Similar to the  imitation switch (ISWI)  subfamily of ATP-dependent chromatin remodelers, CHD complexes regulate the assembly and organization of mature nucleosomes along the DNA. [ 1 ] [ 2 ]   Histones  are removed during DNA replication; following behind the  replisome , histones start to assemble as immature pre-nucleosomes on nascent DNA. With the help of CHD complexes, histone octamers can mature into native nucleosomes. Following nucleosome formation, CHD complexes organize nucleosomes by regularly spacing them apart along the DNA. [ 1 ]


Additionally, CHDs in higher-order organisms can slide/eject nucleosomes or histone dimers to allosterically regulate DNA accessibility. [ 1 ]  Specific CHD complexes, such as the nucleosome remodeling deacetylase ( NuRD ) complex in  C. elegans , can expose binding sites for transcriptional repressors along the chromatin by interacting with highly-modular histone tails; deacetylation of the histone residue  H3K9ac  is an example of how the NuRD complex can downregulate gene expression and affect DNA topology. [ 3 ]


The final mechanism of this subfamily of ATP-dependent remodelers is nucleosome editing. [ 1 ]    Drosophila  dCHD1 can edit nucleosomes by swapping out histone H3 for the variant H3.3. [ 2 ]  Binding of dCHD1 near the nucleosome causes tension in the DNA. To relieve this tension, an upstream H3 dimer is displaced from the nucleosome, allowing for its replacement by histone variant H3.3. [ 4 ]  The addition of H3.3 into the nucleosomes is an  epigenetic  way to keep the chromatin in an accessible, transcription-ready, state. [ 5 ]  Incorporation of alternative histones and post-translational modifications (PTMs) play an integral role in regulating the cell's histone code. [ 6 ]


The unique N-terminal domain of the CDH subfamily contains two  chromodomains . The two lobe domains act in tandem as the translocase domain (Tr) and are connected by a peptide linker (Fig. 01). NegC* (similar to NegC in ISWI) acts as an inhibitor to lobe movement and translocation until proper binding of the  DNA-binding domain  (DBD) occurs. NegC* acts by blocking ATP hydrolysis at the lobes. The DBD also acts as a nucleotide ruler, evenly spacing nucleosomes from one another. Post-DNA binding, the DBD causes tension on the DNA strand resulting in a conformational change that blocks regular NegC* inhibition. This allows for the activation of the TR domain, resulting in DNA translocation. [ 1 ]


The Tr mechanism of DNA translocation is conserved by all ATP-dependent chromatin remodelers; two  RecA -like lobes are mechanistically responsible for translocating the DNA. [ 3 ]  After binding two helical turns away from a nucleosome, the complex causes the shifting of the aforementioned nucleosome upstream one-two base pairs. In this ATP-driven mechanism, energy from hydrolysis causes the lobes to 'crawl' along the DNA towards the nucleosome dyad until the nucleosomes are correctly assembled, accessed or edited.


Proteins in the family:


Computational epigenetics [ 1 ]  uses statistical methods and mathematical modelling in  epigenetic  research. Due to the recent explosion of epigenome datasets, computational methods play an increasing role in all areas of epigenetic research.


Research in computational epigenetics comprises the development and application of bioinformatics methods for solving epigenetic questions, as well as computational data analysis and theoretical modeling in the context of epigenetics. This includes modelling of the effects of histone and DNA CpG island methylation.


Computational methods and next-generation sequencing (NGS) technologies to are being employed to study DNA methylation and histone modifications, which are essential in cancer research. High-throughput sequencing offers valuable insights into epigenetic changes, and the growing volume of these datasets drives the continuous development of bioinformatics techniques for their effective management and analysis. [ 2 ]


There is a need for data integration tools that can merge various types of epigenetic modifications and -omics data (including transcriptomics, genomics, epigenomics, and proteomics) to gain a comprehensive understanding of biological processes. This requires the standardization, annotation, and harmonization of epigenetic data, along with the enhancement of computational and machine learning approaches. [ 3 ]


Understanding the functional implications of epigenetics in diseases can be greatly advanced by using epigenetic editing tools, such as CRISPR-dCas9 technology. These tools enable precise modifications of epigenetic marks at specific loci, allowing researchers to assess the effects of these alterations in cellular and animal models, thus complementing insights obtained from computational analyses. [ 3 ]


Various experimental techniques have been developed for genome-wide mapping of epigenetic information, [ 4 ]  the most widely used being  ChIP-on-chip ,  ChIP-seq  and  bisulfite sequencing . All of these methods generate large amounts of data and require efficient ways of data processing and quality control by bioinformatic methods.


A substantial amount of bioinformatic research has been devoted to the  prediction  of epigenetic information from characteristics of the  genome sequence . Such predictions serve a dual purpose. First, accurate epigenome predictions can substitute for experimental data, to some degree, which is particularly relevant for newly discovered epigenetic mechanisms and for species other than human and mouse. Second, prediction algorithms build statistical models of epigenetic information from training data and can therefore act as a first step toward quantitative modeling of an epigenetic mechanism. Successful computational prediction of DNA and lysine methylation and acetylation has been achieved by combinations of various features. [ 5 ]
 [ 6 ]


The important role of epigenetic defects for  cancer  opens up new opportunities for improved diagnosis and therapy. These active areas of research give rise to two questions that are particularly amenable to bioinformatic analysis. First, given a list of genomic regions exhibiting epigenetic differences between tumor cells and controls (or between different disease subtypes), can we detect common patterns or find evidence of a functional relationship of these regions to cancer? Second, can we use bioinformatic methods in order to improve diagnosis and therapy by detecting and classifying important disease subtypes?


The first wave of research in the field of computational epigenetics was driven by rapid progress of experimental methods for data generation, which required adequate computational methods for data processing and quality control, prompted epigenome prediction studies as a means of understanding the genomic distribution of epigenetic information, and provided the foundation for initial projects on  cancer epigenetics . While these topics will continue to be major areas of research and the mere quantity of epigenetic data arising from epigenome projects poses a significant bioinformatic challenge, several additional topics are currently emerging.


Epigenetics  is the study of changes in  gene expression  that occur via mechanisms such as  DNA methylation ,  histone acetylation , and  microRNA  modification. When these epigenetic changes are  heritable , they can influence  evolution . Current research indicates that epigenetics has influenced evolution in a number of  organisms , including  plants  and  animals . [ 1 ]


DNA methylation is a process by which  methyl groups  are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. Histones are  proteins  found in cell nuclei that package and order the  DNA  into structural units called  nucleosomes . [ citation needed ]


DNA methylation and histone modification are two mechanisms used to regulate gene expression in most organisms which includes plants and animals. DNA methylation can be stable during cell division, allowing for methylation states to be passed to other  orthologous genes  in a genome. DNA methylation can be reversed via enzymes known as DNA de-methylases, while histone modifications can be reversed by removing histone acetyl groups with deacetylases. The process of DNA methylation reversal is known DNA demethylation. [ 2 ]  Interspecific differences due to environmental factors are shown to be associated with the difference between  annual  and  perennial  life cycles. There can be varying adaptive responses based on this. [ 3 ]


Forms of  histone methylation  cause repression of certain genes that are stably inherited through mitosis but that can also be erased during meiosis or with the progression of time. The induction of  flowering  by exposure to low winter temperatures in  Arabidopsis thaliana  shows this effect. Histone methylation participates in repression of expression of an inhibitor of flowering during cold. In annual,  semelparous  species such as Arabidopsis thaliana, this histone methylation is stably inherited through mitosis after return from cold to warm temperatures giving the plant the opportunity to flower continuously during spring and summer until it senesces. However, in perennial,  iteroparous  relatives the histone modification rapidly disappears when temperatures rise, allowing expression of the floral inhibitor to increase and limiting flowering to a short interval. Epigenetic histone modifications control a key adaptive trait in Arabidopsis thaliana, and their pattern changes rapidly during evolution associated with reproductive strategy. [ 3 ]


Another study tested several epigenetic recombinant inbred lines (epiRILs) of Arabidopsis thaliana - lines with similar genomes but varying levels of DNA methylation - for their drought sensitivity and their sensitivity to nutritional stress. It was found that there was a significant amount of heritable variation in the lines in regards to traits important for survival from drought and nutrient stress. This study proved that variation in DNA methylation could result in heritable variation of ecologically important plant traits, such as root allocation,  drought tolerance , and nutrient  plasticity . It also hinted that epigenetic variation alone could result in rapid evolution. [ 4 ]


Scientists found that changes in DNA methylation induced by stress were inherited in  asexual   dandelions . Genetically similar plants were exposed to different ecological stresses, and their offspring were raised in an unstressed environment. Amplified fragment-length polymorphism markers that were methylation-sensitive were used to test for methylation on a genome-wide scale. It was found that many of the environmental stresses caused induction of pathogen and herbivore defenses, which caused methylation in the genome. These modifications were then genetically transmitted to the offspring dandelions. The transgenerational inheritance of a stress response can contribute to the heritable plasticity of the organism, allowing it to better survive environmental stresses. It also helps add to the  genetic variation  of specific lineages with little variability, giving a greater chance of  reproductive success . [ 5 ]


A comparative analysis of  CpG  methylation patterns between  humans  and  primates  found that there were more than 800 genes that varied in their methylation patterns among  orangutans ,  gorillas ,  chimpanzees , and  bonobos . Despite these  apes  having the same genes, methylation differences are what accounts for their  phenotypic variation . The genes in question are involved in development. It is not the protein sequences that account for the differences in physical characteristics between humans and apes; rather, it is the epigenetic changes to the genes. Since humans and the  great apes  share 99% of their DNA, it is thought that the differences in methylation patterns account for their distinction. So far, there are known to be 171 genes that are uniquely methylated in humans, 101 genes that are uniquely methylated in  chimpanzees , 101 genes that are uniquely methylated in  gorillas , and 450 genes that are uniquely methylated in  orangutans . For example, genes involved in blood pressure regulation and the development of the inner ear's semicircular canal are highly methylated in humans, but not in apes. There are also 184 genes that are conserved at the protein level between humans and chimpanzees, but have epigenetic differences. Enrichments in multiple independent gene categories show that regulatory changes to these genes have given humans their specific traits. This research shows that epigenetics plays an important role in the evolution of primates. [ 6 ]
It has also been shown that cis-regulatory elements changes affect the transcription start sites (TSS) of genes. 471 DNA sequences are found to be enriched or depleted in regards to histone trimethylation at the H3K4 histone in chimpanzee, human, and macaque prefrontal cortexes. Among these sequences, 33 are selectively methylated in neuronal  chromatin  from children and adults, but not from non-neuronal chromatin. One  locus  that was selectively methylated was  DPP10 , a  regulatory sequence  that showed evidence of  hominid  adaptation, such as higher  nucleotide substitution  rates and certain regulatory sequences that were missing in other primates. Epigenetic regulation of TSS chromatin has been identified as an important development in the evolution of gene expression networks in the human brain. These networks are thought to play a role in  cognitive processes  and  neurological disorders . [ 7 ]
An analysis of methylation profiles of humans and primate  sperm cells  reveals epigenetic regulation plays an important role here as well. Since mammalian cells undergo reprogramming of DNA methylation patterns during  germ cell  development, the methylomes of human and chimp sperm can be compared to methylation in  embryonic stem cells  (ESCs). There were many hypomethylated regions in both sperms cells and ESCs that showed structural differences. Also, many of the  promoters  in human and chimp sperm cells had different amounts of methylation. In essence, DNA methylation patterns differ between germ cells and  somatic cells  as well as between human and chimpanzee sperm cells. Meaning, differences in promoter methylation could possibly account for the phenotypic differences between humans and primates. [ 8 ]  Research has also shown surprisingly amounts of conserved tissue-specific methylation, in line with phylogenetic relatedness  [ 9 ]


