DNA methylation's critical nature to  corticogenesis  has been shown through knockout experiments in mice. When  DNMT 3b and DNMT1 were  ablated  separately in mouse embryos they died due to impairment of  neural tube  development. DNMT3a silencing did not cause embryonic lethality, but did result in a severe detriment in postnatal neurogenesis. [ 1 ] [ 3 ]  This is largely due to the timing in which these epigenetic mechanisms are active. DNMT3b is expressed in early neural progenitor cells and decrease as neural development proceeds and DNMT3a is barely detectable up until embryonic day 10 (E.10). However, at E.10, DNMT3a expression increases significantly from E13.5 and well into adulthood. In the postnatal forebrain, DNMT3a is expressed in the subventricular zone (SVZ) and the hippocampal  dentate gyrus , the primary locations for adult neurogenesis. [ 1 ] [ 2 ]  The loss of DNMT3a in post natal neural progenitor cells leads to the down-regulation of neuronic genes such as  Dlx2 ,  Neurog2 , and  Sp8 ; but upregulation of genes involved in  astroglial  and  oligodendroglial  differentiation, indicating a role in the cell-fate switch from neurogenesis to  gliogenesis . DNA demethylation, as well as methylation, of certain genes allows for neurogenesis to proceed in a time dependent manner. One such gene is  Hes5 , hypermethylated in E7.5 Embryos but completely demethylated by E9.5, which is one of the target genes in the  Notch Signaling  pathway. GCM1 and GCM2 demethylate the  Hes5  promoter, allowing it to respond to NOTCH signaling and initiating the generation of neural stem cells. [ 1 ]  Another example is the  Gfap  gene which is required for astrocyte differentiation. The ability to differentiate into glial cells is repressed in neural stem cells with a neuronal cell fate. This repression is due largely to an irresponsiveness of neural stem cells towards astrocyte-inducing stimulations. The neural stem cells are non-responsive due to hypermethylated DNA in the promoter regions of astrocyte genes such as  Gfap . The STAT3 binding site in the promoter region of  Gfap   is hypermethylated at E11.5 and barely so at E14.5, at which point it is able to receive astrocyte inducing stimulations and begin cytokine-inducible astrocyte differentiation. [ 3 ]


Studies done by De Pietri Tonelli and Kawase-Koga have shown  conditional knockout  of  Dicer , an enzyme largely used for miRNA synthesis, in mouse  neocortex  resulted in reduced cortical size, increased neuronal apoptosis, and deficient cortisol layering. Neuroepithelial cells and neuroprogenitor cells were not affected until E.14, at which point they also underwent apoptosis. This doesn't show which miRNAs were responsible for the varying factors affected, but it does show that there is a stage-specific requirement for miRNA expression in cortical development. [ 1 ] [ 5 ] [ 6 ] [ 7 ]   miR-124 , the most abundant microRNA in the central nervous system, controls the lineage progression of  subventricular zone  neural progenitor cells into neuroblasts by suppressing protein production by targeting  Sox9 . Another major microRNA player is miR-9/9*. In embryonic neurogenesis miR-9 has been shown to regulate neuronal differentiation and self-renewal. [ 1 ] [ 4 ] [ 5 ]  Ectopic expression of miR-9 in the developing mouse cortex led to premature neuronal differentiation and disrupted the migration of new neurons through targeting  Foxg1 . [ 1 ]


Contrary to the idea that microRNAs are only fine-tuning mechanisms, recent studies have shown that miR-9 and miR-124 can act together to guide  fibroblasts  into neural cells. Transcription factors and regulatory genes, such as  Neurod1 ,  Ascr1 , and  Myt1l , which were previously thought to be responsible for this phenomenon did not transform human fibroblasts in the absence of miR-9 and miR-124, but in the presence of the microRNAs and the absence of the transcription factors human fibroblast transformation proceeded, albeit in a less efficient manner. [ 1 ] [ 4 ] [ 5 ]


Neurogenesis continues after development well through adulthood. [ 2 ]   Growth arrest and DNA-damage-inducible, beta  (GADD45b) is required for the demethylation of promoters of critical genes responsible for new-born neuron development such as  brain-derived neurotrophic factor  (BDNF) and  basic fibroblast growth factor  (FGF2). [ 8 ]  As such, upregulation of GADD45b leads to increased demethylation, increased BDNF and FGF2, and ultimately more neural progenitor cells. [ 1 ] [ 8 ]


