Bone mass and the development of  osteoporosis  have been studied in relation to perinatal nutrition.  An important factor to consider when investigating perinatal nutrition is whether the baby was  breast-fed  or formula-fed.  Studies have shown that breast-fed babies have increased bone mass compared to those were not breast-fed, and that this small increase in bone mass during a period of critical development could potentially program the skeleton to continue along a “healthy” growth trajectory. [ 17 ]   It has also been shown that maternal  vitamin D  insufficiency during late pregnancy is associated with reduced bone size and mineral mass in late childhood. [ 18 ]   Peak bone mass has shown to be a good predictor of risk of fracture and osteoporosis, with even a small increase in peak bone mass resulting in a much lower risk of bone fracture. [ 9 ]  Research shows that genetic markers explain only a small proportion of variation in bone mass and risk of fracture.  Therefore, healthy bone programming is most likely influenced by various epigenetic mechanisms, such as imprinting of the growth promoting genes IGF-2, or changes to the  hypothalamic-pituitary-adrenal axis  (HPA). [ 19 ]


Imbalances in maternal nutrition can also have a significant effect on fetal  neurodevelopment .  Brain development occurs most rapidly during fetal development and infancy, and research has shown that exposure to certain environmental conditions can have long-lasting effects on  cognition .  Specifically, n-3  fatty acids ,  iodine ,  iron  and  choline  have been shown to influence brain development and impact cognitive ability and behavior.  The greatest evidence for a link between nutrition and neurodevelopment comes from studies that show low birth weight associated with low IQ and increased risk of  schizophrenia . [ 20 ] [ 21 ]  Several studies suggest that breast-feeding promotes long-term neurodevelopment by providing the nutrients necessary for proper brain development. [ 22 ]   A study in mice showed that choline-deficient diets during the late gestation period impaired fetal brain development, including decreased cell proliferation and reduced visual-spatial and auditory memory. [ 23 ]   These cognitive changes appeared to be due to altered histone and DNA methylation patterns in the fetal  hippocampus , thus providing a link between maternal nutrition, epigenetics, and early brain development.


It has been postulated that breast-feeding may also protect against type-1 diabetes, with research showing that formula-fed infants are at an increased risk of developing islet  autoantibodies .  Individuals with type-1 diabetes experience a pre-clinical diabetes phase characterized by  autoimmunity  against  pancreatic islets . [ 24 ]   The introduction of certain foods in the first few months of life, such as berries and cereal, is significantly associated with increased risk of islet autoantibody development compared to babies who are exposed to solid foods later in life. [ 25 ]   While the pathogenesis behind development of autoantibodies remains largely unknown, it is very probable that an epigenetic link exists between perinatal diet and risk of type-1 diabetes. [ 9 ]


The majority of research in nutriepigenomics has focused on nutritional imbalances during gestation and lactation periods.  However, foods that are consumed during adulthood can also impact gene expression and disease pathogenesis.  Cancer is the disease most commonly associated with adult nutrition and epigenetic modifications. DNA hypomethelation promotes cancer progression by allowing increased gene transcription, while hypermethylation can silence tumor suppressor genes and further promote uncontrolled  cell division  and  tumor  formation.  Compounds found in foods, such as  genistein  and  tea polyphenols , are able to regulate DNA methyltransferases and histone acetylation in cultured cancer cells and may provide protection against certain types of cancer. [ 13 ]   Other dietary compounds, such as  diallyl disulfide  present in garlic and  sulforaphane  present in cruciferous vegetables, have been associated with cancer prevention in clinical trials. [ 26 ]   This may be due to their ability to inhibit histone deacetylase (HDAC) enzymes and prevent silencing of important regulatory genes.


Many believe epigenetic regulation is cleared during the  fertilization  process, yet more evidence for transgenerational effects (TGEs) are being revealed. [ 1 ]  These TGEs take place when the epigenetic regulatory patterns are not sufficiently erased during fertilization, possibly due to nutrition levels in previous generations. Later generations may be affected from caloric and protein restriction, high-fat interventions and endocrine disruption in earlier generations. [ 1 ]   Differences within the nutritional behavior of the maternal rat are believed to cause malprogramming in the F1 generation and may then be passed to subsequent generations. [ 1 ]
Maternal rats fed a PR-diet during the entire length of pregnancy led to metabolic-related problems in the F1 and F2 generations, even with normal nutrition during the F1 pregnancy. [ 27 ] [ 28 ]  These effects have also been seen in the F3 generation depending on the length of protein restriction. [ 29 ] [ 30 ]   If protein restriction occurred solely during pregnancy, the F1 and F2 offspring had higher systolic blood pressure and lower nephron numbers, possibly predisposing them to hypertension. [ 30 ]  Altered glucose utilization was detected in the grand-offspring of maternal rats fed a PR-diet during pregnancy and lactation, potentially resulting in diabetes later on in life  [ 29 ]


Protein-restriction in the F0 generation led to hypomethylation of promoters involved in metabolism in the F1 and F2 generations, even though the F1 pregnant rat was given a normal diet. [ 31 ]   The exact mechanism of this situation has yet to be elucidated; however, direct transmission is a distinct possibility, meaning the epigenetic marks were preserved during  spermatogenesis  and  oogenesis , when they are normally erased.


Most research to date use common rodent models to investigate the role of nutrition on  phenotype . [ 10 ]  Popular areas to investigate include IUGR studies, whereby rodents, and sometimes sheep, are subjected to a variety of nutritional conditions.  A model for studying IUGR in rodent was developed by Simmons  et al.  (2010) and is used to investigate type II diabetes. [ 32 ]   The maternal rats have their uterine arteries ligated, causing altered use of glucose and insulin in the fetus and can therefore serve as a model for diabetes. These growth-retarded rats were found to be highly similar to human fetuses, as they both display symptoms such as lowered glucose and insulin levels.   Gestational diabetes  may also be studied through chemical induction using  streptozotocin  treatment of the pregnant rats. [ 33 ]  Streptozotocin can cause destruction of the beta cells within the pancreas depending on the concentration given.


The predominant means of investigating nutriepigenetics involves varying the nutritional conditions to which a subject is exposed to and monitoring the effects thereafter.  Restricting caloric and protein intake are the two most common methods. [ 33 ]  A pregnant rodent may have their caloric intake reduced up to 30-50% of normal intake.  Protein restricted rodents are given 8-9%  casein , as opposed to control rats that are fed 20% casein. Micronutrients, such as zinc and iron, may also be restricted to investigate the effects on offspring. Additionally, rats fed diets lacking or including methyl donors are often used to study the effects of diet on epigenomics, as variations within the methylation of DNA are common means of silencing or expressing genes. [ 34 ]   Supplementing maternal mice with folic acid,  vitamin B12 , choline and  betaine  leads to increased levels of DNA methylation at CpG sites and causes a coat color change. [ 35 ]   This is an example of epigenetically modifiable loci called a “metastable epiallele”, of which only a few have been identified.  The above is an example of the “agouti” gene locus, whereby the insertion of a  transposable element  upstream to the Agouti gene is hypermethylated from the supplementation and causes a change in the mice's coat color. Diets containing higher carbohydrate and fat content attempt to mimic typical Western-style diets may also be used in nutriepigenetic studies. [ 10 ] [ 33 ]   Another method used is “catch-up”, where offspring of rats born to mothers subjected to various diets are subsequently cross-fostered to mothers fed normal diets. [ 33 ]


The possibilities of utilizing nutriepigenomics for intervention are quite expansive.  This can include preventative therapies, such as providing an optimal regime for nutrition during pregnancy and lactation. [ 33 ]   It is already common place for pregnant mothers to supplement their diets with choline and folate to prevent the development of neurological disabilities in the fetus.


A highly specific diet, termed an "EpiG diet," may be employed for an individual believed to be at higher risk of developing a metabolic disorder. [ 1 ]  These diets may include supplementation with methyl donors, such as folate. There are also many other natural compounds, such as  resveratrol ,  curcumin  and green tea that have been termed “epigenetic modifiers”, as they have anti-cancer capabilities in addition to being used as treatments for metabolic diseases. [ 36 ]   However, the functions of these compounds still require long-term studies to evaluate their effect over time.


There also exists potential for therapeutic treatments that may correct metabolic disorders, such as type II diabetes. [ 33 ]   Components of garlic and  cruciferous vegetables  are known to possess  HDAC inhibitors  that modify the acetylation of histone proteins and may contain a protection against cancer. [ 26 ]   These same compounds have also been implicated in  irritable bowel syndrome  (IBS) and  colon cancer , as they may modify the histones normally implicated in these diseases. [ 37 ]


Elucidation of disease pathways is another future direction for nutriepigenomic studies.  For example, choline-deficient diets and alcohol metabolism during pregnancy may have very similar metabolic pathways. [ 38 ]  Therefore, animal studies using choline-restricted diets may assist in investigations of  fetal alcohol spectrum disorders .


When compared to studies of maternal transmission, investigations into the role of paternal diets are lacking.  A review demonstrated the nutrition of both parents do in fact play a role in determining the health of their offspring. [ 39 ]  A  germ-line  study reported paternal rats fed a high-fat diet led to insulin dysfunction in the F1 offspring. [ 40 ]   While this likely occurs via epigenetic modifications similar to those postulated in the maternal diets, the exact mechanism remains to be defined.  Assessing the role of epigenetic mechanisms may be easier using paternal inheritance, as sperm transmits epigenetic and genetic information, whereas the female cells also transmit  mitochondrial DNA . [ 39 ]


In  epigenetics , a  paramutation  is an interaction between two  alleles  at a single  locus , whereby one allele induces a heritable change in the other allele. [ 1 ]  The change may be in the pattern of  DNA methylation  or histone modifications. [ 2 ]  The allele inducing the change is said to be paramutagenic, while the allele that has been epigenetically altered is termed paramutable. [ 1 ]  A paramutable allele may have altered levels of  gene expression , which may continue in offspring which inherit that allele, even though the paramutagenic allele may no longer be present. [ 1 ]   Through proper breeding, paramutation can result in siblings that have the same genetic sequence, but with drastically different  phenotypes . [ 3 ]


Though studied primarily in  maize , paramutation has been described in a number of other systems, including animal systems like  Drosophila melanogaster  and mice. [ 1 ] [ 4 ]  Despite its broad distribution, examples of this phenomenon are scarce and its mechanism is not fully understood.


