LITERATURE REVIEW
1.7 Tissue-Specific Differentially Methylated Regions (tDMRs) and Control of Gene Expression
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outlined a clear nonconformity to the philosophy that CGI methylation is restricted to imprinted genes and X inactivation (Shen et al., 2007).
1.7 Tissue-Specific Differentially Methylated Regions (tDMRs) and Control of Gene
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than 38%. Additionally, even though most of the testes-specific gene promoters displayed elevated methylation levels in somatic tissues, a subgroup of four genes deviated from this category. This subgroup of genes did not display differential methylation; promoters of these genes were hypomethylated in all tissues however, interestingly activated solely in testes.
Upon correlation of the data with expression patterns; genes that displayed decreased methylation results of 20% and 19% in brain and monocytes, respectively were expressed specifically in those tissues. Testes-hypomethylated genes, which were the largest group of 38%, were indeed specifically expressed in testes.
A thorough investigation of FLJ40201, ANKRD30A, FTMT, SOHLH2, C12orf12, INSL6 and DPPA5 genes in assessment of cell and tissue-specific CGI promoter methylation was conducted by Shen et al. (2007). Bisulfite pyrosequencing was used to quantitatively analyse testes, sperm, blood, breast, liver, colon, fibroblast and skeletal muscle of human origin. All promoters demonstrated hypermethylation, with exception of testes and sperm tissues. Sperm DNA presented alleles without methylation, and alleles of testes were either almost completely hypo- or hypermethylated. FLJ40201, ANKRD30A, SOHLH2, INSL6 and DPPA5 genes were specifically expressed in testes and the same genes, with exception of SOHLH2, were identified in sperm. To confirm that this group of genes fit into an exclusive class of promoter CGI-associated genes that are methylated and therefore silenced in a tissue- specific manner, final analysis revealed promoter hypomethylation of below 21% for both INSL6 and SOHLH2 placental tissue. The specific hypermethylation/inexpression and demethylation/derepression of somatic and germ-line promoters respectively was attributed to regulatory sequences (Shen et al., 2007). The proposition spurred from previous findings that transcription factors, such as male germ-line specific CTCF-paralogous BORIS (Brother of the Regulator of Imprinted Sites) may initiate demethylation of cancer-testis antigens MAGE-A1 in somatic cells (Kitamura et al., 2007; Schilling and Rehli, 2008; Vatolin et al., 2005).
Eckhardt et al. (2006) piloted DNA methylation profiling of specifically human chromosomes six, 20 and 22. Forty-three samples consisting of twelve various tissues were selected for the study, which reported the methylation status of nearly two million CpG sites.
The pursuit of tDMRs spotted a minimal portion located in the 5ʹ regions and exons, but a third of all non-coding regions were tDMRs. In the attempt to correlate methylation with expression: a total of 53 genes were arbitrarily selected. The intragenic tDMRs did not display any association with expression and the same could be said for 63% of the 43 genes
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that were associated with 5ʹ untranslated regions (UTRs). The rest within this category demonstrated an indirect correlation. The OSM (Oncostatin) gene reflected an inverse relation to expression which was quite interesting as the gene does not contain a CGI in the 5ʹ region.
Rakyan and colleagues (2008) provided a significant contribution to studies of methylation. The group developed the aptly titled Batman program (Bayesian Tool for Methylation Analysis) which provides estimates of absolute levels of methylation following MeDIP profiling (Down et al., 2008) and performed an all-inclusive genome-wide tDMR quest. The methylation profiles that were deciphered by the group, of 13 different somatic tissues including blood, placenta, B cells and lung amongst others; sperm, placenta and the immortalized EBV-transformed cell line GM06990 were included in the initial ENCODE study (ENCODE Project Consortium, 2007; Rakyan et al., 2008). In contrast to observations by Eckhardt et al. (2006) who studied three chromosomes, and a chromosome-wide search by Weber et al. (2007) this study by Rakyan et al. (2008) found that most promoters that were not associated with a CGI displayed unmethylated profiles. An overall negative correlation was detected for methylation and expression, for example in tissues wherein expression was observed, the transcription start site was not methylated. Comparing their results to previous studies with contradictory results led to the unreciprocated query as to why some promoters act irrespective of DNA methylation status, and others depend on methylation for regulation.
For 16 tissues tested, just above 65% of CGIs associated with promoters were also unmethylated. Although located throughout the genome, the promoter-associated tDMRs were positioned mostly within those with mediocre CpG densities. tDMRs represented 18%
of the entire genomic region studied, and conforming to results obtained by many other research studies, a considerable percentage of all tDMRs were sperm-specific. These displayed hypermethylated profiles in all tissues, and low methylation in mature sperm.
Intragenic tDMRs displayed a positive methylation/expression profile; the ICAM3 (Intercellular Adhesion Molecule III) gene presented low methylation at the promoter, but hypermethylation in expressed tissues (Rakyan et al., 2008). Additional tDMRs to distinguish semen from other tissues were identified by Igarashi et al. (2008), who also detected an age- related linear correlation of DNA methylation in the testes tissues.
Rienius et al. (2012) and Lam et al. (2012) established that patterns of methylation in blood display greater variations between cell populations rather than between individuals.
Rienius and colleagues (2012) explored whole blood samples as well as its components.
