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Epigenetics involves the heritable patterns of gene expression that do not involve changes in the sequence of the genome. There are several processes involved in epigenetics including DNA methylation, histone modifications, gene regulation by microRNAs and others. Epigenetic factors contribute to human disease.
<H1>THE HUMAN EPIGENOME PROJECT
The Human Genome Project provided a map of the human genome. However, it did not predict how the genome is packaged into chromatin to ensure differential expression of genes which is essential for normal development and differentiation. The human epigenome project was, therefore, launched to provide better understanding of the human epigenome. Epigenetic processes are now known to be increasingly involved in modulating the phenotype.
The human epigenome project explains the relationships that exist between major epigenetic players and are called the 'epigenetic code'. It helps in plotting comprehensive DNA methylation maps called the 'methylome'. It also provides a complete understanding of histone modifications.
The aim of the human epigenome project was to identify the chemical changes and relationships that exist between chromatin constituents that provide function to the DNA code. This allows better understanding of normal development, aging, abnormal gene control in cancer and other diseases as well as environmental health.
Genomic imprinting is an epigenetic phenomenon by which epigenetic chromosomal modifications drive differential gene expression according to the parent of origin. This means that the expression of the gene is entirely according to the parent of origin. Expression is due to an allele inherited from the mother (as in H19 and CDKN1C genes) or it is because of an allele inherited from the father (such as the IGF2 gene). This inheritance is independent of the classical Mendelian genetics. Usually, imprinted genes are involved in a particular stage of development.
Imprinting is essentially a dynamic process since the profile of imprinted genes varies during development. DNA methylation is believed to be a major mechanism involved in the control of imprinted genes. Imprinted genes are seen to occur in clusters and the control of these genes is by common regulatory elements. The regulatory elements maybe noncoding RNAs or Differentially Methylated Regions (DMR;s). As mentioned, these regulatory elements are clustered together and these regions are called 'Imprinting Control Regions' or ICRs. Any change in the methylation patterns in the ICRs would lead to a loss of imprinting and an abnormal expression of the parental gene.
<H2>Imprinted Genes and Human Genetic Diseases
Expression of imprinted genes is essentially monoallelic. There is only one copy of the gene and that copy is inherited from one parent. So, any problem with that gene would cause a genetic situation like a recessive mutation.
Prader-Willi syndrome (PWS) is a complex genetic condition characterized by mental and physical findings, with obesity being the most significant health problem. PWS is considered the most common genetically identified cause of life-threatening obesity in humans and affects an estimated 350,000-400,000 people worldwide. Prader-Willi syndrome has been estimated to occur in one in 10,000 to 20,000 individuals and present in all races and ethnic groups but reported disproportionately more often in Caucasians. PWS is characterized by infantile hypotonia, early childhood obesity, short stature, small hands and feet, growth hormone deficiency, hypogenitalism/hypogonadism, mental deficiency and behavioural problems including
temper tantrums and skin picking and a characteristic facial appearance with a narrow bifrontal diameter, short upturned nose, triangular mouth, almond-shaped eyes, and oral findings (sticky saliva, enamel hypoplasia) (Fig 6.1).
Butler and Palmer in 1983 were the first to report that the origin of the chromosome 15 deletion was de novo or due to a new event and found that the chromosome 15 leading to the deletion was donated only from the father. In about 70% of subjects with PWS, the 15q11-q13 deletion was present while about 25% of individuals with PWS had either maternal disomy 15 (both 15s from the mother) or defects in the imprinting center controlling the activity of genes in the chromosome 15 region (about 5% of cases) (Fig 6.2). In this last 5% of cases, there would be a defect in the ICR as referred to previously and then there would be a change in the methylation pattern of the gene leading to loss of imprinting. Several paternal genes are expressed in this region and so it is difficult to pinpoint one gene as the cause of all the problems.
Angelman syndrome (AS) which has an entirely different clinical presentation, is characterized by seizures, severe mental retardation, ataxia and jerky arm movements, hypopigmentation, inappropriate laughter, lack of speech, microbrachycephaly, maxillary hypoplasia, a large mouth with protruding tongue, prominent nose, wide spaced teeth, and usually a maternal 15q11- q13 deletion. Although PWS is thought to be a contiguous gene syndrome with several imprinted (paternally expressed) genes as candidates for causing the disorder, AS is caused by a single imprinted (maternally expressed) gene, i.e., UBE3A, a ubiquitin ligase gene involved in early brain development.