The first description of what would come to be called paramutation was given by  William Bateson  and  Caroline Pellew  in 1915, when they described "rogue"  peas  that always passed their "rogue" phenotype onto their progeny. [ 5 ]  However, the first formal description of paramutation was given by  R.A. Brink  at the  University of Wisconsin–Madison  in the 1950s, who did his work in  maize  ( Zea mays ). [ 5 ]  Brink noticed that specific weakly expressed alleles of the  red1   (r1)  locus in maize, which encodes a  transcription factor  that confers red pigment to  corn kernels , can heritably change specific strongly expressed alleles to a weaker  expression  state. [ 1 ]  The weaker expression state adopted by the changed allele is heritable and can, in turn, change the expression state of other active alleles in a process termed secondary paramutation. [ 1 ]  Brink showed that the influence of the paramutagenic allele could persist for many generations. [ 1 ]


The alleles that cause  heritable  changes in the alleles they come into contact which are called paramutagenic, and the alleles modified by them are paramutable. Alleles that do not take part in this interaction are called neutral. When present together in an organism, the paramutable allele is converted to the paramutagenic allele, and retains its paramutagenicity in subsequent generations. No change in DNA sequence accompanies this transformation, but instead epigenetic modifications (e.g. DNA methylation) differentiate the paramutagenic from paramutable alleles. In most cases, it is the paramutable allele that is highly transcribed and the paramutagenic allele that undergoes little to no transcription.


The first described and most extensively researched example is the  r1  locus in maize. The  gene  at this locus, when actively transcribed, codes for a  transcription factor  that promotes  anthocyanin  production, resulting in kernels with a purple color. One  allele  at this locus, referred to as B’, is capable of causing methylation at the other allele, B-I. This methylation results in reduced transcription and, as a result, decreased  anthocyanin  production. These alleles do not differ in DNA sequence, but they do differ in their degree of  DNA methylation . As with other examples of paramutation, this change of the B-I allele to the B’ allele is stable and heritable. Other, similar examples of paramutation exist at other  maize  loci, as well as in other plants such as the model system  Arabidopsis thaliana  and  transgenic   petunias . [ 6 ] [ 7 ] [ 8 ]


Paramutation has also been documented in animals such as  fruit flies ,  C. elegans , and  mice . [ 1 ] [ 4 ] [ 9 ]


Though the specific mechanisms by which paramutation acts vary from organism to organism, all well-documented cases point towards epigenetic modification and RNA-silencing as the underlying mechanism for paramutation. [ 1 ]


In the case of the  r1  locus in maize,  DNA methylation  of a region of  tandem repeats  near the coding region of the gene is characteristic of the paramutagenic B’ allele, and when the paramutable B-I allele becomes paramutagenic, it too takes on the same DNA methylation pattern. [ 10 ]  In order for this methylation to be successfully transferred, a number of genes coding for  RNA-dependent RNA polymerases  and other components of  RNA-silencing  pathways are required, suggesting that paramutation is mediated via endogenous RNA-silencing pathways. [ 1 ]  The transcription of  short interfering RNAs  from the tandem repeat regions corroborates this. In animal systems such as  Drosophila ,  piRNAs  have also been implicated in mediating paramutation. [ 4 ]  In addition to the characteristic  DNA methylation  state changes, changes in  histone modification  patterns in the methylated DNA regions, and/or the requirement of histone modifying proteins to mediate paramutation have also been noted in multiple systems. [ 2 ] [ 9 ]  It has been suggested that these histone modifications play a role in maintaining the paramutated state. [ 2 ]  The previously mentioned  tandem repeat  region in the  r1  locus is also typical of other loci showing paramutation or paramutation-like phenomena. [ 5 ]


However, it has been noted that it is not possible to explain all occurrences and features of paramutation with what is known about RNAi-mediated transcriptional silencing, suggesting that other pathways and/or mechanisms are also at play. [ 7 ]


It has been speculated that in any particular population, relatively few genes would show observable paramutation since the high  penetrance  of paramutagenic alleles (like B’ at the  r1  locus in maize) would drive either the paramutagenic or paramutable allele to  fixation . [ 3 ]  Paramutation at other loci with paramutagenic alleles with lower penetrance may persist, however, which may need to be taken into account by  plant breeders . [ 3 ]


Since there are examples of paramutation, or paramutation-like phenomena, in animals such as fruit flies and mice, it has been suggested that paramutation may explain the occurrence of some human  diseases  that exhibit  non-Mendelian inheritance patterns . [ 11 ]


Pharmacoepigenetics  is an emerging field that studies the underlying  epigenetic  marking patterns that lead to variation in an individual's response to medical treatment. [ 1 ]


Due to  genetic heterogeneity , environmental factors, and  pathophysiological  causes, individuals that exhibit similar disease expression may respond differently to identical drug treatments. Selecting treatments based on factors such as age, body-surface area, weight, gender, or disease stage has been shown to incompletely address this problem, so medical professionals are shifting toward using patient genomic data to select optimal treatments. Now, an increasing amount of evidence shows that  epigenetics  also plays an important role in determining the safety and efficacy of drug treatment in patients. [ 2 ]  Epigenetics is a bridge that connects individual genetics and environmental factors to explain some aspects of gene expression. Specifically, environmental factors have the potential to alter one's epigenetic mechanisms in order to influence the expression of genes. For example, smoking cigarettes can alter the DNA methylation state of genes and thereby expression of genes through different mechanisms.


Epigenetic changes in genes caused by factors such as environment can result in abnormal gene expression and the initiation of diseases. The progression of diseases further alters the epigenetic patterns of the whole  genome . While epigenetic changes are generally long lasting, and in some cases permanent, there is still the potential to change the epigenetic state of a gene. Thus, drugs have been developed to target aberrant epigenetic patterns in cells to either activate or suppress the epigenetically modified gene expression gene expression. This is known as  epigenetic therapy . Besides being drug targets, epigenetic changes are also used as  diagnostic  and  prognostic  indicators to predict disease risk and progression, and this could be beneficial for the improvement of personalized medicine.


The development of the  Human Epigenome Project  and advances in  epigenomics  has given rise to a burgeoning field known as pharmacoepigenetics. Pharmacoepigenetics was initially developed to study how epigenetic patterns of drug transporters, drug-metabolizing enzymes, and nuclear receptors affect individuals’ response to the drug. Now, pharmacoepigenetics has an additional focus: the development of therapeutic epidrugs that can make changes to the epigenome in order to lessen the cause or symptoms of a disease in an individual. Even though a large gap still remains between the knowledge of epigenetic modifications on drug metabolism mechanisms and clinical applications,
pharmacoepigenetics has become a rapidly growing field that has the potential to play an important role in personalized medicine.


In order to develop effective epigenetic therapies, it is important to understand the underlying epigenetic mechanisms and the proteins that are involved. Various mechanisms and modifications play a role in epigenetic remodeling and signaling, including  DNA methylation ,  histone modification ,  covalent  modifications, RNA transcripts,  microRNAs ,  mRNA ,  siRNA , and nucleosome positioning. In particular, scientists have extensively studied the associations of DNA methylation, histone modifications, regulatory microRNA with the development of diseases. [ 3 ] [ 4 ] [ 5 ]


DNA methylation is the most widely studied epigenetic mechanism. Most of them occur at CpG sites.  DNA methyltransferase  is recruited to the site and adds  methyl groups  to the  cytosine  of the CpG dinucleotides. This allows the methyl-CpG binding proteins to bind to the methylated site and cause  downregulation  of genes. [ 6 ]  Histone modification is mainly achieved by modifying the  N-terminal  tails of histones. The mechanisms include  acetylation ,  methylation ,  phosphorylation , unbiquitination, etc. They affect the compaction of chromatin structure, the accessibility of the DNA, and therefore the transcriptional level of specific genes.


Additionally,  microRNA  is a type of noncoding RNA that is responsible for altering gene expression by targeting and marking mRNA transcripts for degradation. Since this process is a posttranscriptional modification, it does not involve changes in DNA sequence. The expression of microRNA is also regulated by other epigenetic mechanisms. Aberrant expression of microRNA facilitates disease development, making them good targets for epigenetic therapies. Epigenetic proteins involved in the regulation of gene transcription fall into three categories-writers, erasers, and readers.  Both writers and erasers have enzymatic activity that allows them to covalently modify DNA or histone proteins. Readers have the ability to recognize and bind to specific sites on chromatin to alter epigenetic signatures.


Once the underlying epigenetic mechanisms are understood, it becomes possible to develop new ways to alter epigenetic marks such as "epidrugs", or  epigenome editing , which is the overwriting of epigenetic patterns using man-made signals to direct epigenetic proteins to target loci. [ 2 ]  Furthermore, based on patients' unique epigenetic patterns, medical professionals can more accurately assign a safe and effective treatment including appropriate epigenetic drugs tailored to the patient.


