Original Article Gene Silencing and DNA Methylation P. Parveen*, K. Deepti Brundavani, K. Mahathi, M.S. Bhavani and SK. Shaheda Sultana Hindu College of Pharmacy, Amaravathi Road, Guntur-522002, Andhra Pradesh, India ABSTRACT This review revealed a new mechanism for gene regulation through “gene silencing” at the transcriptional level (TGS) or at the post- transcriptional level (PTGS), which play a key role in many essential cellular processes. Today dsRNA is used as a powerful tool to experimentally elucidate the function of essentially any gene in a cell. The immense impact of the discovery of RNA interference (RNAi) on biomedical research and its novel medical applications in the future are reviewed in this article, with particular stress on therapeutic applications of radio-labeled antisense oligonucleotides (RASONs) for diagnosis and treatment of various cancers and neurodegenerative diseases by “gene silencing”. Antisense oligonucleotides (ASONs) can also modulate alternative splicing which 74% of all human genes undergo. Epigenetic changes affect Address for chromatin structure and thus regulate processes such as transcription, Correspondence X-chromosome inactivation, allele-specific expression of imprinted genes, and inactivation of tumor suppressor genes. Treatment with Hindu College of inhibitors of DNA methylation and histone deacetylation can Pharmacy, Amaravathi reactivate epigenetically silenced genes and has been shown to Road, Guntur-522002, restore normal gene function. In cancer cells, this results in Andhra Pradesh, India. expression of tumor suppressor genes and other regulatory functions, E-mail: inducing growth arrest and apoptosis. pothukantiparveen Keywords: RNAi, X-chromosome RASONs, DNA methylation, @gmail.com Gene silencing. INTRODUCTION machinery1 in the cell. Genes are regulated Gene silencing is a general term at either the transcriptional or post- describing epigenetic processes of gene transcriptional level. (See fig. 1.) regulation. The term gene silencing is Transcriptional gene silencing is the generally used to describe the "switching result of histone modifications, creating an off" of a gene by a mechanism other than environment of heterochromatin26 around a genetic24 modification. That is, a gene which gene that makes it inaccessible to would be expressed (turned on) under transcriptional machinery (RNA polymerase, normal circumstances is switched off by transcription factors, etc.). American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org Parveen et al________________________________________________ ISSN 2321 – 2748 Post-transcriptional gene silencing is introduced into cells can induce suppression the result of mRNA of a particular gene being of specific genes of interest. RNAi may also destroyed. The destruction of the mRNA be used for large-scale screens that prevents translation to form an active gene systematically shut down each gene in the product (in most cases, a protein). A common cell, which can help identify the components mechanism of post-transcriptional28 gene necessary for a particular cellular process7 or silencing is RNAi. an event such as cell division. Exploitation of Both transcriptional and post- the pathway is also a promising tool in transcriptional gene silencing are used to biotechnology6 and medicine. regulate endogenous genes. Mechanisms of gene silencing also protect3 the organism's DNA methylation genome from transposons and viruses. Gene DNA methylation is a biochemical silencing thus may be part of an ancient process that is important for normal immune system protecting from such development in higher organisms. It involves infectious DNA elements. the addition of through cell a methyl group purine rin to the 5 position of the cytosine RNA interference pyrimidine ring or the number 6 nitrogen of The enzyme dicer trims double the adenineg (cytosine and adenine are two of stranded RNA, to form small interfering RNA the four bases of DNA). This modification or micro RNA. These processed RNAs are can be inherited division. incorporated into the RNA-induced silencing DNA methylation is a crucial part of complex (RISC), which targets messenger normal organismal development2 and cellular RNA to prevent translation. differentiation in higher organisms. DNA RNA interference (RNAi) is a methylation stably alters the gene expression mechanism that inhibits gene expression by pattern in cells such that cells can "remember causing the degradation of specific RNA where they have been" or decrease gene molecules or hindering the transcription of expression; for example, cells programmed to specific genes. The targets are often RNA be pancreatic islets during embryonic from viruses and transposons14 (probably a development remain pancreatic