Red Junglefowl, an ancestor of domestic chickens, show that gene expression and methylation profiles in the  thalamus  and  hypothalamus  differed significantly from that of a domesticated egg-laying breed. Methylation differences and gene expression were maintained in the offspring, depicting that epigenetic variation is inherited. Some of the inherited methylation differences were specific to certain tissues, and the differential methylation at specific loci was not altered much after intercrossing between Red Junglefowl and domesticated laying hens for eight generations. The results hint that domestication has led to epigenetic changes, as domesticated chickens maintained a higher level of methylation for more than 70% of the genes. [ 10 ]


The role of epigenetics in evolution is clearly linked to the selective pressures that regulate that process. As organisms leave offspring that are best suited to their environment, environmental stresses change DNA gene expression that is further passed down to their offspring, allowing for them also to better thrive in their environment. The classic case study of the rats who experience licking and grooming from their mothers pass this trait to their offspring shows that a  mutation  in the DNA sequence is not required for a heritable change. [ 11 ]  Basically, a high degree of maternal nurturing makes the offspring of that mother more likely to nurture their own children with a high degree of care as well. Rats with a lower degree of maternal nurturing are less likely to nurture their own offspring with so much care. Also, rates of epigenetic mutations, such as DNA methylation, are much higher than rates of mutations transmitted genetically [ 12 ]  and are easily reversed. [ 13 ]  This provides a way for variation within a species to rapidly increase, in times of stress, providing opportunity for adaptation to newly arising selection pressures. [ citation needed ]


Lamarckism  supposes that species acquire characteristics to deal with challenges experienced during their lifetimes, and that such accumulations are then passed to their offspring. In modern terms, this transmission from parent to offspring could be considered a method of epigenetic inheritance. Scientists are now questioning the framework of the  modern synthesis , as epigenetics to some extent is Lamarckist rather than  Darwinian . While some evolutionary biologists have dismissed epigenetics' impact on evolution entirely, others are exploring a fusion of epigenetic and traditional genetic inheritance. [ 14 ]


Decreased  DNA Methylation I  ( DDM1 ), is a plant gene that encodes a nucleosome remodeler which facilitates  DNA methylation . The DDM1 gene has been described extensively in  Arabidopsis thaliana  and also in  maize . The protein has been described to be similar to the SWI2/SNF2 chromatin remodeling proteins. [ 1 ]


Since DNA methylation occurs mostly in  transposable elements  (TE), [ 2 ]  DDM1 is thought to be a crucial function in silencing TEs.


DDM1 is required for DNA methylation in highly  heterochromatin  transposable elements. [ 3 ]  DDM1, therefore, often silences transposable elements but the mutation in  Arabidopsis thaliana  is not embryo lethal. [ 3 ]  DDM1 mutants have also


DDM1 is being further explored in maize. Unlike  A. thaliana , maize has two copies of the gene:  CHR101  and  CHR106 . [ 4 ]  In maize, the double mutant of DDM1 is embryo lethal. However, other studies are using the  heterozygote  mutant. The maize genome is 80% transposons [ 5 ]  so DDM1 function is quite important.


Demethylating agents  are chemical substances that can inhibit  methylation , resulting in the expression of the previously hypermethylated silenced  genes  (see  Methylation#Cancer  for more detail).  Cytidine  analogs such as  5-azacytidine  (azacitidine) and 5-azadeoxycytidine ( decitabine ) are the most commonly used demethylating agents. They work by inhibiting  DNA methyltransferases . [ 1 ]  Both compounds have been approved in the treatment of  myelodysplastic syndrome  (MDS) by  Food and Drug Administration  (FDA) in United States.  Azacitidine  and  decitabine  are marketed as Vidaza and Dacogen respectively.  Azacitidine is the first drug to be approved by FDA for treating MDS and has been given  orphan drug  status. [ 2 ] [ 3 ]   Procaine  is a DNA-demethylating agent with growth-inhibitory effects in human cancer cells. [ 4 ]  There are many other demethylating agents that can be used to inhibit the growth of other diseases.  [ 5 ]


There is very little known about the mechanism of action of these drugs. However, it was shown in 2015 that a possible mechanism of action of these drugs in colorectal  cancer-initiating cells  is through activating  dsRNA  expression which leads to the activation of the  MDA5 / MAVS  RNA recognition pathway inducing some sort of viral mimicry inside the cell. [ 5 ]


The silencing of genes created by abnormal DNA methylation is a major contributor to the formation of cancerous tumors. Variations in DNA methylation of normal cells compared to malignant cells shows a prominent mechanism in how cancerous cells proliferate. Those variations are particularly prevalent in cell cycle regulation, DNA repair, and natural tumor suppression mechanisms. A leading therapeutic strategy in treating solid tumors stems from the use of demethylating agents to suppress DNA methylation in cancerous growths. Azacitidine and decitabine are both frequently used demethylating agents while decitabine is significantly more potent in its demethylating abilities. Both of these drugs are inhibitors of DNA Methyltransferases (DNMT) which are enzymes that are responsible for methylating DNA. In the 1970’s, these drugs have shown promising results in hematological cancers in organisms such as mice. The FDA initially rejected the use of azacitidine clinically due to negative side effects caused by elevated toxicity levels. However, in later clinical trials performed on patients with MDS, myelodysplastic syndromes, azacitidine provided effective and exhibited consistent results which led to FDA approval in 2004. The commercial name of azacitidine became Vidaza. Decitabine, with the commercial name Dacogen, followed with FDA approval in 2006. As more research is completed in the field of genetic mutations, specifically involving DNA Methylation, these drugs can be utilized to their maximum efficiency to clinically treat cancerous tumors. As of 2017, there were no approved demethylating agents for the treatment of solid tumors which can be a focus of research in the future. Treatment utilizing demethylating agents can have further clinical use by targeting cancer stem cells and triggering apoptosis. Demethylating agents and their relevance in clinical studies as therapy to treat lymphocytic leukemia can be seen in. [ 6 ]  Procaine can also be used as therapeutic development to inhibit the growth of cancer cells in humans. There is a new world of possibilities of using demethylating agents to treat different diseases such as leukemia and cancer as therapeutic treatment. [ 7 ]


Procaine (PCA) is a demethylating agent considered to be effective in inhibiting the growth of human cancer cells. Several studies have explored and elucidated the effects of procaine on human liver cancer cells and breast cancer cells. Studies have shown that procaine, as an inhibitor of DNA methylation in breast cancer cells, can effectively cause hypomethylation and demethylation of the entire group of breast cancer cell DNA genomes by reducing 5-methylcytosine DNA content. [ 8 ]  In addition, procaine can effectively restore the gene expression of tumor suppressor genes by demethylating densely hypermethylated CpG-enriched DNA. [ 8 ]  For human liver cancer cells, procaine is capable of reducing tumor volume by suppressing the cell viability of HLE, HuH7, and HuH6 cells, and it has shown effective inhibition of S/G2/M transition in HLE cells. [ 9 ]


Developmental origins of health and disease  ( DOHaD ) is an approach to medical research factors that can lead to the development of  human diseases  during early life development.  These factors include  the role of prenatal and perinatal exposure to environmental factors, such as  undernutrition , stress, environmental chemical, etc. [ 1 ] [ 2 ]  This approach includes an emphasis on  epigenetic  causes of adult chronic  non-communicable diseases . [ 1 ] [ 3 ] [ 4 ]  As well as physical human disease, the psychopathology of the foetus can also be predicted by epigenetic factors. [ 5 ]


DOHaD has evolved into its modern understanding from several precursor concepts. In the 19th century the idea of "maternal impressions" was popular. Maternal impression is the idea that anything the mother did before giving birth could affect her offspring. [ 6 ]  Our modern understanding of DOHaD was developed by several studies by  David Barker  and his colleagues, which showed a strong relationship between  infant mortality  rates from 1921 to 1925 and  ischemic heart disease  rates from 1968 to 1978. This led to the  fetal origins hypothesis  of the origins of adult diseases, which proposed that this relationship was caused by differences in early life nutrition, with a supporting theory that birthweight is connected to the development of chronic disease. [ 7 ]


During the  Dutch Hunger Winter Famine  (1944-1945) [ 8 ]  mothers were not able to receive the proper nutrition needed to healthily carry a baby. The babies who were born during this time developed diseases (such as heart disease, schizophrenia, and type 2 diabetes) at increased levels. Researchers were able to determine, decades after the famine, that the babies born during Dutch famine had an increase in methylation in some genes and a decrease in methylation in other genes compared to their siblings who were not born during the famine. The methylation levels explain why these individuals were predisposed to certain diseases. Data collected from the Dutch famine and similar events, such as the one at Leningrad, provided a reliable source of information to scientists studying DOHaD. [ 6 ] [ 9 ] [ 10 ]


These studies in turn led to greater interest in the roles of  developmental plasticity  and early life environmental exposures in adult disease. The World Congress on Fetal Origins of Adult Disease held two meetings – one in 2001 and the other in 2003 – summarizing the new research in these areas. This congress later evolved into the  International Society for Developmental Origins of Health and Disease . [ 1 ]


Between 1944 and 1945, in the western regions of the Netherlands and in Amsterdam, a famine broke out due to a railway strike and German control limiting supplies. The people of these countries were receiving extremely limited calories (around 400-800 a day [ 11 ] ) which had an extreme effect on pregnant women and their children.  Researchers began studying a cohort of middle-aged individuals whose mothers had been pregnant with them during the famine.  The Dutch Hunger Winter study provided significant data to support the DOHaD. Results concluded that the women with low caloric and nutritional intake during pregnancy had children that had greater rates of obesity as opposed to those who were not exposed to famine. [ 11 ]  This is conclusive with the DOHaD theory. The study goes on to investigate at what points in development  DOHaD stood true. It is thought that exposure to famine in early gestational periods has a greater effect on the foetus, however, these theories are still under investigation.