Individual differences in drug metabolism and response can be partially explained by epigenetic changes. [ 7 ] [ 8 ]  Epigenetic changes in genes that encode drug targets, enzymes, or transport proteins that affect the body's ability to absorb, metabolize, distribute and excrete substances that are foreign to the body ( Xenobiotics ) can result in changes in one's toxicity levels and drug response. [ 7 ] [ 8 ]  One of the main effects of drug exposure early in life is altered  ADME  (Absorption, Distribution, Metabolism, and Excretion) gene expression. [ 7 ]  There is evidence that these genes are controlled by DNA methylation, histone acetylation, and miRNAs. [ 9 ] [ 8 ]  A new emerging field, closely related to pharmacoepigenetics, is toxicoepigenetics that captures toxicological epigenetic changes as a result of the exposure to different compounds (drugs, food, and environment). In this field, there is growing interest in mapping changes in histone modifications and their possible consequences. [ 10 ]


More needs to be understood about these mechanisms, but the hope is that it can lead to proper drug selection and dosage. [ 11 ]  Additionally, drug resistance can be acquired through epigenetic mechanisms. This is particularly common in chemotherapy, where cells that develop resistance to treatment continue to divide and survive. [ 7 ]  Pharmacoepigenetic treatment plans can consist of a single epidrug class or combine several in a unique therapy. The following are the examples of how drug response or metabolism related proteins are regulated by epigenetic mechanisms:


Age-related changes to epigenetic modifications on regulatory regions of mouse  Cyp2e1  has been associated with the metabolism mediated by its encoded protein. Cyp2e1 mediated hydroxylation of its probe drug chlorzoxazone to its metabolite, 6-hydroxychlorzoxazone, correlated negatively with DNA methylation and positively with histone acetylation in mouse microsome extracts. [ 12 ]


CXCR4 is a protein that acts as a coreceptor for the entry of  HIV . It has been developed as a drug target for anti-HIV therapy. A study has shown that its expression is dysregulated by abnormal methylation patterns in some cancers. Thus, this could affect the efficiency and drug response to the anti-HIV therapy. [ 1 ]


CYP1A1 is a protein that is well known for its role in chemical compounds and drug metabolism. [ 13 ]  A study in prostate cancer demonstrated that the protein's regulatory region was under the control of the histone modification  H3K4me3 , which typically indicates active gene expression in non-cancerous cells. [ 1 ]  This abnormal methylation typically causes histone modification and changes in chromatin structure at a local level, thus effecting gene expression. [ 14 ]


ABCG2 is a protein that is responsible for multidrug resistance in cancer chemotherapy. Increased expression of ABCG2 is found in different drug resistant cancer cell lines and tumor tissues. One of the microRNA modifications changes its gene and protein expression by destabilizing its mRNA. [ 15 ]


While there is still a lot of work that needs to be done regarding the epigenetic modifications of specific cancers at various steps in tumor development, there is a general understanding of epigenetic modifications in genes that lead to abnormal expression and various types of cancer. These epigenetic biomarkers are being considered in clinical use as a tool to detect disease, classify tumors, and understand drug response to treatments such as target compounds, traditional chemotherapy agents, and epigenetic drugs. Human cancer is generally characterized by hypermethylation of specific promoters, which typically prevents the expression of DNA repair and tumor-suppressing  genes, and the loss of DNA methylation on a global scale, [ 16 ]  which can allow for expression of oncogenes or result in a loss of imprinting. [ 17 ]  Histone modifications play an important role in the regulation of cellular processes, thus epigenetic changes resulting in changed structure can lead to abnormal transcription, DNA repair and replication. [ 17 ]  Below are some examples and then an overview of the ways these epigenetic modifications are being targeted.


Epigenetic changes are highly present in cancer, therefore it is a good model to assess different ways in which epigenetic drugs can be used to make changes that turn up and turn down gene expression. [ 7 ]


DNA methyltransferase inhibitors are being pursued due to the hypermethylation of tumor suppressor genes and increased DNMTs that have been observed in cancer cells. Introduction of these inhibitors can result in reduced promoter methylation and expression of previously silenced tumor suppressor genes.  Azacitidine  and  decitabine , which incorporate into the DNA and covalently trap the methyltransferases, have been approved by the FDA for  myelodysplastic syndrome  (a group of cancers where blood cells from the bone marrow do not mature properly into healthy blood cells) treatment and are currently being investigated for other cancers like leukemia. Other types of drugs are being developed like non-nucleoside analogues, which can covalently bind to DNMTs. [ 18 ]


Some examples include procaine, hydralazine, and procainimide, but they lack specificity and potency making it hard to test them in clinical trials. DNA methyltranferase inhibitors are usually used at a low level due to their lack of specificity and toxic effects on normal cells. HDAC inhibitors are also being used, due to the changes in histone acetylation and the increased HDACs observed. While the mechanism is still under investigation, it is believed that adding the HDAC inhibitors results in increased histone acetylation and therefore the reactivation of transcription of tumor suppressor genes. [ 18 ]


More so, HDACs can also remove acetyl groups from proteins that are not the histone, so it is thought that adding HDAC inhibitors may result in changes in transcription factor activity. There are around 14 different HDAC inhibitors being investigated in clinical trials for haematological and solid tumors, but more research needs to be done on the specificity and mechanisms by which they are inhibiting. Another way to alter epigenetic modifications is through the use of histone methyltransferase inhibitors. [ 18 ]


Loss of function in genes encoding DNA demethylases or the overexpression of DNA methyltransferases can result in the hypermethylation of DNA promoters. [ 18 ]  Loss of function of DNA methyltransferases can lead to hypomethylation. Loss of function in chromosome remodeling, DNA repair, and cell cycle regulation genes can lead to uncontrolled growth of cells giving rise to cancer. [ 18 ]  Histone modification patterns can also lead to changes in genomes that can negatively affect these and other systems, making cancer more likely.


Cells that carry loss-of-function mutations can be targeted by drugs that induce  synthetic lethality , a genetic/protein interaction where the loss of one component induces little change, but the loss of both components results in cell death. In cancer cells where one part of the interaction experiences a loss-of-function mutation, the other part can be interrupted by drug treatment to induce cell death in cancerous cells.  Synthetic lethality is an attractive treatment option in patients with cancer since it there should be minimal / no effect on healthy cells.


For example, with  SWI/SNF  loss of function mutations, DNA replication and repair is negatively affected and can give rise to tumors if cell growth goes unchecked. Mutations of these genes are common causes of cancers. These mutations are not directly targetable, but several synthetic lethal interactions can be exploited by cancer drugs to kill early cancer growth. [ 18 ]


Additionally, loss-of-function mutations can be targeted by using the dynamic states of histone modifications. Loss of function mutations in demethylases, such as  KDMK6A  are common in cancer. [ 18 ]  By inducing upregulation of methyltransferase inhibitors, the effects of the loss-of-function mutation can be mitigated. [ 18 ]


Development of drugs that target or modify epigenetic signatures of target genes is growing, especially as bioinformatic analysis increases our knowledge of the human genome and speeds up the search for synthetic lethal interactions. Most widely used to assess potential synthetic lethal interactions is using siRNA and  CRISPR-Cas9  to modify target genes.  CRISPRi  and  CRISPRa  technology allows researchers to activate or inactivate target genes.


In lung cancer the activation of both dominant and recessive oncogenes and inactivation of tumor suppressor genes has been observed. [ 17 ]  Frequently observed in lung cancer is the methylation of gene promoters that are involved in critical functions like cell-cycle control, repairing DNA, cell adhesion, proliferation, apoptosis, and motility. A few of the common genes frequently observed are  APC ,  CDH1 ,  CDKN2A ,  MGMT , and RASSF1A (a tumor suppressor). [ 16 ]  In the cases of CDKN2A and RASSF1A DNA these genes are methylated, resulting in the loss of tumor suppressor genes. [ 17 ]


Various strategies such as using drugs like  entinostat  and  azacitidine  have been observed in clinical trials of non-small-cell lung carcinoma. The idea being that etinostat, a histone deacetylase inhibitor, can prevent the silencing of genes by allowing them to be accessible to transcription machinery.  Azacitidine  can be metabolized and incorporated into DNA and then recognized as a substrate for DNA methyltransferases, but since the enzyme is bound the methyltransferase cannot add methylation marks and thus silence crucial genes.


Histone modifications, DNA methylation, and microRNAs have been found to play an important role in heart disease. [ 19 ]  Previously, histone tail acetylation has been linked to cardiac hypertrophy or abnormal heart muscle thickening that is usually due to an increase in cardiomyocyte size or other cardiac muscle changes. [ 20 ]  The hypertrophic changes that occur in cardiac muscles cells result from the required acetylation of histone tails via acetyltransferases. In addition to acetyltransferases, histone deacetylases (HDACs) also aid in the regulation of muscle cells. Class II HDACs 5 and 9 inhibit the activity of a factor known as myocyte enhancer factor 2 ( MEF2 ), which unable to bind prevents the expression of genes that produce hypertrophic effects.


Additionally,  loci such as  PECAM1 ,  AMOTL2  and  ARHGAP24  have been seen with different methylation patterns that are correlated with altered gene expression in cardiac tissue. [ 19 ]


There are an increasing number of scientific publications that are finding that miRNA plays a key role in various aspects of heart failure. [ 19 ]   Examples of functions for miRNA include the regulation of the cardiomyocyte cell cycle and regulation of cardiomyocyte cell growth. [ 21 ] [ 22 ]  Knowing the epigenetic modifications allows for the potential use of drugs to modify the epigenetic status of a target sequence. One could possibly target the miRNAs using antagomirs.  Antagomirs  are single strand RNAs that are complementary, which have been chemically engineered oligonucleotides that silence miRNAs so that they cannot degrade the mRNA that is needed for normal levels of expression.


DNA methylation of CpGs can lead to a reduction of gene expression, and in some cases this decrease in gene product can contribute to disease. Therefore, in those instances it is important to have potential drugs that can alter the methylation status of the gene and increase expression levels.  To increase gene expression, one may try to decrease CpG methylation by using a drug that works as DNA methyltransferase inhibitor such as  decitabine  or 5-aza-2'-deoxycytidine. [ 1 ]


On the other hand, some diseases result from a decrease in acetylase activity, which results in a decrease in gene expression. Some studies have shown that inhibiting HDAC activity can attenuate cardiac hypertrophy. [ 19 ]   trichostatin  A and sodium butyrate are two HDAC inhibitors. Trichostatin A is known for its ability to inhibit class I and II HDACs from removing acetylases and decreasing gene expression.  Sodium butyrate  is another chemical that inhibits class I HDACs, thus resulting in the ability for transcription factors to easily access and express the gene.


