching assay, S/RAP1i and

S/TRFi, respectively (Jehi et al., 2014B), were used for this experiment. Induction of RNAi reduced the TbRAP1 and TbTRF protein levels in these cells (Data not shown).

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Figure 4-6 TERRA expression in TbRAP1-depleted cells. S/RAP1i and S/RAP1i+F2H-TbRAP1 cells were induced for 24 and 48 hours. RNA was isolated and 10 ug of RNA is separated on the denaturing gel. To detect TERRA, the c-rich telomere DNA probe was used. After exposing the blot to a phosphorimager, the blot was striped and hybridized with the TbTR probe as a loading control.

We detected an increase in the TERRA signals from 0 hour to 24 hours and

48 hours in TbRAP1 and TbTRF depleted cells (Fig. 4-6 and Fig. 4-7). As a control, we have probed TbTR after stripping the blot and no change in TbTR levels has

120 been observed (Fig. 4-6 and Fig. 4-7). To rule out the off target effects of RNAi, the WT TbRAP1 was expressed from an ectopic location in the S/RAP1i strain

(Similar as used in chapter-3) and it suppressed the phenotype of increased

TERRA levels that were observed after depletion of TbRAP1 (Fig. 4-6). Similarly, we expressed WT TbTRF from an ectopic location in the S/TRFi strain (Jehi et al.,

2014B) and this ectopically expressed TbTRF protein suppressed the phenotype of increased TERRA expression (Fig. 4-7). Upon depletion of either telomere protein, some TERRA molecules exhibited much larger sizes, and this phenotype was reverted when the ectopic WT allele was expressed. (Fig. 4-6, Fig. 4-7).

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Figure 4-7: TERRA expression in TbTRF RNAi condition. S/TRFi and S/TRFi+F2H-TbTRF strains were induced for 24 and 48 hours. RNA was isolated and 10 ug of RNA is separated on the denaturing gel. To detect TERRA, the c-rich strand labelled telomere DNA probe was used. After exposing the blot to a phosphorimager, the blot was striped and hybridized with the TbTR probe as a loading control.

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Northern analysis has revealed an increase in the levels of TERRA upon depletion of TbRAP1 and TbTRF. Next, we did northern slot blot with only 1.5 ug of total RNA per sample to quantify the TERRA levels in TbRAP1 and TbTRF depletion cells (Fig. 4-8, Fig. 4-9). This method allowed us to reduce the amount of materials used, as the signals are concentrated at a spot. As a control we also performed the experiments in the TbRAP1 and TbTRF complementation strains.

We found a ~11-fold increase in the TERRA level upon depletion of TbRAP1, which was reduced to wild type level upon TbRAP1 complementation (Fig. 4-8). In

TbTRF knockdown cells, a ~5 fold increase in the TERRA level was observed (Fig.

4-9). Ectopic expression of a WT TbTRF allele in the TbTRF RNAi cells suppressed this phenotype (Fig. 4-9). Collectively, depletion of TbRAP1 and

TbTRF both result in higher TERRA levels. Therefore, T. brucei telomere proteins regulate TERRA expression. Increase in the TERRA levels by depletion of TbTRF suggests that, derepression of ES is not the only reason for higher amount of

TERRA, as expected earlier. We expect that removal of TbTRF or TbRAP1 would leave the telomere DNA less occupied and protected, which could allow the RNA pol I to continue to transcribe the telomeres for much longer. However, this hypothesis requires further analysis. We have not observed a significant change in the telomere length upon TbRAP1 depletion (Pandya et al., unpublished).

However, the growth arrest is acute when TbRAP1 is depleted, which prevented us to examine telomere length changes that usually only occur after a long period of cell culture. Hence, it is also possible that, the variation in telomere length may have direct or indirect role in TERRA expression.

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Figure 4-8: TERRA levels increase upon TbRAP1 depletion. (A) Northern slot blot analysis to detect changes in TERRA levels. TbRAP1 was depleted for 24 and 48 hours. 1.5 ug of RNA was loaded onto the slot blot, which was hybridized with the C-rich telomeric probe. Tubulin probe was used as a loading control. Complementation of TbRAP1 was achieved by ectopic expression of F2H- TbRAP1. (B) Quantification of the signals detected in (A). Results from at least three experiments were used to calculate the average.

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Figure 4-9: Depletion of TbTRF results in an increase in TERRA levels. (A) Northern slot blot analysis to detect changes in TERRA levels. TbTRF was depleted for 24 and 48 hours. 1.5 ug of RNA was loaded onto the slot blot, which was hybridized with the C-rich telomeric probe. Tubulin probe was used as a loading control. Complementation of TbTRF was achieved by ectopic expression of F2H-TbTRF. (B) Quantification of TERRA signals detected in (A). Results from at least three experiments were used to calculate the average.

We observed an increase in TERRA levels in TbRAP1 depleted cells.

However, it is not known whether TERRA are still transcribed from the active BES-

125 adjacent telomere or also from the derepressed BES-adjacent telomeres upon depletion of TbRAP1. To determine where was TERRA transcribed in TbRAP1 depleted cells, we performed the same RT-PCR experiment as described above

(4.2.2).

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Figure 4-10: Transcription of TERRA in the presence and absence of TbRAP1. EtBr-stained gels with RT-PCR products are shown. TELC20 and TELG20 are primers used in the RT reaction. Locus-specific primers used in PCR reactions are labeled on top of each lane. Random Using random hexamer and no primer in RT served as a positive and negative control, respectively. (A) VSG2 is active. VSG3 and VSG9 are silent ES associated VSGs. (B) TbRAP1 protein is depleted for 24 hours with addition of DOX. VSG3 and VSG9 were derepressed upon TbRAP1 depletion.

The S/RAP1i cells were used for the RT-PCR based TERRA origin analysis.

The active ES in this strain is the VSG2-containing ES. Before induction of

TbRAP1 RNAi, TERRA originates from the active ES as VSG2 RT-PCR product was detected with the TELC20 primer (Fig. 4-10A, similar to Fig. 4-5). After

TbRAP1 was depleted for 24 hours, we found that TERRA was still transcribed from the active ES, as only primers specific to VSG2 yielded PCR product after RT was done with TELC20 (Fig. 4-10B). No product was detectable when PCR was performed on cDNA prepared with TELG20 as RT primer or and no RT primer was included (Fig. 4-10). Upon TbRAP1 depletion, VSG3 and VSG9 should be derepressed (Yang et al., 2009). Indeed, we detected VSG3 and VSG9 RT-PCR products in S/RAP1i induced cells, when cDNA was prepared using the random hexamer as the RT primer (Fig. 4-10B, random hexamer). We also detected amplified products with 70 bpR associated primers in TEC20 primer prepared cDNA (Fig. 4-10B, random hexamer). This suggests that the 70 bp repeat region is transcribed and the transcript is stabilized upon removal of TbRAP1 (Compare, fig. 4-10 A & B, random hexamer). Also, it is possible that, the increase in the length of the TERRA moiety, we have observed in the northern blot (Fig. 4-7) is due to increased unspliced products. Hence, we conclude that T. brucei telomere

128 proteins regulate TERRA levels in a negative manner and TERRA is transcribed from the active ES.

TbTRF and TbTIF2 are two other telomere proteins that did not affect VSG silencing (Pandya et al., 2013, Jehi et al., 2014A). Hence, we do not expect TERRA to be originated from different ES upon deletion of TbTRF. However, that needs to be validated and we have not done that in this thesis.

4.2.4 Increased TERRA levels results in more RNA:DNA hybrids (R-loops) at the telomeres, in TbRAP1 deletion condition.

Depletion of telomere proteins TbRAP1 and TbTRF increases TERRA levels in T. brucei cells. However, the role of TERRA in T. brucei is still unknown.

In yeast and human cells, strong induction of TERRA has been shown to increase senescence, reduce telomere length, and form telomere associated RNA:DNA hybrids (R-loops) (Balk et al., 2013, Azzalin et al, 2011). In the R-loops, an RNA strand displaces one DNA strand of the double stranded DNA and hybridizes with the complementary strand of DNA, leaving the other DNA strand single stranded

(Hamperl et al., 2014). R-loops are often observed at the regions that are highly transcribed and where non-template strand is G-rich (Such as telomeric sequences TTAGGG). R-loops have been shown to increase DSBs, specifically on the free DNA strand and increase homologous recombination (Hamperl et al.,

2014). A replication fork can encounter the R-loop containing SSB in either the same or the opposite orientation. If the fork can progress over the RNA:DNA hybrids than separation from the parental strain may result in DNA breaks in the

129 lagging strand of DNA polymerase complex (Wellinger et al., 2006, Azvolinsky et al., 2009).

We have detected an increase in the amount of DSBs at the VSG and telomere loci upon removal of TbRAP1 (chapter 3). In the above section (4.2.3.), we found that TERRA levels increased in TbRAP1-depleted cells. Therefore, we hypothesize that under the TbRAP1 depletion condition, TERRA forms RNA:DNA hybrids at the telomere in T. brucei, resulting in more DSBs at the telomere and telomere-adjacent VSG loci. To test our hypothesis, we first examined the presence of R-loops at the telomeres in TbRAP1 RNAi cells.

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Figure 4-11: Depletion of TbRAP1 leads to increased amount of telomeric RNA:DNA hybrid. (A) Dot blot southern analysis of input (diluted 20-fold), IgG immunoprecipitated, and S9.6 immunoprecipitated DNA samples in S/RAP1i cells before and after induction of TbRAP1 RNAi. Samples were either treated or not treated with 20U of RNase H (Thermo fisher Scientific) before IP. A TTAGGG repeat probe was used in the hybridization. (B) Quantification of the dot blot hybridization signals. Average values were calculated from four independent experiments. Standard deviations are shown as error bars. Unpaired t-test P values are indicated for selected pairs of values. To identify R-loops, we performed DNA:RNA immunoprecipitation (DRIP, also known as R-loop assay) in the S/RAP1i strain at 0 or 24 hours after the induction of TbRAP1 RNAi. We isolated 100 µg of genomic DNA and sonicated it so that the average DNA fragments became ~500 bp. Samples were immunoprecipitated with various antibodies overnight (~14 hours) and eluted from the magnetic beads the next day. Eluted samples were blotted on nylon membranes and southern blot was performed using the telomere probe (Detailed protocol in chapter-2). We used a monoclonal R-loop antibody s9.6, which specifically recognizes RNA:DNA hybrids (Boguslawski et al.,1986) (a generous gift from Dr. Stephen Leppla, NIH). Mouse normal IgG was used as a control.

