Additional Information

Mechanisms underlying epigenetics

As explained, every cell in the organism carries an identical genome, however, despite the stability of these instructions, the terminal phenotype within an organism is not fixed and deviation is caused by gene expression changes in response to environmental cues. DNA methylation, histone modification and RNA-associated silencing are the major ways these changes are controlled, which are described in more detail below.

DNA methylation

The methylome is the genomic distribution of methylated DNA sequence present in a cell and is capable of undergoing modification with respect to the environment or the developmental stage. DNA methylation involves the covalent addition of a methyl group at position 5 of the pyrimidine ring of cytosine that is represented as 5-methyl C or CMe. Transcription of most protein coding genes in mammals is initiated at promoters rich in CG sequences, where cytosine is positioned next to a guanine nucleotide linked by a phosphate called a CpG site. Such short stretches of CpG-dense DNA are known as CpG islands. In the human genome 60–80% of 28 million CpG dinucleotides are methylated (Lister et al., 2009; Ziller et al., 2013). Chromatin structure adjacent to CpG island promoters facilitates transcription, while methylated CpG islands impart a tight compaction to chromatin that prevents onset of transcription and therefore, gene expression.
In CpG islands active C’s are normally unmethylated and when an unmethylated cytosine spontaneously deaminates to uridine, it is converted back to cytosine by DNA repair mechanisms, thus preserving CpG sequences through evolution. The presence of 5-methyl C in a CpG island denotes an inactive promoter owing to the condensation of chromatin triggered by DNA methylation.

CpG sites are methylated by one of three enzymes called DNA methyltransferases (DNMTs). A variety of DNMTs are responsible for DNA methylation patterns established during embryogenesis. One type of DNA methyltransferase, DNMT1, is responsible for maintaining normal methylation patterns by copying them exactly between cell generations during replication. DNMT2 is associated with embryonic stem cells and potential RNA methylation. DNMT3a and DNMT3b are involved in de novo DNA methylation at CpG sites (Clouaire and Stancheva,2008; Singh and Li, 2012).

Histone modification

Histones are the core protein components of chromatin complexes, and they provide the structural backbone around which DNA wraps at regular intervals generating chromatin. The nucleosome represents the first level of chromatin organization and is composed of two of each of histones H2A, H2B, H3, and H4, assembled in an octameric core with DNA tightly wrapped around the octamer (Luger et al., 1997). Histones regulate DNA packaging with immense
influence on the degree of chromatin compaction, influencing transcriptional activity as well as
transcriptional silencing.

Histone modifications are post-translational changes on the histone tails, that are flexible stretches of N or C terminal residues extending from the globular histone octamer. Modifications of histones include acetylation of lysine residues, methylation of lysine and arginine residues, phosphorylation of serine and threonine residues, and ubiquitination of lysine residues present on histone tails, as well as sumoylation and ADP ribosylation. All of these changes influence DNA transcription. Addition or removal of methyl groups on DNA (see above) and histones and acetyl groups on histones are the prime mechanisms of changing the epigenetic landscape (Cedar and Bergman,2009)

Histone acetylation is carried out by enzymes called histone acetyltransferases (HATs), that are responsible for adding acetyl groups to lysine residues on histone tails while histone deacetylases (HDACs) are those that remove acetyl groups from acetylated lysines. Generally, presence of acetylated lysine on histone tails leads to a relaxed chromatin state that promotes transcriptional activation of selected genes; in contrast, deacetylation of lysine residues leads to chromatin compaction and transcriptional inactivation.

RNA silencing

RNA-associated silencing is a type of post-transcriptional gene modification during which the expression of one or more genes is downregulated or suppressed by small non-coding stretches of RNA, sometimes called microRNAs (miRNA) and small interfering RNAs (siRNA). Although microRNAs only represent 1% of the genome they have been estimated to target 30% of genes (Lewis et al., 2005). These RNAs can act as switches and modulators, exerting extensive influence within the cell and beyond. These RNAs fine-tune the gene expression as
they act as specific modulators based on cell-type specificity of the organism during development as well as pathological conditions (Giraldez et al., 2005; Girardot et al., 2012; Baer et al., 2013). Also, miRNAs have been known to play a role in tumor suppression, apoptosis,
cellular proliferation and cell movement which suggests that they can be manipulated in treating
epigenetic diseases like cancer (Kala et al., 2013).

Putative mechanisms of RNA silencing include the ability of non-coding RNA to negatively regulate expression of target genes at the posttranscriptional level by binding to 3′-untranslated regions of target mRNAs resulting in their degradation (Singh et al., 2008).

All genes in every cell type are activated or silenced by an underlying interplay between these described epigenetic mechanisms. And as explained in the Introduction, exogenous epigenetic forces modify the endogenous inherited epigenetic pattern.

Endogenous and exogenous epigenetic regulation of genes

In order to illustrate the endogenous epigenetic regulation of a gene, we will use the example of OCT4. OCT4 is the master pluripotency gene, which is regulated through different stages of human development, and its activation is necessary for maintaining pluripotency, whereas it must be silenced in order for a cell to differentiate (Kellner and Kikyo, 2010) (Figure ​(Figure2).2). OCT4 is thus active in embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) as well as in cancer cells, but is silenced in differentiated cell types. The three types of epigenetic modifications explained above i.e., DNA methylation, histone modification and RNA silencing are responsible for such regulation of OCT4 gene expression. This has been illustrated in detail along with the various epigenetic tags involved in its regulation in ​Figure22.


