EPIGENETICS DEFINITION

The term “epigenetics” was first introduced by Conrad Waddington in 1942 (Waddington, 1956) who referred to the study of epigenesis, namely how phenotypes derive from genotypes during development (Casadesús & Noyer‐Weidner, 2013; Dupont et al., 2009). He defined epigenetics as “the branch of biology that studies the causal interactions between genes and their products which bring the phenotype into being” (Noble, 2015; Waddington, 1956). With the rapid growth of genetics, the meaning of the term has undergone several transitions and is now used to describe the study of changes in gene function that are heritable and are not attributed to alterations in the DNA sequence (Deans & Maggert, 2015). While the sequence of DNA remains constant, the epigenetic state of the genome (i.e., epigenome) changes dynamically in the cells of different tissues and, by using many coordinating enzymes, regulates the expression of genes to alter the phenotypes in different cell types (Lacal & Ventura, 2018). Epigenetic mechanisms can influence genes at the transcriptional and post‐transcriptional levels and/or at the translational and post‐translational level (Gibney & Nolan, 2010).

Epigenetic and genetic information are both inherited, but contrary to genetic information, epigenetic data are reversible (called epigenetic plasticity) and may be affected by various stimuli or environmental factors (Bohacek & Mansuy, 2013). These changes may persist through multiple cell divisions, even for all of a cell’s life, and may also be transferred to the offspring, lasting for multiple generations via trans‐generational epigenetic inheritance (Wei, Huang, Yang, & Kang, 2017). The knowledge of the mechanism by which epigenetic information is transmitted is poor, but it is known to be transferred differently between sexes. More specifically, an exposure to an environmental factor, such as a toxicant, to a gestating F0 female individual promotes an epigenetic alteration in the germ cell programming of the F1 generation fetus (Figure 2.1). The altered germ cell epigenetics is then transmitted in the second (2) and the third (3) generations (Lacal & Ventura, 2018; Nilsson, Ben Maamar & Skinner, 2022), and consequently, only the fourth generation can be considered as “event free”. An exposure to an environmental factor in the father, it can only modify his sperm, producing an epigenetic change in the F1 generation, effecting a reliable non‐genetic inheritance in the third generation (Donkin & Barrès, 2018; Lacal & Ventura, 2018).

EPIGENETIC MECHANISMS

The fundamental molecular mechanisms that mediate epigenetic modulation are DNA methylation, histone posttranslational modification, and non‐coding RNA regulation (Dupont et al., 2009) (Figure 2.2). The above‐mentioned epigenetic mechanisms are controlled by several enzymes, the writers that add chemical residues to DNA and histones, the erasers that remove them, and the readers, specialized domain‐containing proteins that identify and interpret those modifications (Biswas & Rao, 2018).

The most‐studied epigenetic mechanism is DNA methylation, which includes the addition of a methyl group (CH₃) at the nucleotide cytosine (Dupont et al., 2009). The most common DNA methylation process is the covalent addition of the methyl group at the 5‐carbon of the cytosine ring resulting in 5‐methylcytosine (5‐mC). In mammals, DNA methylation occurs at cytosines in any context of the genome; in somatic cells methylation occurs almost exclusively in a cytosine–guanine site (CpGs), while in embryonic stem cells (ESCs) as much as a quarter of all methylation appears in a non‐CpG context. When methyl groups are present on a gene, that gene is turned off or silenced, and a protein is not produced from that gene.

Furthermore, histone modifications are key epigenetic regulators that control chromatin structure and gene transcription, thereby affecting various cellular phenotypes (Molina‐Serrano, Kyriakou, & Kirmizis, 2019). Histone modification includes multiple, mostly reversible, chemical alterations (e.g., acetylation, methylation, and phosphorylation) of nucleosome core histones, particularly at their N‐terminal sequences, and of the linker histone H1. Due to this characteristic, epigenetic modifications are reversible and thus able to dynamically modulate chromatin structure to activate or silence gene expression (Bannister & Kouzarides, 2011).

Novel insights have highlighted the important role of non‐coding RNA transcripts in epigenetic gene regulation (Mattick & Makunin, 2006; Wei et al., 2017). The term non‐coding RNA (ncRNA) is commonly used for RNA that does not encode a protein, however, many of these RNAs do contain information and are functional. Non‐coding RNA helps control gene expression by attaching to coding RNA, along proteins that break down the coding RNA and prevent it from making proteins. Non‐coding RNA may also recruit proteins that modify histones to turn genes on or off by making them more or less accessible, respectively, to RNA‐making machinery.