IP products were analyzed by the dot blot hybridization using a telomere repeat probe (TTAGGG)n to specifically examine the amount of telomeric

RNA:DNA hybrids. We observed a significant increase in the signal corresponding to telomeric R-loops in TbRAP1-depleted cells when compared to that from uninduced S/RAP1i cells (Fig. 4-11A). Treating genomic DNA with 20U of RNase

H (Thermo fischer Scientific) before s9.6 IP reduced the precipitated telomeric R- loop signals to the background level, confirming that the detected signals are from an RNA:DNA hybrid structure. Quantification of the hybridization signals indicates

131 that the amount of telomeric R-loops in TbRAP1-depleted cells was ~16-fold of that in uninduced S/RAP1i cells (Fig. 4-11B). Therefore, depletion of TbRAP1 results in increased amount of telomeric RNA:DNA hybrids.

We attempted to further examine whether the highly transcribed active VSG mRNA and the derepressed VSG mRNA in TbRAP1-depleted cells can also form the RNA:DNA hybrid structure at subtelomeric loci. We performed the same IP using s9.6 antibody and estimated the level of RNA:DNA hybrids specifically at different VSG loci by quantitative PCR using primers specific to different VSG genes. However, we did not detect any VSG mRNA-containing RNA:DNA hybrids in S/RAP1i cells before or after induction of RNAi, indicating that the amount of R- loops formed with the VSG mRNA, if any, is very low.

4.2.5 Identification of RNAseH1 gene and generation of ectopic expressing

TbRNaseH1 strain

To further determine the role of telomeric R-loops in VSG switching, we would like to develop a method to suppress R-loop levels in T. brucei cells. R-loop has been shown to be sensitive to RNaseH activities. In T. brucei, TbRnaseH1

(Tb427.07.4930) was cloned and characterized previously (Kobil and Campbell,

2000). However, the detailed function of this gene has not been studied previously.

The sequence alignment for RNaseH1 homologues in T. brucei, S. cerevisiae, and

H. sapiens is shown in figure 4-12. TbRNAseH1 is ~31% identical with hRNaseH1 overall, and 68% identical at the c-terminal catalytic domain, while it is 28% identical with ScRNaseH1 full-length protein and 58% identical in the HBD domain

(ClustalX, EMBL-EBI). A weaker similarity in the central connection domain is also

132 seen among these proteins (Fig. 4-12, * marked amino acids).

Figure 4-12: Protein sequence alignment using ClustalX for RNaseH1 homologues. S. cerevisiae (budding yeast), H. sapiens (human) and T. brucei RNaseH1 sequences are compared. Identical amino acids are marked as (*), similarly charged amino acids are marked as (:) and non-charged amino acids are marked as (.). Highly conserved regions are seen in N and C terminus of the RNaseH proteins. We cloned this TbRNaseH1 gene in the pLew100v5 plasmid, which can be used for conditional expression in T. brucei under tight regulation (Cestari et al.,

2015). The resulting construct was introduced in S/EV and S/RAP1i strains.

TbRNaseH1 was tagged with two HA epitopes at the C-terminus. After obtaining the expected strains, we first checked the cell growth upon induction by

133 doxycycline. Addition of doxycyclin induces ectopic expression of TbRNaseH1 and it induces TbRAP1 RNAi. Ectopic expression of TbRNAseH1 did not have any effect on cell growth as no difference in population doublings was observed in S/Ev

+ TbRNAseH1 strain when induced with doxycycline (Fig 4-13A). Ectopic expression of TbRNaseH1 in TbRAP1 RNAi cells partially suppressed the growth defects seen in S/RAP1i strains. It delayed growth arrest by 24 hours (Fig. 4-13A).

However, continuous knockdown of TbRAP1 in S/RAP1i +TbRNaseH1 cells still arrested cell growth at a later time point (Fig. 4-13A). This indicated that the

TbRAP1 depletion defects cannot be completely rescued by ectopic expression of

TbRNaseH1. We also confirmed the knockdown of the TbRAP1 protein and expression of the HA-tagged TbRNaseH1 protein by western analysis in these cells (Fig. 4-13B). No change in the TbRAP1 protein level was observed in the

S/EV + TbRNaseH1 strain (Fig. 4-13B). Minor leaky expression of TbRNaseH1 was detected before induction in S/RAP1i +TbRNaseH1 strain. Nevertheless, we detected increase in the TbRNaseH1 protein level in both S/RAP1i +TbRNaseH1 and S/ev +TbRNaseH1 strains.

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Figure 4-13: Ectopic expression of TbRNaseH1 delays TbRAP1 RNAi growth phenotypes. (A) Growth curve analysis for S/RAP1i, S/RAP1i + F2H-TbRAP1, S/ev + TbRNaseH1 and S/RAP1i + TbRNAseH1 cells under induced and uninduced conditions. (B) Western blot analysis, total cell extract was used. TbRAP1 protein was detected with rabbit anti-TbRAP1 antibody 598 (Yang et al., 2009), Expression of TbRNaseH1-2HA was detected with anti-HA antibody (F-7 probe, Santa Cruz Biotechnologies). Detection of EF-2 protein by anti-EF2 antibody (Santa Cruz Biotechnologies) was used as a loading control.

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Figure 4-14: Ectopic expression of TbRNAseH1 does not affect the VSG derepression phenotype upon TbRAP1 depletion. RNA was isolated at different time points after inducing TbRAP1 RNAi. Steady state mRNA levels of different VSGs were examined by q-RT-PCR. Three repeats were performed and the average results are plotted. To confirm that TbRAP1’s phenotypes are unaltered due to ectopic expression of TbRNaseH1, we performed RTPCR and confirmed expression of the active and silent VSGs 24 hours post induction of TbRAP1 RNAi. We observed the similar VSG derepression phenotypes as that in TbRAP1-depleted cells (Yang et al., 2009, Pandya et al., 2013), indicating that expression of an ectopic allele of

TbRNAseH1 did not rescue the VSG derepression defect caused by TbRAP1 knockdown.

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4.2.6 Ectopic expression of TbRNaseH1 reduces telomeric R-loop levels and partially suppresses the elevated TERRA levels in TbRAP1 depleted cells

After generating the S/RAP1i + TbRNaseH1 strain, we first performed R- loop assays (explained in 4.2.4 & chapter -2) after inducing it for 24 hours. Addition of doxycycline induces TbRAP1 RNAi and ectopic expression of TbRNaseH1 simultaneously. We harvested genomic DNA at 0 and 24 hours after induction.

Results for the telomere R-loop were detected after performing southern blotting using the telomere DNA probe (Fig. 4-15). In S/RAP1i + TbRNAseH1 cells after 24 hours induction, we did not detect an increase in the telomeric R-loop levels as we observed in TbRAP1i-depleted cells (fig. 4-15A, right blot). Rather, the levels of telomeric R-loops were similar to that in the wild type cells.

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Figure 4-15: Ectopic expression of TbRNaseH1 in TbRAP1 RNAi strain suppresses the amount of telomeric RNA:DNA hybrid. (A) Dot blot Southern analysis of input (diluted 20-fold), IgG immunoprecipitated, and S9.6 immunoprecipitated DNA samples in S/RAP1i and S/RAP1i + TbRNAseH1 cells before and after induction of TbRAP1 RNAi. Samples were either treated or not treated with 20 U of RNase H (Thermofischer Scientific) before IP. A TTAGGG repeat probe was used in the hybridization. (B) Quantification of the hybridization signals. Average values were calculated from four independent experiments.

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Standard deviations are shown as error bars. Unpaired t-test P values are indicated for selected pairs of values. Our result showed that the telomeric R-loop is indeed sensitive to the

RNaseH1 gene product. More importantly, we detected reduction in the R-loop levels with ectopic expression of TbRNaseH1 in S/RAP1i (Fig. 4-15). The reduction in the R-loop levels we have detected is presumably due to the removal of the RNA strand from the R-loop by ectopically expressed TbRNAseH1. However, it is also important to check the expression of TERRA levels in the S/RAP1i + TbRNAseH1 strain

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Figure 4-16: Changes in TERRA levels upon ectopic expression of TbRNAseH1 in TbRAP1 depletion cells. (A) Northern slot blot analysis to detect changes in TERRA levels. TbRAP1 was depleted for 24 and 48 hours in S/RAP1i, S/RAP1i +

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F2H-TbRAP1, S/EV + TbRNAseH1 and S/RAP1i + TbRNAseH1 cells. 1.5 µg of RNA was blotted onto nylon membranes. The blot was hybridized with a C-rich telomeric probe and tubulin probe was used as a loading control. Complementation of TbRAP1 was achieved by ectopic expression of F2H-TbRAP1. (B) Quantification of the signals detected in (A). The average value was calculated from at least three experiments. Partial suppression of TERRA levels was observed when TbRNAseH1 was ectopically expressed in the TbRAP1 RNAi strain. To examine the effect of RNaseH1 on TERRA levels, we induced TbRAP1

RNAi in S/RAP1i + TbRNaseH1 cells, which also induced the ectopic expression of TbRNaseH1-2HA, and performed northern slot blot analysis of total RNA by hybridization with the C-rich telomere DNA probe (described in chapter 4, Fig. 4.8).