The  thrifty phenotype hypothesis  was proposed by C. Nicholas Hales and David Barker in a study published in 1992. [ 12 ]  This hypothesis suggested that poor growth during the fetal and infant stages can cause a development of type 2 diabetes later in life. Hales and Barker suggested that this poor growth was due to maternal malnutrition which leads to low birth weight. As previously stated, low infant birth weight usually leads to an increased risk of obesity which, if not acted on, can lead to type 2 diabetes. [ 13 ]


The paper suggests that due to maternal malnutrition, infants can have lower birth weights. Since this is occurring during the plastic stage of development, this can cause the foetus to be "programmed" to conserve energy and store fat, thus leading to a lower metabolism. If there is a surplus of food during adulthood their body simply stores this abundance as more fat. This can then lead to not only diabetes but other metabolic diseases, too. [ 14 ]


There are two different types of plasticity responses when dealing with human development. The first is an immediate adaptive response. This response will alter the development that is needed to survive if there have been changes in the environment. [ 15 ]  An example of this type of response is when oxygen deprivation causes a change in blood flow that would normally help with the expense of less critical tissues.  Predictive adaptive response  (PAR) is the second type of plasticity response. PARs are cues that happen in early life development that cause phenotype development to change that are normally adapted to environmental cues in later life. [ 15 ]  This can be a benefit only  when the predicted and actual postnatal environment match.


This is also referred to as the  mismatch theory . Foetal malnutrition followed by excess nutrition in adults is a key example of environmental mismatches. This is very common among people born in impoverished societies. For pregnant women who are living in this type of circumstance the foetuses which are in utero are more likely to sense a low-protein condition and alter their development to survive. Therefore, the foetus will predict that getting food will be very difficult which then will set its metabolism for a "thrifty phenotype". [ 15 ]  By doing this it will use every calorie to its most efficient use for survival. This can be the same cause for a society with plenty of food available. The embryo will develop insulin resistance and enzyme levels will convert the food that is not being used into fat, which in later life can lead to obesity and diabetes.  Overall the environmental mismatch hypothesis is an advantage for human development but not without some disadvantage in later life.


Epigenetic alterations of gene expression are closely related with developmental origins of the health and disease hypothesis. DNA methylation, histone modifications and non-coding RNAs are altered by the environment in the womb and potentially go on to produce higher rates of adult disease later in life. [ 16 ]


The process of DNA methylation is the most studied epigenetic response as it relates to the developmental origins of health and disease. The methylation of chief regulatory cytosines changes the DNA's hydrophobicity and begins to inhibit interactions with transcription factors responsible for the expression of the gene. Certain metabolic disorders, cancer and neurodegenerative disorders can be attributed to DNA methylation events. [ 16 ]


Modifications of histones are important processes in developmental programming, with studies showing that maternal and paternal stress can induce histone modifications. These histone modifications alter phenotypes of organisms and can be the mechanism behind the  predisposition of some metabolic disorders. [ 16 ]  Unbalanced diets increase the thioredoxin-interacting protein expressions causing blastocysts to form incorrectly because of altered histone methylation. [ 16 ]  High-fat diets are shown to alter histone methylation and acetylation and potentially lead to changes in gene expression within fetal adipose, liver, and skeletal muscle tissues. [ 16 ]


Recent studies have shown the importance in non-coding RNA's ability to regulate cell differentiation and organismal development. There are many types of non-coding RNA that are present and play a role in differentiating stem cells. There are many non-coding RNAs responsible for differentiating cells for human brain and muscle development. These RNA molecules have also been implicated in having a role in cardiovascular development. Long non-coding RNA called lncRNA Fendrr has shown to be an important regulator of heart development and it modifies chromatin and controls developmental signaling of genetically modified mice hearts. [ 17 ] [ 16 ]  There is still a vast amount of research needed to fully understand this mechanism and its relation to DOHaD. [ 16 ]


In a study done by Barker, he found a strong connection between poor prenatal environments and increased possibilities of  cardiovascular diseases  in adults. He found a direct correlation between infant mortality in 1921-1925 and mortality rates in 1968-1978 because of heart diseases in England. In areas where pregnant mothers had to face poor nutritional states, their newborn children were at a high risk of death. If they survived the early stages of life, they developed a higher risk of cardiovascular diseases. [ 7 ]


Studies on rats found that maternal nutrient restriction resulted in damage to the cardiac renin-angiotensin system (which regulates blood pressure and volume). Additionally, these studies have shown a decrease in the number of nephrons produced by the offspring of these mothers. These differences have been found to affect males and females differently, at least in the early stages. [ 6 ]  The exact mechanism of action is unknown but it is believed that it is epigenetic. [ 6 ]


A study done at UC Irvine looked at the impact that maternal stress has on foetal development and overall foetal health. The researchers determined that a mother's stress and adverse pregnancy outcomes (APOs) related to the length of gestation and growth of the foetus along with impacts on the endocrine and immune systems of the foetus. [ 18 ]


Early life influences, both prenatal and postnatal, have important effects on children later in life. It was determined that breastfed infants have significantly lower risks of obesity later in life than infants that were formula-fed. [ 19 ]


Nutrition and growth during the early years of life can be related to the growth of diseases in humans later in their lives. For example, a study done in Jamaica showed that the blood pressure of children was associated with the mother's hemoglobin levels and body fat during pregnancy. [ 20 ]  Another example of this is shown in an article from the New England Journal of Medicine which took place during the Dutch famine. This study concluded that those who were in utero at the time of the famine were at a greater risk of obesity, hypertension, and heart disease than those who were born before or after the famine. [ 21 ]


A maternal high-fat diet was used to help investigate how saturated fats cause unrestrained gestational weight gain and maternal obesity of offspring. [ 22 ]  When there is high-fat feeding during pregnancy, there are effects on the maternal metabolism and body composition. Some of the effects are insulin resistance, increased circulating lipids, increased adiposity, and hyperinsulinemia (high insulin in blood). When the fat intake was increased it started to adjust consumption of other macronutrients in the diet, which reduced the carbohydrate and protein consumption to match the increase of fatty acid. On another note, people who have obesity and a diet that is high in fat can develop a condition called dyslipidemia. It can lead to fibrosis of the liver, which is the cause for about 45% of deaths in the world.  [ 23 ]


During the Dutch famine doctors found that under-nutrition during the gestation period related to reduced glucose tolerance and raised insulin concentrations between the ages 50 and 58. There were 120 minute glucose and insulin concentrations that were known to be higher in people that were exposed to the famine at any stage during foetal development than those who were not exposed. [ 13 ]  The effect could be explained due to the lower birth rates of babies that were born during the famine and the low weight gain of their mothers. People that were born before the famine had a larger increase in glucose and insulin concentration. That was due to them experiencing starvation during the early stage of their life which might have impacted their postnatal growth pattern.


Autism spectrum disorder (ASD) is a condition that typically manifests during the first three years of a person's life. It is a developmental disorder that impairs the brain's ability to develop the typical social and communication skills that are necessary for everyday life. People with ASD may experience difficulties with social interactions, verbal and nonverbal communication, and repetitive or restricted behaviors. The degree to which ASD affects an individual can vary widely, with some people experiencing mild symptoms while others may face more significant challenges.


The precise cause of ASD is still unknown, but it is believed that a combination of factors may contribute to its development. Research suggests that genetics may play a role, as ASD can run in families. In addition, certain medications taken during pregnancy may increase the risk of a child developing ASD. While some theories have been proposed, they have yet to be proven. For instance, some scientists believe that damage to a specific region of the brain, the amygdala, may be linked to ASD, while others are examining the possibility that a viral infection may trigger the disorder.


There has been some controversy regarding whether vaccines can cause ASD, but numerous studies have shown that there is no evidence of a link between vaccines and ASD. Major medical and government organizations have also confirmed this finding. The increase in ASD diagnoses in recent years may be attributed to better awareness and more comprehensive definitions of the disorder. Treatment for ASD involves a highly structured schedule of constructive activities that build on the child's interests and various techniques. It is important to avoid unproven treatments and seek advice from ASD specialists. [ 24 ]


Large-scale famines offer insights into the effects prenatal malnutrition has on developing foetuses. A team led by Mingqing Xu investigated this possible connection between prenatal malnutrition and  schizophrenia  by analyzing medical records of people born between 1960 and 1961 during the  Great Chinese Famine . This study found that there was a two-fold risk for someone who was born in a rural area during this period of famine to later develop schizophrenia than someone who was born either before or after. [ 25 ]  However, being born in an urban area during this time was not associated with an increased risk of schizophrenia. [ 25 ] [ 26 ]  This is probably due to factors that exacerbated the impact of the famine in rural areas such as grain procurement and lack of large-scale grain storage. [ 26 ]


Similar results have been found in studying the effects of the  Dutch famine  from 1944 to 1945. A study by Hoek compared the levels of schizophrenia for those born between August 15 and December 31, 1945, to those born after the famine had ended. This study found that those born during the time period were between two and two and a half times more likely to have schizophrenia than those born after the famine had ended. [ 27 ]


The exact mechanism which connects undernutrition to an increased chance of schizophrenia later in life is not fully understood. Famines may lead to an enhanced risk of schizophrenia because they deprive the developing foetus of key micronutrients. Some of the leading candidates are folate, essential fatty acids, retinoids, vitamin D, and iron. [ 28 ]  Of these micronutrients, folate, iron and vitamin D seem to be the most promising. [ 28 ] [ 29 ]   Folate jumps out as a key candidate as the occurrence of  neural tube defects  raised alongside schizophrenia during the Dutch famine. [ 28 ]   A lack of folate could cause DNA methylation which may affect the expression of genes crucial to neurodevelopment, and it could impede the conversion of homocysteine to methionine, causing homocysteine to accumulate and cause problems in the developing brain. [ 30 ]  Iron has been put forward as a leading candidate as low levels of hemoglobin, a molecule created by iron, during development was highly associated with developing schizophrenia later on in life. [ 31 ]  This connection may be due to iron having a key role in the creation and function of dopamine transmission. [ 32 ]


