The Epigenetics And The Epigenome Biology

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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 so as to ensure a differential expression of genes at different times 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 us to understand the physiology of normal development, aging, abnormal gene control in cancer and other diseases as well as environmental health.

<H1>GENOMIC IMPRINTING

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 (DMRs). 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.

H1>DNA METHYLATION

Some genes are constitutive genes and are expressed all the time. However, there are some genes which are expressed only at certain times. These genes can be expressed only in specific tissues (Spatial expression) or they maybe expressed at specific times (temporal expression). 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. These CpG islands are responsible for turning gene expression on and off. Chromatin containing CpG islands is generally heavily acetylated, lacks histone H1, and includes a nucleosome-free region. The configuration is thus that of an open chromatin and 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 i.e. if the gene is methylated, it is not expressed and vice versa.

The enzymes that transfer methyl groups to the cytosine ring are called cytosine 5-methyltransferases, or DNA methyltransferases (DNA-MTase). There are currently three known,

catalytically active DNMTs, DNMT1, 3a, and 3b and each one appears to play a distinct and critical role in the cell. 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. Several transcription factors are known including AP-2, cMyc/Myn, E2F and NFκB. It is likely that these transcription factors bind to sequences in the CpG islands. Binding of these factors to the CpG islands has been shown to be inhibited by methylation.

The second mechanism includes the direct binding of specific transcriptional repressors to methylated DNA. Two such factors are MeCP-1 and MeCP-2 (methyl cytosine binding proteins 1 and 2). These factors bind to the CpG islands and cause the genes to be methylated.

The third mechanism of methylation 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. Methylation of the CpG islands serves as a locking mechanism that may follow or precede other events that turn a gene on or off. Once the methylation mechanism is in place, it can prevent activation even if the nuclear environment is optimum 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. Neoplastic cells may show hypermethylation of tumour suppressor genes or there may be hypomethylation of oncogenes. This leads to repression of tumour suppressor genes and development of cancer.

<H3>DNA hypomethylation in cancer

DNA may show hypomethylation in cancer. Decreased level of overall genomic methylation is a common finding in tumorigenesis. This decrease in global methylation appears to begin early, much before the development of frank tumor formation. Specific oncogenes are hypomethylated. This leads to an increase in the expression of oncogenes and development of cancer. 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

In recent years, several attempts have been made to use methylation in a therapeutic scenario. 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. Since there is an inhibition of methylation, genes which were previously silenced can now be expressed. Among these genes is the fetal γ globin gene. 5-azacytidine can thus cause an increase in the expression of the γ globin gene which can restore the imbalance between the α chains and the non α chains. Unfortunately 5 azacytidine is mutagenic. 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, it may be possible to detect the presence of micro metastasis by looking for the presence of methylated genes.

<H1>HISTONES AND EPIGENETIC REGULATION OF GENE EXPRESSION

Histones form the protein backbone of chromatin and are an important component of epigenetics. They act as important translators between genotypes and phenotypes. They are known to have a dynamic function. As compared to DNA methylation, not much work has been done on the study of histones..

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. However, in addition, they may also perform important functions in gene regulation. .

The core histones are highly conserved basic proteins with globular domains. DNA is wrapped around these globular domains. The histones also contain a relatively unstructured flexible tail which protrudes from the nucleosome. These tails are subject to a variety of post translational modifications (PTMs) such as methylation, acetylation and phosphorylation. The other changes which can take place in the tail are 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 histones influence the higher order chromatin structure by affecting contacts between different histones and between histones and DNA. Specific histone modifications take place which are responsible for compartmentalization of the genome into two parts. The first part is the transcriptionally silent heterochromatin and the second portion is the transcriptionally active euchromatin. Thus, these histone - histone and histone - DNA interactions decide if a gene is to be transcriptionally active or inactive. Thus, they regulate nuclear processes like replication, transcription, DNA repair and chromosome condensation. The common changes that take place in the histone molecule and perhaps also 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 MICRORNAs

MicroRNAs (miRNAs) were discovered in the early 1990s by Victor Ambros and colleagues. They found that miRNAs act as gene regulators. Gene hunters at that time were mainly interested in long mRNA molecules because the long mRNA molecules were the ones which were translated to proteins. The small fragments of mRNA or the microRNAs were disregarded since at that time it was believed that they did not have any function. This has now been proved wrong.

MicroRNAs are approximately 22 nucleotides in length. They are single stranded and they inhibit the expression of specific mRNA targets. They do this by binding to sequences usually located in the 3' untranslated regions or UTRs. The portion of miRNA which binds to the 3'UTR is called the 'seed region'. The human genome is believed to code for up to 1000 miRNAs.

miRNA coding sequences can be found in introns or exons of a protein-coding gene. It can also be found in intergenic regions. Several miRNA genes can be clustered along the genome and they may share the same promoter. They can also be present individually. miRNA genes are transcribed into large non coding mRNA strands which is called the primary miRNA transcript. Primary miRNA is then processed and then exported across the nuclear membrane.

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