Histone  acetylation ,  deacetylation , as well as the inhibition of histone deacetylation mechanisms also play large roles in the proliferation and self-renewal of post-natal neural stem cells.  DNA , which encodes genes within the genome, including those involved in adult neurogenesis, is packaged into  chromatin . Chromatin itself is made up of  nucleosome  subunits, each consisting of two copies each of histone proteins  H2A ,  H2B ,  H3  and  H4 . One of the primary roles acetylation plays in the regulation of gene expression is through the inhibition of adjacent nucleosome interactions. When the H4 histones are not acetylated, they are basic in nature, and insert into the acidic pocket of the H2A-H2B protein dimers in adjacent nucleosomes, leading to tight association between nucleosomes and further packing of the chromatin. Thus, acetylation causes the H4 histone to lose its basicity, and prevents nucleosome cross-linking. [ 9 ]  This acetylation of histone tails additionally increase the affinity of  chromatin remodeling enzyme complexes  such as SWI–SNF and ISWI, which utilize ATP to produce nucleosome-free regions at promoter and enhancer sites. [ 10 ]  This allows for greater ability of recognition of these sites by transcription factors, particularly  TFIID  which is the major transcription factor involved in  transcription initiation . Furthermore, the acetylation of  lysine  residues on histone tails can be recognized by the  TAF1  component of TFIID, and when bound, TAF1 becomes a  histone acetyltransferase  (HAT), further acetylating adjacent H3 and H4 histones and recruiting more HATs in the process. [ 11 ]  DNA wraps around the histones of chromatin, and the acetylation of these histone tails leads to reduction of the positive charge associated with the histones. This results in the negatively-charged DNA to lose affinity for the histone, allowing for more space for  transcription factors  to bind promoter regions and further facilitate expression.


Eventually, the processes of histone acetylation and consequent chromatin remodeling allow for greater expression of target genes, including those involved in adult neurogenesis. The most prominently studied and well-understood regulators of chromatin remodeling, which play an important role in adult neurogenesis are histone acetyltransferases (HATs) and  histone deacetylases  (HDACs). HATs add acetyl groups to nucleosomes, while HDACs remove them. The acetylation of histones leads to decreased condensation of the nucleosomes to target DNA, and increases the likelihood that gene expression may occur by freeing up the DNA targets to bind to their respective transcriptional factors. This process is involved in neural proliferation regulation, as different neuronal cell genes are expressed and repressed. Deacetylation of histones leads to the reverse, and increases the likelihood for the repression of gene expression. [ 12 ]


HDAC inhibitors (HDACi), such as  valproic acid  (VPA) and  trichostatin A  can promote proliferation of adult neurogenesis through the inhibition of HDAC activity, inducing differentiation of adult progenitor cells. [ 12 ]  Neural-expressed HDACs interact with Tlx, an essential neural stem cell regulator, to suppress TLX target genes. This includes the cyclin-dependent kinase inhibitor P21 and the tumor suppressor gene Pten to promote neural stem cell proliferation. [ 1 ]  Inhibition of HDACs by the antiepileptic drug valproic acid induces neuronal differentiation as in embryonic neurogenesis, but also inhibits glial cell differentiation of adult neural stem cells. This is likely mediated through upregulation of neuronal specific genes such as the neurogeneic basic helix-loop-helix transcription factors NEUROD, NEUROGENENIN1, and Math1. Conditional loss of HDAC1 and HDAC2 in neural progenitor cells prevented them from differentiating into neurons and their loss in  oligodendrytic  progenitor cells disrupted oligodendrocyte formations, suggesting that histone deacetlyation plays important but varying roles in different stages of neuronal development. [ 1 ]


MicroRNAs ( miRNAs ) are small noncoding RNAs that play a significant role in eukaryotic epigenetic regulation. miRNAs function to modulate protein expression levels of their mRNA targets without affecting the sequences of the genes of interest. While miRNAs play a large role in the modulation of epigenetic mechanisms, they are also modified and regulated by other epigenetic factors including DNA methylation, histone modifications and other  RNA modifications . [ 13 ]  Together, miRNAs create an epigenetic feedback loop with other epigenetic factors to affect the expression levels of specific genes. A number of specific miRNAs have been implicated as agents of epigenetic regulation in adult neurogenesis. miR-9 targets the nuclear receptor  TLX  in adult neurogenesis to promote neural differentiation and inhibit neural stem cell proliferation. It also influences neuronal subtype specification and regulates axonal growth, branching, and targeting in the central nervous system through interactions with  HES1 , a neural stem cell homeostasis molecule. miR-124 promotes cell cycle exit and neuronal differentiation in adult neurogenesis. Mouse studies have shown that ectopic expression of miR-124 showed premature neural progenitor cell differentiation and exhaustion in the  subventricular zone .