We only observed partial reduction in the TERRA levels in S/RAP1i + TbRNaseH1 cells, when compared to S/RAP1i cells (Fig. 4-16). However, TERRA levels were still significantly higher than the WT level (Fig. 4-16). The partial reduction we observe in TERRA levels in S/RAP1i + TbRNaseH1-2HA strain at 24 and 48 hours is because enzymatic activity of TbRNaseH1, which will cleave TERRA strand from the telomeric RNA:DNA hybrid, hence, the final signals of TERRA we detect were lower compared to that in TbRAP1-depleted cells (Fig. 4-16). However, ectopic expression of TbRNAseH1 in S/ev cells caused no change in TERRA expression levels, and the TERRA levels in these cells are comparable to that in uninduced samples with same strain. These observations allowed us to conclude that in the wild type condition, TERRA levels are significantly lower and only higher levels of

TERRA forms R-loop at the telomeres (Fig. 4-15, Fig. 4-16) (Balk et al., 2013,

Pfeiffer et al., 2013).

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4.2.7 Consequence of ectopic expression of TbRNaseH1 in suppression of subtelomeric DNA DSBs.

In Chapter 3, we detected increased amount of DNA DSBs at the ES promoters, VSG loci, and telomere repeats upon removal of TbRAP1.

Simultaneously, we also detected increased TERRA (Fig. 4-8) and telomeric R- loop (Fig. 4-11) levels in TbRAP1-depleted cells. These observations led us to hypothesize that more telomeric R-loop causes more DSBs at the telomere vicinity.

If this is true, removal of telomeric R-loops with ectopic expression of TbRNAseH1 in S/RAP1i + TbRNaseH1 cells should reduce the amount of DSBs at telomeres/subtelomeres. In addition, the reduction in DSBs at subtelomeric ESs should lead to a decrease in the VSG switching frequency, as breaks in the active

ES (at 70 bp repeat regions, and between VSG and telomere locus) have been shown to induce VSG switching efficiently (Glover et al., 2013, Devlin et al., 2016,

Boothroyd et al., 2009, Hovel-miner et al., 2016). To check whether this hypothesis is correct, we first analyzed the association of TbRAD51 and γH2A to the subtelomeric regions by ChIP-qPCR as TbRAD51 and γH2A are good markers for

DSBs, and we have used them earlier to determine the DNA DSBs (Chapter -3).

We performed ChIP in a similar manner explained in chapter- 3 using anti-

TbRAD51 and anti-γH2A antibodies. The ChIP products were analyzed by qPCR

(Fig. 4-18) and Southern slot blot (Fig. 4-17).

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143

Figure 4-17: TbRAP1 depletion leads to an increase in DSBs at telomeres/subtelomeres, but the ectopic expression of TbRNaseH1 in the TbRAP1 RNAi background suppresses the amount of DSBs at telomeres/subtelomeres. Data for S/ev and S/RAP1i is the same as shown in figure 3-10. (A) Southern slot blot analysis of ChIP products. ChIP was performed on S/ev, S/RAP1i and S/RAP1i+TbRNaseH1 cells. 0 hour sample represents uninduced cells, 24 hour sample represents cells induced with doxycycline. The telomere probe and a 50 bp repeat probe were hybridized to the blot. The signals were detected using phosphorimagers. (B) Quantification of hybridization signals using the telomeric probe. (C) Quantification of hybridization signals using the 50 bp repeat probe. Multiple repeats were performed. The average of three or more experiments was calculated. Standard deviation is displayed in the form of error bars. A difference with P-values of 0.05 or lower is considered as significant. Color codes for B and C are same.

Through telomeric slot blot analysis we detected significant reduction in the association of TbRAD51 and γH2A with the telomere chromatin in the

S/RAP1i+TbRNaseH1 strain when compared to the S/RAP1i strain (Fig. 4-17).

Ectopic expression of TbRNaseH1 clearly suppressed the DSB levels at the telomeres (Fig. 4-17). No change in the TbRAD51 and γH2A signal was detected at telomeres and 50 bp repeats, when compared uninduced and induced cells in

S/RAP1i + TbRNAseH1 (Fig. 4-17). The amount of chromatin associated

TbRAD51 and γH2A was not changed at the 50 bp repeat region between uninduced and induced S/RAP1i + TbRNAseH1 cells. Hence, we conclude that removal of R-loop from the telomeres due to expression of TbRNaseH1 poses telomeres for less DNA DSBs.

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Figure 4-18: TbRAP1 depletion increased DSBs at the subtelomeric VSG loci but ectopic expression of TbRNAseH1 suppresses this phenotype. ChIP was performed using anti-TbRAD51 antibody (Top), anti-γH2A antibody (Bottom), and IgG (as a control) in S/ev, S/RAP1i and S/RAP1i + TbRNaseH1 cells at 0 and 24 hours after induction with doxycycline. ChIP products were quantified by quantitative PCR using primers specific to VSG2 (the active VSG), VSG21 (a Silent VSG), BES promoters, rRNA, and tubulin as indicated. The enrichment of ChIP

145 product over the input was calculated. Averages calculated from at least three experiments are shown in the bar graph, with the Standard deviations shown as error bars. P-values are calculated using unpaired t-test. By ChIP-qPCR experiments, we found that expression of TbRNaseH1 in

S/RAP1i significantly reduces the association of TbRAD51 and γH2A with the chromatin at the VSG (active and silent) and the ES promoter regions (Fig. 4-18).

There was no change at the rDNA and tubulin loci, which served as controls. The data here clearly suggests that removal of R-loops at the telomere with the help of ectopic expression of TbRNaeH1 further reduces DSBs at the VSG and telomere loci (Fig. 4-17, Fig. 4-18).

Figure 4-19: A transient depletion of TbRAP1 increased VSG switching frequency. Ectopic expression of TbRNAseH1 in the TbRAP1 RNAi background reduces VSG switching frequency back to that in control cells such as S, S/ev, uninduced S/RAP1i, and S/RAP1i + F2H-TbRAP1 cells. The total number of switchers collected in each strain is written on top of each bar in the chart. Four individual repeats were performed and the average switching frequency is plotted.

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Next we performed switching assays in S/ev + TbRNaseH1 and S/RAP1i

+ TbRNaseH1 strains in the same way as we explained earlier in the chapter - 3.

There was no difference in VSG switching frequency between S/ev + TbRNAseH1 and S/RAP1i + TbRNAseH1, suggesting ectopic expression of TbRNaseH1 does not contribute in VSG switching alone (Fig. 4-19). Also no difference was detected between TbRNAseH1 ectopically expressing strains to control strains, all having

VSG switching frequencies close to ~0.5 - 1.5 x10-5 (Fig. 4-19). Our data indicated that removal of R-loops at the telomeres led to a decrease in VSG switching frequency. Therefore, upon TbRAP1 depletion, the increase in VSG switching frequency phenotype depends on elevated TERRA levels and formation of telomeric R-loops. Formation of telomeric R-loop generates more DNA DSBs at the active and/or silent ES, which is potent for cell survival and known to increase

VSG switching frequency, leading to higher VSG switching events. We also determined that increased breaks at the VSG loci (chapter - 3, Fig. 3-8 to 3-12) generates more VSG associated gene conversion events in the switchers. If this is true than we expect to see reduction in VSG GC events in the switchers collected from S/RAP1i + TbRNAseH1 strain.

147

148

Figure 4-20: Ectopic expression of TbRNaseH1 in TbRAP1 RNAi strain reverted

TbRAP1 RNAi switching pattern to that in wild type cells. The total number of switchers analyzed for each individual strain is labeled on top of each bar in the chart. Percentages of each mechanism among all switchers are indicated and plotted.

We characterized the switching mechanism on the switchers collected from the S/ev + TbRNAseH1 and S/RAP1i + TbRNAseH1. We examined each switcher for its genotype and phenotype to identify their switching mechanisms (Fig. 4-20).

Most of the switchers were ES GC/ES loss+ In-situ switchers (Fig. 4-20). Observed switching events for new collected switchers from in S/ev + TbRNaseH1 and

S/RAP1i + TbRNaseH1 was found to be similar to all the control strains we have used in this study (S, S/ev, and S/RAP1i + F2H-TbRAP1) and opposite to what we have observed in TbRAP1 RNAi cells (S/RAP1i-clone B1 and S/RAP1i-clone C1), as most of their switchers arose through VSG GC (Fig. 4-20). It is also important to note that no change in VSG switching frequency or switching mechanisms was observed in the S/ev + TbRNaseH1 strain, indicating that ectopic expression of

TbRNaseH1 alone does not affect VSG switching. Previously it has been demonstrated that breaks at the 70 bpR region to VSG GC for the (~9% total) surviving switcher (Glover et al., 2013). However, outcome of the DNA DSB inside the VSG gene itself has not been characterized yet. Our data is in line with the observations that increased DNA DSBs at VSG locus, either inside VSG or between VSG and telomeres increases VSG GC in TbRAP1 depleted cells, and increase in DSBs is depended on increased TERRA and R-loop at the telomeres.

149

Hence, we conclude from these results that the increase in VSG switching frequency observed in S/RAP1i strain can be reverted by removal of R-loop at the telomeres.

4.3 Discussion

Telomere transcription has been identified in multiple organisms. It has been shown to play an important role in telomere biology in yeast and human cells

(Azzalin et al., 2007, Schoeftner and blasco, 2008). In T. brucei telomere transcription has been shown a long time ago (Rudenko, 1989). In both bloodstream form and procyclic form T. brucei cells, TERRA molecules of heterogeneous sizes (0.5-4.0 kb) were reported (Rudenko, 1989).