There are a number of challenges with the developing epigenetic therapies for widespread medical use. While laboratory results indicate relationships between genes and potential drug interactions that could mitigate the effects of mutations, the complexity of the human genome and epigenome makes it difficult to develop therapies that are safe, efficient, and consistent. Epigenetic alteration may affect more systems than the target genes, which gives potential for deleterious effects to rise out of treatment. Additionally, epigenetic mutations can be a result of lineage. [ 5 ]


As tissue gene expression is largely regulated by epigenetic interactions, certain tissue-specific cancers are difficult to target with epigenetic therapies. Additionally, genes that encode for elements that prevent one type of cancer in a cell, may have altered function in another and lead to another type of cancer. Trying to modify these proteins, such as EZH2, may give rise to other types of cancer. Selectivity is another hurdle in the development of therapies. Since many proteins are structurally similar, especially within the same protein family, Broad-spectrum inhibitors can't always be used since modifying the regulation of one protein may do the same to others in the family. [ 5 ]


Based on the differences in these epigenetic patterns, scientists and physicians can further predict the drug response of each patient. One of the most compelling examples is methylation of the tumor suppressor gene at promoter sequence that codes for MGMT. [ 23 ]   MGMT  is a DNA repair protein responsible for transferring methyl groups from O(6)-alkylguanine in DNA to itself to fight against mutagenesis and the buildup of toxic compounds that result from alkylating agents. [ 24 ]


Therefore, MGMT is responsible for the repair of areas that have been damaged by toxins. This MGMT promoter region has been found to be highly methylated, and thereby repressed, in patients with various types of cancer. [ 24 ]  Several drugs such as  procarbazine ,  streptozotocin , BCNU ( carmustine ), and  temozolamide  are designed to remodel DNA to reverse this abnormal methylation modification so that MGMT may be normally expressed and repair DNA. The methylation status of the promoter become the best predictor of responses to BCNU and temozolamide in patients with brain cancer.


Proteins containing  bromodomains  recognize and bind  acetylated   lysine  residues in  histones , causing chromatin structure modification and a subsequent shift in levels of gene expression. Bromodomain and extra-terminal (BET) proteins bind acetyl groups and work with  RNAPII  to help with  transcription  and elongation of chromatin.  BET inhibitors  have been able to prevent successful interaction between BET proteins and acetylated histones. [ 18 ]  Using a BET inhibitor can reduce the over expression of bromodomain proteins, which can cause aberrant chromatin remodeling, transcription regulation, and histone acetylation. [ 18 ]


Several studies have shown that  histone acetyltransferase  (HAT) inhibitors are useful in re-inducing expression of tumor suppression genes by stopping histone acetyltransferase activity to prevent chromatin condensation. [ 25 ]


Protein  methyltransferase  (PMT) inhibitors: PMT's play a key role in methylating lysine and arginine residues to affect transcription levels of genes. It has been suggested that their enzymatic activity plays a role in cancer, as well as neurodegenerative and inflammatory diseases. [ 25 ]


Using  Histone deacetylase  (HDAC)  inhibitors  allows for genes to remain transcriptionally active. HDACi's have been used in various Autoimmune Disorders, such as systemic lupus erythematosus, rheumatoid arthritis, and systemic onset juvenile idiopathic arthritis. [ 7 ]  They have also proven useful for treating cancer, since they are structurally diverse and only effect 2-10% of expressed genes. [ 25 ]  Using HDAC Inhibitors for the treatment of psychiatric
and neurodegenerative diseases has shown promising results in early studies. [ 25 ]  Additionally, studies have demonstrated that HDACi are useful in minimizing damage after a stroke, and encouraging angiogenesis and myogenesis in embryonic cells. [ 25 ]


One of the common characteristics of various types of cancer is hypermethylation of a tumor suppressing gene. Repression of this methyltransferase action at targeted loci can prevent recurring transfer of methyl groups to these sites and keep them open to transcriptional machinery, allowing more tumor-suppression genes to be made. These drugs are typically  cytidine  derivatives. These drugs tether DNMT to the DNA and prevent their continued action. Treatments that inhibit DNMT function without attachment to DNA (which can cause toxic effects) show they could be effective treatment options but they are not developed enough to see widespread use. [ 7 ]


Population epigenetics  is a field that examines the extent (degree of variation) and dynamics (change over time) of  epigenetic  variation in natural  populations , especially in response to changing environmental conditions. [ 1 ]  This emerging discipline integrates concepts from  population genetics , epigenetics, and  ecology  to explore how epigenetic modifications, such as  DNA methylation , contribute to  phenotypic  diversity and  adaptation  in wild populations. [ 1 ]  Unlike traditional population genetics, which focuses on variations in  DNA sequences , population epigenetics studies heritable changes in gene expression that occur without alterations to the DNA sequence itself. [ 2 ]


Since epigenetic changes can be influenced by environmental factors, have a potentially higher rate of  spontaneous mutation  than genetic sequences, and may be passed down through  non-Mendelian inheritance , they play a significant role in adaptation. [ 1 ]  Population epigenetics therefore offers new insights into the mechanisms of  evolution ,  disease susceptibility , and the ability of organisms to adapt to changing environments. [ 1 ] [ 2 ]


Position effect  is the effect on the  expression  of a  gene  when its location in a  chromosome  is changed, often by  translocation .  This has been well described in  Drosophila  with respect to eye color and is known as  position effect variegation  (PEV). [ 1 ]


The  phenotype  is well characterised by unstable expression of a gene that results in the red eye coloration.  In the  mutant  flies the eyes typically have a mottled appearance of white and red  sectors .  These phenotypes are often due to a chromosomal translocation such that the color gene is now close to a region of  heterochromatin .  Regions of heterochromatin can spread and influence transcription, which may result in the cessation of gene expression and subsequently, white eye sectors.


Position effect  is also used to describe the variation of expression exhibited by identical  transgenes  that insert into different regions of a  genome .  In this case the difference in expression is often due to  enhancers  that regulate neighboring genes.  These local enhancers can also affect the expression pattern of the transgene.  Since each  transgenic organism  has the transgene in a different location each transgenic organism has the potential for a unique expression pattern.


Probabilistic epigenesis  is a way of understanding human behavior based on the relationship between experience and biology. [ 1 ]  It is a variant form of  epigenetics , proposed by American psychologist  Gilbert Gottlieb  in 1991. [ 1 ]  Gottlieb’s model is based on  Conrad H. Waddington 's idea of developmental  epigenesis . [ 2 ]  Both theories examine the complexity of the ways in which the brain develops and explore factors that occur outside the genome. [ 2 ]  However, probabilistic epigenesis differs from Waddington’s model as it relies much more heavily on the potential developmental impacts of experience and environment and how they interact with an individual’s genes. [ 2 ]  Probabilistic epigenesis takes into account developmental, hormonal, environmental, neuropsychological, and genetic factors in order to explain various forms of behavior. [ 3 ]


In developmental psychology, probabilistic epigenesis is a theory of human behavior that assumes that different neural structures develop and activate either based on an individual's biology or interactions with their environment. [ 3 ]  It relies on the idea that there are multi-directional interactions between biological and psychological factors, meaning probabilistic epigenesis is a non-hierarchical model of understanding development. [ 3 ]


The biological factors, also known as genetic determinants, involve an individual's genetic makeup, and how it influences their behavior. [ 3 ]  These factors must be looked at alongside environment variables, however, as the number of genes in a human brain is insufficient in regards to explaining all aspects of the human mind–there is simply not enough storage. [ 3 ]  According to the theory of probabilistic epigenesis, the environment (socioeconomic, household, etc.) in which a person lives and interacts with contributes to overall behavioral development by triggering certain genetic information to activate. [ 3 ]


The relationship between socioeconomically disadvantaged youth and poor physical health is a topic notably evaluated by James Hamblin. [ 4 ]  Research has proven there is a correlation between disadvantaged youth and high blood pressure, excess body fat, and high levels of cortisol. [ 4 ]  The pressures associated with a low socioeconomic background have proven to produce chronic stress in individuals who strive for upward mobility, likely due to the increased familial and social pressures. [ 4 ]  Subsequently, chronic stress has been linked to a breakdown in bodily functions, and is thus a stimulant for disease. [ 4 ]  In other words, this means that the extrinsic factor of socioeconomic class can cause an individual to be less physically capable of handling stress and heavy workloads than someone born into a wealthier environment. [ 4 ]  This means that their self-control and work ethic comes with the price of their health, as people from low socioeconomic backgrounds are more likely to age faster at the cellular level. [ 4 ]


An unpredictable and chaotic household structure can be linked to socioemotional development. [ 5 ]  Socioemotional development, if occurring in an environment that lacks a regular pattern, can result in behavioral difficulties and symptoms of internalization. [ 5 ]


An example of this can be seen in a study conducted by Urie Bronfenbrenner, in which he examines how the exchange of energy between the developing child and the persons and objects in their close settings effects their development. [ 5 ]  His research ultimately suggests that the more regular and positive these household interactions are, the better the child will perform academically and the less likely they will be to internalize problems. [ 5 ]


Gottlieb's model of probabilistic epigenesis is based on the idea that certain areas of the brain are operational before they are completely developed. [ 3 ]  This means that, while the development process is occurring, neural structures are prone to influence–either internal or external. [ 3 ]  The multi-directional relationship between these influences is again important when regarding brain development into maturation, as brain maturation and functional experience are both the cause and effect of one another. [ 1 ]


“ Nature versus nurture ,” a term coined by  Francis Galton  in the late 1800s, was an early and simple way of explaining human behavior. [ 6 ]  In this model, child development into adolescence and adulthood can be explained either by intrinsic aspects of the child or by extrinsic factors influencing the child. [ 6 ]


Probabilistic epigenesis draws from this model in that it emphasizes the importance of factors that could be categorized as nature and/or nurture. [ 7 ]  However, it expands upon and complicates the idea that it is either nature or nurture that causes a person to act a certain way. [ 7 ]  In probabilistic epigenesis, nature and nurture interact so that every variable is both a cause and an effect. [ 7 ]  As developmental and neurological understandings have progressed, the idea that intrinsic and extrinsic factors interact with one another rather than independently, as suggested in the probabilistic epigenesis model, has become the predominant way of understanding behavior. [ 6 ]


In  epigenetics ,  proline isomerization  is the effect that  cis-trans  isomerization  of the  amino acid   proline  has on the  regulation of gene expression . Similar to  aspartic acid , the  amino acid   proline  has the rare property of being able to occupy both  cis  and  trans   isomers  of its  prolyl peptide bonds  with ease.  Peptidyl-prolyl isomerase , or PPIase, is an  enzyme  very commonly associated with proline isomerization due to their ability to catalyze the isomerization of prolines. PPIases are present in three types: cyclophilins, FK507-binding proteins, and the parvulins. [ 1 ]  PPIase enzymes  catalyze  the transition of proline between  cis  and  trans  isomers and are essential to the numerous biological functions controlled and affected by prolyl isomerization (i.e.  cell signalling ,  protein folding , and epigenetic modifications) [ 2 ]  Without PPIases, prolyl peptide bonds will slowly switch between  cis  and  trans  isomers, a process that can lock proteins in a nonnative structure that can affect render the protein temporarily ineffective. Although this switch can occur on its own, PPIases are responsible for most isomerization of prolyl peptide bonds. The specific amino acid that precedes the prolyl peptide bond also can have an effect on which conformation the bond assumes. For instance, when an  aromatic  amino acid is bonded to a proline the bond is more favorable to the  cis  conformation.  Cyclophilin A uses an "electrostatic handle" to pull proline into  cis  and  trans  formations. [ 3 ]  Most of these biological functions are affected by the isomerization of proline when one isomer interacts differently than the other, commonly causing an activation/deactivation relationship. As an amino acid, proline is present in many  proteins . This aids in the multitude of effects that isomerization of proline can have in different biological mechanisms and functions.