Variations were found between the low methylated CpGs of the myeloid cell population
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(monocytes, neutrophils and eosinophils) and the highly methylated lymphoid population (B, C, NK cells). In whole blood as well as individual populations, distributions of differentially methylated patterns were mostly intragenic. The unmethylated cell populations were subjected to gene ontology enrichment analysis to determine cell-specific functions. Even though enrichment for the eosinophils was more typical of general cell functions instead of specific, the peripheral blood mononuclear cells (PBMCs) demonstrated cell-specific enrichment pathways. This included T cells involved in leukocyte and lymphocyte activation and NK cells in molecular signalling cascades. Functions of genes exhibiting variation in methylation were highlighted more by the cell-specific profiles of methylation of a set of genes according to surface expression. For example, membranous expressions of the CD14 and CD3 genes which demonstrated elevated methylation profiles in the cells that they were unexpressed. Strangely, B cells demonstrated overall largest variations in methylation. As the B cells evidently epitomize, methylation patterns found in the study were well correlated to cell specific functions. They are involved in numerous critical pathways and roles such as the humoral immune response, presentation of antigens, and internalization amongst others, which estranges them from T cells. Eighty-five percent of genes that were selected during inferences to inflammatory diseases such as asthma, atopic dermatitis, inflammatory bowel disease, rheumatoid arthritis, and diabetes were differentially methylated. The TNF and LTA genes (Tumour Necrosis Factor and Lymphotoxin Alpha, respectively), which are both associated with asthma, proved differentially methylated in the promoter regions only. In contrast, TCF7L2 (Type 2 Diabetes Candidate Gene Transcription Factor 7-Like II), positioned on the 10q25.3 chromosome, exhibited an analogous methylation profile throughout the gene however methylation toward the promoter islands tended to decline.
Some CpG sites of CD14+ monocytes were specifically unmethylated (Rienius et al., 2012).
A thorough expression study consisting of 13 assays by Prokunina-Olsson et al. (2009) proved that the ex7–8 isoform of TCF7L2 is activated specifically in these monocytes with lowest levels of expression in activated T cells and B cells.
A particular example of research that did not find an association between DNA methylation and gene expression was by Grunau and colleagues (2000). The group performed a detailed methylation analysis of three X linked genes, namely MSSK1 (Muscle-specific Serine Kinase), CDM, SLC6A8 (Creatine Transporter) and the pseudogene ψSLC6A8 in eight tissues. The MSSK1 gene presented low methylation patterns in prostate, heart and brain and intermediate methylation in kidney, muscle, pancreas and lung tissues but was only
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specifically expressed in muscle and heart. In spite of overall strong methylation in brain and heart, and hypomethylation in the prostate the CDM gene was expressed in all tissues.
Similar to MSSK1 and CDM, there was no distinct pattern of methylation and gene expression in SLC6A8. Hypomethylation was observed in prostate and the additional tissue tested, i.e. testis. A fascinating finding was that even though the methylation profiles of liver and muscle were nearly indistinguishable, the gene is inhibited in the latter and specifically expressed in the former. The autosomal pseudogene ψSLC6A8 was the only gene that showed an affiliation between methylation and gene expression. In addition to the nine tissues tested, methylation of white cerebral matter from seven more participants were profiled. High intensities of methylation were observed in all, except testis which was completely free of methylation. High methylation rendered the pseudogene silenced in all somatic tissues, and demethylation enabled testis-specific expression. In somatic cells, the pseudogene ψSLC6A8, as well as the PDHA-2 and PGK-2 genes (Pyruvate Dehydrogenase (Lipoamide) Alpha II and Phosphoglycerate Kinase II, respectively) are all silenced due to methylation, yet unmethylated and active in male germ-line cells. A plausible offering was that transcription of testis-specific pseudogenes may be a by-product of the transient demethylation during the course of spermatogenesis (Ariel et al., 1991; Grunau et al., 2000; Iannello et al., 1997). It is conceivable that lack of correspondence with expression would simply be that the studied tDMRs may not have been situated in regions that govern expression. Or rather another rational explanation, as suggested later by Illingworth and Bird (2009) the intragenic methylation status encumbers gene body transcription with subsequent hindering of parent genes.
Recent studies have identified unorthodox non-CpG methylation patterns in the brain (Lister et al., 2013), embryonic stem cells (Lister et al., 2009) and germ cells (Kobayashi et al., 2013). Schultz et al. (2015) also detected this occurrence in human post-mortem tissue samples including lung, pancreas, sigmoid colon and liver. A negative relationship between the DMRs and gene expression was observed in MYH10, which is linked to blood vessel function. Hypomethylated DMRs in aorta overlapped with aorta-specific enhancers, indicating that decreased methylation levels corresponded with tissue-specific functions.
Non-CpG methylation was most evident in purified glia, brain neurons and H1 embryonic stem cells. Non-CpG methylation correlated with positive expression in H1 cells in the study by Lister et al. (2009) but both Lister et al. (2013) and Schultz et al. (2015) found a negative correlation, thus the function of non-CpG methylation is not known. (Schultz et al., 2015).
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Such studies of non-CpG methylation and unknown roles in expression and development only highlight the poorly understood role of DNA methylation.
1.8 Tissue-Specific Differentially Methylated Regions (tDMRs) and its Application