Fig 6.1 - A patient with Prader Willi syndrome. Note the dysmorphic facial features: high prominent forehead, narrow bifrontal diameter, downturned corners of the mouth, micrognathia.
Fig 6.2 - The karyotype of Prader Willi syndrome showing a deletion of chromosome 15
The most widely studied epigenetic modification is the cytosine methylation of DNA within the CpG dinucleotide. The CpG dinucleotide is a sequence of 5'-CG-3'. During evolution, the dinucleotide CpG has been progressively eliminated from the genome of higher eukaryotes and is present at only 5% to 10% of its predicted frequency. In the genome, there are smaller regions of DNA, called CpG islands ranging from 0.5 to 5 kb and occurring on an average after every 100 kb. CpG islands are usually found in the promoter region of genes. Chromatin containing CpG islands is generally heavily acetylated, lacks histone H1, and includes a nucleosome-free region. This is an open chromatin configuration and it allows for interaction of transcription factors with gene promoters.
Approximately half of all genes in mouse and humans (i.e., 40,000 to 50,000 genes) contain CpG islands. These are mainly housekeeping genes that have a broad tissue pattern of expression, but approximately 40% of genes with a tissue-restricted pattern of expression are also represented. Usually methylation is inversely correlated with the transcriptional status of the genes.
The enzymes that transfer methyl groups to the cytosine ring are called cytosine 5-methyltransferases, or DNA methyltransferases (DNA-MTase). A role of DNA methylation in the differential regulation of gene expression was hypothesized many years ago. Three possible mechanisms have been proposed to account for transcriptional repression by DNA methylation. These mechanisms are as follows:
Direct interference with the binding of specific transcription factors to their recognition sites in their respective promoters. Transcription factors include AP-2, c-Myc/Myn, the cyclic AMP dependent activator CREB, E2F, and NF-kB. These recognize sequences that contain CpG residues, and binding to each has been shown to be inhibited by methylation.
A second potential mechanism for methylation induced silencing is through the direct binding of specific transcriptional repressors to methylated DNA. Two such factors, MeCP-1 and MeCP-2 (methyl cytosine binding proteins 1 and 2), have been identified and shown to bind to methylated CpG residues in any sequence context.
A third mechanism by which methylation may mediate transcriptional repression is by altering chromatin structure. Experiments show that methylation inhibits transcription only after chromatin is assembled. Once chromatin has assumed its inactive state after DNA methylation, it cannot be counteracted even by strong transcriptional agents. Therefore, in addition to stabilizing the inactive state, methylation also prevents activation by blocking the access of transcription factors.
It is important to realise that methylation turns off genes. It appears that methylation, particularly of CpG-rich genes, may serve as a locking mechanism that may follow or precede other events that turn a gene off, but that once in place, can prevent activation despite an optimum nuclear environment for transcription.
<H2>DNA demethylation during development and tissue specific differentiation
After implantation, most of the genomic DNA is usually in the methylated state, whereas, tissue-specific genes undergo demethylation in their tissues of expression. This essentially means that some genes can be expressed, whereas, the other genes are repressed. This allows the body a step-wise development which accounts for the perfect structure of the tissues of the human body. If this system of methylation did not exist, tissues would develop randomly and the human body would never reach the perfect form.
<H2>DNA methylation in cancer
Role of DNA methylation in oncogenesis has been hypothesized since many years. Numerous studies have suggested aberrations in DNA methyltransferase activity in tumor cells. Transformed cells often have increased total DNA-MTase activity, widespread loss of methylation from normally methylated sites, and more regional areas of hypermethylated DNA.
<H3>DNA hypomethylation in cancer
Decreased level of overall genomic methylation is common finding in tumorigenesis. This decrease in global methylation appears to begin early, much before the development of frank tumor formation. Specific oncogenes have been observed to be hypomethylated in human tumor cells. A good inverse correlation between methylation and gene expression was observed in the antiapoptotic bcl-2 gene in B-cell chronic lymphocytic leukemia and the k-ras proto-oncogene in lung and colon carcinomas.
<H3>Hypermethylation of tumor-suppressor genes
An additional means of inactivating tumour suppressor genes is by hypermethylation of the promoter sequences of the tumour suppressor genes in cancer. The retinoblastoma gene (Rb) was the first classic tumor-suppressor gene in which CpG island hypermethylation was detected.
