**4. Detection of the methylation region**

Any DNA region containing a CpG sequence may potentially undergo methylation. For this reason, any gene may be subjected to methylation; however, methylation most commonly occurs in the promoter region of genes. That is quite reasonable given the fact that the promoter region is the recognition site for RNA polymerases and therefore of critical importance

**Figure 3.** QUMA Web-based program interface.

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**Figure 3.** QUMA Web-based program interface.

Despite the understanding of these methylation changes in mitochondrial genome, the function of methylated mtDNA has not been fully understood; however, Monique et al. have revealed a different situation. Contrary to what is expected with the methylation of CpGs and GpCs in mtDNA, they have demonstrated that methylation of CpG base pairs had no effect on expression while methylated GpCs were associated with decreased expression [25].

Furthermore, since the mitochondria in humans are entirely of maternal origin, life style of the mother may also have effects at mitochondrion level. Habitual behaviour of the mother, her diet and excessive consumption of fats and sugars trigger obesity, which may affect epigenetics, including that of mitochondria in subsequent generations. A study conducted in mice revealed increased methylation leading to alterations in gene expression and suppression, particularly in the respiratory tract of the offspring of mice that were fed high-fat diets [26]. Furthermore, because the structure of mitochondrion is highly similar to that of bacteria,

Any DNA region containing a CpG sequence may potentially undergo methylation. For this reason, any gene may be subjected to methylation; however, methylation most commonly occurs in the promoter region of genes. That is quite reasonable given the fact that the promoter region is the recognition site for RNA polymerases and therefore of critical importance

some genetic factors and structures may also be the same.

**4. Detection of the methylation region**

**Figure 2.** Gene structure of mitochondrial genome [22].

6 Chromatin and Epigenetics

for gene expression. Although more rarely, methylation may also be observed in exon 1 and other exons of certain genes. One of the ways of finding out whether a gene or DNA region may undergo methylation is to investigate that region with sequence analysis programs specifically developed for this task. There are paid programs by companies such as Fermentas (Methyl Primer Express® Software v1.0) serving this purpose as well as Web-based free access programs such as 'MethPrimer' developed by Li LC and Dahiya R. (available at http:// www.urogene.org/methprimer/) [27]. There is also a Web-based program 'DiseaseMeth' (available at http://202.97.205.78/diseasemeth/Analyze.html#form3), which offers researchers who is interested in using a study of which disease or cancer is associated with a desired gene methylation. Another Web-based program is SMS (Sequence Manipulation Suite; available at http://www.bioinformatics.org/sms2/) [28]. One of many useful programs at this address is program which shows CpG islands in a desired DNA region. Another program is a Webbased program QUMA (Quantification tool for Methylation Analysis; available at http://quma. cdb.riken.jp/) (**Figure 3**) [29]. These programs allow methylation region mapping, designing methylation-specific primers, determining bisulphite sequencing primers, identification of CpG islands and determination of DNA sequences that are altered or newly formed due to bisulphite modifications.

In order to do this, the initial step should be obtaining the FASTA sequence of the sequence in question. The Ensembl genome browser 92, available at https://www.ensembl.org/, is a very

**Figure 5.** Enter the sequence into Methyl Primer Express. The selection is made for bisulphite sequestration or MSP.

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**Figure 6.** The possible gene region resulting from the analysis.


**Figure 4.** Survivin gene sequence on Ensembl genome browser 92.

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**Figure 5.** Enter the sequence into Methyl Primer Express. The selection is made for bisulphite sequestration or MSP.

for gene expression. Although more rarely, methylation may also be observed in exon 1 and other exons of certain genes. One of the ways of finding out whether a gene or DNA region may undergo methylation is to investigate that region with sequence analysis programs specifically developed for this task. There are paid programs by companies such as Fermentas (Methyl Primer Express® Software v1.0) serving this purpose as well as Web-based free access programs such as 'MethPrimer' developed by Li LC and Dahiya R. (available at http:// www.urogene.org/methprimer/) [27]. There is also a Web-based program 'DiseaseMeth' (available at http://202.97.205.78/diseasemeth/Analyze.html#form3), which offers researchers who is interested in using a study of which disease or cancer is associated with a desired gene methylation. Another Web-based program is SMS (Sequence Manipulation Suite; available at http://www.bioinformatics.org/sms2/) [28]. One of many useful programs at this address is program which shows CpG islands in a desired DNA region. Another program is a Webbased program QUMA (Quantification tool for Methylation Analysis; available at http://quma. cdb.riken.jp/) (**Figure 3**) [29]. These programs allow methylation region mapping, designing methylation-specific primers, determining bisulphite sequencing primers, identification of CpG islands and determination of DNA sequences that are altered or newly formed due to

In order to do this, the initial step should be obtaining the FASTA sequence of the sequence in question. The Ensembl genome browser 92, available at https://www.ensembl.org/, is a very

bisulphite modifications.

8 Chromatin and Epigenetics

**Figure 4.** Survivin gene sequence on Ensembl genome browser 92.

**Figure 6.** The possible gene region resulting from the analysis.

**Figure 7.** Methyl and unmethyl primer sequences and product length for the gene region of interest.

good resource that can be utilised for this step. Ensembl is a database that allows access to DNA sequences in formats such as BLAST and BLAT for comparative genomic studies, evolution studies, sequence variants and transcriptional variants across vertebrate genomes. The relevant DNA sequence obtained from such a database is added to the methylation primer program, the sites of interest are labelled, and the program is run (**Figures 4**–**8**).

**5. Methylation-specific PCR (MSP)**

[30, 31].

MSP is an established and the most commonly utilised method to determine the presence or absence of methylation in a gene region of interest as well as the extent of methylation, if any

**Figure 8.** Upper row: original sequence. Lower row: bisulphite modified sequence. (For display, assume all CpG sites are methylated) ++, CpG sites; ::::, non-CpG 'C' converted to 'T'; M>>>>>>, left methylated-specific primer; M<<<<<<, right methylated-specific primer; U>>>>>>, left unmethylated-specific primer; U<<<<<<, right unmethylated-specific primer.

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In this method, DNA is initially subjected to total bisulphite treatment. In this way, all of the unmethylated cytosines in the DNA sequence are transformed into thymine. However, the methylated cytosines remain unchanged (**Figure 5**). This results in a motif change in the methylated region. Subsequently, spectrophotometric DNA quantification is conducted with DNA samples. For this, measurements are made at wavelengths of 260/280 nm and multiplied by the

good resource that can be utilised for this step. Ensembl is a database that allows access to DNA sequences in formats such as BLAST and BLAT for comparative genomic studies, evolution studies, sequence variants and transcriptional variants across vertebrate genomes. The relevant DNA sequence obtained from such a database is added to the methylation primer program, the

**Figure 7.** Methyl and unmethyl primer sequences and product length for the gene region of interest.

sites of interest are labelled, and the program is run (**Figures 4**–**8**).

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**Figure 8.** Upper row: original sequence. Lower row: bisulphite modified sequence. (For display, assume all CpG sites are methylated) ++, CpG sites; ::::, non-CpG 'C' converted to 'T'; M>>>>>>, left methylated-specific primer; M<<<<<<, right methylated-specific primer; U>>>>>>, left unmethylated-specific primer; U<<<<<<, right unmethylated-specific primer.