In addition to miR-9 and miR-124, other miRNAs play essential roles in regulation of adult neurogenesis. miR-137, miR-184 and miR-195 regulate adult neural stem cell proliferation, with their over-expression leading to up-regulated proliferation while their down-regulation leads to a decrease in neuronal proliferation. [ 14 ]   Methyl-CpG  binding protein 1 ( MBD1 ) represses miR-184, which is a microRNA responsible for proliferation of adult neural stem/progenitor cells (aNSCs) along with the inhibition of differentiating these cells. miR-184 regulates embryonic brain development by binding to the mRNA for the Numblike (Numbl) protein and altering its expression. MBD1, Numbl, and miR-184 all work together to regulate the proliferation and differentiation of aNSCs. [ 15 ]  In addition, miR-195 works closely with MBD1 to regulate aNSC proliferation and differentiation. mIR-194 and MBD1 form a negative regulatory loop in aNSCs and work to repress the expression of each other. Inhibition of miR-195 promotes aNSC differentiation. Once differentiation has occurred, levels of miR-195 decreases. [ 16 ]


Astrocytes  are  glial  cells that forms the blood brain barrier, support synapses as well as guide axons. [ 17 ]  Unlike neurons, these specialized glial cells are able to alter their cell fate prior to reaching full maturation and “ dedifferentiate ,”  in large part due to epigenetic factors. This dedifferentiation allows astrocytes to potentially reach a different  cell fate  entirely, so long as this dedifferentiation occurs before complete maturation occurs, and can lead to their consequent differentiation and conversion from glial cells into neurons in the adult brain. [ 18 ]  Prior to complete maturation while dedifferentiation is still possible, the expression of genes Mash1,  NeuroG1  and  NeuroG2  can reprogram astrocytes into neurons. [ 19 ]  In addition to the expression of these genes in brain cells, multiple epigenetic factors play a role in these gene's expression patterns. An  up-regulation  of acetylation at H3K9 and H3K14 residues adjacent to both NeuroG1 and NeuroG2 genes has been shown to accompany astrocyte dedifferentiation, over-expression and/or forced expression of these genes can directly induce the differentiation of astrocytes.


In addition, silencing of methylation mechanisms, specifically the silencing of many classes of DNA methyltransferases, which themselves are involved in silencing expression, inhibits the  progenitor  cells of astrocytes from differentiating back to their original fate as glial cells. [ 20 ]  Despite this knowledge of the mechanism of methylation repression, the identity of these silenced genes is not yet fully known. While this overall repression of methylation is necessary to prevent expression of specific genes needed to allow an astrocyte to fully mature and reach an astrocytic cell fate, it was found that the over-expression of one specific histone methyltransferase, Ezh2, which catalyzes the tri-methylation of H3K27, represses genes needed for astrocyte maintenance, thus allowing the cell to retain its neural stem cell morphology. This demonstrates that differential methylation by distinct methyltransferases and their consequent repression or over-expression have differing roles in the dedifferentiation of astrocytes to form neurons. Furthermore, while not sufficient to induce astrocyte dedifferentiation alone, Ezh2 is necessary for astrocytes to dedifferentiate as they are inhibited from reaching complete maturity into their original cell fate. Once in this inhibited stage, expression of the gene NeuroD4 in these specified glial cells has been shown to lead to neuronal formation, and thus neurogenesis, from the dedifferentiated astrocytes in adult mammalian brains. [ 18 ]


The Growth Arrest and DNA Damage inducible 45 (Gadd45) gene family plays a large role in the hippocampus. Gadd45 facilitates hippocampal long-term potentiation and enhances persisting memory for motor performance, aversive conditioning, and spatial navigation. [ 21 ]  Additionally, DNA methylation has been shown to be important for activity-dependent modulation of adult neurogenesis in the hippocampus, which is mediated by GADD45b. GADD45b seems to act as a sensor in mature neurons for environmental changes which it expresses through these methylation changes. [ 1 ]  This was determined by examining the effects of applying an electric stimulus to the hippocampal dentate gyrus (DG) in normal and GADD45b knockout mice. In normal mice application of electrical stimulation to the DG increased neurogenesis by increasing BDNF. However, in GADD45b deficient mice the electrical stimulus had less of an effect. Further examination revealed that around 1.4% of  CpG islands  in DG neurons are actively methylated and demethylated upon electric shock. This shows that the post-mitotic methylation states of neurons are not static and given that electric shock equipment such as that used in the study has been shown to have therapeutic effects to human patients with depression and other psychiatric disorders, the possibility remains that epigenetic mechanisms may play an important role in the pathophysiology of neuropsychiatric disorders. [ 2 ] [ 8 ]  DNMT1 and DNMT3a are both required in conjunction for learning, memory, and synaptic plasticity. [ 8 ]