<H2>Clinical and therapeutic implications of DNA methylation
The vertebrate globin genes were among the target for clinical intervention based on drugs that affect methylation. Treatment with 5-azacytidine has been attempted. This drug is an irreversible inhibitor of DNA methyltransferase and therefore inhibits methylation. This inhibition has been shown to increase expression of the fetal γ globin gene in nonhuman primates and subsequently in patients with β thalassemia and sickle cell anemia. Because of its mutagenicity and the observation that the other S-phase active cytotoxic agents that do not inhibit DNA methylation could induce similar increase in γ globin gene expression, 5-azacytidine has not been widely used for this application. This points to the limitations of the use of agents that cause global DNA methylation.
The recent advances in understanding of altered DNA methylation in cancer also have potential clinical implications. Because methylation of many involved genes may represent a process specific to neoplastic cells, this may be a sensitive index of micrometastases.
<H1>HISTONES AND EPIGENETIC REGULATION OF GENE EXPRESSION
Histones, the protein backbone of chromatin, are also important in epigenetics. Today, they are recognized as being important translators between genotypes and phenotypes, having a dynamic function in the regulation of chromatin structure and gene activity. Understanding the importance of histones in a normal cell and how its role changes in neoplasia, is still in its infancy compared with that of DNA methylation.
In eukaryotic cells, DNA and histone proteins form chromatin, and it is in this context that transcription takes place. As mentioned earlier, the basic unit of chromatin is the nucleosome, and consists of an octamer of two molecules of each of the four histone molecules (H2A, H2B, H3 and H4), around which is wrapped 147 bp of DNA. Histones help package DNA so that it can be contained in the nucleus but more recently, their involvement in regulating gene expression has also been shown.
The core histones are highly conserved basic proteins with globular domains (around which the DNA is wrapped) and relatively unstructured flexible 'tails' that protrude from the nucleosome. The tails are subject to a variety of post-translational modifications (PTMs) like methylation, acetylation and phosphorylation. Other modifications include ubiquitination, sumoylation, ADP ribosylation and deimination, and the non-covalent proline isomerization that occurs in histone H3. Most histone PTMs are dynamic and are regulated by families of enzymes that promote or reverse the modifications.
How do histones influence transcription? The histone code influences higher-order chromatin structure by affecting contacts between different histones and between histones and DNA. Specific histone modifications are responsible for the compartmentalization of the genome into distinct domains, such as transcriptionally silent heterochromatin and transcriptionally active euchromatin. The ability of the histone code to dictate the chromatin environment allows it to regulate nuclear processes, such as replication, transcription, DNA repair, and chromosome condensation.
The common changes to take place in the histone molecule and perhaps the best studied are histone acetylation and methylation. Ranking next to DNA methylation, histone acetylation and histone methylation are well-characterized epigenetic markers. Methylation at some of the histones (H3K4, H3K36 or H3K79) results in an open chromatin configuration and is, therefore, characteristic of euchromatin. Acetylation mediated by histone acetyl transferase (HAT) also results in an open chromatin pattern or euchromatin. On the contrary, histone deacetylases remove these changes and result in transcriptional repression.
An analogy of the relationship between DNA and histones can be found in any 'C' grade movie. The histones are akin to the big brother and their job is to protect the DNA or the younger sister. Histones allow access to the DNA only under certain circumstances and prevent access under a different set of circumstances. Since these changes are independent of the genetic code, they come under the ambit of epigenetic changes.
Essentially, three general principles are thought to be involved in histone modifications and gene expression. These principles are:
PTMs directly affect the structure of chromatin, regulating its higher order conformation and thus acting in cis to regulate transcription;
PTMs disrupt the binding of proteins that associate with chromatin (trans effect);
PTMs attract certain effector proteins to the chromatin (trans effect).
<H1>THE ROLE OF MICRO RNAs
MicroRNAs (miRNAs) were discovered in the early 1990s by Victor Ambros and colleagues. They found that miRNAs act as gene regulators and had perhaps escaped detection till that time because of their size, as gene hunters were mainly interested in long mRNAs and disregarded very short RNAs.