islets form of innate immune response), although it throughout the life of the organism without also plays a role in regulating16 development continuing signals telling them that they need and genome maintenance. Key to the RNAi to remain islets. DNA methylation is typically processes are small interfering RNA strands removed during zygote formation and re- (siRNA), which have complementary established through successive cell divisions nucleotide sequences to a targeted RNA during development. However, the latest strand. The siRNA "guides" proteins5 within research shows that hydroxylation of methyl the RNAi pathway to the targeted messenger group occurs rather than complete removal of RNA (mRNA) and "cleaves" them, breaking methyl groups in zygote. Some methylation them down into smaller portions that can no modifications that regulate gene expression longer be translated24 into protein. A type of are inheritable and are referred to as RNA transcribed from the genome itself, epigenetic regulation. miRNA, and works in the same way. In addition, DNA methylation The selective and robust effect of suppresses the expression of viral genes42 and RNAi on gene expression makes it a valuable other deleterious elements that have been research tool, both in cell culture and in living incorporated into the genome of the host over organisms because synthetic dsRNA time. DNA methylation also forms the basis AJPCT[2][5][2014]544-555 Parveen et al________________________________________________ ISSN 2321 – 2748 of chromatin structure, which enables cells to transcriptional state of the gene. Exceptions to form the myriad characteristics necessary for this rule, however, can be found in multi cellular life from a single immutable mammalian cells where these regions are sequence of DNA. Methylation also plays a methylated to maintain transcriptional crucial role in the development of nearly all inactivation. Thus, CpG islands in promoters types of cancer.DNA methylation at the 5 of genes located on the inactivated X position of cytosine has the specific effacing chromosome of females are methylated, as gene expression and has been found in every are certain imprinted genes in which only the vertebrate examined. In adult ct of redu maternal or paternal allele is expressed. somatic tissues, DNA methylation typically Epigenetic effects such as occurs in a CpG dinucleotide context; non- hypermethylation can also induce inevitable CpG methylation9 is prevalent in embryonic alterations in gene xpression. Methylation of stem cells. the DNA repair genes MLH1 and MGMT can lead to their inactivation, methylation may Mechanism of DNA methylation affect the transcription of genes in two ways. In mammalian DNA, 5-methylcyto- First, the methylation of DNA itself may sine is found in approximately 4% of genomic physically impede the binding of DNA, primarily at cytosine-guanosine transcriptional proteins to the gene, and dinucleotides (CpGs). Such CpG sites occur second, and likely more important, at lower than expected frequencies throughout methylated DNA may be bound by proteins the human genome but are found more known as methyl-CpG-binding domain frequently at small stretches of DNA called proteins (MBDs). MBD proteins then recruit CpG islands. These islands are typically additional proteins to the locus, such as found in or near promoter regions of genes, histone deacetylases and other chromatin where transcription is initiated. In contrast to remodeling proteins that can modify histones, the bulk of genomic DNA, in which most thereby forming compact, inactive chromatin, CpG sites are heavily methylated, CpG termed heterochromatin. This link between islands in germ-line tissue and promoters of DNA methylation and chromatin structure is normal somatic cells remain un-methylated, very important. In particular, loss of methyl- allowing gene expression to occur. (See fig. CpG-binding protein 2 (MeCP2) has been 2.) implicated in Rett syndrome; and DNA methylation helps to maintain medimethyl-CpG-binding domain protein 2 transcriptional silence in non expressed or (MBD2) ates the transcriptional silencing of non coding regions of the genome. For hypermethylated genes in cancer. Research example, pericentromeric11 heterochromatin, has suggested that long-term memory storage which is condensed and transcriptionally in humans may be regulated by DNA inactive, is heavily methylated. Hyper methylation. methylation thus ensures this DNA is late- replicating and transcriptionally quiescent, Regulation of DNA methylation and suppresses the expression of any DNA methylation is controlled at potentially harmful viral sequences or several different levels in normal and tumor transposons that may have integrated into cells. The addition of methyl groups