Here in this study, we re-confirmed the telomere transcription by northern blot analysis. Indeed, similar to previous reports, we found heterogeneous molecules of TERRA in both BF and PF cells. Both the forms of T. brucei cells generate TERRA molecules of size from 0.4 kb to 5.0 kb long. In PF cells, TERRA was detected to be originating from both the telomeres as we have detected them with both the telomere DNA strand specific probes. Heterogeneous size of TERRA in PF developed fainter signals, but one obvious strong signals were detected ~800 bps originating in C-rich and G-rich telomere DNA probes. This type of band was undetected in previously published report. It is possible that TERRA may be generated from multiple telomeres in PF cells, giving rise to heterogeneity, but one specific locus can transcribe ~800 bp long telomere transcript. Presence of both the types of TERRA in PF forces to ask the question that may be TERRA is involved in telomere silencing. Non-coding RNAs have been shown to silence a

150 locus in the mouse cells silencing genes responsible for sex determination. Hence, it is possible that in PF cells presence of TERRA promotes VSG silencing.

However, further studies are required to further comment on the role of TERRA in

PF cells.

Conversely, in BF cells we detected TERRA molecules originating only from

C-rich telomere DNA strand giving rise to 5’-UUAGGG-3’ transcript. Results detected in BF cells are comparable to previously published report. T. brucei

TERRA transcription is not sensitive to α-amanitin treatment, suggesting that it is transcribed by RNA Pol I (Rudenko, 1989). In the same paper authors have speculated that TERRA in BF cells, can be transcription read-through post VSG on the active ES. To confirm this hypothesis, we first performed reverse transcription using a primer that can anneal to the 5’-UUAGGG-3’ transcript and found that, indeed, TERRA is a read-through of RNA pol I. As we only detected

PCR product with the active VSG adjacent to telomeres, but not with the silent

VSG (Fig. 4-3. Fig. 4-4 and Fig. 4-5). We also confirmed same by using different strains with different VSG expressers and similar results were obtained. It is puzzling how TERRA expression is still limited from the active ES in TbRAP1- depleted cells. These results indicate that; TERRA transcription is controlled by a mechanism independent of TbRAP1 presence. It has been reported that although all the VSGs are derepressed in TbRAP1 RNAi cells, transcription of active ES remains almost similar, hence, it is possible that TERRA transcription continues to originate from the originally active ES. However, further experiments are required to identify the roots of this conundrum. In budding yeast and human cells, TERRA

151 is transcribed by RNA pol II. (Bah et al., 2011, Schoeftner and blasco, 2008). Also, yeast and human telomeres are not controlled for its subtelomere transcription in monoallelic manner, suggesting TERRA may originate from multiple telomeres. In fission yeast, various telomere RNA molecules are transcribed by RNA Pol II, some having and some other not having telomere sequences (Greenwood and

Cooper, 2011). However, we only identify one G-rich transcript as TERRA in T. brucei. These observations suggest that although conserved from yeast to mammals, TERRA may behave and regulated differently in different organisms.

Telomere proteins have been discussed earlier in this thesis for their role in

TERRA regulation and maintenance in several organisms. Here, first we checked the TERRA levels by depleting TbRAP1 protein. We have detected ~11 fold increase in TERRA levels in TbRAP1 depletion condition by northern slot blot analysis (Fig. 4-8). Also, by northern blot analysis, we found shift in the signal intensity towards the high molecular weight, suggesting removal of TbRAP1 allows telomere transcription further in telomere (Fig. 4-6). In T. brucei, it was shown earlier that depletion of TbRAP1 disrupts telomeric silencing and increases subtelomeric ES expression, which is transcribed by RNA Pol I (Yang et al., 2009).

Depletion of TbRAP1 does not affect the active VSG expression significantly, suggesting that the extra amount of TERRA in TbRAP1-depleted cells are transcribed from derepressed ESs. However, it is possible that removal of TbRAP1 from the telomere chromatin may allow further read-through from the active BES transcription into the downstream telomeric region. Upon deletion of TbRAP1, originally silent VSGs were derepressed as shown in Fig. 4-10B. However, TERRA

152 still originated from the active ES. Moreover, this suggests that removal of TbRAP1 allows stronger and longer read through of the telomeres. The increase in the length of the TERRA molecule observed in TbRAP1 RNAi strain by northern blot

(Fig. 4-6), could be due to unspliced 70bp repeats, as we could also detect region

70 bp repeats amplified in the RTPCR experiment (Fig. 4-10B). However, defects in splicing needs to be evaluated in TbRAP1 RNAi condition. Our data demonstrate that TbRAP1 is an important regulator of the TERRA levels, but further studies needs to be done to identify molecular mechanism for this regulation. Similarly, budding yeast RAP1 is essential for telomeric silencing and suppresses TERRA levels, indicating that the function of RAP1 in TERRA expression regulation is conserved between T. brucei and budding yeast, however, detailed mechanism of the TERRA regulation in yeast is unknown. A report has been provided suggesting the modification of the chromatin at the telomeres in RAP1 depleted budding yeast cells (Iglesias, et al., 2011), but no significant difference at the chromatin structure was observed when TbRAP1 was depleted in BF cells (Pandya et al., 2013) On top of that we also found a ~5 fold increase in the TERRA amount upon removal of the TbTRF protein (Fig. 4-7, Fig. 4-9). We have not performed northern analysis in TbTIF2 RNAi strains, as it is shown that depletion of TbTIF2 affects TbTRF protein stability, we do expect an increase in TERRA level similar to that in TbTRF depleted cells (Jehi et al., 2016). In general, T. brucei telomere proteins have direct or indirect role in the regulation of the TERRA expression.

R-loops are three-stranded structure formed by an RNA:DNA hybrid plus a displaced single-stranded DNA, which is sensitive to RNaseH activity and can be

153 recognized specifically by the S9.6 antibody. Excessive R-loops formed co- transcriptionally at highly transcribed regions are an important source of DNA breaks as they potentially block DNA replication (Hamperl et al., 2014). Increased levels of TERRA can form the R-loop structure with telomere DNA in mammalian and yeast cells, which promotes telomere recombination (Bah et al., 2013, Arora et al., 2014). Here we detected a significant amount of telomeric RNA:DNA hybrids upon TbRAP1 depletion (Fig.4-11), indicating that the telomeric RNA:DNA hybrids can be formed in T. brucei cells and that telomeric R-loop is a conserved feature from Kinetoplastids to mammals. In yeast, when individual telomere transcription is induced, the highly transcribed telomere is rapidly shortened, indicating that

TERRA exerts its function in cis. Since R-loops are formed co-transcriptionally, it is expected that TERRA can only form R-loops with the telomere from which it is transcribed. We have not performed any experiment to solve this mystery and further investigation is essential to further deduce the involvement of TERRA to form R-loop in cis or trans. Our observations suggest that increased TERRA levels is the reason for more telomeric R-loops in TbRAP1-depleted cells. However, it is possible that increased TERRA level may not be the exclusive factor for higher levels of telomeric RNA:DNA hybrid. Unprocessed DNA replication intermediates such as also contain RNase H-sensitive RNA:DNA hybrids.

Therefore stalled DNA replication at telomeres could also contribute to the increased amount of telomeric RNA:DNA hybrids. However, it is currently unknown whether TbRAP1 is important for telomere/subtelomere DNA replication and whether DNA replication is stalled upon TbRAP1 depletion. Although, a recent

154 report suggests that highly transcribed regions are more prone to early replicate in the BF cells (Devlin et al., 2016), suggesting potential encounter between transcription and replication machineries collides on the active ES.

We found that TbRAP1 depletion led to increased levels of the telomere transcript and increased amount of telomeric RNA:DNA hybrids. Since telomeric

RNA:DNA hybrids have been shown to induce more telomeric DNA recombination events, we hypothesize that RNA:DNA hybrids are an important factor contributing to more DSBs at subtelomeric VSG loci and the telomere/subtelomere junctions in

TbRAP1-depleted cells. These breaks in turn leads to increased VSG gene conversion and more frequent VSG switching in TbRAP1-depleted cells. True to our hypothesis, we observed more DSBs at both the active and the silent subtelomeric VSG loci upon TbRAP1-depletion (Fig. 3-8, Fig. 3-9, Fig. 3-11, Fig.

3-12). Hence, we conclude that in TbRAP1 depleted cells, higher amount of

TERRA levels, increase R-loop levels which will ultimately generates more DNA

DSBs.

Ribonuclease H is an enzyme that specifically cleaves the RNA strand of the RNA:DNA hybrid (Hamperl et al., 2014). R-loops are sensitive to RNaseH, as our data suggested that treatment with in-vitro RNaseH reduced the amount of

IPed telomeric R-loops and brought R-loop levels similar to IgG pulldown in

S/RAP1i strain (Figure 4-11). Also, in yeast and human telomeres RNaseH protein has been shown to reduce R-loop levels at the telomeres (Balk et al., 2014, Luke et al., 2013) and overexpression of RNaseH reduces the recombination of telomeres (Balk et al., 2014, Luke et al., 2013). To do the similar type of

155 experiments we ectopically expressed TbRNaseH1 gene. Ectopic expression ofTbRNaseH1 in TbRAP1 RNAi background delayed the growth arrest by 24 hours but did not affect TbRAP1’s transcription associated phenotypes. This evidence suggests that, ectopic expression of TbRNaseH1 and possibly R-loop are not involved in ES transcription silencing.

Previously we concluded that increase in the DNA DSBs in TbRAP1 RNAi strains are caused by increase in TERRA and R-loop levels. Hence removal of R- loop should suppress DNA DSBs. As expected, reduction in R-loop levels were observed in S/RAP1i + TbRNAseH1 strain, and DNA DSB break levels were similar to wild type at the VSG loci as well as at telomeres when TbRNaseH1 was ectopically expressed (Fig. 4-17, Fig. 4-18). DNA breaks in S/RAP1i +

TbRNAseH1 strain were analyzed by loading of TbRAD51 and γH2A. Although they both serve as DNA DSBs associated markers, different levels of loading were observed. No change in TbRAD51 loading was observed when we compared the signals between uninduced and induced S/RAP1i + TbRNaseH1 strain, however, there was significant reduction in γH2A at the same locus was observed (Fig. 4-

17C). γH2A loading can sometimes be stretched up to 2M bp long span (Kinner

A., 2008). Therefore, we detected higher amount of γH2A loading at 50 bp repeats and EsPr in S/RAP1i strains, but a significant reduction was observed at the subtelomeres in ectopically expressing TbRNAseH1 strain. As the breaks are less in TbRNaseH1 expressing strain compared to S/RAP1i strain, that leads to less spreading of γH2A, hence we detect significant reduction in γH2A association at

50 bp repeat region (Fig. 4-17C).