Another, less accepted, theory that explains the connection between famine and schizophrenia is protein-calorie malnutrition. Protein-calorie malnutrition has been associated with increased dopamine and serotonin release and malfunctions in the hippocampus such as reduced dendritic branching and a lower cell count, which are also found in people with schizophrenia. [ 28 ]


The effect stress has on expecting women may not only affect them, but their child as well. Studies have shown a link between child mental health and behavioral problems to maternal stress during pregnancy. Stress in the body leads to an increase in the cortisol levels. Maternal stress, therefore, exposes the foetus to high cortisol levels. These levels have been linked to neurological and behavioral regulation issues in the child later in life. [ 33 ]  For example, cognitive performance at 5 years of age was impaired in kids who mother had experienced stress from a catastrophic ice storm. Investigators found that schizophrenia was frequent in offspring that mothers endured the ice storm during pregnancy. Prenatal stress has been shown to increase reactivity in infants. [ 33 ]  Infants that are from pregnancy with a lot of stress are harder to soothe rather than those that mothers didn't experience any stress during pregnancy. Human and animal studies did show that infants from stressed pregnancies have a poor immune function and are more likely to contract childhood illnesses as well as mental disorders. [ 33 ]


There was a study published in the Journal of Pediatrics of August 19, 2020, which looked at how  maternal recognition and stress can affect infant health after or during pregnancy. [ 34 ]  The study consisted of women from low income areas, and those that were overweight which were at the age of 28 years old.  The women had to rate how they felt during challenges they had to endure. The infants born during this study had to get their medical records looked at from the first year of their lives. There was a 38 percent increase for having infectious illnesses. There was a 73 percent increase from not having non-infectious illnesses with an increase of 53 percent in other illnesses amid the infants. [ 34 ]  It seems that the effects were more likely later in the pregnancy, but after the study was completed they realized that stress and depression in mothers is not associated with increase in illnesses amongst the infants. [ 34 ]


Differentially methylated regions  (DMRs) are  genomic  regions with different  DNA methylation  status across different biological samples and regarded as possible functional regions involved in  gene transcriptional  regulation. The biological samples can be different cells/tissues within the same individual, the same cell/tissue at different times, cells/tissues from different individuals, even different alleles in the same cell. [ 1 ]


DNA is mostly methylated at a  CpG site , which is a  cytosine  followed by a  guanine . The “p” refers to the phosphate linker between them. DMR usually involves adjacent sites or a group of sites close together that have different methylation patterns between samples.  CpG islands  appear to be unmethylated in most of the normal tissues, however, are highly methylated in cancer tissues. [ 1 ]


There are several different types of DMRs. These include tissue-specific DMR (tDMR), cancer-specific DMR (cDMR), development stages (dDMRs), reprogramming-specific DMR (rDMR), allele-specific DMR (AMR), and aging-specific DMR (aDMR). [ 1 ]  DNA methylation is associated with  cell differentiation  and  proliferation . [ 2 ]


DNA condensation  refers to the process of compacting  DNA  molecules  in vitro  or  in vivo . [ 1 ]  Mechanistic details of DNA packing are essential for its functioning in the process of  gene regulation  in living systems. Condensed DNA often has surprising properties, which one would not predict from classical concepts of dilute solutions. Therefore, DNA condensation  in vitro  serves as a  model system  for many processes of  physics ,  biochemistry  and  biology . [ 2 ]  In addition, DNA condensation has many potential applications in  medicine  and  biotechnology . [ 1 ]


DNA diameter is about 2 nm, while the length of a stretched single molecule may be up to several dozens of centimetres depending on the organism. Many features of the DNA double helix contribute to its large stiffness, including the mechanical properties of the sugar-phosphate backbone, electrostatic repulsion between  phosphates  (DNA bears on average one elementary negative charge per each 0.17 nm of the  double helix ), stacking interactions between the bases of each individual strand, and strand-strand interactions. DNA is one of the stiffest natural polymers, yet it is also one of the longest molecules. The persistence length of double-stranded DNA (dsDNA) is a measure of its stiffness or flexibility, which depends on the DNA sequence and the surrounding environment, including factors like salt concentration, pH, and temperature. Under physiological conditions (e.g., near-neutral pH and physiological salt concentrations), the persistence length of dsDNA is generally around 50 nm, which corresponds to approximately 150 base pairs. [ 1 ]  This means that at large distances DNA can be considered as a flexible rope, and on a short scale as a stiff rod. Like a garden hose, unpacked DNA would randomly occupy a much larger volume than when it is orderly packed. Mathematically, for a non-interacting flexible chain randomly diffusing in 3D, the end-to-end distance would scale as a square root of the polymer length. For real polymers such as DNA, this gives only a very rough estimate; what is important, is that the space available for the DNA  in vivo  is much smaller than the space that it would occupy in the case of a free diffusion in the solution. To cope with volume constraints, DNA can pack itself in the appropriate solution conditions with the help of ions and other molecules. Usually, DNA condensation is defined as "the collapse of extended DNA chains into compact, orderly particles containing only one or a few molecules". [ 3 ]  This definition applies to many situations in vitro and is also close to the definition of DNA condensation in bacteria as "adoption of relatively concentrated, compact state occupying a fraction of the volume available". [ 4 ]  In  eukaryotes , the DNA size and the number of other participating players are much larger, and a DNA molecule forms millions of ordered  nucleoprotein  particles, the  nucleosomes , which is just the first of many levels of DNA packing. [ 1 ]


In  viruses  and  bacteriophages , the DNA or RNA is surrounded by a protein  capsid , sometimes further enveloped by a lipid  membrane . Double-stranded DNA is stored inside the capsid in the form of a spool, which can have different types of coiling leading to different types of  liquid-crystalline  packing. This packing can change from  hexagonal  to  cholesteric  to  isotropic  at different stages of the phage functioning. Although the double helices are always locally aligned, the DNA inside viruses does not represent real  liquid crystals , because it lacks fluidity. On the other hand,  DNA condensed  in vitro , e.g., with the help of polyamines also present in viruses, is both locally ordered and fluid. [ 1 ]


Bacterial DNA is packed with the help of  polyamines  and proteins called  nucleoid-associated proteins . Protein-associated DNA occupies about 1/4 of the intracellular volume forming a concentrated viscous phase with liquid crystalline properties, called the nucleoid. Other research also indicated that the genome of bacteria occupies approximately 10-15% of the bacteria's volume. [ 5 ]  Similar DNA packaging exists also in  chloroplasts  and  mitochondria . Bacterial DNA is sometimes referred to as the  bacterial chromosome . Bacterial nucleoid evolutionary represents an intermediate engineering solution between the protein-free DNA packing in viruses and protein-determined packing in eukaryotes. [ 1 ]


Sister chromosomes  in the bacterium  Escherichia coli  are induced by stressful conditions to condense and undergo pairing. [ 6 ]   Stress-induced condensation occurs by a non-random, zipper-like convergence of sister chromosomes.  This convergence appears to depend on the ability of identical double-stranded  DNA  molecules to specifically identify each other, a process that culminates in the proximity of homologous sites along the paired chromosomes.  Diverse stress conditions appear to prime bacteria to effectively cope with severe  DNA damages  such as double-strand breaks.  The apposition of homologous sites associated with stress-induced chromosome condensation helps explain how repair of double-strand breaks and other damages occurs. [ 6 ]


Eukaryotic DNA with a typical length of dozens of centimeters should be orderly packed to be readily accessible inside the micrometer-size nucleus. In most eukaryotes, DNA is arranged in the cell nucleus with the help of histones. In this case, the basic level of DNA compaction is the nucleosome, where the double helix is wrapped around the histone octamer containing two copies of each  histone   H2A ,  H2B ,  H3  and  H4 . Linker  histone H1  binds the DNA between nucleosomes and facilitates packaging of the 10 nm "beads on the string" nucleosomal chain into a more condensed 30 nm fiber. Most of the time, between cell divisions,  chromatin  is optimized to allow easy access of transcription factors to active  genes , which are characterized by a less compact structure called  euchromatin , and to alleviate protein access in more tightly packed regions called  heterochromatin . During the cell division, chromatin compaction increases even more to form  chromosomes , which can cope with large mechanical forces dragging them into each of the two daughter cells. [ 1 ]  Many aspects of transcription are controlled by chemical modification on the histone proteins, known as the  histone code .


Chromosome scaffold  has important role to hold the chromatin into compact chromosome. Chromosome scaffold is made of proteins including  condensin , topoisomerase IIα and kinesin family member 4 (KIF4) [ 7 ]


Dinoflagellates  are very divergent eukaryotes in terms of how they package their DNA. Their chromosomes are packed in a liquid-crystalline state. [ 8 ]  They have lost many of the conserved histone genes, using mostly  dinoflagellate viral nucleoproteins  (DVNPs) or bacteria-derived  dinoflagellate histone-like proteins  (HLPs) for packaging instead. It is unknown how they control access to genes; those do retain histone have a special  histone code . [ 9 ] [ 10 ]


Depending on the organism, an archaeon may use a bacteria-like HU system or a eukaryote-like nucleosome system for packaging. [ 11 ]


DNA condensation can be induced  in vitro  either by applying external force to bring the double helices together, or by inducing attractive interactions between the DNA segments. The former can be achieved e.g. with the help of the osmotic pressure exerted by crowding neutral polymers in the presence of monovalent salts. In this case, the forces pushing the double helices together are coming from entropic random collisions with the crowding polymers surrounding DNA condensates, and salt is required to neutralize DNA charges and decrease DNA-DNA repulsion. The second possibility can be realized by inducing attractive interactions between the DNA segments by multivalent cationic charged ligands (multivalent  metal ions , inorganic  cations ,  polyamines ,  protamines ,  peptides ,  lipids ,  liposomes  and  proteins ). [ 1 ]


Condensation of long double-helical DNAs is a sharp  phase transition , which takes place within a narrow interval of condensing agent concentrations.[ref] Since the double helices come very closely to each other in the condensed phase, this leads to the restructuring of water molecules, which gives rise to the so-called  hydration forces .[ref] To understand attraction between negatively charged DNA molecules, one also must account for  correlations  between counterions in the solution.[ref] DNA condensation by proteins can exhibit hysteresis, which can be explained using a modified  Ising model . [ 12 ]


Nowadays descriptions of gene regulation are based on the approximations of  equilibrium binding  in  dilute solutions , although it is clear that these assumptions are in fact violated in  chromatin . The dilute-solution approximation is violated for two reasons. First, the chromatin content is far from being dilute, and second, the numbers of the participating molecules are sometimes so small, that it does not make sense to talk about the bulk concentrations. Further differences from dilute solutions arise due to the different binding affinities of proteins to condensed and uncondensed DNA. Thus in condensed DNA both the reaction rates can be changed and their dependence on the concentrations of reactants may become nonlinear. [ 1 ]


For  molecular biology  in mammals, DNA  demethylation  causes replacement of  5-methylcytosine  (5mC) in a DNA sequence by  cytosine  (C) (see figure of 5mC and C).  DNA demethylation  can occur by an active process at the site of a 5mC in a DNA sequence or, in replicating cells, by preventing addition of methyl groups to DNA so that the replicated DNA will largely have cytosine in the DNA sequence (5mC will be diluted out).