Cell signaling involves many different processes and proteins. One of the most studied cell signaling phenomena involving proline is the interactions with  p53  and prolyl isomerases, specifically  Pin1 . The protein p53, along with p63 and  p73 , are responsible for ensuring that alterations to the  genome  are corrected and for preventing the formation and growth of  tumors . proline residues are found throughout the p53 proteins and without the phosphorylation and isomerization of specific Serine/Threonine-Proline motifs within p53, they cannot exhibit control over their target genes. The main signalling processes that are affected by p53 are  apoptosis  and cell cycle arrest, both of which are controlled by specific isomerization of the prolines in p53. [ 4 ]


Although isomerization of proteins has been known about since 1968 when it was discovered by C. Tanford, proline isomerization and its use as a noncovalent histone tail modification was not discovered until 2006 by Nelson and his colleagues. [ 1 ] [ 5 ]


One of the most well known epigenetic mechanisms that proline isomerization plays a role in is the modification of histone tails, specifically those of  histone  H3. Fpr4 is a PPIase, in the FK507BP group, that exhibits catalytic activity at the proline positions 16, 30, and 38 (also written P16, P30, and P38 respectively) on the N-terminal region of histone H3 in  Saccharomyces cerevisiae . [ 1 ] [ 5 ] [ 6 ]  Fpr4's binding affinity is strongest at the P38 site, followed by P30 and then P16. However the catalytic efficiency, or the increase in isomerization rates, is highest at P16 and P30 equally, followed by P38 which exhibits a very small change in isomerization rates with the binding of Fpr4. [ 6 ]  Histone H3 has an important  lysine  residue at the 36 position (also written K36) on the  N-terminal tail  which can be  methylated  by Set2, a  methyltransferase . Methylation of K36 is key to normal  transcription elongation . [ 5 ]  Due to P38's proximity to K36, cross-talk between P38 isomerization and K36 methylation can occur. [ 1 ] [ 5 ] [ 7 ]  This means that isomer changes at the P38 position can affect methylation at the K36 position. In the  cis  position, P38 shifts the histone tail closer to the DNA, crowding the area around the tail. This can cause a decrease the ability of proteins to bind to the DNA and to the histone tail, including preventing Set2 from methylating K36. Also, this tail movement can increase the number interactions between the histone tail and the DNA, increasing likelihood of  nucleosome  formation and potentially leading to the creation of  higher-order chromatin structure . In  trans , P38 leads to the opposite effects: allowing for Set2 to methylate K36. Set2 is only affected by isomerization of P38 when creating a trimethylated K36 (commonly written as K36me3), however, and not K36me2. [ 1 ] [ 8 ]  Fpr4 also binds to P32 in H4, though its effects are minimal. [ 9 ]


In mammalian cells, the isomerization of H3P30 interacts with the  phosphorylation  of H3S28 (serine in the 28 position of histone H3) and the methylation of H3K27. [ 1 ] [ 7 ]  hFKBP25 is a PPIase that is a  homolog  for Fpr4 in mammalian cells and is found to commonly be associated with the presence of  HDACs . Cyp33 is a cyclophilin that has the ability to isomerize H3 proline residues at P16 and P30 positions. [ 9 ] [ 10 ]  Histones H2A and H2B also have multiple proline residues near amino acids that when modified affect the activity surrounding the histone.


The isomerization of the peptide bond between histone H3's alanine 15 and proline 16 is affected by the  acetylation  at K14 and can control the  methylation  states of K4. [ 3 ] [ 11 ]  K4me3 represses gene transcription and depends upon the Set1  methyltransferase  complex subunit Spp1 being balanced with the Jhd2  demethylases  for proper function. Acetylation of K14 allows for a state change in P16 and primarily promotes the  trans  state of P16. This  trans  isomer of P16 reduces K4 methylation, which results in transcription repression. [ 5 ] [ 11 ]  Isomerization of P16 has downstream effects of controlling protein binding to acetylated K18. [ 9 ]  When P16 is in the  trans  conformation, Spt7 is allowed to bind to K18ac, increasing transcription.


Proline isomerization of certain prolines in RNA polymerase II is key in the process of recruiting and placing processing factors during transcription. [ 12 ]  PPIases target  RNA polymerase II  by interacting with the Rpb1  carboxy terminal domain , or CTD. [ 9 ] [ 12 ]  Proline isomerization is then used as part of the mechanism of the CTD to recruit  co-factors  required for co-transcriptional  RNA processing , regulating RNA polymerase II activity.  Nrd1  is a protein that is responsible for many of the transcriptional activities of RNAP II, specifically through the  Nrd1 - dependent termination pathway. [ 12 ]  This pathway requires the parvulin Ess1, or Pin1 depending on the organism, to isomerize the pSer5-Pro6 bond in the CTD. Without the  cis  conformation of the pSer5-Pro6 bond, created by Ess1/Pin1, Nrd1 cannot bind to RNAP II. Any variation from this process leads to a decrease in Nrd1 binding affinity, lowering the ability of RNAP II to process and degrade  noncoding RNAs .


Cyp33 in mammals causes isomerization in  MLL1 . [ 13 ]  MLL1 is a multiprotein complex that regulates gene expression and chromosomal translocations involving this gene often lead to leukemia. [ 10 ]  MLL's target genes include HOXC8, HOXA9, CDKN1B, and C-MYC. MLL also has two binding domains: a Cyp33  RNA-recognition motif  domain (RRM), and a  PHD3  domain that binds to H3K4me3 or Cyp33 RRM. Cyp33 has the ability to downregulate the expression of these genes through proline isomerization at the peptide bond between His1628 and Pro1629 within MLL. [ 13 ]  This bond lies in a sequence between the PHD3 finger of MLL1 and the bromeodomain of MLL1, and its isomerization mediates the bonding of the PHD3 domain and the Cyp33 RRM domain. When these two domains are bonded transcription is repressed through recruitment of histone deacetylases to MLL1 and inhibition of H3K4me3. [ 9 ] [ 13 ]


Phosphorylated amino acids are crucial for the modulation of the binding of  transcription factors  and other gene  regulatory proteins . Pin1's effect on isomerization of proline residues leads to an increase or decrease in recruitment of phosphatases, namely Scp1 and Ssu72 and their recruitment to the RNAP II CTD. [ 13 ]  The  cis- Pro formation is associated with an increase in Ssu72. Scp1 on recognizes  trans -Pro formations, and is not affected by such isomerization. Pin1 also triggers the activation of the  DSIF  complex and  NELF , which are responsible for pausing RNAP II in mammalian cells, and their conversion into positive elongation factors, facilitating elongation. [ 9 ]  This potentially could be an isomerization dependent process.


Pin1 , a parvulin, regulates  mRNA stability  and expression in certain eukaryotics mRNAs. [ 13 ]  These mRNAs are GM-CSF, P th , and TGFβ and each of them have AREs, or  AU-rich cis-elements . The ARE binding protein KSRP has a Pin1 binding site. Pin1 binds to this site and dephosphorylates the serine and isomerizes the peptide bond between Ser181 and Pro182. This isomerization causes the decay of P th  mRNA. KSRP, and other ARE binding proteins like AUF1, are thought to affect the other mRNAs through mechanisms similar to P th , with the requirement of a phosphorylated serine bonded to a proline in a specific conformation. Pin1 also triggers proline isomerization of  Stem-Loop Binding Protein (SLBP) , allowing it to control the dissociation of  SLBP  from histone mRNA. This leads to Pin1 being able to affect histone mRNA decay. Pin1 affects many other genes in the form of gene silencing through the disruption of cell pathways, making it important in mRNA turnover by modulating RNA binding protein activity.


Currently there are no existing compounds that can mimic the peptide bond of proline to other amino acids while maintaining only a  cis  or  trans  configuration because most mimics found will eventually change from one isomer to another. This makes research on the direct effect of each of the isomers on biological mechanisms more difficult. [ 14 ]  Also, the actual isomerization of proline is a slow process, meaning that any studying of the effects of the different isomers of proline takes a large amount of time to complete. [ 15 ]


Protein methylation  is a type of  post-translational modification  featuring the addition of  methyl groups  to  proteins . It can occur on the nitrogen-containing side-chains of  arginine  and  lysine , [ 1 ] [ 2 ]  but also at the amino- and carboxy-termini of a number of different proteins. In biology,  methyltransferases  catalyze the methylation process, activated primarily by  S-adenosylmethionine .
Protein  methylation  has been most studied in  histones , where the transfer of methyl groups from S-adenosyl methionine is catalyzed by  histone methyltransferases . Histones that are methylated on certain residues can act  epigenetically  to repress or activate gene expression. [ 3 ] [ 4 ]


Multiple sites of proteins can be methylated. For some types of methylation, such as N-terminal methylation and prenylcysteine methylation, additional processing is required, whereas other types of methylation such as arginine methylation and lysine methylation do not require pre-processing.