156

Break in the active ES was found to be the cause of increase in VSG switching, when we depleted TbRAP1. As removal of R-loop from the telomeres by ectopic expression of TbRNaseH1 reduced DNA DSBs. We also observed reduction in VSG switching frequency and it was similar to wild type. On top of that, all the switched clones had similar switching events patterns very similar to wild type cells switchers.

We failed to detect any R-loops at the highly transcribed VSG locus by

DRIP-qPCR. This suggests that may be many R-loop processing factors are playing role at these regions and do not allow RNA pol I complex to stall to form

RNA:DNA hybrids. Also, it is possible that these regions are simply unfavorable to form R-loops, as it requires more repetitive sequences with high G-rich in a non- template region (Hamperl et al., 2014). On top of that, we do not rule out the technical limitations of DRIP-qPCR. R-loop formation at the telomeres can abruptly stop the transcription, as the stability of R-loop at telomere is much stronger

(Hamperl et al., 2014) and RNAP complex stalls leaving a single strand DNA unprotected, this can generate breaks in close region to telomeres and 3UTR of

VSGs. Break in the active ES will increase the VSG switching frequency.

Understanding the mechanisms of antigenic variation and its molecular regulators was the ultimate goal of this thesis. Ectopic expression of TbRNaseH1 in the S/RAP1i cells reverted the increased VSG switching frequency and VSG GC switching events, either usually observed in S/RAP1i strain. This observation led us to conclude that the increase in the VSG switching frequency and the increase in the VSG associated gene conversion events in TbRAP1-depleted cells was due

157 to the increased TERRA levels and more telomeric R-loops. Conclusions from the observed results allowed to generate a model and it is presented above in figure

4-21.

Figure 4-21: A working model of the function of TbRAP1 linking TERRA to DSB- associated VSG switching. In the wild type cells, there is only one type of VSG expressed on the cell surface. A basal level of TERRA is detectable. DSBs are mostly detected at the 70 bp repeat regions (Boothroyd et al., 2009, Hovel-miner et al., 2016). TbRAP1 depletion leads to derepression of all silent ESs, higher levels of TERRA, and more telomeric R-loops. More DSBs are detected close to the telomeres and VSGs. This leads to more frequent VSG switching and most switchers arise through VSG GC events. However, ectopic expression of an extra TbRNAseH1 allele reverts the increase in telomeric R-loop levels and reduces the amount of DSBs at the VSG loci and telomeres. This in turn reduces the VSG switching frequency and the switching pattern is again similar to that in wild type cells. Red arrows indicate fully transcribing ES. Black arrow indicated transcription attenuation in the silent ES.

158

CHAPTER V

TbRAP1 INTERACTING CANDIDATES

5.1 Introduction

RAP1 is a conserved protein from trypanosomes to mammals (Longtime et al., 1989, Li et al., 2000, de Lange, 2010). At telomeres RAP1 is required for many different functions, such as telomere length maintenance, telomere end protection, and telomeric silencing (Marcand et al., 1997, Kanoh and Ishikawa, 2001,

Vodenicharov et al., 2010, Miller et al., 2005). In S. cerevisiae, RAP1 interacts with

Rif1/Rif2 and together they regulate telomere length. Also, RAP1 interacts with

Sir3/Sir4 to maintain subtelomere gene silencing. In S. pombe RAP1 interacts with

TAZ1 to maintain telomere length and it is an indispensable factor for spindle pole body formation at the premeiotic horsetail stage (Chikashige and Hiraoka, 2001).

In mammals, RAP1 is an essential protein in humans and a non-essential component in mouse, also it is important for the telomere length regulation. Most importantly it inhibits NHEJ at the human telomeres suggesting that, it suppresses telomere recombination (Li et al., 2000, Bae and Baumann, 2007, Sarthy et al.,

2009). RAP1 functions in DNA damage repair and homologous recombination, and

159 molecular steps for these processes are much more complicated. RAP1 is also involved in telomeric silencing and represses transcription of the subtelomeric genes. All these evidence provided the proof that RAP1 is an intrinsic factor of telomere complex and is essential for telomere end protection and subtelomeric gene silencing.

Although RAP1’s role at telomeres has been studied extensively, it was initially identified as transcriptional activator and a repressor in S. cerevisiae

(Shore & Nasmyth, 1987). RAP1 has been reported to be involved in non-telomeric functions. RAP1 controls transcription of glycolytic enzymes and ribosomal genes

(Shore, 1994). Recent ChIP-seq analysis revealed that, RAP1 binds to extra- telomeric sites in the mouse and yeast (Martinez et al., 2010). Gene set enrichment analysis (GSEA) of RAP1 null mouse embryonic fibroblasts (MEFs) revealed deregulated pathways involved in cell adhesion and metabolism, including the peroxisome proliferator-activated receptor (PPAR) pathway (Martinez et al., 2010).

PPAR belongs to the major regulator hormone receptor family and plays a crucial role in nutrient homeostasis (Kersten et al., 2000). RAP1 binds to PPRα and

PGC1α and regulates its transcription activation, ultimately linking RAP1’s role in obesity (Martinez et al., 2013, Yeung et al., 2013). RAP1 was also found to have non-telomeric functions as a transcriptional and a regulator of NF-κB pathway by interacting with IKB kinase (IKK) (Kabir et al., 2010). Overall, these observations suggest that RAP1 interacts with many telomeric and non-telomeric proteins and is involved in multiple cellular functions.

160

In T. brucei, TbRAP1 is reported to interact with TbTRF, and it is required for VSG silencing (Yang et al., 2009). It also helps maintain the subtelomeric heterochromatic structure in PF T. brucei cells (Pandya et al., 2013). Now, we have shown that it suppresses telomere transcription, telomeric R-loops, telomeric and subtelomeric DSBs, and VSG switching. All these observation establishes that,

TbRAP1 protects T. brucei telomeres and plays an important role in subtelomeric gene silencing in T. brucei. To further study TbRAP1’s role in T. brucei telomere biology and antigenic variation, we need to identify TbRAP1-interacting proteins.

To do so we have done several IP and mass spectrometry analyses.

5.2 Results & Discussion

5.2.1 Large-scale immunoprecipitation and mass-spectrometry analysis of

TbRAP1 protein complex

We tagged one endogenous TbRAP1 allele at its N-terminus with the FLAG-

HA-HA epitope in WT427 procyclic form cells. The other allele of WT TbRAP1 is replaced by a hygromycin resistance gene. We performed a large scale immunoprecipitation in this strain in a sequential manner where we first pulled- down the TbRAP1 protein complex with anti-flag M2 beads (Sigma). After eluting the first IP products by the FLAG peptide, we used anti-HA antibody to perform the second IP. Final IP products were further separated on a 20%-60% sucrose gradient by centrifugation. Protein samples were collected from different fractions and analyzed by 10% PAGE gel. Western blotting was performed using anti-RAP1 antibody 598 and anti-HA antibody. Results from these experiments are reported in Fig. 5-1 & Fig. 5-2.

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Fig. 5-1: Western analysis of immunoprecipitation of the TbRAP1 protein complex. Samples collected at each stage of the immunoprecipitations are numbered 1 to 8. Where, 1- whole cell extract, 2-supernatant, post high salt treatment, 3- pellete post high salt treatment, 4- Input, 5-supernatant, anti-flag M2 beads IP, 6- pellete, anti-flag M2 beads IP, 7- supernatant, anti-HA-12CA5 IP, 8- pellete, anti-HA12CA5 IP. Sample-9 is the sample from the pooled fraction 9-12 of 20% to 60% sucrose gradient (Fig. 5-2). Top blot, anti-TbRAP1 antibody 598. Bottom blot, anti-HA antibody, 12CA5.

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Fig. 5.2 – Western analysis of protein samples collected from 20% - 60% sucrose gradient. IPed sample (Pellete fraction of 12CA5 IP, Fig.5.1) was loaded on 20%- 60% sucrose gradient. Samples are centrifuges for 19 hours at 4C at 41000 rpm in the Beckmann ultracentrifuge with a SWTi55 rotor. Samples were collected from top to bottom, manually, with 20% on top and 60% at the bottom. 20 equal samples are collected and purified with strata-clean resins (Agilent technologies). Protein samples were separated on a 10% PAGE; fraction numbers are labelled on top of each figure. Whole cell extract samples were used as a control.

We detected TbRAP1 protein spreading from fraction 5 to fraction 20 when the whole cell extract was loaded on the 20%-60% sucrose gradient. However, sequential IPed samples were separated between fractions 9-12. Hence, we pooled these fractions in one, and ran them on 10% pre-cast PAGE gel (Bio-rad).

The gel was stained with 0.25% coommassie blue and the entire IP product lane was sent for the mass-spec analysis. We have performed this experiment twice

163 and coommassie blue stained gels are shown in the Fig. 5-3. As a negative control,

Dr. Xiafong Yang, a former postdoc fellow in our lab, performed the same sequential IP with anti-FLAG and anti-HA antibodies using WT427 cells, where

TbRAP1 protein is not tagged. She also did one large-scale IP and mass-spec analysis without sucrose gradient analysis. Based on the mass-spec data from the control strain, we will be able to remove any contaminants binding FLAG and HA antibodies non-specifically.

Fig. 5-3 Coommassie-blue staining of TbRAP1 IPed protein complex. Fraction 9- 12 were pooled together and loaded on 10% PAGE gel and stained with 0.25% Coommassie-blue stain. Entire lane was subjected to mass-spectrometry analysis.