MicroRNAs are ~22-nucleotide single-stranded RNAs that inhibit the expression of specific mRNA targets through Watson-Crick base pairing between the miRNA 'seed region' and sequences commonly located in the 3′ untranslated regions (UTRs). The human genome is estimated to encode up to 1,000 miRNAs, which are either transcribed as standalone transcripts, frequently encoding several miRNAs, or generated by the processing of introns of protein-coding genes. The integration of miRNAs into introns of protein-coding genes serves to coordinate the expression of the miRNA with the mRNA encoded by that gene, without the necessity for a separate set of cis-regulatory elements to drive expression of the miRNA. It is not uncommon for intronic miRNAs to modulate the same biological processes as the protein encoded by the host gene microRNAs.
Bioinformatics and cloning studies have estimated that miRNAs may regulate 30% of all human genes and each miRNA can control hundreds of gene targets. miRNAs are highly conserved between distantly related organisms, indicating their participation in essential biological processes. It is well known today that miRNAs have very important regulatory functions in such basic biological processes as development, cellular differentiation, proliferation and apoptosis.
<H2>MicroRNA biology and function
MicroRNAs are transcribed by RNA polymerase II or III as long primary microRNAs termed pri-microRNA. This molecule is then modified in the nucleus through capping and polyadenylation and subsequently cleaved into smaller segments by Drosha, an RNAse III enzyme. This forms a hairpin precursor of approximately 60-70 nucleotides, termed pre-microRNA, which is exported to the cytoplasm and modified by another enzyme, the RNAse II endonuclease, Dicer, to form a duplex of mature microRNA. One of the microRNA strands of the duplex is loaded onto the RNA-induced silencing complex (RISC), where it is then able to either cleave RNA targets or repress protein translation dependent upon its complementarity to the target mRNA.
Through their binding to target mRNA sequences, microRNAs have a large number of biologically diverse functions. They have the capacity to control the expression of many downstream genes which can affect several cell regulatory pathways, such as cell growth, differentiation, mobility and apoptosis. miRNAs have been studied most intensively in the field of oncological research, and emerging evidence suggests that altered miRNA regulation is involved in the pathogenesis of cancers - mainly by regulating the translation of oncogenes and tumor suppressors. Changes in the expression of miRNAs have been observed in a variety of human tumors.
<H2>The Detection of MicroRNA Expression
Several techniques have been developed to examine microRNA expression. One of the most predominant methods in the literature is by use of microRNA microarrays. Microarray technology offers a powerful high-throughput tool to monitor the expression of thousands of microRNAs at once.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is another reliable and highly sensitive technique for microRNA detection, which is simple and robust, and only requires very small amounts of input total RNA.
Standard Northern blotting has also been employed to detect and validate microRNA expression levels.
In addition, techniques are available to detect microRNAs by in situ hybridization. MicroRNAs can be isolated form tissues as well as from blood.
<H2>The Role of MicroRNAs in Cancer
The expression patterns, function and regulation of microRNAs in normal and neoplastic human cells are largely unknown but emerging data about their loss of heterozygosity and their frequent location at fragile sites, common break-points or regions of amplification reveal that they may play significant role in human carcinogenesis. The abnormal expression of several microRNAs has been observed in Burkitt's lymphomas, B cell chronic lymphocytic leukemia (CLL) and many solid cancer types, such as breast, liver, lung, ovarian, cervical, colorectal and prostate. Functional analysis has revealed the downregulation of PTEN by miR-21, the tumor suppressor function of the LET-7 family and the oncogenic function of the miR17-92 cluster. The biological and clinical relevance of microRNA expression patterns have been established in human B cell CLL and solid tumors, including breast cancers.
The altered expression of microRNAs in cancer can be a causative factor or perhaps a consequence of the disease state. Dependent upon the nature of their target gene(s), microRNAs may function as tumor suppressors by downregulating target oncogenes (e.g. LET -7 g, miR-15/16 and miR-34) or as oncogenes by negatively controlling genes that regulate tumor cell differentiation and apoptosis (e.g. miR-155 and miR-21). Alternatively, changes in microRNA expression may be a downstream effect of potent oncogenes or tumor suppressors in the carcinogenesis process such as the modulation of miR- 34 by p53. MicroRNAs have also been shown to play a role in cancer progression through the modulation of cellular adhesion, cell matrix and signaling activities. In addition, microRNAs also play important role in regulating the expression of hypoxia-related genes.
<H2>Clinical Applications of MicroRNAs
Since the expression of microRNAs is altered in cancers, it is thought that they may function as suitable biomarkers for disease state and progression. Recent studies indicate that expression profiling of microRNAs is a superior method for cancer subtype classification and prognostication.