Arginine can be methylated once (monomethylated arginine) or twice (dimethylated arginine). Methylation of arginine residues is catalyzed by three different classes of protein arginine methyltransferases (PRMTs):  Type I PRMTs (PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8) attach two methyl groups to a single terminal nitrogen atom, producing  asymmetric dimethylarginine  (N G,N G-dimethylarginine). In contrast, type II PRMTs (PRMT5 and PRMT9) catalyze the formation of symmetric dimethylarginine with one methyl group on each terminal nitrogen (symmetric N G,N' G-dimethylarginine). Type I and II PRMTs both generate N G-monomethylarginine intermediates; PRMT7, the only known type III PRMT, produces only monomethylated arginine.  [ 5 ]


Arginine-methylation usually occurs at glycine and arginine-rich regions referred to as "GAR motifs", [ 6 ]  which is likely due to the enhanced flexibility of these regions that enables insertion of arginine into the PRMT active site. Nevertheless, PRMTs with non-GAR consensus sequences exist. [ 5 ]  PRMTs are present in the nucleus as well as in the cytoplasm. In interactions of proteins with nucleic acids, arginine residues are important hydrogen bond donors for the phosphate backbone — many arginine-methylated proteins have been found to interact with DNA or RNA. [ 6 ] [ 7 ]


Enzymes that facilitate  histone  acetylation  [ citation needed ]  as well as histones themselves can be arginine methylated. Arginine methylation affects the interactions between proteins and has been implicated in a variety of cellular processes, including protein trafficking, signal transduction and transcriptional regulation. [ 6 ]  In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial. [ 5 ]


Lysine can be methylated once, twice, or three times by lysine methyltransferases (PKMTs). [ 8 ]  Most lysine methyltransferases contain an evolutionarily conserved  SET domain , which possesses  S-adenosylmethionine -dependent methyltransferase activity, but are structurally distinct from other S-adenosylmethionine binding proteins. Lysine methylation plays a central part in how histones interact with proteins. [ 9 ]  Lysine methylation can be reverted by lysine  demethylases  (PKDMs). [ 8 ]


Different SET domain-containing proteins possess distinct substrate specificities. For example, SET1, SET7 and MLL methylate lysine 4 of histone H3, whereas Suv39h1, ESET and G9a specifically methylate lysine 9 of histone H3. Methylation at lysine 4 and lysine 9 are mutually exclusive and the epigenetic consequences of site-specific methylation are diametrically opposed: Methylation at lysine 4 correlates with an active state of transcription, whereas methylation at lysine 9 is associated with transcriptional repression and heterochromatin. Other lysine residues on  histone H3  and  histone H4  are also important sites of methylation by specific SET domain-containing enzymes. Although histones are the prime target of lysine methyltransferases, other cellular proteins carry N-methyllysine residues, including elongation factor 1A and the calcium sensing protein  calmodulin . [ 9 ]


Many eukaryotic proteins are post-translationally modified on their N-terminus. A common form of N-terminal modification is N-terminal methylation (Nt-methylation) by N-terminal methyltransferases (NTMTs). Proteins containing the consensus motif H 2 N-X-Pro-Lys- (where X can be Ala, Pro or Ser) after removal of the initiator methionine (iMet) can be subject to N-terminal α-amino-methylation. [ 10 ]  Monomethylation may have slight effects on α-amino nitrogen nucleophilicity and basicity, whereas trimethylation (or dimethylation in case of proline) will result in abolition of nucleophilicity and a permanent positive charge on the N-terminal amino group. Although from a biochemical point of view  demethylation of amines  is possible, Nt-methylation is considered irreversible as no N-terminal demethylase has been described to date. [ 10 ]
Histone variants  CENP-A  and  CENP-B  have been found to be Nt-methylated in vivo. [ 10 ]


Eukaryotic  proteins with C-termini that end in a CAAX motif are often subjected to a series of posttranslational modifications. The CAAX-tail processing takes place in three steps: First, a  prenyl lipid anchor is attached  to the cysteine through a  thioester  linkage. Then endoproteolysis occurs to remove the last three amino acids of the protein to expose the prenylcysteine α-COOH group. Finally, the exposed prenylcysteine group is methylated. The importance of this modification can be seen in targeted disruption of the methyltransferase for mouse CAAX proteins, where loss of isoprenylcysteine carboxyl methyltransferase resulted in mid-gestation lethality. [ 11 ]


The biological function of prenylcysteine methylation is to facilitate the targeting of CAAX proteins to membrane surfaces within cells. Prenylcysteine can be demethylated and this reverse reaction is catalyzed by isoprenylcysteine carboxyl methylesterases. CAAX box containing proteins that are prenylcysteine methylated include  Ras , GTP-binding proteins,  nuclear lamins  and certain  protein kinases .  Many of these proteins participate in cell signaling, and they utilize prenylcysteine methylation to concentrate them on the cytosolic surface of the plasma membrane where they are functional. [ 11 ]


Methylations on the C-terminus can increase a protein's chemical repertoire [ 12 ]  and are known to have a major effect on the functions of a protein. [ 1 ]


In eukaryotic cells,  phosphatases  catalyze the removal of phosphate groups from tyrosine, serine and threonine phosphoproteins. The catalytic subunit of the major serine/threonine phosphatases, like  Protein phosphatase 2  is covalently modified by the reversible methylation of its C-terminus to form a  leucine  carboxy methyl ester. Unlike CAAX motif methylation, no C-terminal processing is required to facilitate methylation. This C-terminal methylation event regulates the recruitment of regulatory proteins into complexes through the stimulation of protein–protein interactions, thus indirectly regulating the activity of the serine-threonine phosphatases complex. [ 13 ]  Methylation is catalyzed by a unique protein phosphatase methyltransferase.  The methyl group is removed by a specific protein phosphatase methylesterase. These two opposed enzymes make serine-threonine phosphatases methylation a dynamic process in response to stimuli. [ 13 ]


Damaged proteins accumulate  isoaspartyl  which causes protein instability, loss of biological activity and stimulation of autoimmune responses. The spontaneous age-dependent degradation of L-aspartyl residues results in the formation of a succinimidyl intermediate, a  succinimide  radical. This is spontaneously hydrolyzed either back to L-aspartyl or, in a more favorable reaction, to abnormal L-isoaspartyl. A methyltransferase dependent pathway exists for the conversion of L-isoaspartyl back to L-aspartyl. To prevent the accumulation of L-isoaspartyl, this residue is methylated by the protein  L-isoaspartyl methyltransferase , which catalyzes the formation of a methyl ester, which in turn is converted back to a succinimidyl intermediate. [ 14 ]
Loss and gain of function mutations have unmasked the biological importance of the L-isoaspartyl O-methyltransferase in age-related processes: Mice lacking the enzyme die young of fatal epilepsy, whereas flies engineered to over-express it have an increase in life span of over 30%. [ 14 ]


A common theme with methylated proteins, as with phosphorylated proteins, is the role this modification plays in the regulation of  protein–protein interactions . The arginine methylation of proteins can either inhibit or promote protein–protein interactions depending on the type of methylation. The asymmetric dimethylation of arginine residues in close proximity to proline-rich motifs can inhibit the binding to  SH3 domains . [ 15 ]   The opposite effect is seen with interactions between the survival of motor neurons protein and the snRNP proteins SmD1, SmD3 and SmB/B', where binding is promoted by symmetric dimethylation of arginine residues in the snRNP proteins. [ 16 ]


A well-characterized example of a methylation dependent protein–protein interaction is related to the selective methylation of lysine 9, by  SUV39H1  on the N-terminal tail of the  histone H3 . [ 9 ]  Di- and tri-methylation of this lysine residue facilitates the binding of  heterochromatin protein 1  (HP1). Because HP1 and Suv39h1 interact, it is thought the binding of HP1 to histone H3 is maintained and even allowed that to spread along the chromatin. The HP1 protein harbors a  chromodomain  which is responsible for the methyl-dependent interaction between it and lysine 9 of histone H3. It is likely that additional chromodomain-containing proteins will bind the same site as HP1, and to other lysine methylated positions on histones H3 and  Histone H4 . [ 13 ]


C-terminal protein methylation regulates the assembly of protein phosphatase. Methylation of the  protein phosphatase 2A  catalytic subunit enhances the binding of the regulatory B subunit and facilitates holoenzyme assembly. [ 13 ]


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.


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 ).


In humans, about 70% of  promoters  located near the  transcription  start site of a gene (proximal promoters) contain a  CpG island . [ 1 ] [ 2 ]   CpG islands are generally 200 to 2000 base pairs long, have a C:G  base pair  content >50%, and have regions of  DNA  where a  cytosine   nucleotide  is followed by a  guanine  nucleotide and this occurs frequently in the linear  sequence  of  bases  along its  5′ → 3′ direction . [ 3 ] [ 4 ]


Genes may also have distant promoters (distal promoters) and these frequently contain CpG islands as well.  An example is the promoter of the DNA repair gene  ERCC1 , where the CpG island-containing promoter is located about 5,400 nucleotides upstream of the coding region of the  ERCC1  gene. [ 5 ]   CpG islands also occur frequently in promoters for  functional noncoding RNAs  such as  microRNAs . [ 6 ]


In humans,  DNA methylation  occurs at the 5′ position of the  pyrimidine  ring of the cytosine residues within  CpG sites  to form  5-methylcytosines .  The presence of multiple methylated CpG sites in CpG islands of promoters causes stable inhibition (silencing) of genes. [ 7 ]   Silencing of transcription of a gene may be initiated by other mechanisms, but this is often followed by methylation of CpG sites in the promoter CpG island to cause the stable silencing of the gene. [ 7 ]


In cancers, loss of  expression of genes  occurs about 10 times more frequently by transcription silencing (caused by promoter hypermethylation of CpG islands) than by mutations.  As Vogelstein et al. point out, in a colorectal cancer there are usually about 3 to 6  driver  mutations and 33 to 66  hitchhiker  or passenger mutations. [ 8 ]   In contrast, in colon tumors compared to adjacent normal-appearing colonic mucosa, there are about 600 to 800 heavily methylated CpG islands in promoters of genes in the tumors while these CpG islands are not methylated in the adjacent mucosa. [ 9 ] [ 10 ] [ 11 ]


