INVESTIGATING HUMAN L1 RETROTRANSPOSON ACTIVITY IN ORAL SQUAMOUS CELL CARCINOMA PATIENTS

Abstract

Mobile genetic elements also known as jumping genes or transposable elements were discovered by Barbara McClintock in 1956 during her experiments with maize crop. These are the piece of DNA sequences which can move around; can jump from one place to another place of the genome in the same cell. During that time this was a very unconventional concept opposing the classical genetics theory that genes are static and have a particular fixed locus in a specific chromosome like beads in a string. Over subsequent years the same kind of phenomenon discovered in other organism including mouse and human, changes the concept that some DNA sequences can move within a genome and thus can cause mutation if it disrupts an essential gene. Genome sequencing of human, mouse and other higher eukaryotic organisms revealed that around 1-2% of the total genome encodes proteins whereas different kind of repetitive sequences occupied more than half of the genome. These repeats are mainly belong to the transposable element which can be classified as 1) DNA transposons which move as such either by cut and paste or copy and paste mechanism and 2) Retrotransposons which use RNA as an intermediate to jump from one place of the genome to another place. Retrotransposons are further divided into LTR and Non-LTR retrotransposons on the basis of long terminal repeats (LTR) present or not. In human genome the DNA transposons (~3% of total genome) and the LTR transposons (~9%) are no longer active. The non-LTR transposons are of great interest as they are actively transposing in the human genome. There are two types of non-LTR transposons, autonomous (transposes using their own machinery) and nonautonomous (required machinery of autonomous elements for their transposition). Long Interspersed Element 1 or LINE1 are the only active autonomous non-LTR retrotransposon and occupies around 20% of the human genome. Transposable elements have been studied in many organisms since its discovery by Barbara McClintock but in human it was in 1988 when Kazazian et al. (Nature 1988, V332, 164-166) noticed a haemophilia A patient resulting from de-novo insertion of LINE-1 sequence without any pedigree for the disease. Sequencing of the factor VIII gene from patient showed LINE1 inserted in the exon 14 and which was the actual cause for the disease. Subsequently, Kazazian laboratory cloned the L1 sequence which disrupted factor VIII gene from patient DNA and then showed the element is highly Abstract ii active in cell culture based retrotransposon assay. These observations confirmed for the first time that the transposable element is active in the recent day human genome. An active L1 is 6.0 kb in length, containing a 900 base pairs (bp) 5´- untranslated region (5’-UTR) with an internal promoter activity, two open reading frames designated as ORF1p and ORF2p separated by a small 63 bp inter ORF spacer sequence and followed by a ~200 bp 3’-UTR. Although the functions of ORFs are poorly understood, both proteins are critical in the process of retrotransposition. ORF1 encoded protein showed in vitro single stranded nucleic acid binding and nucleic acids chaperone activities whereas ORF2 encodes a protein with reverse transcriptase (RT) and endonuclease (EN) activities. It was a general believe that LINE-1 retrotransposons are only active in germ cells (sperm and ovum) and at early stage of development. It is also believed that LINE-1 as a parasite is active in germ cells for its propagation to the next generation. But recent high throughput sequencing analysis revealed that L1 is also active in certain parts of normal brain and in few cancers. The activity of L1 is high in those cancers which are epithelial origin. Although it is known that L1 is highly active in certain cancers, its role towards the development or progression of cancer is completely unknown. Oral cancer a subtype of head and neck is very deadly and highly prevalent in India due to excessive use of tobacco. No study has been performed to see the activity of L1 retrotransposons in oral cancer samples. In this study, L1 retrotransposon activity has been investigated in oral cancer samples obtained from Indian patient. The thesis has been divided into four chapters. Chapter 1 includes the introduction and detailed literature review about transposable elements specifically about mammalian LINE1 retrotransposons structure, mechanism of retrotransposition and its role in health and disease. The chapter also focuses about LINE1 activity in different types of cancer along with literature about oral cancer is included. Chapter 2 comprises the materials and methods used in the research work, Those includes recipes for reagents, solutions, protocols for cloning, expression and purification of proteins, protocols for antibody generation, immunohistochemistry, western blotting and methylation studies and others. Chapter 3 contains details of the results obtained in the study. The main objective of the study was "To find out human L1 retrotransposon activity in oral Abstract iii cancer samples obtained from Indian patient". The main objective was answered by performing following sub-objectives. i) To make antibody against human LINE1 proteins (ORF1p and ORF2p) and characterization of reverse transcriptase (RT) activity encoded by RT domain of ORF2p ORF2 protein of human LINE1 contains three domains:- Endonuclease (EN) domain, Reverse transcriptase (RT) domain and CCHC domain. In the present study different size L1ORF2 fragments containing RT domain was cloned in a bacterial expression vector and its expression was checked in E.coli expression cells. The results showed that RT domain protein was expressed enough in bacterial expression system and due to mis-folding the protein formed inclusion bodies. Next, the RT domain protein was solubilized using urea and purified by Ni-agarose chromatography. After purification the refolding of the protein showed formation of inclusion