In the IPed TbRAP1 complex, we identified TbTRF and TbTIF2, two known telomere proteins, suggesting that we have successfully pulled-down the TbRAP1- containing telomere protein complex. More than 30 hypothetical proteins have been identified, among which are RNA processing factors, chromatin remodelers,

164 histone proteins, metabolic pathway associated proteins, and several proteins without any known domains/functions. We also identified a number of housekeeping proteins such as tubulin, heat shock proteins, and translation machinery subunits.

To confirm the identified candidates indeed interact with TbRAP1, we tagged 10 most abundant hits with the FLAG-HA-HA epitope at the C-terminus of their endogenous allele through PCR based targeting or with the TY1 or GFP epitope at the N-terminus by transfecting a targeting construct. After this, we performed Co-IP experiment and found two candidates interact with TbRAP1 (Fig.

5-4). Among them one is TbNOT1, which is an adaptor protein for a large protein complex that often contains CCR4 and NOT4 that are involved in mRNA decay and mRNA export (Farber et al., 2013). Nine other proteins have been found in the

TbNOT1 protein complex (Esbestan et al., 2013). We tagged TbNOT1 at the N- terminus with TY1 epitope and replace the other allele with the hygromycin resistance gene. To check its interaction with TbRAP1, we performed co-IP and IP results were analyzed by western blotting (Fig. 5-4). After co-IP, we can detect

TbRAP1 in the IPed TbNOT1 protein complex, but we could not detect TbNOT1 in

IPed TbRAP1 protein complex in this experiment, although we identified TbNOT1 in TbRAP1 IPed complexes in the large-scale IP/Mass spectrometry analysis. It is possible that the interaction between TbNOT1 and TbRAP1 is transient. It is also possible that TbRAP1 forms many different protein complexes and TbNOT1 is only on one of the sub complexes. Further investigation is necessary to test which scenario is true. Initial identification of CCR4-NOT1 complex suggests that it has

165 important functions in the cytoplasm. However, several studies in yeast also suggest that TbNOT1 may have an important function in the nucleus and regulate transcription initiation (Collart M., 2013). Why TbRAP1 interacts with TbNOT1 is an interesting question to pursue. In yeast and human cells, NOT1 complex is present in many diverse functions (Collart, 2013). Among them two roles which can stand out here in T. brucei with its interaction to TbRAP1. First, TbRAP1 and

TbNOT1 interaction may be important for transcription elongation of the active ES, as TbRAP1 may potentially bind to silent ES in TbNOT1 interaction dependent manner but not at the active ES. Therefore, presence of TbNOT1 to the silent ES may not allow them to be fully transcribed. Second, telomeres are usually located close to nuclear lamina, hence, interaction of TbNOT1 and TbRAP1 may assist transcripts originating from active ES to be transported, but do not allow transcripts from the silent ES to be transported. Hence upon depletion of TbRAP1 may affect either or both the possible functions allowing VSGs to derepress. TbNOT1 is an essential protein in PF cells and depletion of TbNOT1 is shown to increase global transcript stabilization (Esbestan et al., 2013).

166

Fig. 5-4: Western analysis of CO-IP products done in cells expressing Ty1- TbNOT1. IP was performed using whole cell lysate using mouse anti-TY1 antibody BB2 (MSKCC antibody core) and anti-TbRAP1 antibody 598 (Yang et al., 2009). IgG was used as a negative control. WCE, whole cell extract.

Since TbRAP1 is essential for VSG silencing and suppresses VSG switching, we speculate that TbNOT1 may play a role in antigenic variation, too.

Therefore, we would like to examine phenotypes in cells lacking functional

TbNOT1. However, we were not able to knockdown the TbNOT1 protein by RNAi, possibly because the RNAi construct does not work efficiently. We have recently made new RNAi constructs, which we will use in the immediate future.

The second protein that interacts with TbRAP1 is TbPIP5

(phosphatidylinositol 5-phosphatase). It is an inositol phosphate pathway associated enzyme which converts diphosphate molecules of phosphatidylinositol pathway intermediates into monophosphate (Such as, PI-(4,5)-P2 to PI-(4)-P) by removing a phosphate group. PIP5 interacts with TbRAP1, it is present in the nucleus and has a very similar effect on VSG silencing as TbRAP1 (Cestari et al.,

2015). Why TbPIP5 interacts with TbRAP1? Answer to this question is still a mystery, and further experiments are required for further understanding on the

TbRAP1 and TbPIP5 interaction.

The recent report confirms that TbRAP1 is indeed associated with other unknown partners to regulate VSG silencing. However, we have only confirmed the interaction between TbRAP1 and two of the candidates among many. Our current IP products appear to include many proteins that do not interact with

167

TbRAP1 tightly. To increase the specificity of the IP, we plan to repeat TbRAP1 IP using nuclear extracts followed by sucrose gradient separation.

168

CHAPTER VI

FUTURE PERSEPECTIVES

6.1 TbRAP1’s role in antigenic variation

TbRAP1 was identified to interact with TbTRF in vivo (Yang et al., 2009). It is also reported to be involved in VSG silencing (Yang et al., 2009, Pandya et al.,

2013). Here, we have identified that TbRAP1 is involved in VSG switching regulation, as transient depletion of TbRAP1 led to 6-7 fold increase in VSG switching frequency. Further, we also found that depletion of TbRAP1 increase

DNA DSBs at the telomeres and subtelomeres. The effect of TbRAP1 depletion on telomere/subtelomere integrity is similar to its effect on VSG silencing in that more severe defects have been observed at loci closer to telomeres. As reported earlier, breaks in the active ES are responsible for increase in VSG switching frequency and the site of the break can decide the switching mechanism (Glover et al., 2013). Increase in the amount of DSBs at subtelomeres led to an increase in VSG GC switchers in TbRAP1-depleted cells. TbRAP1 is the first telomeric protein that when depleted still prefers VSG GC as the switching mechanism,

169 which is different from that in WT cells. Depletion of TbTIF2 and TbTRF, two other telomere proteins, although also increased VSG switching frequency, did not change the preferred VSG switching mechanism (most switchers still arose through ES GC/ES loss + in situ). Upon close analysis of the DNA break sites in the ES in TbRAP1 depleted cells suggests that breaks mostly are inside the VSG or between VSG 3’UTR and telomeres. Break at that site has been reported to generate more VSG GC associated switchers. On the contrary, DNA break profile to other two telomeric protein is different, TbTRF, where no significant increase in the DNA breaks was detected in active ES and TbTIF2, where breaks are significantly higher in the entire ES as well as at EsPr. Breaks at the EsPr rarely allows switching to conclude to ES loss or GC type of phenotypes (Glover et al.,

2013). That’s why, switchers in TbTRF and TbTIF2 generates switcher from WT associated switching mechanism. Hence, we provided further evidence for the role of TbRAP1 beyond VSG silencing and in telomere/subtelomere DNA protection.

We attempted to identify new TbRAP1 interacting candidates, out of many hits, we have confirmed two proteins (TbPIP5 and TbNOT1) that interact with

TbRAP1. Among them TbPIP5 has been reported by another group to play essential role in VSG silencing (Cestari et al., 2015). Further investigation regarding involvement of TbRAP1 with TbPIP5 is very essential to decipher the functional importance of VSG silencing in T. brucei. Similarly, TbNOT1 is a protein with multiple diverse cellular functions. We need to dissect TbNOT1’s functions that specifically require its interaction with TbRAP1, which is challenging. Finally, we will perform TbRAP1 IP using the nuclear extract to identify more specific

170

TbRAP1 interacting candidates. It is also important to study levels of TERRA and formation of R-loop at the telomere in TbPIP5 depleted cells, as depletion of

TbPIP5 showed derepression of all the silent VSGs in similar way. Identifying the role of this interaction for the role of R-loop maintenance will be the first of its kind.

It is still unknown whether TbRAP1 associates with the telomere chromatin through TbTRF or through binding telomere DNA directly. TbRAP1 has one central myb domain, while Myb domains are known to form a helix-turn-helix structure and can bind to double-stranded DNA when homodimerized. However, sequence analysis of the TbRAP1 myb domain suggests that this domain is not a typical

DNA-binding myb domain. Still, our preliminary structural analysis suggests that the region of TbRAP1 between the central myb domain and the C-terminal RCT domain may harbor a short region that could interact with dsDNA directly. Hence, it is important to express different domains of TbRAP1 as recombinant proteins and check their interactions with dsDNA by gel shift experiments. In addition, different TbRAP1 mutants lacking various domains will be expressed in T. brucei cells, and the association of these TbRAP1 mutants with the telomere chromatin can be examined by ChIP and IF analyses. Because TbTRF binds duplex telomere

DNA and TbRAP1 interacts with TbTRF (Li et al., 2005, Yang et al., 2009), it is likely the interaction between TbTRF and TbRAP1 helps to recruit TbRAP1 to the telomere. Therefore, it is also important to identify the exact positions on TbRAP1 and TbTRF that are required for their interaction. This will allow us to generate

TbRAP1 mutants that do not interact with TbTRF so that we can determine whether TbRAP1’s localization at the telomere depends on TbTRF. This will also

171 allow us to study TbRAP1’s role at telomeres independent of its interaction with

TbTRF even if TbTRF is not essential for recruiting TbRAP1 to the telomere.

RAP1, also has been shown to interact with non-telomeric sites in yeast and human cells (Martinez et al., 2013). Initial identification of RAP1 was due to its function as a repressor at the sites other than telomeres (Kyuon et al., 1989). Also, it has been identified to be responsible for the transcription of the genes related to glucose metabolism. RAP1 null mice although viable are have predominance to obesity (Yeung et al., 2013, Martinez et al., 2013). Therefore, we want to identify that besides VSGs, what are the other transcripts are affected by RNA-seq analyses in TbRAP1 depleted cells. This will allow us to determine whether

TbRAP1 can function as a transcription regulator for non-VSG genes. In T. brucei, glucose metabolism pathway is highly active and discovery of TbRAP1 involved in these pathway regulation may potentially open new horizon to further understand the role of TbRAP1 in antigenic variation.