Methylated  cytosine  is frequently present in the linear  DNA sequence  where a cytosine is followed by a  guanine  in a  5' → 3' direction  (a  CpG site ). In mammals,  DNA methyltransferases  (which add  methyl groups  to DNA bases) exhibit a strong sequence preference for cytosines at  CpG sites . [ 1 ]  There appear to be more than 20 million CpG dinucleotides in the human genome (see  genomic distribution ). In mammals, on average, 70% to 80% of CpG cytosines are methylated, [ 2 ]  though the level of methylation varies with different tissues.  Methylated cytosines  often occur in groups or  CpG islands  within the  promoter regions of genes , where such methylation may reduce or silence gene expression (see  gene expression ). Methylated cytosines in the gene body, however, are positively correlated with expression. [ 3 ]


Almost 100% DNA demethylation occurs by a combination of passive dilution and active enzymatic removal during the  reprogramming  that occurs in  early embryogenesis  and in  gametogenesis . Another large demethylation, of about 3% of all genes, can occur by active demethylation in neurons during formation of a strong memory. [ 4 ]  After surgery, demethylations are found in peripheral blood mononuclear cells at sites annotated to immune system genes. [ 5 ]  Demethylations also occur during the formation of cancers. [ 6 ]  During global DNA hypomethylation of tumor genomes, there is a minor to moderate reduction of the number of methylated cytosines (5mC) amounting to a loss of about 5% to 20% on average of the 5mC bases. [ 7 ]


The mouse  sperm   genome  is 80–90%  methylated  at its  CpG sites  in DNA, amounting to about 20 million methylated sites. [ citation needed ]  After  fertilization , the paternal chromosome is almost completely demethylated in six hours by an active process, before DNA replication (blue line in Figure).


Demethylation of the maternal genome occurs by a different process. In the mature  oocyte , about 40% of its CpG sites in DNA are methylated. While somatic cells of mammals have three main DNA methyltransferases (which add methyl groups to cytosines at CpG sites),  DNMT1 ,  DNMT3A , and  DNMT3B , in the pre-implantation embryo up to the blastocyst stage (see Figure), the only methyltransferase present is an isoform of DNMT1 designated DNMT1o. [ 8 ]  DNMT1o has an alternative oocyte-specific promoter and first exon (exon 1o) located 5' of the somatic and spermatocyte promoters. As reviewed by Howell et al., [ 8 ]  DNMT1o is sequestered in the cytoplasm of mature oocytes and in 2-cell and 4-cell embryos, but at the 8-cell stage is only present in the nucleus. At the 16 cell stage (the  morula ) DNMT1o is again found only in the cytoplasm. It appears that demethylation of the maternal chromosomes largely takes place by blockage of the methylating enzyme DNMT1o from entering the nucleus except briefly at the 8 cell stage. The maternal-origin DNA thus undergoes passive demethylation by dilution of the methylated maternal DNA during replication (red line in Figure). The  morula  (at the 16 cell stage), has only a small amount of  DNA methylation  (black line in Figure).


DNMT3b begins to be expressed in the blastocyst. [ 9 ]  Methylation begins to increase at 3.5 days after fertilization in the  blastocyst , and a large wave of methylation then occurs on days 4.5 to 5.5 in the  epiblast , going from 12% to 62% methylation, and reaching maximum level after implantation in the uterus. [ 10 ]  By day seven after fertilization, the newly formed  primordial germ cells  (PGC) in the implanted  embryo  segregate from the remaining  somatic cells . At this point the PGCs have about the same level of methylation as the somatic cells.


The newly formed primordial germ cells (PGC) in the implanted embryo devolve from the somatic cells. At this point the PGCs have high levels of methylation. These cells migrate from the epiblast toward the  gonadal ridge . As reviewed by Messerschmidt et al., [ 11 ]  the majority of PGCs are arrested in the G2 phase of the cell cycle, while they migrate toward the hindgut during embryo days 7.5 to 8.5. Then demethylation of the PGCs takes place in two waves. [ 11 ]  At day 9.5 the primordial germ cells begin to rapidly replicate going from about 200 PGCs at embryo day 9.5 to about 10,000 PGCs at day 12.5. [ 12 ]  During days 9.5 to 12.5 DNMT3a and DNMT3b are repressed and DNMT1 is present in the nucleus at a high level. But DNMT1 is unable to methylate cytosines during days 9.5 to 12.5 because the  UHRF1  gene (also known as  NP95 ) is repressed and UHRF1 is an essential protein needed to recruit DNMT1 to replication foci where maintenance DNA methylation takes place. [ 12 ]  This is a passive, dilution form of demethylation.


In addition, from embryo day 9.5 to 13.5 there is an active form of demethylation. As indicated below in "Molecular stages of active reprogramming," two enzymes are central to active demethylation. These are a ten-eleven translocation  methylcytosine dioxygenase (TET)  and  thymine-DNA glycosylase  (TDG). One particular TET enzyme, TET1, and TDG are present at high levels from embryo day 9.5 to 13.5, [ 12 ]  and are employed in active demethylation during gametogenesis. [ 11 ]  PGC genomes display the lowest levels of DNA methylation of any cells in the entire life cycle of the mouse at embryonic day 13.5. [ 13 ]


Learning and memory have levels of permanence, differing from other mental processes such as thought, language, and consciousness, which are temporary in nature. Learning and memory can be either accumulated slowly (multiplication tables) or rapidly (touching a hot stove), but once attained, can be recalled into conscious use for a long time. Rats subjected to one instance of  contextual fear conditioning  create an especially strong long-term memory. At 24 hours after training, 9.17% of the genes in the genomes of rat hippocampus neurons were found to be  differentially methylated . This included more than 2,000 differentially methylated genes at 24 hours after training, with over 500 genes being demethylated. [ 4 ]  Similar results to that in the rat hippocampus were also obtained in mice with contextual fear conditioning. [ 14 ]


The hippocampus region of the brain is where contextual fear memories are first stored (see figure of the brain, this section), but this storage is transient and does not remain in the hippocampus. In rats contextual fear conditioning is abolished when the hippocampus is subjected to hippocampectomy just one day after conditioning, but rats retain a considerable amount of contextual fear when hippocampectomy is delayed by four weeks. [ 15 ]  In mice, examined at 4 weeks after conditioning, the hippocampus methylations and demethylations were reversed (the hippocampus is needed to form memories but memories are not stored there) while substantial differential CpG methylation and demethylation occurred in  cortical  neurons during memory maintenance. There were 1,223 differentially methylated genes in the anterior cingulate cortex of mice four weeks after contextual fear conditioning. Thus, while there were many methylations in the hippocampus shortly after memory was formed, all these hippocampus methylations were demethylated as soon as four weeks later.


The human genome contains about 28 million CpG sites, and roughly 60% of the CpG sites are methylated at the 5 position of the cytosine. [ 16 ]  During formation of a cancer there is an average reduction of the number of methylated cytosines of about 5% to 20%, [ 7 ]  or about 840,00 to 3.4 million demethylations of CpG sites.


DNMT1  methylates CpGs on hemi-methylated DNA during DNA replication. Thus, when a DNA strand has a methylated CpG, and the newly replicated strand during semi-conservative replication lacks a methyl group on the complementary CpG, DNMT1 is normally recruited to the hemimethylated site and adds a methyl group to cytosine in the newly synthesized CpG. However, recruitment of DNMT1 to hemimethylated CpG sites during DNA replication depends on the  UHRF1  protein. If UHRF1 does not bind to a hemimethylated CpG site, then DNMT1 is not recruited and cannot methylate the newly synthesized CpG site. The arginine methyltransferase  PRMT6  regulates DNA methylation by methylating the arginine at position 2 of  histone 3  (H3R2me2a). [ 17 ]  (See  Protein methylation#Arginine .) In the presence of H3R2me2a UHRF1 can not bind to a hemimethylated CpG site, and then DNMT1 is not recruited to the site, and the site remains hemimethylated. Upon further rounds of replication the methylated CpG is passively diluted out. PRMT6 is frequently overexpressed in many types of cancer cells. [ 18 ]  The overexpression of PRMT6 may be a source of DNA demethylation in cancer.


Three molecular stages are required for actively, enzymatically reprogramming the  DNA methylome . Stage 1: Recruitment. The enzymes needed for reprogramming are recruited to genome sites that require demethylation or methylation. Stage 2: Implementation. The initial enzymatic reactions take place. In the case of methylation, this is a short step that results in the methylation of cytosine to 5-methylcytosine. Stage 3: Base excision DNA repair. The intermediate products of demethylation are catalysed by specific enzymes of the base excision DNA repair pathway that finally restore cystosine in the DNA sequence.