bodies. Checking soluble fraction showed less 1% induced RT was still in soluble fraction. The purified protein from soluble fraction showed significant RT activity on L1 RNA template. To get the antibody against ORF2p, the partially purified RT domain protein was separated in denatured SDS-PAGE gel and the band corresponds to RT domain protein was injected to rabbit. Immunoblot analysis using partially purified RT domain protein didn't show any band suggesting that injected protein was not immunogenic to rabbit. Since human L1 RT domain protein formed inclusions bodies and didn't make any antibody in rabbit, next I tried to clone, express and purify human L1 ORF1p to generate antibody against it. The human ORF1 fragment was sub-cloned in bacterial expression vector and expression studies showed significant expression of ORF1p (~40kDa) in bacterial system. Although, the protein was expressed in significant amount, it was not purified in homogeneity both in Ni-agarose, as well as in gel filtration chromatography. Simultaneously, antibody against RRM domain (30 kDa) of ORF1p was being tried in the laboratory and RRM domain antigen showed good antibody response in rabbit. So detection of ORF1p in OSCC samples (described below) was performed using ORF1p RRM domain specific antibody [α-hORF1p (RRM)]. Abstract iv ii) To investigate the L1 promoter methylation status and LINE1 retrotransposon activity in OSCC samples. OSCC samples were collected from Acharya Tulsi Regional Centre for Cancer and Treatment Bikaner, Rajasthan. All the experiments using cancer samples were performed as per institute human ethics committee approval and guidelines. The neoplastic nature of all cancer samples used in this study was confirmed by hematoxylene and eosin staining. Next, the samples were proceeded to make paraffin block. Slides made from these blocks were proceeded for immunohistochemistry with ORF1p RRM domain specific antibody [α-hORF1p (RRM)]. Around 60% samples showed ORF1p positive suggesting human L1 retrotransposon pathway is highly active in OSCC samples in the cancer tissues compared to normal. Epigenetic silencing of the L1 5’-UTR by DNA methylation is a common means to inactivate L1 expression and ultimately retrotransposition. Epigenetic alterations are frequent in cancers; indeed, several studies have reported L1 promoter Hypomethylation in a variety of cancers. To date, the methylated state of the L1 5’- UTR in OSCC remained unexamined; therefore bisulfite conversion analysis of genomic DNA across nine paired normal-cancer tissues followed by PCR, subcloning of amplicons, and Sanger sequencing to ascertain the methylation level of the L1 promoter were performed. Specifically, a 363 bp region of the L1 promoter (nucleotide sequence 209-572, L1HS from Repbase) was amplified which contains 20 CpG sites and the resultant amplicons were sequenced. Investigating L1 promoter methylation status, showed significant hypomethylation of L1 promoter in cancer tissues compared to its normal counterpart. Overall, the data shows very high L1 retrotransposon activity in OSCC which might have some significant role in the onset and progression of this particular type of cancer. Chapter 4 includes the discussion part of the thesis which concludes the inferences obtained from the results. Further conclusion and future prospectives of the work has been discussed. Acknowledgements v Acknowledgements I would like to express my profound gratitude to all the wonderful people I had the opportunity to work with during my Ph.D. work. First and foremost I would like to take immense pleasure in expressing my deep sense of gratitude to my supervisor Dr. Prabhat K. Mandal for providing me the opportunity to work in his lab. I also want to thank his constant and untiring support for developing my scientific skills. I am grateful for his constructive criticism and invaluable advice whenever needed while his bounded optimism has always kept my spirits high. I would like to thank Professor R. Prasad for the constant support for doing my experiments. I also express my sincere gratitude towards Professor Partha Roy and Dr. A. K. Sharma for their kind and valuable suggestions. I want to express my gratitude to the current head of the department Prof Partha Roy, who has always been helpful and available for having an open door and encouraging attitude. I take this opportunity to sincerely acknowledge the Department of Biotechnology (DBT), Government of India for providing me the financial support. Most importantly I am thankful to Dr. Jitendra Nangal (Oncologist, Bikaner, Rajasthan) for providing me with important research material, which was very essential to complete the study. I would like to thank Dr. Shilpi Saxena (Pathologist, Military hospital, Roorkee) for helping me with the suggestions whenever needed and providing her lab facilities for the experimental work. I would like to express my thanks and hearty wishes to my labmates Sofia, Debpali, Koel and Keyur for their help and support during the experiments. My warmest thanks to my colleagues and friends at IIT Roorkee and outside it, specially Dr. Manju, Swati Choudhary, Jyoti, Pooja, Anchal, Himanshu, Kaushik, Apurva, Anjlika, Raj kishore, Rashmi, Poonam, Manish, Krishankant and Dr Ashwani for making my tenure at IIT Roorkee memorable. I am specially thankful for my husband Dr. Vinay Tomar for his constant encouragement and support. He has supported me in the most stressful times and Acknowledgements vi always cheered me to make me strong. I also extend my thanks to each member of my in-laws family, father in law, Shri Om Singh, Mother in law, Smt. Jaswati Devi, Brother in laws Mr. Vipin Tomar and Dr. Amit Tomar, sister in laws Smt. Sushma and Dr. Payal who supported me in every possible way to see the completion of this work. Finally I dedicate my thesis to my lovely parents Sh. Devraj Budania and Smt. Durga Rani who always believed me and supported me in every way they could. I thank my younger siblings Pradeep and Kavita for their unconditional love and affection.