Using  gene set enrichment  analysis, 569 out of 938  gene sets  were hypermethylated and 369 were hypomethylated in cancers. Hypomethylation of CpG islands in promoters results in increased transcription of the genes or gene sets affected. [ 11 ]


One study [ 12 ]  listed 147 specific genes with colon cancer-associated hypermethylated promoters and 27 with hypomethylated promoters, along with the frequency with which these hyper/hypo-methylations were found in colon cancers.  At least 10 of those genes had hypermethylated promoters in nearly 100% of colon cancers.  They also indicated 11  microRNAs  whose promoters were hypermethylated in colon cancers at frequencies between 50% and 100% of cancers.  MicroRNAs (miRNAs) are small endogenous RNAs that pair with sequences in  messenger RNAs  to direct  post-transcriptional repression .  On average, each microRNA represses or inhibits transcriptional expression of several hundred target genes. Thus microRNAs with hypermethylated promoters may be allowing enhanced transcription of hundreds to thousands of genes in a cancer. [ 13 ]


For more than 20 years,  microRNAs  have been known to act in the cytoplasm to degrade transcriptional expression of specific target gene  messenger RNAs  (see  microRNA history ).  However, recently, Gagnon et al. [ 14 ]  showed that as many as 75% of microRNAs may be shuttled back into the nucleus of cells.  Some nuclear microRNAs have been shown to mediate transcriptional gene activation or transcriptional gene inhibition. [ 15 ]


DNA repair genes are frequently repressed in cancers due to hypermethylation of CpG islands within their promoters.  In  head and neck squamous cell carcinomas  at least 15 DNA repair genes have frequently hypermethylated promoters; these genes are  XRCC1, MLH3, PMS1, RAD51B, XRCC3, RAD54B, BRCA1, SHFM1, GEN1, FANCE, FAAP20, SPRTN, SETMAR, HUS1,  and  PER1 . [ 16 ]   About seventeen types of cancer are frequently deficient in one or more DNA repair genes due to hypermethylation of their promoters. [ 17 ]   As summarized in one review article, promoter hypermethylation of the DNA repair gene  MGMT  occurs in 93% of bladder cancers, 88% of stomach cancers, 74% of thyroid cancers, 40%-90% of colorectal cancers and 50% of brain cancers. [ citation needed ]   Promoter hypermethylation of  LIG4  occurs in 82% of colorectal cancers.  This review article also indicates promoter hypermethylation of  NEIL1  occurs in 62% of  head and neck cancers  and in 42% of  non-small-cell lung cancers ; promoter hypermetylation of  ATM  occurs in 47% of  non-small-cell lung cancers ; promoter hypermethylation of  MLH1  occurs in 48% of  squamous cell carcinomas; and promoter hypermethylation of  FANCB  occurs in 46% of  head and neck cancers . [ citation needed ]


On the other hand, the promoters of two genes,  PARP1  and  FEN1 , were hypomethylated and these genes were over-expressed in numerous cancers.   PARP1  and  FEN1  are essential genes in the error-prone and mutagenic DNA repair pathway  microhomology-mediated end joining .  If this pathway is over-expressed, the excess mutations it causes can lead to cancer.   PARP1  is over-expressed in tyrosine kinase-activated leukemias, [ 18 ]  in neuroblastoma, [ 19 ]  in testicular and other germ cell tumors, [ 20 ]  and in Ewing's sarcoma, [ 21 ]   FEN1  is over-expressed in the majority of cancers of the breast, [ 22 ]  prostate, [ 23 ]  stomach, [ 24 ] [ 25 ]  neuroblastomas, [ 26 ]  pancreatic, [ 27 ]  and lung. [ 28 ]


DNA damage appears to be the primary underlying cause of cancer. [ 29 ] [ 30 ]   If accurate DNA repair is deficient, DNA damages tend to accumulate. Such excess DNA damage can increase  mutational  errors during  DNA replication  due to error-prone  translesion synthesis . Excess DNA damage can also increase  epigenetic  alterations due to errors during DNA repair. Such mutations and epigenetic alterations can give rise to  cancer  (see  malignant neoplasms ).  Thus, CpG island hyper/hypo-methylation in the promoters of DNA repair genes are likely central to progression to cancer. [ 31 ] [ 32 ]


In biology,  reprogramming  refers to erasure and remodeling of  epigenetic  marks, such as  DNA methylation , during mammalian development or in cell culture. [ 1 ]  Such control is also often associated with alternative covalent modifications of  histones .


Reprogrammings that are both large scale (10% to 100% of epigenetic marks) and rapid (hours to a few days) occur at three life stages of mammals.  Almost 100% of epigenetic marks are reprogrammed in two short periods early in development after  fertilization  of an  ovum  by a  sperm .  In addition, almost 10% of  DNA methylations  in  neurons  of the hippocampus can be rapidly altered during formation of a strong fear memory.


After fertilization in mammals,  DNA methylation  patterns are largely erased and then re-established during early embryonic development. Almost all of the methylations from the parents are erased, first during early  embryogenesis , and again in  gametogenesis , with demethylation and remethylation occurring each time.  Demethylation during early embryogenesis occurs in the preimplantation period.  After a sperm fertilizes an  ovum  to form a  zygote , rapid  DNA demethylation  of the paternal DNA and slower demethylation of the maternal DNA occurs until formation of a  morula , which has almost no methylation.  After the  blastocyst  is formed, methylation can begin, and with formation of the  epiblast  a wave of methylation then takes place until the  implantation  stage of the embryo.  Another period of rapid and almost complete demethylation occurs during gametogenesis within the primordial  germ cells  (PGCs).  Other than the PGCs, in the post-implantation stage, methylation patterns in somatic cells are stage- and  tissue -specific with changes that presumably define each individual  cell type  and last stably over a long time. [ 2 ]


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).  In the mature  oocyte , about 40% of its CpG sites are methylated.  Demethylation of the maternal chromosome largely takes place by blockage of the methylating  enzymes  from acting on maternal-origin DNA and 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).  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. [ 3 ]   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 . Now the cells are rapidly proliferating and beginning demethylation in two waves.  In the first wave, demethylation is by replicative dilution, but in the second wave demethylation is by an active process. The second wave leads to demethylation of specific  loci . At this point the PGC genomes display the lowest levels of DNA methylation of any cells in the entire  life cycle  [at embryonic day 13.5 (E13.5), see the second figure in this section]. [ 4 ]


After fertilization some cells of the newly formed embryo migrate to the germinal ridge and will eventually become the  germ cells  (sperm and oocytes) of the next generation. 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 the process of  gametogenesis  the 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 demethylated in order to erase their epigenetic signatures and acquire  totipotency . There is asymmetry at this point: the male pronucleus undergoes a quick and active demethylation.  Meanwhile the female pronucleus is demethylated passively during consecutive cell divisions.  The process of  DNA demethylation  involves  base excision repair  and likely other DNA-repair-based mechanisms. [ 5 ]    Despite the global nature of this process, there are certain sequences that avoid it, such as  differentially methylated regions  (DMRS) associated with imprinted genes,  retrotransposons  and  centromeric   heterochromatin . Remethylation is needed again to differentiate the embryo into a complete organism. [ 6 ]


In vitro  manipulation of pre-implantation embryos has been shown to disrupt methylation patterns at imprinted loci [ 7 ]  and plays a crucial role in cloned animals. [ 8 ]


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 h after training, 9.17% of the genes in the rat genomes of  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. [ 9 ]   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 1 day after conditioning, but rats retain a considerable amount of contextual fear when a long delay (28 days) is imposed between the time of conditioning and the time of hippocampectomy. [ 10 ]


Three molecular stages are required for 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.


The Figure in this section indicates the central roles of ten-eleven translocation  methylcytosine dioxygenases (TETs)  in the demethylation of 5-methylcytosine to form cytosine. [ 12 ]   As reviewed in 2018, [ 12 ]  5mC is very often initially oxidized by TET dioxygenases to generate  5-hydroxymethylcytosine  (5hmC). 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). In an alternative oxidative deamination pathway, 5hmC can be oxidatively deaminated by  APOBEC  (AID/APOBEC) deaminases to form 5-hydroxymethyluracil (5hmU) or 5mC can be converted to  thymine  (Thy). 5hmU can be cleaved by TDG,  SMUG1 ,  NEIL1 , or  MBD4 . AP sites and T:G mismatches are then repaired by base excision repair (BER) enzymes to yield cytosine (Cyt).


The isoforms of the  TET enzymes  include at least two isoforms of TET1, one of  TET2  and three isoforms of  TET3 . [ 13 ] [ 14 ]  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 was 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. [ 15 ]  TET2 does not have an affinity for 5-methylcytosine in DNA. [ 16 ]  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 . [ 14 ]


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 demthylation) [ 17 ]  and  EGR1 . [ 18 ]


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. [ 19 ]   OGG1 very rapidly finds 8-OHdG.  OGG1 proteins bind to oxidatively damaged DNA with a half maximum time of about 6 seconds. [ 20 ]   When OGG1 finds 8-OHdG it changes conformation and complexes with 8-OHdG in the binding pocket of OGG1. [ 21 ]   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 , [ 22 ]  or about 11 minutes in the livers of  irradiated  mice. [ 23 ]   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. [ 24 ]   TET1 binds (is recruited to) the OGG1 bound to 8-OHdG (see figure). [ 17 ]   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 large scale demethylation of 5-methylcytosine to about 20% of its initial level in DNA. [ 17 ]


The gene  early growth response protein 1  ( EGR1 ) is an  immediate early gene  (IEG).  The defining characteristic of IEGs is the rapid and transient up-regulation—within minutes—of their mRNA levels independent of protein synthesis. [ 25 ]   EGR1 can rapidly be induced by neuronal activity. [ 26 ]   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 . [ 25 ]   This expression is linked to control of cognition, emotional response, social behavior and sensitivity to reward. [ 25 ]   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. [ 26 ]   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. [ 26 ]   EGR1 recruits TET1s to genomic regions flanking EGR1 binding sites. [ 26 ]  In the presence of EGR1, TET1s is capable of locus-specific demethylation and activation of the expression of downstream genes regulated by EGR1. [ 26 ]