IEpigenetic regulation of OCT4 in stem cells, cancer cells and somatic cells.

IThe figure represents the various epigenetic mechanisms involved in regulating the gene expression of OCT4. (A) This section represents the structure of the OCT4 gene.

In a similar way, let us consider the effect of exogenous epigenetic forces on the expression of OCT4. Vitrification, which is a commonly used method for cryopreservation, has been documented to alter the methylation patterns of the OCT4 gene. In two such cases, vitrification resulted in decreased methylation of the OCT4 promoter causing reduced gene expression in mouse blastocysts and the same was observed for mouse oocytes that underwent vitrification followed by in vitro maturation (Milroy et al., 2011; Zhao et al., 2012). This explains how the external environment, in this case temperature, can lead to alteration of the epigenetic profile of one or many genes, ultimately causing differential gene expression.

How do cellular biochemical changes cause epigenetic changes? a hypothetical mechanism

The effects of an epigenetic factor can be manifested as a global change in DNA methylation affecting multiple genes, or modified expression of very specific genes. The mechanisms and cellular pathways that are involved in the creation of these global or specific epigenetic changes are currently obscure. Below we describe a working hypothesis on how these changes might occur.

We propose that an epigenetic factor can act through either a direct or(Figure 3). A direct effect could happen in two ways; which we term Type 1 and Type 2. Type 1 direct effect occurs when the epigenetic factor directly alters the state of epigenetic
enzymes—either by binding to them and preventing them from carrying out their normal function, damaging them in some other way, or upregulating them. The altered bioavailability of epigenetic enzymes then results in aberrant recruitment of epigenetic tags to promoters and enhancers on a genome-wide scale. Such a direct effect would be effective across the entire genome, not affecting any specific gene but resulting in a randomly altered epigenome. An example of a directly acting epigenetic factor is the antihypertensive hydralazine, which inhibits DNA methylation.


Figure 3

The direct and indirect epigenetic pathway.

The figure represents two different routes through which an epigenetic factor can modify the epigenome leading to altered gene expression. Epigenetic effects exerted by an external factor or intrinsic environment…

Type 2 direct effects occur when an epigenetic factor causes a change in a biochemical process that results in an altered availability of a substrate, intermediate, by-product or any other metabolite participating in the biochemical pathway, that is used to make up epigenetic tags (for example acetate). This in turn leads to altered availability of epigenetic tags, in this case acetyl groups on histones, which leads non-specific modification of the epigenome (Figure 33.)

The second major way that factors can cause epigenetic changes is by what we term indirect mechanisms (Figures ​(Figures3,3, ​,4).4). A biphasic mechanism is postulated for indirect effects in which acute exposure to a factor influences cellular signaling pathways that leads to altered expression of growth factors, receptors and ion channels, which in turn alter transcription factor activity at gene promoters. With more chronic exposure, the transcription factors and other gene regulatory proteins, in addition to altering gene expression activity, actually recruit or repel epigenetic enzymes to/from the associated chromatin, resulting in the addition or removal of epigenetic tags (Figure ​(Figure4).4). In this way cells adapt to the persistent gene expression changes by causing permanent modifications to DNA methylation and chromatin structure, leading to enduring alteration of the affected epigenetic network (Figures ​(Figures3,3, ​,4).4). An example of an indirectly acting factor is the drug isotretinoin, which has transcription factor activity.


Figure 4

The indirect epigenetic pathway.

An epigenetic factor operating through an indirect pathway interferes with transcriptional machinery. Chronic exposure to an epigenetic factor can lead to the retention of an already altered state of transcriptional machinery. …

Epigenetic factors known to cause such direct and indirect effects are well-documented but their exact mechanism has not been accurately elucidated. For example, nutritional interventions in adulthood like caloric restriction can induce epigenetic changes that have the potential to alleviate age-related diseases (Bacalini et al., 2014). In caloric restriction food intake is intentionally reduced by 30–50% (Zakhari, 2013) and has been shown to delay the aging process in mice by decreasing the levels of histone deacetylase 2 (HDAC2) which otherwise increases during the normal aging process (Chouliaras et al., 2013). Thus, this is an example of direct effect by an epigenetic influence (caloric restriction).

Another example is curcumin, a polyphenol found in turmeric, which can be regarded as a dietary epigenetic factor. It is an inhibitor of the Histone acetlytransferase p300/CBP (co-activator) and GATA4 (a zinc finger transcription factor), which leads to decrease in nuclear acetylation induced during myocardial cell hypertrophy (Morimoto et al., 2008). The possible mechanisms through which curcumin exerts such an effect might be through allosteric regulation of p300 or through interfering with nuclear signaling pathways like transcriptional activation by NF-κB (Morimoto et al., 2008). Hence the epigenetic effect of curcumin is an example of a combined direct and indirect effect.