EPIGENETICS AND DISEASE EPIDEMIOLOGY

Alterations in epigenetic marks such as modification of the wrong gene or the failure to add a chemical group to a particular gene or histone, could lead to abnormal gene activity or inactivity (Casadesús & Noyer‐Weidner, 2013). Such epigenetic mechanisms could result in a vast spectrum of cell differentiations, morphogenesis, variability, and adaptability. Epigenetic modifications can directly cause disease, such as Beckwith Weidemann syndrome (BWS), an imprinting disorder caused by abnormal DNA methylation (Bakulski & Fallin, 2014). Epigenetics can also be the mechanism linking environmental exposures and disease, and classic example of this interaction is oxidative DNA damage caused by toxicants. Epigenetics may also influence the relationships between exposure and disease or genotype and disease via effect modification (i.e., exposure to air pollution and increased risk of cardiovascular disease). When targeting treatment, epigenetic factors can serve as useful biomarkers of disease status or disease subtype.

Incorrect epigenetic marks can result in birth defects, childhood diseases, or diseases during a person’s lifespan. In this regard, epigenetic errors are associated with multiple NCDs, including cancer, cardiovascular, respiratory, and neurodegenerative diseases (Romani, Pistillo, & Banelli, 2015). Indeed, epigenetic modifications may be behind middle to late life functional declines. Early studies on epigenetic functions mainly focused on fetal development, aging, and cancer. As epigenetic research has progressed and epigenetic mechanisms have become better understood, possible epigenetic pathways have been implicated in complex diseases etiology, such as autoimmune diseases, inflammation, allergy, obesity, insulin resistance, type 2 diabetes (T2D), neurodegenerative diseases, autism spectrum disorders, bipolar disorder, and schizophrenia (Dupont et al., 2009; Ramsay, 2015).

Epigenetic errors are also implicated in imprinting disorders, such as Silver–Russell syndrome (SRS) and Beckwith–Weidemann syndrome (BWS), for individuals conceived by assisted reproductive technologies (ART) (Peral‐Sanchez et al., 2021). ART conception is associated with an approximately two‐fold increased risk of preterm birth, low birth weight, being small for gestational age or perinatal mortality, some of which may be attributable to epigenetic variation induced in the periconceptional period. Genome‐wide DNA methylation profiling in cord blood and placenta revealed differences in the methylation status between ART and spontaneous conceiving populations (Novakovic et al., 2019).

Across epigenomic sites, particular regions show change in methylation within the same person over time (Bakulski & Fallin, 2014). Furthermore, it was recently demonstrated that some epigenetic marks can be reversible, a trait that it is as challenging as it is promising (Bohacek & Mansuy, 2013; Foley et al., 2008). This characteristic makes the genome flexible to respond and adapt to external stimuli, such as nutrition, stress, exercise, pollutants, and drugs, since epigenetic modifications are dynamic and intertwined with environmental and lifestyle cues.

MATERNAL UNDERNUTRITION

Very interesting scientific data concerning the effect of nutrition during gestation arose from a classic epidemiological study during the Dutch famine of 1944–1945 (World War II), which demonstrated that individuals conceived during the famine were at a greater risk of CVD and metabolic disorders, such as obesity, diabetes, impaired glucose tolerance, and hyperlipidemia in adulthood (Lumey, 1992). Decades later, a significant reduction in DNA methylation in famine‐exposed offspring was observed at the promoter of the insulin‐like growth factor 2 (IGF2) gene, which regulates fetal development and growth. Most importantly, the Dutch famine data highlight that nutritional deprivation can have adverse effects on health beyond a person’s lifespan and leave a lasting epigenetic imprint on offspring. Similarly, epidemiological data from other famine studies (Hult et al., 2010; Li et al., 2010) demonstrate that adults exposed in utero or early in life to energy deficits had an increased risk for developing hypertension, glucose intolerance, and obesity compared to individuals conceived after the famine.