The first person to successfully demonstrate reprogramming was  John Gurdon , who in 1962 demonstrated that differentiated somatic cells could be reprogrammed back into an embryonic state when he managed to obtain swimming tadpoles following the transfer of differentiated intestinal epithelial cells into enucleated frog eggs. [ 27 ]  For this achievement he received the 2012  Nobel Prize in Medicine  alongside  Shinya Yamanaka . [ 28 ]   Yamanaka was the first to demonstrate (in 2006) that this somatic cell nuclear transfer or oocyte-based reprogramming process (see below), that Gurdon discovered, could be recapitulated (in mice) by defined factors ( Oct4 ,  Sox2 ,  Klf4 , and  c-Myc ) to generate  induced pluripotent stem cells  (iPSCs). [ 29 ]  Other combinations of genes have also been used, including LIN25 [ 30 ]  and  Homeobox protein NANOG . [ 30 ] [ 31 ]


With the discovery that cell fate could be altered, the question of what progression of events occurs signifies a cell undergoing reprogramming. As the final product of iPSC reprogramming was similar in  morphology , proliferation,  gene expression ,  pluripotency , and  telomerase  activity, genetic and morphological markers were used as a way to determine what phase of reprogramming was occurring. [ 32 ]  Reprogramming is defined into three phase: initiation, maturation, and stabilization. [ 33 ]


The initiation phase is associated with the downregulation of cell type specific genes and the upregulation of pluripotent genes. [ 33 ]  As the cells move towards pluripotency, the  telomerase  activity is reactivated to extend  telomeres . The cell morphology can directly affect the reprogramming process as the cell is modifying itself to prepare for the gene expression of pluripotency. [ 34 ]  The main indicator that the initiation phase has completed is that the first genes associated with pluripotency are expressed. This includes the expression of  Oct-4  or  Homeobox protein NANOG , while undergoing a  mesenchymal–epithelial transition  (MET), and the loss of  apoptosis  and  senescence . [ 35 ]


If the cell is directly reprogrammed from one  somatic cell  to another, the genes associated with each cell type begin to be upregulated and downregulated accordingly. [ 33 ]  This can either occur through direct cell reprogramming or creating an intermediate, such as a iPSC, and differentiating into the desired cell type. [ 35 ]


The initiation phase is completed through one of three pathways:  nuclear transfer ,  cell fusion , or defined factors ( microRNA ,  transcription factor , epigenetic markers, and other small molecules). [ 30 ] [ 35 ]


An  oocyte  can reprogram an adult nucleus into an embryonic state after  somatic cell nuclear transfer , so that a new organism can be developed from such cell. [ 36 ]


Reprogramming is distinct from development of a  somatic epitype , [ 37 ]  as somatic epitypes can potentially be altered after an organism has left the developmental stage of life. [ 38 ]   During somatic cell nuclear transfer, the oocyte turns off tissue specific genes in the somatic cell nucleus and  turns back on embryonic specific genes. This process has been shown through cloning, as seen through  John Gurdon  with the tadpoles [ 27 ]  and  Dolly the Sheep . [ 39 ]  Notably, these events have shown that cell fate is a reversible process.


[ 35 ]
 Cell fusion  is used to create a multi nucleated cell called a  heterokaryon . [ 35 ]  The fused cells allow for otherwise silenced genes to become reactivated and expressive. As the genes are reactivated, the cells can re-differentiate. There are instances where transcriptional factors, such as the Yamanaka factors, are still needed to aid in  heterokaryon  cell reprogramming. [ 40 ]


Unlike nuclear transfer and cell fusion, defined factors do not require a full genome, only reprogramming factors. These reprogramming factors include  microRNA ,  transcription factor , epigenetic markers, and other small molecules. [ 35 ]  The original transcription factors, that lead to iPSC development, discovered by Yamanaka include  Oct4 ,  Sox2 ,  Klf4 , and  c-Myc  (OSKM factors). [ 29 ] [ 32 ]  Although the OSKM factors have been shown to induce and aid in pluripotency, other transcription factors such as  Homeobox protein NANOG , [ 41 ]  LIN25, [ 30 ]  TRA-1-60, [ 41 ]  and C/EBPα [ 42 ]  aid in the efficiency of reprogramming. The use of  microRNA  and other small molecule-driven processes has been utilized as a means of increasing the efficiency of the differentiation from somatic cells to pluripotency. [ 35 ]


The maturation phase begins at the end of the initiation phase, when the first pluripotent genes are  expressed. [ 33 ]  The cell is preparing itself to be independent from the defined factors, that started the reprogramming process. The first genes to be detected in iPSCs are  Oct4 ,  Homeobox protein NANOG , and Esrrb, followed later by  Sox2 . [ 35 ]  In the later stages of maturation,  transgene  silencing marks the start of the cell becoming independent from the induced  transcription factor . Once the cell is independent, the maturation phase ends and the stabilization phase begins.


As reprogramming efficiency has proven to be a variable and low efficiency process, not all the cells complete the maturation phase and achieve  pluripotency . [ 42 ]  Some cells that undergo reprogramming still remain under  apoptosis  at the beginning of the maturation stage from  oxidative stress  brought on by the stresses of gene expression change. The use of  microRNA , proteins, and different combinations of the OSKM factors have started to lead towards a higher efficiency rate of reprogramming.


The stabilization phase refers to the processes in the cell that occur after the cell reaches  pluripotency . One genetic marker is the expression of  Sox2  and  X chromosome   reactivation , while epigenetic changes include the  telomerase  extending the  telomeres [ 30 ]  and loss of the cell’s epigenetic memory. [ 33 ]  The epigenetic memory of a cell is reset by the changes in DNA methylation, [ 43 ]  using  activation-induced cytidine deaminase  (AID),  TET enzymes  (TET), and  DNA methyltransferase  (DMNTs), starting in the maturation phase and into the stabilization stage. [ 33 ]  Once the epigenetic memory of the cell is lost, the possibility of differentiation into the three germ layers is achieved. [ 32 ]  This is considered a fully reprogrammed cell as it can be passaged without reverting to its original somatic cell type. [ 35 ]


Reprogramming can also be induced artificially through the introduction of exogenous factors, usually  transcription factors . In this context, it often refers to the creation of  induced pluripotent stem cells  from mature cells such as adult  fibroblasts . This allows the production of  stem cells  for  biomedical research , such as research into  stem cell therapies , without the use of embryos. It is carried out by the  transfection  of stem-cell associated genes into mature cells using  viral vectors  such as  retroviruses .


One of the first transacting factors discovered to change a cell was found in a myoblast when the  complementary DNA  (cDNA) coding for  MyoD  was expressed and converted a  fibroblast  to a myoblast. Another transacting factor that directly transformed a  lymphoid cell  into a  myeloid cell  was C/EBPα. MyoD and C/EBPα are examples of a small number of single factors that can transform cells. More often, a combination of transcription factors work in conjunction to reprogram a cell.


The OSKM factors ( Oct4 ,  Sox2 ,  Klf4 , and  c-Myc ) were initially discovered by Yamanaka in 2006, by the induction of a mouse fibroblast into an  induced pluripotent stem cell  (iPSCs). [ 29 ]  Within the following year, these factors were used to induce human fibroblasts into iPSCs. [ 32 ]


Oct4  is part of the core regulatory genes needed for pluripotency, as it is seen in both  embryonic stem cells  and tumors. [ 44 ]  The use of Oct4 even in small increases allows for the start differentiation into pluripotency. Oct4 works in conjecture with Sox2 for the expression of  FGF4  which could aid in differentiation.


Sox2  is a gene used in maintaining pluripotency in stem cells. Oct4 and Sox2 work together to regulate hundreds of genes utilized in pluripotency. [ 44 ]  However, Sox2 is not the only possible Sox family member to participate in gene regulation with Oct4 –  Sox4 ,  Sox11 , and  Sox15  also participate, as the Sox protein is redundant throughout the stem cell  genome .


Klf4  is a transcription factor used in  proliferation ,  differentiation ,  apoptosis , and  somatic cell  reprogramming. When being utilized in cellular reprogramming, Klf4 prevents cell division of damaged cells using its apoptotic ability, and aids in  histone acetyltransferase  activity. [ 32 ]


c-Myc  is also known as an  oncogene , and in certain conditions can become cancer causing. [ 45 ]  In cellular reprogramming, c-Myc is used for  cell cycle  progression,  apoptosis , and cellular transformation for further differentiation.


Homeobox protein NANOG  (NANOG) is a transcription factor used to aid in the efficiency of generating iPSCs by maintaining  pluripotency [ 46 ]  and suppressing  cell determination factors . [ 47 ]  NANOG works by promoting  chromatin  accessibility through repression of  histone  markers, such as  H3K27me3 . NANOG aids recruitment of  Oct4 ,  Sox2 , and Esrrb used in  transcription , while also recruiting  Brahma-related gene-1  (BRG1) for  chromatin  accessibility.


CEBPA  is a commonly used factor when reprogramming cells into not only iPSCs, but also other cells. C/EBPα has shown itself to be a single transacting factor during direct reprogramming of a lymphoid cell into a myeloid cell. [ 42 ]  C/EBPα is considered a 'path breaker' to aid in preparing the cell for intake of the OSKM factors and specific transcription events. [ 41 ]  C/EBPα has also been shown to increase the efficiency of the reprogramming events. [ 33 ]


The properties of cells obtained after reprogramming can vary significantly, in particular among iPSCs. [ 48 ]   Factors leading to variation in the performance of reprogramming and functional features of end products include genetic background, tissue source, reprogramming factor stoichiometry and stressors related to cell culture. [ 48 ]


A  sex-determination system  is a  biological  system that determines the development of  sexual characteristics  in an  organism . [ 1 ]  Most  organisms  that create their  offspring  using  sexual reproduction  have two common sexes,  males  and  females , and in other species, there are  hermaphrodites , organisms that can function reproductively as either female or male, or both. [ 2 ]


There are also some species in which only one sex is present, temporarily or permanently. This can be due to  parthenogenesis , the act of a female reproducing without  fertilization . In some plants or algae the  gametophyte  stage may reproduce itself, thus producing more individuals of the same sex as the parent.