Demethylation of 5-methylcytosine to generate  5-hydroxymethylcytosine  (5hmC) very often initially involves oxidation of 5mC (see Figure in this section) by ten-eleven translocation methylcytosine dioxygenases ( TET enzymes ). [ 20 ]  The molecular steps of this initial demethylation are shown in detail in  TET enzymes . In successive steps (see Figure)  TET enzymes  further hydroxylate 5hmC to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC).  Thymine-DNA glycosylase  (TDG) recognizes the intermediate bases 5fC and 5caC and excises the glycosidic bond resulting in an  apyrimidinic site  (AP site). This is followed by base excision repair (stage 3). In an alternative oxidative deamination pathway, 5hmC can be oxidatively deaminated by  APOBEC  (AID/APOBEC) deaminases to form 5-hydroxymethyluracil (5hmU). Also, 5mC can be converted to  thymine  (Thy). 5hmU can be cleaved by TDG,  MBD4 ,  NEIL1  or  SMUG1 . AP sites and T:G mismatches are then repaired by base excision repair (BER) enzymes to yield cytosine (Cyt). The TET family of dioxygenases are employed in the most frequent type of demethylation reactions. [ 20 ]


TET dioxygenase isoforms  include at least two isoforms of TET1, one of  TET2  and three isoforms of TET3. [ 21 ] [ 22 ]  The full-length canonical TET1 isoform appears virtually restricted to early embryos, embryonic stem cells and primordial germ cells (PGCs). The dominant TET1 isoform in most somatic tissues, at least in the mouse, arises from alternative promoter usage which gives rise to a short transcript and a truncated protein designated TET1s. The isoforms of TET3 are the full length form TET3FL, a short form splice variant TET3s, and a form that occurs in oocytes and neurons designated TET3o. TET3o is created by alternative promoter use and contains an additional first N-terminal exon coding for 11 amino acids. TET3o only occurs in oocytes and neurons and is not expressed in embryonic stem cells or in any other cell type or adult mouse tissue tested. Whereas TET1 expression can barely be detected in oocytes and zygotes, and TET2 is only moderately expressed, the TET3 variant TET3o shows extremely high levels of expression in oocytes and zygotes, but is nearly absent at the 2-cell stage. It is possible that TET3o, high in neurons, oocytes and zygotes at the one cell stage, is the major TET enzyme utilized when very large scale rapid demethylations occur in these cells.


The TET enzymes do not specifically bind to  5-methylcytosine  except when recruited. Without recruitment or targeting, TET1 predominantly binds to high CG promoters and CpG islands (CGIs) genome-wide by its CXXC domain that can recognize  un-methylated  CGIs. [ 23 ]  TET2 does not have an affinity for 5-methylcytosine in DNA. [ 24 ]  The CXXC domain of the full-length TET3, which is the predominant form expressed in neurons, binds most strongly to CpGs where the C was converted to 5-carboxycytosine (5caC). However, it also binds to  un-methylated CpGs . [ 22 ]


For a  TET enzyme  to initiate demethylation it must first be recruited to a methylated  CpG site  in DNA. Two of the proteins shown to recruit a TET enzyme to a methylated cytosine in DNA are  OGG1  (see figure Initiation of DNA demethylation at a CpG site) [ 25 ]  and  EGR1 . [ 26 ]


Oxoguanine glycosylase  (OGG1) catalyses the first step in base excision repair of the oxidatively damaged base  8-OHdG . OGG1 finds 8-OHdG by sliding along the linear DNA at 1,000 base pairs of DNA in 0.1 seconds. [ 27 ]  OGG1 very rapidly finds 8-OHdG. OGG1 proteins bind to oxidatively damaged DNA with a half maximum time of about 6 seconds. [ 28 ]  When OGG1 finds 8-OHdG it changes conformation and complexes with 8-OHdG in its binding pocket. [ 29 ]  OGG1 does not immediately act to remove the 8-OHdG. Half maximum removal of 8-OHdG takes about 30 minutes in HeLa cells  in vitro , [ 30 ]  or about 11 minutes in the livers of irradiated mice. [ 31 ]  DNA oxidation by reactive oxygen species preferentially occurs at a guanine in a methylated CpG site, because of a lowered ionization potential of guanine bases adjacent to 5-methylcytosine. [ 32 ]  TET1 binds (is recruited to) the OGG1 bound to 8-OHdG (see figure). [ 25 ]  This likely allows TET1 to demethylate an adjacent methylated cytosine. When human mammary epithelial cells (MCF-10A) were treated with H 2 O 2 , 8-OHdG increased in DNA by 3.5-fold and this caused about 80% demethylation of the 5-methylcytosines in the MCF-10A genome. [ 25 ]


The gene  early growth response protein 1  ( EGR1 ) is an  immediate early gene  (IEG). EGR1 can rapidly be induced by neuronal activity. [ 33 ]  The defining characteristic of IEGs is the rapid and transient up-regulation—within minutes—of their mRNA levels independent of protein synthesis. [ 34 ]  In adulthood, EGR1 is expressed widely throughout the brain, maintaining baseline expression levels in several key areas of the brain including the medial prefrontal cortex, striatum, hippocampus and amygdala. [ 34 ]  This expression is linked to control of cognition, emotional response, social behavior and sensitivity to reward. [ 34 ]  EGR1 binds to DNA at sites with the motifs 5′-GCGTGGGCG-3′ and 5'-GCGGGGGCGG-3′ and these motifs occur primarily in promoter regions of genes. [ 33 ]  The short isoform TET1s is expressed in the brain. EGR1 and TET1s form a complex mediated by the C-terminal regions of both proteins, independently of association with DNA. [ 33 ]  EGR1 recruits TET1s to genomic regions flanking EGR1 binding sites. [ 33 ]  In the presence of EGR1, TET1s is capable of locus-specific demethylation and activation of the expression of downstream genes regulated by EGR1. [ 33 ]


As indicated in the Figure above, captioned "Demethylation of 5-methylcytosine," the first step in active demethylation is a TET oxidation of 5-methylcytosine (5mC) to  5-hydroxymethylcytosine  (5hmC). The demethylation process, in some tissues and at some genome locations, may stop at that point. As reviewed by Uribe-Lewis et al., [ 35 ]  in addition to being an intermediate in active DNA demethylation, 5hmC is often a stable DNA modification. Within the genome, 5hmC is located at transcriptionally active genes, regulatory elements and chromatin associated complexes. In particular, 5hmC is dynamically changed and positively correlated with active gene transcription during  cell lineage  specification, and high levels of 5hmC are found in  embryonic stem cells  and in the  central nervous system . [ 36 ]  In humans, defective 5-hydroxymethylating activity is associated with a phenotype of lymphoproliferation, immunodeficiency and autoimmunity. [ 37 ]


The third stage of DNA demethylation is removal of the intermediate products of demethylation generated by a TET enzyme by  base excision repair . As indicated above in  Stage 2 , after 5mC is first oxidized by a TET to form 5hmC, further oxidation of 5hmC by TET yields 5fC and oxidation of 5fC by TET yields 5caC. Both 5fC and 5caC are recognized by a  DNA glycosylase ,  TDG , a  base excision repair  enzyme, as an abnormal base. As shown in the Figure in this section, TDG removes the abnormal base (e.g. 5fC) while leaving the sugar-phosphate backbone intact, creating an apurinic/apyrimidinic site, commonly referred to as an  AP site . In this Figure, the 8-OHdG is left in the DNA, since it may have been present when  OGG1  attracted TET1 to the CpG site with a methylated cytosine. After an AP site is formed,  AP endonuclease  creates a nick in the  phosphodiester  backbone of the  AP site  that was formed when the TDG  DNA glycosylase  removed the 5fC or 5caC. The human AP endonuclease incises DNA 5′ to the AP site by a hydrolytic mechanism, leaving a 3′-hydroxyl and a 5′-deoxyribose phosphate (5' dRP) residue. [ 38 ]  This is followed by either short patch or long patch repair. In short patch repair, 5′ dRP lyase trims the 5′ dRP end to form a phosphorylated 5′ end. This is followed by DNA  polymerase β  (pol β) adding a single cytosine to pair with the pre-existing guanine in the complementary strand and then  DNA ligase  to seal the cut strand. In long patch repair, DNA synthesis is thought to be mediated by  polymerase δ  and  polymerase ε  performing displacement synthesis to form a flap. Pol β can also perform long-patch displacement synthesis. Long-patch synthesis typically inserts 2–10 new nucleotides. Then  flap endonuclease  removes the flap, and this is followed by  DNA ligase  to seal the strand. At this point there has been a complete replacement of the 5-methylcytosine by cytosine (demethylation) in the DNA sequence.


Physical exercise has well established beneficial effects on learning and memory (see  Neurobiological effects of physical exercise ).  BDNF  is a particularly important regulator of learning and memory. [ 39 ]  As reviewed by Fernandes et al., [ 40 ]  in rats, exercise enhances the  hippocampus  expression of the gene  Bdnf , which has an essential role in memory formation.  Enhanced expression  of  Bdnf  occurs through demethylation of its  CpG island promoter  at  exon IV [ 40 ]  and this demethylation depends on steps illustrated in the two figures. [ 19 ]


In a panel of healthy adults, negative associations were found between total DNA methylation and exposure to traffic related air pollution. DNA methylation levels were associated both with recent and chronic exposure to Black Carbon as well as benzene. [ 41 ]


After injury,  neurons  in the adult  peripheral nervous system  can switch from a dormant state with little  axonal  growth to robust  axon regeneration . DNA demethylation in mature mammalian neurons removes barriers to axonal regeneration. [ 42 ]  This demethylation, in regenerating mouse peripheral neurons, depends upon  TET3  to generate  5-hydroxymethylcytosine  (5hmC) in DNA. [ 42 ] [ 43 ]  5hmC was altered in a large set of regeneration-associated genes (RAGs), including well-known RAGs such as  Atf3 ,  Bdnf , and  Smad1 , that regulate the axon growth potential of neurons. [ 43 ]


DNA methylation  is a biological process by which  methyl groups  are added to the  DNA  molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene  promoter , DNA methylation typically acts to repress gene  transcription . In mammals, DNA methylation is essential for normal development and is associated with a number of key processes including  genomic imprinting ,  X-chromosome inactivation , repression of  transposable elements ,  aging , and  carcinogenesis .


As of 2016, two nucleobases have been found on which natural, enzymatic DNA methylation takes place:  adenine  and  cytosine . The modified bases are N 6 -methyladenine, [ 1 ]   5-methylcytosine [ 2 ]  and N 4 -methylcytosine. [ 3 ]


Cytosine methylation  is widespread in both  eukaryotes  and  prokaryotes , even though the rate of cytosine DNA methylation can differ greatly between species: 14% of cytosines are methylated in  Arabidopsis thaliana , 4% to 8% in  Physarum , [ 4 ]  7.6% in  Mus musculus , 2.3% in  Escherichia coli , 0.03% in  Drosophila ; methylation is essentially undetectable in  Dictyostelium ; [ 5 ] [ 6 ]  and virtually absent (0.0002 to 0.0003%) from  Caenorhabditis [ 7 ]  or fungi such as  Saccharomyces cerevisiae  and  S. pombe  (but not  N. crassa ). [ 8 ] [ 9 ] : 3699    Adenine methylation  has been observed in bacterial, plant, and recently in mammalian DNA, [ 10 ] [ 11 ]  but has received considerably less attention.


