As DNA methylation patterns are maintained post-replication, these modifications contribute to the regulation of transcription of normal biological functions of cells such as inactivation of the X-chromosome, imprinting, suppression of transcription of parasite sequences, differentiation and tissue specific gene expression (Jones and Takai, 2001) which are discussed briefly below. In addition, as DNA methylation influences protein-DNA interactions and as many pathways are regulated through DNA-protein interactions, the vital role of DNA methylation in many biological functions is apparent (Doerfler, 1983).
1.3.4.1. Imprinting
DNA methylation is involved in imprinting (Gronbaek et al., 2007). Imprinting or parent-of- origin specific-gene expression is the expression of one of the two alleles of a gene, either the maternal or the paternal allele, and complete suppression of the other allele. This causes non- equivalence between mammalian parents in autosomal genetic material and prevents parthenogenesis (Reik and Walter, 2001; Hore et al., 2007). Imprinting has only been confirmed in mammals, with approximately 100 imprinted genes identified in humans and mice. Imprinted genes are usually located in clusters, partly as a result of a phenomenon known as epigenetic spreading (Reik and Walter, 2001).
In addition to preventing parthenogenesis and possible correction of gene duplications, it is hypothesised that imprinting mechanisms have evolved in mammals to prevent conflict between offspring and mother over food resources. This is referred to as the “parent conflict hypothesis”. Some of the paternal genes increase foetal growth at the expense of the mother, whilst it is in the interest of the mother to limit this growth. Therefore, imprinting is a way of balancing foetal growth rate. For example in mice IGF2, a growth enhancer, is imprinted in the maternal allele while the IGF2 receptor, a growth suppressor, is methylated in the paternal allele. However, due to the spreading of the epigenetic changes some of the imprinted genes are “innocent bystanders” in this conflict (Hore et al., 2007).
As mentioned in section 1.3.1, mammalian genomes undergo two extensive epigenetic reprogramming events, one during embryogenesis and the other during gametogenesis. In the latter epigenetic markers, including DNA methylation and chromatin modifications are re-set
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in a sex-specific manner in imprinted genes which are protected from the second wave of reprogramming during embryogenesis (Hore et al., 2007; Godmann et al., 2009). This results in inheritance of imprinted patterns in a parent-of-origin manner in the somatic cells (Reik and Walter, 2001). Therefore, as imprinted genes are only represented and expressed monoallelically, methylation or deletion of the active allele has serious consequences. For example Prader-Willi and Angelman syndrome are both consequence of deletion of the active paternal and maternal alleles in the 15q11-13 region of the neuronal tissue, respectively (Hore et al., 2007).
1.3.4.2. X-chromosome inactivation
DNA methylation is involved in X chromosome inactivation (Gronbaek et al., 2007). During this process, in order to compensate for the 2-fold higher occurrence of the genes located on the X chromosomes in females compared to males, one of the X chromosomes in females is inactivated (Xi) (Li, 2002). The X inactivation occurs during embryogenesis after
implantation, with equal inactivation probabilities for the maternal and paternal X chromosome (Avner and Heard, 2001; Li, 2002).
The inactivation process is initiated by X (inactive)-specific gene (Xist) located at the X inactivation centre (XIC). The Xist gene encodes a large non-coding RNA, expressed at a background level from both alleles prior to X chromosome inactivation. During inactivation a marked increase in expression and accumulation of Xist gene of the allele selected for inactivation is observed. This leads to cis coating of the X chromosome with the Xist RNA and triggers a cascade of events leading to inactivation of the entire X-chromosome (Avner and Heard, 2001).
X chromosome inactivation is a synergy between DNA methylation, histone deacetylation, chromatin remodeling and Xist gene expression. Methylation of the Xist gene of the active X chromosome (Xa) prevents its inactivation (Norris et al., 1994). In addition, following
initiation of the inactivation via Xist, DNA methylation contributes to the inactivation process by spreading the inactivation to all CGIs of the housekeeping genes in the X chromosome and stabilising and ensuring maintenance of a silence phenotype throughout cell division (Avner and Heard, 2001). The significance of the contribution of other epigenetic mechanisms is extremely clear. This is because the Xi resembles a highly condensed heterochromatin
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comprised of CGI regions containing methylated CpG dinucleotides and deacetylated histones in the promoters of the house keeping genes (Csankovszki et al., 2001). In addition, it has been shown that mutation in DNMT3b gene, without altering the activity of Xist gene, results in hypomethylation and activation of several X-linked genes (Li, 2002).
1.3.4.3. Differentiation and tissue specific gene expression
The DNA sequence of the genetic material is the same for all tissue types, thus it cannot directly explain the differences observed in expression patterns and phenotypes of diverse categories of tissues. Phenotype-specific gene expression in differentiated cells is achieved mainly by altering the proportion and location of hetero- and eu-chromatin regions. As epigenetic markers regulate gene expression by altering the protein-DNA interactions and DNA packaging, epigenetic mechanisms have been proposed as the key components for establishing tissue-specific gene expression profiles (Godmann et al., 2009; Enrlich, 2005; Momparler and Bovenzi, 2000). In addition, epigenetic markers in contrast to gene sequences are flexible. Therefore, the epigenomes of different cells can differ depending on the microenvironment of differentiating cells (Godmann et al., 2009). Indeed, tissue-specific expression of genes with altered methylation profiles (hypo- or hyper-methylation) based on the tissue type, have been identified in humans (Christensen et al., 2009). For example, testes and uterus exclusively express a testis-specific lactate dehydrogenase gene and a myometrium-specific oxytocin receptor respectively and these are controlled through methylation of their regulatory regions (Enrlich, 2005). Although, it has to be stated that, due to the complex nature of the relationship between DNA methylation and gene expression (discussed in section 1.3.2.3), the contribution of epigenetic mechanisms is not always clear.
1.3.4.4. Transposons
Transposons, scattered throughout the genome, comprise more than 40% of the mammalian genome (compared to exonic regions comprising <2%) (Jones and Takai, 2001). Due to evolution-induced alterations, most transposable elements are inactive. However, the remaining small proportions of active transposons are a threat to the genome by inducing mutations, chromosome instability, translocations and formation of chimeric mRNAs. Nevertheless, generally, little damage is caused by these potentially hazardous elements. This
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has been mainly attributed to DNA methylation-induced inactivation of transposons. This theory, known as the genome defence model, corresponds with the CpG dinucleotide richness of the promoter regions of transposons (Yoder et al., 1997; Gronbaek et al., 2007; Esteller, 2008). Indeed, the mammalian genome is found to be globally methylated (Suzuki and Bird, 2008) with the most highly methylated regions corresponding to transposons (Bird, 2002). In contrast to high levels of methylated transposable elements in somatic cells, transposons are un-methylated and transcriptionally active in germ cells, where they can cause long-term damage compared with somatic cells (Bird, 2002). Therefore, in addition to the genome defence model proposed by Yoder et al., (1997), it has been suggested that in general, methylation of un-necessary promoters may have evolved as a mechanism to reduce the background transcriptional noise (Bird, 2002).