STUDIES ON HUMAN LINE-1 RETROTRANSPOSON ENCODED ORF2 PROTEIN

Abstract

Almost two-third of the human genome is derived from the repetitive DNA sequences which are believed to play significant role in shaping the human genome in the course of evolution. One such repeat called retrotransposons occupied around one-third of the human genome. Retrotransposons are sequences which move from one place of the genome to another place in the same cell using RNA as an intermediate and the process is called retrotransposition. The major consequence of retrotransposition is, it disrupts the sequence where it inserts and thus cause insertion mutation. The Long Interspersed Elements (LINEs or L1s) is the most abundant retrotransposon in the human genome with almost 500,000 copies occupied around 17% of the human genome. Although most of the copies are inactive around 100-150 copies are actively jumping in the recent day human genome. It was first reported in 1988 when Kazazian et al. (Nature 1988; V332; 164-166) found hemophilia A patient resulting from de-novo insertion of LINE-1 sequence without any trace of pedigree for the disease. Sequencing of the factor VIII gene from patient showed LINE1 inserted in exon 14 and disrupt the factor VIII gene. Subsequently, the disease causing L1 sequence showed active retrotransposition in cell culture based retrotransposition 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 kb long with around 906 bp 5’UTR with internal promoter activity, two open reading frames (ORF1 and ORF2) separated by 63 bp spacer sequence and a 3’UTR with around 205 bp long. The element end with Poly A signal sequences at the 3' end and the complete element is flanked by around 20-100 bp target site duplication (TSD) sequences.ORF1p encodes a 40 kDa protein with single stranded nucleic acid binding activity. The ORF2p encoded protein (150 kDa) showed endonuclease (EN) and reverse transcriptase (RT) activities. Although, both proteins are critical in the process of retrotransposition, the exact function in the process of retrotransposition remains unclear. The 5'-UTR of L1 also contains a potent antisense promoter (L1ASP) which transcribes the 5’ flanking sequences and thus form chimeric transcripts. L1 is believed to jump by a mechanism called target primed reverse transcription (TPRT) where EN activity of Abstract ii L1ORF2p introduce a nick at the chromosomal target which creates a 3’OH group. The RT of ORF2p then primes the nick and synthesizes cDNA using L1 mRNA as a template. Human L1 ORF2p is the key protein in the process of retrotransposition and is extremely difficult to express in-vitro. The expression of this protein is used as an marker to find the active L1 retrotransposition in cells and tissues. Although few antibody against L1-ORF2p is available with some L1 researcher, their quality is not good; many non-specific bands found in immunoblotting. Prior it was believed that LINE-1 retrotransposons are only active in germ cells (sperm and ovum) and early stage of development. But recent transgenic animal models and high throughput sequencing analysis revealed that L1 is also active in certain parts of normal brain and in few cancers. The studies also showed that cancer of epithelial origin showed more L1 insertion compared to other types of cancer. 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. Very limited study has been performed to see the activity of L1 retrotransposons in oral cancer samples. In this study I have tried to synthesize an human L1 ORF2p (hL1ORF2p) antibody. I have also explored hL1ORF2p expression in oral squamous cell carcinoma (OSCC) samples obtained from Indian patients. The thesis has been divided into three 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 ORF2 protein activity in retrotransposition and its aberrant expression in different types of cancer along with literature about oral cancer is included. Chapter 2 comprise the materials and methods used in the research work, Those includes recipes for reagents, solutions, protocols for cloning, expression and purification Abstract iii of proteins, Site directed mutagenesis, biophysical characterization of purified proteins, protocols for in house antibody generation, tissue processing, immunohistochemistry, animal cell culture, western blotting along with other general techniques. Chapter 3 embodies details of the results obtained in the study. The main objectives of the study I) To clone and express fragments of hL1-ORF2p for antibody generation II) To generate an in house antibody specific to hL1-ORF2p III) Detection of hL1-ORF2p expression in OSCC samples. I) To clone and express fragments of hL1-ORF2p for antibody generation ORF2 protein of human LINE1 contains three domains: - N-terminal endonuclease (EN) domain, central reverse transcriptase (RT) domain and C- terminal CCHC domain. In the present study fragments from CCHC and EN domains were cloned in a bacterial expression vector and its expression was checked in E.coli expression cells. Both the domains showed very less expression and the amount was not sufficient to make antibody. Hence, a peptide was designed using bioinformatics tool and a peptide antibody was generated. Although, the peptide antigen showed antibody response, the antibody showed some cross reaction. Next, enhanced green fluorescent protein (EGFP) fused ORF2p sequence was used as antigen for making antibody against ORF2p. The first bleed show good response and the process is ongoing. II) To generate an in house antibody specific to hL1-ORF2p Prior generating antibody against hL1-ORF2p, the protocol was standardized using EGFP as an antigen and generated EGFP specific antibody and the results obtained showed specific immune response against EGFP. By following the same procedure the peptide stretch from EN along with carrier protein was injected in to rat for generation of antibody. Abstract iv III) To detect hORF2p expression in Oral Squamous Cell Carcinoma (OSCC) samples. OSCC samples were collected from Acharya Tulsi Regional Centre for Cancer and Treatment Bikaner, Rajasthan as all experiments 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 Hematoxylin and Eosin staining. Next the samples were proceeded to make paraffin block. Slides made from these blocks were then proceeded for Immunohistochemistry with anti human L1 ORF1p antibody [α-hORF1p (RRM)]. The ORF1p positive samples were examined with RT domain specific antibody [α-hORF2p (RT)] which is available in the laboratory. Around 50% samples showed ORF2p positive suggesting human L1 retrotransposon pathway is highly active in OSCC samples in the cancer tissues compared to normal. Data showed 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 prospective of the work has been discussed.