Additionally, maternal malnutrition along with low‐protein diets from the pre‐implantation period to late lactation, is associated with the onset of metabolic diseases in adult offspring, which are mediated by epigenetic modifications (Koemel and Skilton, 2022; Peral‐Sanchez et al., 2021). Undernutrition during pregnancy may alter the fetal epigenome through reduced intake and bioavailability of protein, amino acids, and key micro‐nutrients involved in one‐carbon metabolism, such as folate, methionine, choline, betaine, vitamin B12, vitamin B6, antioxidants, and other bioactive compounds (Cai et al., 2021; Jankovic‐Karasoulos et al., 2021). The above‐mentioned nutrients are involved in one carbon metabolism and subsequently in DNA‐methylation process as co‐factors or methyl donors. More specifically, methionine, choline, and folate are substrates needed for the methylation process (methyl‐donor nutrients); the vitamins B2, B6, and B12 are co‐factors needed for methyltransferases, the enzymes involved in the methylation process (Kohil, Al‐Asmakh, Al‐Shafai, & Terranegra, 2021). Vitamin B6 serves as a coenzyme to serine hydroxymethyltransferase, the key enzyme in the folate cycle converting tetrahydrofolate (THF) to 5,10‐methyleneTHF. Riboflavin (vitamin B2) is a precursor for flavin adenine dinucleotides, which is a cofactor to methylenetetrahydrofolate reductase (MTHFR) in the conversion of 5,10‐methylene THF to 5‐methyl THF. Vitamin B12 is the coenzyme of methionine synthase, which catalyzes the reaction of homocysteine involved in the production of methionine from homocysteine and betaine (Figure 2.5) (Anderson, Sant, & Dolinoy, 2012).

Folate‐mediated one‐carbon metabolism refers to a complex network of interconnected metabolic pathways, which ultimately result in a supply of methyl groups for DNA, RNA, or protein methylation. Folate, in particular, is a key source of the methyl group required for DNA and histone methylation, and sub‐optimal intake during gestation may alter methylation patterns that are vital for fetal development and the offspring’s health (Lintas, 2019). Most importantly, folate deficiency is correlated with methylation changes in brain tissue and may alter epigenetic marks implicated in neural tube defects, thus neurocognitive and neurobehavioral deficits (Joubert et al., 2016).

Interestingly, there is little evidence that nutritional interventions during early or mid‐pregnancy, like providing under‐nourished mothers with protein, energy, and micronutrient supplements, reduces the cardio‐metabolic disease risk in offspring. Data from animal studies suggest that it may be more crucial to intervene earlier in the pregnancy, when the developmental window is more critical, or even pre‐conception, to modify placentation and organogenesis (which occur mainly in the first trimester) as well as peri‐conceptional epigenetic changes (Fall & Kumaran, 2019).

MATERNAL OVERNUTRITION

Excess fat and caloric consumption during gestation as well as a mother’s pre‐pregnancy obesity are also linked to epigenetic changes (Figure 2.6). Maternal metabolic disorders (e.g., poor glycemic control, hypertension) are associated with epigenetic alterations of the offspring too. Notably, genes that regulate energy and glucose metabolism as well as adipogenesis, leptin encoding, and transcription factors are controlled by epigenetic mechanisms (Elshenawy & Simmons, 2016). Maternal overnutrition may modify the expression of hypothalamic genes that can increase fetal and neonatal energy intake (Şanlı & Kabaran, 2019). Moreover, considering that lipids act as both transcriptional activators and signaling molecules, excess lipid exposure may regulate genes involved in lipid sensing and metabolism. It was suggested that the prevalence of an obese phenotype is closely related to epigenetic marks, originating from in utero stimuli and passing on through trans‐generational transmission (Heerwagen, Miller, Barbour, & Friedman, 2010).

More specifically, leptin and insulin signals modify the development of eating behaviors by affecting the neuronal circuits in the hypothalamus. According to these neuroendocrine factors, increased fetal appetite, energy intake, and adiposity alter the food preferences and body composition after birth (Lawlor et al., 2006). Overall, maternal obesity and overnutrition can lead to permanent changes in metabolic and epigenetic mechanisms, and their offspring are predisposed to be overweight or with obesity themselves. Siblings born to mothers with obesity before and after the mother’s bariatric surgery had different obesity risks; the children born after weight loss had a lower risk of being overweight or with obesity (Şanlı & Kabaran, 2019). Therefore, considering the public health burden of obesity and metabolic diseases across generations, perinatal weight control interventions including lifestyle modifications through a healthy and balanced dietary pattern and exercise in mothers with obesity should be considered (Figure 2.7).