6.2 Role of TERRA in wild type cells

We have identified that depletion of TbRAP1 leads to excessive amount (10 fold higher) of TERRA which contributes to more (16 fold higher) telomeric R-loops that in turn causes more DSBs at the telomere vicinity. However, what are the roles of TERRA in wild type cells is still unknown. In human cells, TERRA has been shown to interact with TRF2 and ORC to form a stable complex (Deng et al., 2009).

TERRA has also been shown to play an important role in telomere length regulation in telomerase dependent and independent manner in yeast and human cells (Arora et al., 2014, Balk et al., 2013). Overall, studies of TERRA in human,

172 mouse, and yeast cells suggest that TERRA has both conserved and diverse cellular functions (Arora et al., 2011).

We found different moieties of TERRA in two different life cycle stages in T. brucei. In PF cells TERRA originates from both the telomeres giving rise to C-rich and G-rich TERRA. Subtelomeres are not transcribed in PF cells, suggesting a stronger heterochromatin structure present at the PF telomeres. Why TERRA is of a particular size in PF cells? In PF cells subtelomeric VSGs are not expressed, so

TERRA is not expected to be transcribed from any ES-adjacent telomeres. Where is it transcribed from in PF cells then? Does TERRA arise from multiple telomeres in PF cells? Studying the role of TERRA in PF may answer about TERRA’s function in heterochromatin formation. Depletion of TbRAP1 in PF cells opened more chromatin at the subtelomeres. Should removal of TbRAP1 in these cells increase

TERRA expression levels than, it may increase our knowledge regarding the role of TERRA in strong heterochromatin formation.

Conversely, in BF only one subtelomeres are transcribed and they give rise to TERRA molecule from the active ES only. It is important to find out the significance of this observation in WT cells. If, TERRA is involved in heterochromatin structure at formation at T. brucei telomeres than it may be possible that TERRA interacts in trans to silent telomeres and hence keeping only one ES fully transcribed.

We would also like to perform RNA-IP to identify proteins that bind to

TERRA. As in human cells, TERRA may interact with multiple telomeric proteins.

Identification of these proteins will help reveal functions of TERRA. Our preliminary

173 data (Experiments performed by Dr. Ranjodh Sandhu a previous graduate student at Dr. Bibo Li’s lab, and currently doing post doc at UC Davis & Dr. Unnati Pandya, another graduate student, and currently a post doc at New York University School of Medicine) suggest that TbTRF interacts with TERRA but TbTbRAP1 does not.

However, further studies are required to confirm these findings. If interaction of

TbTRF and TERRA is stable than may be combine they form strong heterochromatin structure at the silent telomeres but not at the active telomeres.

Similar interactions need to be evaluated in PF cells as well. Studies of TERRA in both the form of cells will provide further evidence that may be non-coding RNA and its transcription is associated to control transcription of procyclins, mVSGs and

VSGs in procyclic form, metacyclic form and bloodstream form of T. brucei.

Depletion of TERRA by siRNA showed increase in telomere transcription and shortened telomeres. It also induced increase telomere dysfunction foci (TIFs)

(Deng et al., 2009). Also, depletion of TERRA leads to reduction in telomere heterochromatin structure, as it reduces ORC loading and decreased H3K9 timethylation at the telomeres (Deng et al., 2009). Here, we found that excessive amount of TERRA forms telomeric R-loops, hence we tried initially to stop the expression of TERRA by RNAi using several different RNAi vectors. We tried to express different lengths of double stranded telomere RNA and tried a stem-loop formation construct. However, we could not achieve efficient knockdown of

TERRA. Because TERRA are transcribed from the active ES-adjacent telomere, we will try to interrupt the transcription after the active VSG by introducing a lac operator immediately upstream of the telomere repeats and expressing the Lac

174 repressor in the cells under the induced condition. This technique may allow us to stop the transcription of TERRA.

We also plan to overexpress TERRA under the inducible condition. After achieving that, first we want to confirm the increase in R-loop level phenotypes at the telomeres as we have observed here. It is completely possible that, we may not detect more R-loops, even after overexpressing TERRA, as we do not know whether TERRA works in cis or trans in the cell. Also, it is important to note that so far we observed more telomeric R-loops only in TbRAP1-depleted cells. It is possible that TbRAP1 may hinder R-loop formation or stability, thus removal of

TbRAP1 allows TERRA to form the telomeric R-loop structure. If this is true, then overexpression of TERRA in the presence of WT TbRAP1 protein may not lead to more telomeric R-loops. Therefore, by performing manipulating the expression of

TERRA we may potentially be able to identify previously unknown function of

TERRA in T. brucei and importantly, enhance our knowledge about antigenic variation.

Finally, further investigation in T. brucei telomere biology will potentially help us to advance our knowledge in telomere biology and telomere’s function in parasite’s pathogenesis. We may be able to identify new targets for anti-parasite drugs. Collectively, this thesis explains that how much antigenic variation is a very complex process and T. brucei is a master organism which has devoted significant number of proteins and cellular machinery for the regulation of it. We have made new discoveries in the field of T. brucei biology and knowledge obtained here

175 regarding the functions of TbRAP1 in antigenic variation can be further applied for the eradication of T. brucei.

176

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APPENDICES

212

Note for this and following tables: a Confirmed by PFGE and Southern data not shown b Confirmed by PFGE and Southern, shown in Fig. 3-7 c switcher lost while recovering for PFGE analysis d Some ES GC/ES loss+in situ (ES GC/ESLIS) switchers showed weak BSD

(100 µg/ml) resistance, with cells growing extremely slow. PCR analysis

using BSD specific primers showed that these switchers only retained part

of the BSD gene.

S, sensitive; R, resistant

Transcription from silent BES promoters is weakly active and quickly

attenuated [65]. Therefore, in situ switchers weakly express the BES

promoter adjacent BSD and are resistant to 5 µg/ml blasticidin but

sensitive to 100 µg/ml blasticidin.

213

Appendix - A

Table-6: Phenotype and genotype characterization for switcher from S +Dox cells.

Phenotype Genotype Puro BSD BSD Switching 2 100ug ug 5ug/m BSD VSG2 mechanisms /m /m l l l 1 S S S - - ES GC / ESLIS 2 S S S - - ES GC / ESLIS 3 S S S - - ES GC / ESLIS 4 S S S - - ES GC / ESLIS 5 S R R + - VSG GC 6 S R R + - VSG GC 7 S R R + - VSG GC 8 S R R + - VSG GC 9 S S S - - ES GC / ESLIS 10 S S S - - ES GC / ESLIS 11 S S S - - ES GC / ESLIS Weak ES GC / ESLIS 12 d S R - - R 13 S S S - - ES GC / ESLIS Weak ES GC / ESLIS 14 d S R - - R 15 S S S - - ES GC / ESLIS 16 S R R + - VSG GC 17 S S S - - ES GC / ESLIS 18 S R R + - VSG GC 19 S S S - - ES GC / ESLIS

214

Appendix - B

Table - 7: Phenotype and genotype characterization for switcher from S/ev + Dox cells.

Phenotype Genotype BSD Puro Switching BSD 100ug 2ug/ BSD VSG2 mechanisms 5ug/ml /m ml l 1 S S S - - ES GC / ESLIS 2 S S S - - ES GC / ESLIS 3 S S S - - ES GC / ESLIS 4 S S S - - ES GC / ESLIS Weak 5 d S R - - ES GC / ESLIS R 6 S R R + - VSG GC 7 S S S - - ES GC / ESLIS 8 S S S - - ES GC / ESLIS 9 S S S - - ES GC / ESLIS 10 S R R + - VSG GC 11 S R R + - VSG GC 12 S S S - - ES GC / ESLIS 13 S S S + + In-situ 14 S R R + - VSG GC 15 S S S - - ES GC / ESLIS 16 S S S - - ES GC / ESLIS 17 S S S - - ES GC / ESLIS

215

Appendix - C

Table – 8: Phenotype and genotype characterization for switcher from S/RAP1i -

Dox cells.

Phenotype Genotype Switching

Puro BSD BSD mechanism 2ug/ 100ug/ BSD VSG2 5ug/ml s ml ml ES GC / 1 S S S - - ESLIS ES GC / 2 S S S - - ESLIS ES GC / 3 S S S - - ESLIS ES GC / 4 S S S - - ESLIS ES GC / 5 S S S - - ESLIS ES GC / 6 S S S - - ESLIS 7 S S S + + In-situ ES GC / 8 S S S - - ESLIS ES GC / 9 S S S - - ESLIS ES GC / 10 S S S - - ESLIS ES GC / 11 S S S - - ESLIS ES GC / 12 S S S - - ESLIS ES GC / 13 S S S - - ESLIS ES GC / 14 S S S - - ESLIS

216

ES GC / 15 S S S - - ESLIS ES GC / 16 S S S - - ESLIS ES GC / 17 S S S - - ESLIS Weak ES GC / 18 d S R - - R ESLIS ES GC / 19 S S S - - ESLIS 20 S R R + - VSG GC 21 S R R + - VSG GC 22 S R R + - VSG GC ES GC / ES 23 S S S - - LOSS + In-situ ES GC / 24 S S S - - ESLIS 25 S S S + + In-situ ES GC / 26 S S S - - ESLIS 27 S R R + - VSG GC ES GC / 28 S S S - - ESLIS ES GC / 29 S S S - - ESLIS 30 S R R + - VSG GC ES GC / 31 d S S S - - ESLIS ES GC / 32 S S S - - ESLIS

217

Appendix - D

Table – 9: Phenotype and genotype characterization for switcher from

S/RAP1i+F2H-TbRAP1 + Dox cells.