Methylation of cytosine to form  5-methylcytosine  occurs at the same 5 position on the  pyrimidine  ring where the DNA base  thymine 's methyl group is located; the same position distinguishes thymine from the analogous RNA base  uracil , which has no methyl group. Spontaneous  deamination  of  5-methylcytosine  converts it to thymine. This results in a T:G mismatch. Repair mechanisms then correct it back to the original C:G pair; alternatively, they may substitute A for G, turning the original C:G pair into a T:A pair, effectively changing a base and introducing a mutation. This misincorporated base will not be corrected during DNA replication as thymine is a DNA base. If the mismatch is not repaired and the cell enters the cell cycle the strand carrying the T will be complemented by an A in one of the daughter cells, such that the mutation becomes permanent. The near-universal use of  thymine  exclusively in DNA and  uracil  exclusively in RNA may have evolved as an error-control mechanism, to facilitate the removal of uracils generated by the spontaneous deamination of cytosine. [ 12 ]   DNA methylation as well as many of its contemporary DNA methyltransferases have been thought to evolve from early world primitive RNA methylation activity and is supported by several lines of evidence. [ 13 ]


In plants and other organisms, DNA methylation is found in three different sequence contexts: CG (or  CpG ), CHG or CHH (where H correspond to A, T or C). In mammals however, DNA methylation is almost exclusively found in CpG dinucleotides, with the cytosines on both strands being usually methylated. Non-CpG methylation can however be observed in embryonic  stem cells , [ 14 ] [ 15 ] [ 16 ]  and has also been indicated in  neural development . [ 17 ]  Furthermore, non-CpG methylation has also been observed in  hematopoietic  progenitor cells, and it occurred mainly in a CpApC sequence context. [ 18 ]


The DNA methylation landscape of vertebrates is very particular compared to other organisms. In mammals, around 75% of CpG dinucleotides are methylated in  somatic cells , [ 19 ]  and DNA methylation appears as a default state that has to be specifically excluded from defined locations. [ 16 ] [ 20 ]  By contrast, the genome of most plants, invertebrates, fungi, or protists show "mosaic" methylation patterns, where only specific genomic elements are targeted, and they are characterized by the alternation of methylated and unmethylated domains. [ 21 ] [ 22 ]


High CpG methylation in mammalian genomes has an evolutionary cost because it increases the frequency of spontaneous mutations. Loss of amino-groups occurs with a high frequency for cytosines, with different consequences depending on their methylation. Methylated C residues spontaneously deaminate to form T residues over time; hence CpG dinucleotides steadily deaminate to TpG dinucleotides, which is evidenced by the under-representation of CpG dinucleotides in the human genome (they occur at only 21% of the expected frequency). [ 23 ]  (On the other hand, spontaneous deamination of unmethylated C residues gives rise to U residues, a change that is quickly recognized and repaired by the cell.)


In mammals, the only exception for this global CpG depletion resides in a specific category of GC- and CpG-rich sequences termed CpG islands that are generally unmethylated and therefore retained the expected CpG content. [ 24 ]  CpG islands are usually defined as regions with: 1) a length greater than 200bp, 2) a G+C content greater than 50%, 3) a ratio of observed to expected CpG greater than 0.6, although other definitions are sometimes used. [ 25 ]  Excluding repeated sequences, there are around 25,000 CpG islands in the human genome, 75% of which being less than 850bp long. [ 23 ]  They are major regulatory units and around 50% of CpG islands are located in gene promoter regions, while another 25% lie in gene bodies, often serving as alternative promoters. Reciprocally, around 60-70% of human genes have a CpG island in their promoter region. [ 26 ] [ 27 ]  The majority of CpG islands are constitutively unmethylated and enriched for permissive  chromatin modification  such as  H3K4  methylation. In somatic tissues, only 10% of CpG islands are methylated, the majority of them being located in intergenic and intragenic regions. [ citation needed ]


DNA methylation was probably present at some extent in very early eukaryote ancestors. In virtually every organism analyzed, methylation in promoter regions correlates negatively with gene expression. [ 21 ] [ 28 ]  CpG-dense promoters of actively transcribed genes are never methylated, but, reciprocally, transcriptionally silent genes do not necessarily carry a methylated promoter. In mouse and human, around 60–70% of genes have a CpG island in their promoter region and most of these CpG islands remain unmethylated independently of the transcriptional activity of the gene, in both differentiated and undifferentiated cell types. [ 29 ] [ 30 ]  Of note, whereas DNA methylation of CpG islands is unambiguously linked with transcriptional repression, the function of DNA methylation in CG-poor promoters remains unclear; albeit there is little evidence that it could be functionally relevant. [ 31 ]


DNA methylation may affect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of  transcriptional proteins  to the gene, [ 32 ]  and second, and likely more important, methylated DNA may be bound by proteins known as  methyl-CpG-binding domain  proteins (MBDs).  MBD  proteins then recruit additional proteins to the locus, such as  histone deacetylases  and other  chromatin remodeling  proteins that can modify  histones , thereby forming compact, inactive chromatin, termed  heterochromatin . This link between DNA methylation and chromatin structure is very important. In particular, loss of  methyl-CpG-binding protein 2  (MeCP2) has been implicated in  Rett syndrome ; and  methyl-CpG-binding domain protein 2 (MBD2)  mediates the transcriptional silencing of hypermethylated genes in "cancer." [ citation needed ]


DNA methylation is a powerful transcriptional repressor, at least in CpG dense contexts. Transcriptional repression of protein-coding genes appears essentially limited to very specific classes of genes that need to be silent permanently and in almost all tissues. While DNA methylation does not have the flexibility required for the fine-tuning of gene regulation, its stability is perfect to ensure the permanent silencing of  transposable elements . [ 33 ]  Transposon control is one of the most ancient functions of DNA methylation that is shared by animals, plants and multiple protists. [ 34 ]  It is even suggested that DNA methylation evolved precisely for this purpose. [ 35 ]


DNA methylation of transposable elements has been known to be related to genome expansion. However, the evolutionary driver for genome expansion remains unknown. There is a clear correlation between the size of the genome and CpG, suggesting that the DNA methylation of transposable elements led to a noticeable increase in the mass of DNA. [ 36 ]


A function that appears even more conserved than transposon silencing is positively correlated with gene expression. In almost all species where DNA methylation is present, DNA methylation is especially enriched in the body of highly transcribed genes. [ 21 ] [ 28 ]  The function of gene body methylation is not well understood. A body of evidence suggests that it could regulate  splicing [ 37 ]  and suppress the activity of intragenic transcriptional units (cryptic promoters or transposable elements). [ 38 ]  Gene-body methylation appears closely tied to H3K36 methylation. In yeast and mammals, H3K36 methylation is highly enriched in the body of highly transcribed genes. In yeast at least,  H3K36me3  recruits enzymes such as histone deacetylases to condense chromatin and prevent the activation of cryptic start sites. [ 39 ]  In mammals,  DNMT3a  and  DNMT3b  PWWP domain binds to H3K36me3 and the two enzymes are recruited to the body of actively transcribed genes. [ citation needed ]


DNA methylation patterns are largely erased and then re-established between generations in mammals. Almost all of the methylations from the parents are erased, first during  gametogenesis , and again in early  embryogenesis , with demethylation and  remethylation  occurring each time. Demethylation in early embryogenesis occurs in the preimplantation period in two stages – initially in the  zygote , then during the first few embryonic replication cycles of  morula  and  blastula . A wave of methylation then takes place during the implantation stage of the embryo, with CpG islands protected from methylation. This results in global repression and allows  housekeeping genes  to be expressed in all cells. In the post-implantation stage, methylation patterns are stage- and tissue-specific, with changes that would define each individual cell type lasting stably over a long period. [ 40 ]  Studies on rat limb buds during embryogenesis have further illustrated the dynamic nature of DNA methylation in development. In this context, variations in global DNA methylation were observed across different developmental stages and culture conditions, highlighting the intricate regulation of methylation during organogenesis and its potential implications for regenerative medicine strategies. [ 41 ]


Whereas DNA methylation is not necessary  per se  for transcriptional silencing, it is thought nonetheless to represent a "locked" state that definitely inactivates transcription. In particular, DNA methylation appears critical for the maintenance of mono-allelic silencing in the context of  genomic imprinting  and  X chromosome inactivation . [ 42 ] [ 43 ]  In these cases, expressed and silent alleles differ by their methylation status, and loss of DNA methylation results in loss of imprinting and re-expression of Xist in somatic cells. During embryonic development, few genes change their methylation status, at the important exception of many genes specifically expressed in the germline. [ 44 ]  DNA methylation appears absolutely required in  differentiated cells , as knockout of any of the three competent DNA methyltransferase results in embryonic or post-partum lethality. By contrast, DNA methylation is dispensable in undifferentiated cell types, such as the inner cell mass of the blastocyst, primordial germ cells or embryonic stem cells. Since DNA methylation appears to directly regulate only a limited number of genes, how precisely DNA methylation absence causes the death of differentiated cells remain an open question.


Due to the phenomenon of  genomic imprinting , maternal and paternal genomes are differentially marked and must be properly  reprogrammed  every time they pass through the germline. Therefore, during  gametogenesis , primordial germ cells must have their original biparental DNA methylation patterns erased and re-established based on the sex of the transmitting parent. After fertilization, the paternal and maternal genomes are once again demethylated and remethylated (except for differentially methylated regions associated with imprinted genes). This reprogramming is likely required for totipotency of the newly formed embryo and erasure of acquired epigenetic changes. [ 45 ]


In many disease processes, such as  cancer , gene promoter  CpG islands  acquire abnormal hypermethylation, which results in  transcriptional silencing  that can be inherited by daughter cells following cell division. [ 46 ]  Alterations of DNA methylation have been recognized as an important component of cancer development. Hypomethylation, in general, arises earlier and is linked to chromosomal instability and loss of imprinting, whereas hypermethylation is associated with promoters and can arise secondary to gene (oncogene suppressor) silencing, but might be a target for  epigenetic therapy . [ 47 ]  In developmental contexts, dynamic changes in DNA methylation patterns also have significant implications. For instance, in rat limb buds, shifts in methylation status were associated with different stages of chondrogenesis, suggesting a potential link between DNA methylation and the progression of certain developmental processes. [ 41 ]


Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms. [ 48 ]  Typically, there is hypermethylation of  tumor suppressor genes  and hypomethylation of  oncogenes . [ 49 ]


Generally, in progression to cancer, hundreds of genes are  silenced or activated . Although silencing of some genes in cancers occurs by mutation, a large proportion of carcinogenic gene silencing is a result of altered DNA methylation (see  DNA methylation in cancer ). DNA methylation causing silencing in cancer typically occurs at multiple  CpG sites  in the  CpG islands  that are present in the  promoters  of protein coding genes. [ citation needed ]


Altered expressions of  microRNAs  also silence or activate many genes in progression to cancer (see  microRNAs in cancer ). Altered microRNA expression occurs through  hyper/hypo-methylation  of  CpG sites  in  CpG islands  in promoters controlling transcription of the  microRNAs .


