br Introduction Before planning population based genetic

Introduction Before planning population-based genetic analysis or GWAS, it is important to annotate the sequence of the gene because these sequences represent the major sites such as promoter and transcription factor BHQ which may ultimately affect the expression of the gene. Similarly, a single nucleotide polymorphism (SNP) is a source of variance in the genome and significantly influences the variability of genetic predisposition and susceptibility of disease among different populations (Smith et al., 2003; Chasman and Adams, 2001). Reckoning the vast genomic data and its clinical implications computational tools with different algorithms might help to overcome the difficulty of prioritizing the pathogenic variant from a pool of data and also help in understanding the level of specificity of the different sequence towards different proteins such as transcription factors. To increase the confidence in prediction of functional and deleterious SNPs (ncSNPs and cSNPs), we have applied most commonly used computational methods like RegulomeDB, PolymiRTS, Sorting of Intolerant From Tolerant (SIFT), Polymorphism Phenotyping (Polyphen 2.0), I-Mutant v2.0 and Screening for Non Acceptable Polymorphism (SNAP2) (Ng and Henikoff, 2003; Bromberg et al., 2008; Capriotti et al., 2008; Adzhubei et al., 2010). In the present study, we have incorporated the results of polymorphism and annotation approach (sequence analysis with respect to polymerase II, III binding and transcription factor binding) in conjunction with protein modeling and molecular dynamics for predicting disease-causing mutation in EZH2 (Enhancer of Zeste homolog-2) gene. EZH2 encodes for polycomb protein that exhibits the catalytic activity of trimethylation of lysine residue of histone 3 (H3K27me3), a chromatin repressive mark (Simon et al., 2012; Deb et al., 2013a, Deb et al., 2013b). In recent years, there has been considerable interest in understanding the genetic basis of EZH2 association with various human cancers (Bachmann et al., 2006). EZH2 is one of the most commonly amplified genes involved in oncopathology which regulates inactivation of various sets of genes involved in a number of pathways (Cheng et al., 2011). Previous studies have demonstrated that the over-expression of EZH2 gene, results in a silenced/repressive state of several genes involved in DNA repair, cell-cell adhesion and tumor suppression (Simon et al., 2012). To best of our knowledge, this is the foremost comprehensive computational study targeting the regulatory as well as structural aspects of the EZH2 gene.
Materials and methods In the present study, all the information related to the EZH2 gene was obtained from NCBI- Entrez, GeneCards and dbSNP databases. From this information, data regarding regulatory sequences (5’UTR, CpG islands, 3’UTR) and structural sequences (coding and non-coding) of the gene was collected. The collected dataset was subjected to various computational analyses in order to annotate the gene and to predict the regulatory and structural sequences. The strategy followed is shown in Fig. 1.
Results Entrez and GeneCards are sequence databases which contain all the information regarding human genes. In the present study we have targeted human EZH2 gene (regulatory and structural sequence analysis). The regulatory sequence of EZH2 was computationally analyzed by Promoter 2.0 Prediction server, Proscan v1.7, TSSG (5’UTR); CpG Finder (CpG islands) and POLYAH (3’UTR).
Discussion Enhancer of Zeste Homolog 2 (EZH2) is a histone lysine N-methyltransferase enzyme and it act as an important subunit of polycomb protein which is responsible for healthy embryonic development through epigenetic maintenance of genes responsible for regulating development and differentiation (Morey and Helin, 2010). Studies have indicated that the up-regulation of EZH2 gene has caused aggressiveness in various cancers such as cutaneous melanoma, prostate cancer, breast cancer and endometrial cancers (Venkitaraman, 2002; Narod and Foulkes, 2004; Lakhani et al., 2005; Turner et al., 2007; Gonzalez et al., 2011). Hence, it is indispensable to study regulatory and structural aspects of the gene. In the present study, we have tried to annotate the regulatory sequence of the EZH2 gene and we found the information regarding promoter region of the targeted gene in terms of promoter strength (Fig. 2) and concluded that 38% region of the 5’ UTR flanking sequence predicted to exhibit polymerase II binding site with 95% accuracy.