Phenotype Genotype Puro BSD Switching BSD 2ug/ 100ug/ BSD VSG2 mechanisms 5ug/ml ml ml 1 S R R + - VSG GC 2 S R R + - VSG GC ES GC / 3 S S S - - ESLIS ES GC / 4 S S S - - ESLIS 5 S R R + - VSG GC 6 S R R + - VSG GC 7 S R R + - VSG GC ES GC / 8 S S S - - ESLIS ES GC / 9 S S S - - ESLIS 10 S S S + + In-situ ES GC / 11 S S S - - ESLIS 12 S R R + - VSG GC 13 S R R + - VSG GC ES GC / 14 S S S - - ESLIS ES GC / 15 S S S - - ESLIS ES GC / 16 S S S - - ESLIS

218

Appendix - E

Table – 10: Phenotype and genotype characterization for switcher from

S/RAP1i clone B1 + Dox cells.

Phenotype Genotype Puro BSD BSD Switching

2ug/m 5ug/m 100ug/m BSD VSG2 mechanisms l l l 1 b S S S - - ES GC / ESLIS 2 S R R + - VSG GC 3 b S S S - - ES GC / ESLIS 4 b S S S - - ES GC / ESLIS 5 S R R + - VSG GC 6 c S R R + - VSG GC 7 c S R R + - VSG GC 8 S R R + - VSG GC 9 S R R + - VSG GC 10 S S S - - ES GC / ESLIS 11 S R R + - VSG GC 12 S S S - - ES GC / ESLIS b 13 S R R + - VSG GC 14 S R R + - VSG GC 15 S R R + - VSG GC b 16 S R R + - VSG GC 17 S R R + - VSG GC 18 S R S + + In-situ b 19 S R R + - VSG GC 20 S R R + - VSG GC

219

21 S R R + - VSG GC 22 S R R + - VSG GC 23 S R R + - VSG GC 24 S R R + - VSG GC 25 S R R + - VSG GC 26 S S S - - ES GC / ESLIS a 27 S R R + - VSG GC 28 S R R + - VSG GC 29 S R R + - VSG GC 30 S R R + - VSG GC a 31 S R R + - VSG GC 32 S R R + - VSG GC 33 S R R + - VSG GC 34 S R S + + In-situ a 35 S R S + + In-situ a 36 S S S - - ES GC / ESLIS 37 S R R + - VSG GC VSG LOSS + In- 38 S R S + - situ 39 S R R + - VSG GC 40 S R R + - VSG GC 41 S R R + - VSG GC 42 S R R + - VSG GC 43 S R R + - VSG GC 44 S R R + - VSG GC 45 S R R + - VSG GC 46 S S S - - ES GC / ESLIS b 47 S R R + - VSG GC

220

48 S R R + - VSG GC c 49 S R R + - VSG GC 50 S S S - - ES GC / ESLIS 51 S R R + - VSG GC 52 S R R + - VSG GC 53 S S S - - ES GC / ESLIS 54 S R R + - VSG GC 55 S R R + - VSG GC 56 S S S - - ES GC / ESLIS 57 S R R + - VSG GC 58 S R R + - VSG GC 59 S R R + - VSG GC 60 S S S - - ES GC / ESLIS 61 S R R + - VSG GC 62 S S S - - ES GC / ESLIS 63 S R S + + In-situ a 64 S S S - - ES GC / ESLIS 65 S R R + - VSG GC 66 S R R + - VSG GC 67 S R R + - VSG GC 68 S R R + - VSG GC 69 S R R + - VSG GC 70 S R R + - VSG GC 71 S R R + - VSG GC 72 S R R + - VSG GC 73 S R R + - VSG GC 74 S S S - - ES GC / ESLIS 75 S R R + - VSG GC 76 S R R + - VSG GC

221

77 S R R + - VSG GC 78 S R R + - VSG GC

Appendix - F

Table – 11: Phenotype and genotype characterization for switcher from

S/RAP1i clone C1 + Dox cells.

Phenotype Genotype Puro BSD BSD Switching 2ug/ 5ug/ 100ug/m BSD VSG2 mechanisms m m l l l 79 S R R + - VSG GC

80 S R R + - VSG GC

81 S R R + - VSG GC

82 a S S S - - ES GC / ESLIS

83 S S S - - ES GC / ESLIS

84 S S S - - ES GC / ESLIS

85 S S S - - ES GC / ESLIS

86 c S R R + - VSG GC

VSG LOSS + In- 87 a S R S + - situ

222

88 S R R + - VSG GC

89 S R R + - VSG GC

90 S R R + - VSG GC

91 S S S - - ES GC / ESLIS

92 S S S - - ES GC / ESLIS

93 a S S S - - ES GC / ESLIS

94 S S S - - ES GC / ESLIS

95 S S S - - ES GC / ESLIS

96 S S S - - ES GC / ESLIS

97 S R R + - VSG GC

98 S R R + - VSG GC

99 S S S - - ES GC / ESLIS

100 b S S S - - ES GC / ESLIS

101 S S S - - ES GC / ESLIS

102 S S S - - ES GC / ESLIS

103 S R R + - VSG GC

104 S R R + - VSG GC

223

105 S R R + - VSG GC

106 S R R + - VSG GC

107 S S S - - ES GC / ESLIS

108 S R R + - VSG GC

109 S R R + - VSG GC

110 S R R + - VSG GC

111 S R R + - VSG GC

112 S S S - - ES GC / ESLIS

113 S R R + - VSG GC

114 S R R + - VSG GC

115 S R R + - VSG GC

116 S R R + - VSG GC

117 b S S S - - ES GC / ESLIS

118 S R R + - VSG GC

119 S R R + - VSG GC

120 S R R + - VSG GC

121 S R R + - VSG GC

224

122 S S S - - ES GC / ESLIS

123 S R R + - VSG GC

124 S R R + - VSG GC

125 a S R S + + In-situ

126 S R R + - VSG GC

127 S S S - - ES GC / ESLIS

128 S S S - - ES GC / ESLIS

129 S R R + - VSG GC

130 S R R + - VSG GC

131 S R R + - VSG GC

132 S R R + - VSG GC

133 S R R + - VSG GC

134 S S S - - ES GC / ESLIS

135 S R R + - VSG GC

136 S S S - - ES GC / ESLIS

137 S R R + - VSG GC

138 S R R + - VSG GC

225

139 S R R + - VSG GC

140 S S S - - ES GC / ESLIS

141 S R R + - VSG GC

142 S R R + - VSG GC

143 S R R + - VSG GC

144 S R R + - VSG GC

145 d S R Weak R - - ES GC / ESLIS

146 S S S - - ES GC / ESLIS

147 S R R + - VSG GC

148 S R R + - VSG GC

149 d S R Weak R - - ES GC / ESLIS

150 S S S - - ES GC / ESLIS

151 S R R + - VSG GC

152 S R R + - VSG GC

153 S S S - - ES GC / ESLIS

154 S R R + - VSG GC

155 S R R + - VSG GC

226

156 S S S - - ES GC / ESLIS

157 S R R + - VSG GC

158 S S S - - ES GC / ESLIS

159 S S S - - ES GC / ESLIS

160 S R R + - VSG GC

161 S S S - - ES GC / ESLIS

162 S R R + - VSG GC

163 S R R + - VSG GC

164 S R R + - VSG GC

165 S S S - - ES GC / ESLIS

166 S R R + - VSG GC

167 S R R + - VSG GC

168 S S S - - ES GC / ESLIS

169 S R R + - VSG GC

170 S R R + - VSG GC

171 S R R + - VSG GC

172 S R R + - VSG GC

227

173 S S S - - ES GC / ESLIS

174 S R S + + In-situ

175 S S S - - ES GC / ESLIS

176 S R R + - VSG GC

177 S R R + - VSG GC

178 S S S - - ES GC / ESLIS

179 S S S - - ES GC / ESLIS

180 S S S - - ES GC / ESLIS

181 S R R + - VSG GC

182 S R R + - VSG GC

183 S S S - - ES GC / ESLIS

184 S S S - - ES GC / ESLIS

185 S R R + - VSG GC

186 S S S - - ES GC / ESLIS

187 S R R + - VSG GC

188 S R R + - VSG GC

189 S R R + - VSG GC

228

190 S R R + - VSG GC

191 S R R + - VSG GC

192 S R R + - VSG GC

193 S R R + - VSG GC

194 S R R + - VSG GC

195 S R R + - VSG GC

Appendix - G

Table – 12: Phenotype and genotype characterization for switcher from S/RAP1i

+ TbRNaseH1-2HA cells.

Phenotype Genotype Puro BSD BSD Switching 2ug/ 5ug/ 100ug/m BSD VSG2 mechanisms m m l l l 1 S S S - - ES GC / ESLIS 2 S R R + - VSG GC 3 S S S - - ES GC / ESLIS 4 R R S + + In-situ 5 S S S - - ES GC / ESLIS 6 S R R + - VSG GC 7 S R R + - VSG GC 7 S S S - - ES GC / ESLIS 9 S R R + - VSG GC

229

10 S S S - - ES GC / ESLIS 11 S S S - - ES GC / ESLIS 12 S S S - - ES GC / ESLIS 13 S S S - - ES GC / ESLIS 14 S R S + + In-situ 15 S S S - - ES GC / ESLIS 16 S S S - - ES GC / ESLIS 17 S S S - - ES GC / ESLIS 18 S S S - - ES GC / ESLIS 19 R R R + + In-situ 20 S S S - - ES GC / ESLIS 21 R R R + + In-situ 22 S S S - - ES GC / ESLIS 23 S R R + - VSG GC 24 S R S + + In-situ 25 S R R + - VSG GC 26 S S S - - ES GC / ESLIS

Appendix - H

Table – 13: Phenotype and genotype characterization for switcher from S/ev

+ TbRNaseH1-2HA cells.

Phenotype Genotype

Puro BSD Switching

BSD VSG 2ug/ 5ug/ BSD mechanisms m m 100ug/ml 2 l l 1 S S S - - ES GC / ESLIS 2 S R R + - VSG GC

230

ES GC / ESLIS 3 S S S - - ES GC / ESLIS 4 S S S - - ES GC / ESLIS 5 S S S - - 6 S R S + + In-situ ES GC / ESLIS 7 S S S - - ES GC / ESLIS 7 S S S - - 9 S R R + - VSG GC

231

232