Silencing of DNA repair genes through methylation of CpG islands in their promoters appears to be especially important in progression to cancer (see  methylation of DNA repair genes in cancer ). [ citation needed ]


Epigenetic modifications such as DNA methylation have been implicated in cardiovascular disease, including  atherosclerosis . In animal models of atherosclerosis, vascular tissue, as well as blood cells such as mononuclear blood cells, exhibit global hypomethylation with gene-specific areas of hypermethylation. DNA methylation polymorphisms may be used as an early biomarker of atherosclerosis since they are present before lesions are observed, which may provide an early tool for detection and risk prevention. [ 50 ]


Two of the cell types targeted for DNA methylation polymorphisms are monocytes and lymphocytes, which experience an overall hypomethylation. One proposed mechanism behind this global hypomethylation is elevated  homocysteine  levels causing  hyperhomocysteinemia , a known risk factor for cardiovascular disease. High plasma levels of homocysteine inhibit DNA methyltransferases, which causes hypomethylation. Hypomethylation of DNA affects genes that alter smooth muscle cell proliferation, cause endothelial cell dysfunction, and increase inflammatory mediators, all of which are critical in forming atherosclerotic lesions. [ 51 ]  High levels of homocysteine also result in hypermethylation of CpG islands in the promoter region of the  estrogen receptor  alpha (ERα) gene, causing its down regulation. [ 52 ]  ERα protects against atherosclerosis due to its action as a growth suppressor, causing the smooth muscle cells to remain in a quiescent state. [ 53 ]  Hypermethylation of the ERα promoter thus allows intimal smooth muscle cells to proliferate excessively and contribute to the development of the atherosclerotic lesion. [ 54 ]


Another gene that experiences a change in methylation status in atherosclerosis is the  monocarboxylate transporter  (MCT3), which produces a protein responsible for the transport of lactate and other ketone bodies out of many cell types, including vascular smooth muscle cells. In atherosclerosis patients, there is an increase in methylation of the CpG islands in exon 2, which decreases MCT3 protein expression. The downregulation of MCT3 impairs lactate transport and significantly increases smooth muscle cell proliferation, which further contributes to the atherosclerotic lesion. An ex vivo experiment using the demethylating agent  Decitabine  (5-aza-2 -deoxycytidine) was shown to induce MCT3 expression in a dose dependent manner, as all hypermethylated sites in the exon 2 CpG island became demethylated after treatment. This may serve as a novel therapeutic agent to treat atherosclerosis, although no human studies have been conducted thus far. [ 55 ]


In addition to  atherosclerosis  described above, specific epigenetic changes have been identified in the failing human heart. This may vary by disease etiology. For example, in ischemic heart failure DNA methylation changes have been linked to changes in gene expression that may direct gene expression associated with the changes in heart metabolism known to occur. [ 56 ]  Additional forms of heart failure (e.g. diabetic cardiomyopathy) and co-morbidities (e.g. obesity) must be explored to see how common these mechanisms are. Most strikingly, in failing human heart these changes in DNA methylation are associated with racial and socioeconomic status which further impact how gene expression is altered, [ 57 ]  and may influence how the individual's heart failure should be treated.


In humans and other mammals, DNA methylation levels can be used to accurately estimate the age of tissues and cell types, forming an accurate  epigenetic clock . [ 58 ]


A  longitudinal study  of  twin  children showed that, between the ages of 5 and 10, there was divergence of methylation patterns due to environmental rather than genetic influences. [ 59 ]  There is a global loss of DNA methylation during aging. [ 49 ]


In a study that analyzed the complete DNA methylomes of CD4 +   T cells  in a newborn, a 26 years old individual and a 103 years old individual were observed that the loss of methylation is proportional to age. [ 60 ]  Hypomethylated CpGs observed in the centenarian DNAs compared with the neonates covered all genomic compartments (promoters,  intergenic ,  intronic  and  exonic  regions). [ 60 ]  However, some genes become hypermethylated with age, including genes for the  estrogen receptor ,  p16 ,  insulin-like growth factor 2 , [ 49 ]   ELOVL2 [ 61 ]  and  FHL2 [ 62 ]


High intensity exercise has been shown to result in reduced DNA methylation in skeletal muscle. [ 63 ]   Promoter  methylation of  PGC-1α  and  PDK4  were immediately reduced after high intensity exercise, whereas  PPAR-γ  methylation was not reduced until three hours after exercise. [ 63 ]  At the same time, six months of exercise in previously sedentary middle-age men resulted in increased methylation in  adipose tissue . [ 64 ]  One study showed a possible increase in global genomic DNA methylation of  white blood cells  with more physical activity in non-Hispanics. [ 65 ]


A study that investigated the methylome of  B cells  along their differentiation cycle, using whole-genome  bisulfite sequencing  (WGBS), showed that there is a hypomethylation from the earliest stages to the most differentiated stages. The largest methylation difference is between the stages of germinal center B cells and memory B cells. Furthermore, this study showed that there is a similarity between B cell tumors and long-lived B cells in their DNA methylation signatures. [ 18 ]


Two reviews summarize evidence that DNA methylation alterations in brain neurons are important in learning and memory. [ 66 ] [ 67 ]  Contextual  fear conditioning  (a form of associative learning) in animals, such as mice and rats, is rapid and is extremely robust in creating memories. [ 68 ]  In mice [ 69 ]  and in rats [ 70 ]  contextual fear conditioning, within 1–24 hours, it is associated with altered methylations of several thousand DNA cytosines in genes of  hippocampus  neurons. Twenty four hours after contextual fear conditioning, 9.2% of the genes in rat  hippocampus  neurons are differentially methylated. [ 70 ]  In mice, [ 69 ]  when examined at four weeks after conditioning, the hippocampus methylations and demethylations had been reset to the original naive conditions. The  hippocampus  is needed to form memories, but memories are not stored there. For such mice, at four weeks after contextual fear conditioning, substantial differential  CpG  methylations and demethylations occurred in  cortical  neurons during memory maintenance, and there were 1,223 differentially methylated genes in their anterior cingulate cortex. [ 69 ]  Mechanisms guiding new DNA methylations and new  DNA demethylations  in the  hippocampus  during memory establishment were summarized in 2022. [ 71 ]   That review also indicated the mechanisms by which the new patterns of methylation gave rise to new patterns of  messenger RNA  expression.  These new  messenger RNAs  were then transported by  messenger RNP  particles (neuronal granules) to synapses of the neurons, where they could be translated into proteins. [ 71 ]   Active changes in neuronal DNA methylation and demethylation appear to act as controllers of  synaptic scaling  and  glutamate receptor  trafficking in  learning  and  memory  formation. [ 66 ]


In mammalian cells, DNA methylation occurs mainly at the C5 position of CpG dinucleotides and is carried out by two general classes of enzymatic activities – maintenance methylation and  de novo  methylation. [ 72 ]


Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle. Without the  DNA methyltransferase  (DNMT), the replication machinery itself would produce daughter strands that are unmethylated and, over time, would lead to passive demethylation. DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells. [ citation needed ]


It is thought that DNMT3a and DNMT3b are the  de novo  methyltransferases that set up DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the  de novo  methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Mice and rats have a third functional  de novo  methyltransferase enzyme named DNMT3C, which evolved as a paralog of  Dnmt3b  by tandem duplication in the common ancestral of Muroidea rodents. DNMT3C catalyzes the methylation of promoters of transposable elements during early spermatogenesis, an activity shown to be essential for their epigenetic repression and male fertility. [ 73 ] [ 74 ]  It is yet unclear if in other mammals that do not have DNMT3C (like humans) rely on DNMT3B or DNMT3A for  de novo  methylation of transposable elements in the germline. Finally,  DNMT2 (TRDMT1)  has been identified as a DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 (TRDMT1) does not methylate DNA but instead methylates cytosine-38 in the anticodon loop of aspartic acid transfer RNA. [ 75 ]


Since many tumor suppressor genes are silenced by DNA methylation during  carcinogenesis , there have been attempts to re-express these genes by inhibiting the DNMTs. 5-Aza-2'-deoxycytidine ( decitabine ) is a  nucleoside analog  that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, thus resulting in the enzymes' degradation. However, for decitabine to be active, it must be incorporated into the  genome  of the cell, which can cause mutations in the daughter cells if the cell does not die. In addition, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their  mRNAs  and preventing their  translation . However, it is currently unclear whether targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation. [ citation needed ]


Significant progress has been made in understanding DNA methylation in the model plant  Arabidopsis thaliana . DNA methylation in plants differs from that of mammals: while DNA methylation in mammals mainly occurs on the cytosine nucleotide in a  CpG site , in plants the cytosine can be methylated at CpG, CpHpG, and CpHpH sites, where H represents any nucleotide but not guanine. [ 76 ]  Overall,  Arabidopsis  DNA is highly methylated,  mass spectrometry  analysis estimated 14% of cytosines to be modified. [ 9 ] : abstract   Later, bisulfite sequencing data estimated that around 25% of  Arabidopsis  CG sites are methylated, but these levels vary based on the geographic location of  Arabidopsis  accessions (plants in the north are more highly methylated than southern accessions). [ 77 ]


The principal  Arabidopsis  DNA methyltransferase enzymes, which transfer and covalently attach methyl groups onto DNA, are DRM2, MET1, and CMT3. Both the DRM2 and MET1 proteins share significant homology to the mammalian methyltransferases DNMT3 and DNMT1, respectively, whereas the CMT3 protein is unique to the plant kingdom. There are currently two classes of DNA methyltransferases: 1) the  de novo  class or enzymes that create new methylation marks on the DNA; 2) a maintenance class that recognizes the methylation marks on the parental strand of DNA and transfers new methylation to the daughter strands after DNA replication. DRM2 is the only enzyme that has been implicated as a  de novo  DNA methyltransferase. DRM2 has also been shown, along with MET1 and CMT3 to be involved in maintaining methylation marks through DNA replication. [ 78 ]  Other DNA methyltransferases are expressed in plants but have no known function (see the  Chromatin Database ).