Epigenetic modification process

Epigenetic modifications are the processes that involve changes in gene expression without modifying the DNA sequence (Shaw, 2006).

The main processes are DNA methylation, histone modification, alterations of factors involved in nucleosomes assembly and remodeling, and gene regulation by non-coding RNAs (Rodríguez-Paredes & Esteller, 2011).

DNA methylation is the addition of methyl groups in a CpG dinucleotide by DNA methyltransferases (DNMTs), resulting in the regulation of the expression of that gene (Sharma et al., 2010). DNA methylation usually affects the gene promoter, leading to the inhibition of gene transcription (Lee et al., 2020). In mammals, the DNMT family includes five proteins: DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L (DNMT3-like) (Bestor, 2000).

The other important epigenetic process is histone modifications. Histones are proteins involved in DNA packaging into nucleosomes. The modifications on the tails of histones modify the chromatin formation, affecting gene accessibility (Greer & Shi, 2012). In particular, eukaryotic cells have 5 main families of histones, and histone H3 has an important role in the epigenetics (Shechter et al., 2007). Histones can undergo methylation, acetylation or phosphorylation (Ilango et al., 2020). Methylation of histones is carried out by histone methyltransferases and demethylation by histone demethylases. Based on the different lysine that is methylated, there could be the activation or the repression of transcription. For example, if lysine 4, 36 or 79 undergo methylation there will be as consequence the activation of transcription (Kouzarides, 2007), whereas lysines 9 or 27 methylation is linked to transcriptional repression (Raha et al., 2011). Furthermore, also the number of methyl groups is an important factor in the transcriptional effect. Lysine 9 di- and tri-methylation is associated with repression, whereas lysine 4 mono-methylation is associated with transcriptional activation (Lachner et al., 2001; Rosenfeld et al., 2009).

Modification of the acetylation status of histones is under the control of histone acetyltransferases (HATs) that carry out acetylation, which can be reverted by histone deacetylases (HDACs). Histone acetylation instead is usually associated to an increased transcription, due to the removal of positive charges and therefore the relaxion of the chromatin (euchromatin), whereas histone deacetylation is related to transcription repression, due to the formation of heterochromatin (Watson et al., 2014).

There is also emerging evidence of a crosstalk between methylated DNA regions and histone deacetylation (Lee et al., 2020). Indeed, histone deacetylases are part of a multiprotein transcription repression complex and they are associated to Sin3A and methyl-CpG binding proteins (MeCPs). MeCPs are able to bind to CpG islands which are methylated and to interact with Sin3A, that in turns interacts with histone deacetylases, leading to transcriptional repression (Bird & Wolffe, 1999, Cho et al., 2004; Song et al., 2013).

Finally, non-coding RNAs, like lncRNA, sncRNA, miRNA, siRNA and piRNA, are some of the main players in epigenetics (Rajagopal et al., 2020). They are RNA transcripts that will not be translated into proteins, but they modulate gene expression by inhibiting target mRNA transcripts or by favoring their degradation (Esteller, 2011). They are involved in the development of several diseases, including cancer (Esteller, 2011). Between them, lncRNAs are characterized by different actions, such as histone modification and transcriptional regulation (Jiang et al., 2020).

Histone methylation

Histone methylation can be detected directly by measuring histone methyltransferase activity/inhibition on specific histones using commercially available kits or indirectly as detection of specific histone methylation levels by western blotting with antibodies raised against specific histone modifications.

DNA methylation (Urdinguio et al, 2009)

Site-specific DNA methylation

Bisulfite genomic sequencing of multiple clones is a DNA-methylation assay that entails initial modification of DNA by sodium bisulphite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent genomic sequencing. This assay constitutes one of the gold standards for DNA-methylation analysis, but is time consuming and expensive.

Methylation-specific PCR uses bisulfite modified DNA and subsequent amplification with primers that are specific for methylated versus unmethylated DNA. This method is fast and cheap, but is not quantitative and requires previous knowledge of the DNA-methylation pattern.

MethyLight is a high-throughput quantitative methylation assay that uses fluorescence-based real-time PCR technology. This technique also uses bisulfite modified DNA.

Pyrosequencing is a DNA-sequencing method in which light is emitted as a result of an enzymatic reaction each time a nucleotide is incorporated into the growing DNA chain.

Methylation-dependent DNA sequence variation, after sodium bisulphite treatment, is treated as a single nucleotide polymorphism.

Genome-wide DNA methylation

DNA-methylation arrays involve the modified DNA being hybridisied after bisulfite treatment to previously designed arrays and each methylation data point is represented by fluorescent signals from the M (methylated) and U (unmethylated) alleles printed in the platform. The arrays are imaged using a BeadArray Reader. This technique is reliable and automatised, but is limited to the CpG sites present in the array.

Methylated DNA immunoprecipitation (methyl-DIP) involves immunoprecipitation with anti-5- methylcytosine antibodies followed by hybridisation to genomic microarrays or ultrasequencing, allowing the identification of methyl-CpG rich sequences. This method allows wide coverage of the human genome, but is expensive and requires a caerful bioinformatic analysis.

Global DNA-methylation quantification

High-performance capillary electrophoresis (HPCE) provides reliable measure of the total 5- methylcytosine DNA content starting with small amounts of sample, but requires specialised machinery.

High performance liquid chromatography (HPLC) provides a reliable measure of the total 5- methylcytosine DNA content but requires important amounts of sample and specialised machinery.

In situ DNA-methylation imaging

Immunostaining with anti-5-methylcytosine antibodies requires the use of other markers to map the stained regions and has variability depending on the antibody badge.

miRNAs and LncRNA

A broad spectrum of methods is available in miRNAs lncRNA research, ranging from computational annotation of lncRNA genes to molecular and cellular analyses of the function of individual lncRNA. Description of these methods and methodological details are provided in a series of methods and protocols published in Methods in Molecular Biology by Humana Press (2006 and 2021)

In Vivo measurements of epigenetic biomarkers

Zebrafish has been proposed as a model for gene expression assessment of epigenetic biomarkers (Torres et al. 2021). In this model, morphological abnormalities and epigenetic changes were assessed at 80 hours-post fertilization, including DNA global methylation and gene expression of both DNA and histone epigenetic modifications

Epigenetic modulation is widely involved in developmental processes, differentiation, and reprogramming of stem cells, as well as a variety of human diseases. Dysregulation of epigenetic control is involved in several pathologies (Feinberg, 2007). Large amount of data accumulated in cancer epigenetics where epigenetic alterations contribute to cancer initiation and progression (Jones and Baylin, 2007; Esteller, 2008)

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