MicroRNA expression profiles have been used to distinguish tumor cells from normal samples, to identify tissue of origin in tumors of unknown origin and in poorly differentiated tumors, and to distinguish between different subtypes of tumors. Sample datasets have been stratified to show that certain alterations of microRNAs occur in patients at an early stage of cancer and thus may be quite useful for early detection. Large tissue specimens are not needed for accurate microRNA detection since their expression can be easily measured in biopsy specimens. MicroRNAs can be measured in formalin fixed paraffin embedded (FFPE) tissues.
Recent studies have also shown that microRNAs can be detected in serum. These studies offer the promise of utilizing microRNA screening via less invasive blood based mechanisms. Mature microRNAs are relatively stable. These phenomena make microRNAs superior molecular markers and as such, microRNA expression profiling can be utilized as a tool for cancer diagnosis.
MicroRNAs are useful indicators of clinical outcome in a number of cancer types. In addition, microRNAs have been shown to play a predictive role in determining the tendency for recurrence and metastasis. These microRNA alterations have not only been found in tumor specimens, but have also been observed in surrounding non-cancerous tissue, indicating that microRNAs may also serve to detect alterations in the cancer microenvironment. MicroRNAs have also been shown to indicate the patient groups that respond better to a particular treatment regimen.
<H2>Therapeutic Application of MicroRNAs
As noted above, several microRNAs have been shown to be altered in disease states when compared to normal specimens. Whether this differential expression occurs as a consequence of the pathological state, or, whether the disease is a direct cause of this differential expression, is currently unknown. Nonetheless, since microRNAs are deregulated in cancer, it is thought that normalization of their expression could be a potential method of intervention.
One way of regulating the action of micro RNAs is to use anti-microRNA oligonucleotides (AMOs) which have been generated to directly compete with endogenous microRNAs. However, the ability of AMOs to specifically inactivate endogenous targets is quite inefficient. Thus, several modifications of AMOs have been generated to improve their effectiveness and stability such as the addition of 2'-O methyl and 2'-O-methoxyethyl groups to the 5' end of the molecule. AMOs conjugated to cholesterol (antagomirs) have also been generated and described to efficiently inhibit microRNA activity in-vivo. In addition, locked nucleic acid antisense oligonucleotides (LNAs) have been designed to increase stability and have been shown to be highly aqueous and exhibit low toxicity in-vivo.
Another method for reducing the interaction between microRNAs and their targets is the use of microRNA sponges. These sponges are synthetic mRNAs that contain multiple binding sites for an endogenous microRNA. Sponges designed with multimeric seed sequences have been shown to effectively repress microRNA families sharing the same seed sequence. Although the in-vitro performance of microRNA sponges is similar to that of chemically modified AMOs, their efficacy in-vivo remains to be determined.
Although these oligonucleotide-based methods have been shown to work, they do elicit off-target side effects and unwanted toxicity. This is due to the capability of microRNAs to regulate hundreds of genes. A strategy called miR-masking is an alternative strategy designed to combat this effect. This method utilizes a sequence with perfect complementarity to the target gene such that duplexing will occur with higher affinity than that between the target gene and its endogenous microRNA. Another strategy to increase specificity of effects is the use of small molecule inhibitors against specific microRNAs. Azobenzene, for example, has been identified as a specific and efficient inhibitor of miR-21. Although the effectiveness of such inhibitors awaits exploration in-vivo, they are potentially promising tools for cancer therapy.
<H3>Strategies to overexpress microRNAs
Elevating the expression of microRNAs with tumor suppressive roles is a strategy to restore tumor inhibitory functions in the cell. This can be achieved through the use of viral or liposomal delivery mechanisms. Several microRNAs have been introduced to the tumor cells via this methodology. These include miR-34, miR-15, miR-16 and LET-7. This approach reduces toxicity since AAV vectors do not integrate into the host genome and are eventually eliminated. The non-viral methods of gene transfer include cationic liposome mediated systems. These lipoplexes lack tumor specificity and have relatively low efficiency when compared to viral vectors.
MicroRNA mimics have also been used to increase microRNA expression. These small, chemically modified double-stranded RNA molecules mimic endogenous mature microRNA. These mimics are now commercially available. They do not have vector-based toxicity and are therefore promising tools for therapeutic treatment of tumors.