Epigenetic dysregulation, or alterations in epigenomic machinery, can cause DNA methylation and histone acetylation processes to go rogue. The epigenetic machinery influences neural differentiation regulation (i.e. neurogenesis)  [ 22 ]  and are also involved in processes related to memory consolidation and learning in healthy individuals. [ 23 ]  Increasing age can produce various epigenetic changes such as reduced global heterochromatin, nucleosome remodeling, altered histone marks, and changes in DNA methylation. For instance, nucleosome loss occurs due to aging because core histone proteins are lost and less protein synthesis occurs. [ 24 ]  As aging is the main risk for many neurological disorders, epigenetic dysregulation can in turn lead to alterations on the transcriptional level of genes involved in the  pathogenesis  of neural degenerative diseases such as  Parkinson's disease ,  Alzheimer's disease ,  Huntington's disease ,  schizophrenia , and  bipolar disease . [ 1 ] [ 25 ]


MicroRNA expression is critical for neurogenesis. In patients with Alzheimer's disease miR-9 and miR-128 is upregulated, while miR-15a is downregulated. [ 4 ]   Alzheimer's patients also show decreases in brain-derived neurotrophic factor, which has been shown to be repressed through DNA methylation. [ 8 ]  Although what has been argued as the most evidence for epigenetic influence in Alzheimer's is the gene which controls the protein responsible for  amyloid plaque  formation,  App . This gene has very high GC content in its  promoter region , meaning that it is highly susceptible to DNA methylation. This promoter site has been shown to naturally reduce methylation with aging, exemplifying the parallels between aging and Alzheimer's already well known. [ 26 ] [ 27 ]  Heavy metals also seem to interfere with epigenetic mechanisms. Specifically in the case of APP, lead exposure earlier in life has been shown to cause a marked over-expression of the APP protein, leading to more amyloid plaque later in life in the aging brain. [ 27 ]


DNA methylation's age relation has been further investigated in the promoter regions of several Alzheimer's related genes in the brains of  postmortem  late-onset Alzheimer's disease patients. The older patients seem to have more abnormal epigenetic machinery than the younger patients, despite the fact that both had died from Alzheimer's. Though this in of itself is not conclusive evidence of anything, it has led to an age-related epigenetic drift theory where abnormalities in epigenetic machinery and exposure to certain environmental factors which occur earlier in life lead to aberrant DNA methylation patterns far later, contributing to sporadic Alzheimer's Disease predisposition. [ 27 ]


Histone modifications may also have an impact in Alzheimer's disease, but the differences between HDAC effects in rodent brains compared to human brains have researchers puzzled. [ 27 ]  As the focus for neurodegenerative diseases begins to shift towards epigenetic pharmacology, it can be expected that the interactions of histone modifications with respect to neurogenesis will become more clear.


Histone acetylation  has received increasing support over the years as a proposed mechanism through which epigenetic dysregulation leads changes in gene expression that contribute to HD. [ 28 ]  Studies that look at mice with HD versus the wild type (WT) have shown that specific gene loci (Drd2, Penk1, Actb, and Grin1) decrease in histone acetylation levels, suggesting that a mutation of the Huntington (HTT) gene and its overexpression may be the cause of this epigenetic dysregulation.


It has been thought that  HDAC inhibitors  (HDACi's) could partially reverse the low acetylation levels seen in patients with HD. Preclinical studies have been performed using various HDACi's [such as  suberoxylanilide hydroxamic acid  (SAHA), Trichostatin A (TSA), phenylbutyrate, and  sodium butyrate  (NaB)] that target HDACI and HDACII. Although these inhibitors improve some phenotypes of HD in mice, such as  neuropathology  and  motor function , these beneficial effects do not lead towards a conclusion for the definitive need for increasing acetylation levels in HD patients. However, inactivation of a target of SAHA, Hdac 4, alleviates neurodegenerative complications in mice with HD through a transcription-independent mechanism which acts upon mutant Htt aggregation processes-which may indicate that there is a mechanism involving non-histone proteins. [ 29 ]  The proposed mechanism through which SAHA is speculated to act is through a RANBP2-mediated  proteasome degradation  model-which likely comes about as a general outcome of HDAC inhibitor actions. In this mechanism, SAHA is shown to down-regulate Hdac 4 through an increase in  sumoylation , which is then followed up with the activation of degradation through a proteasomal pathway. This mechanism reveals the connectivity between acetylation, deacetylation, and sumoylation processes. [ 30 ]


As of 2014, HDACi treatment has not been shown to restore normal expression of neuronal-identity genes. [ 31 ]  However clinical studies using HDACi are currently ongoing and the results are pending, with the Phase II studies showing promise for safe and tolerable use of several compounds such as phenylbutyrate.


Non-histone-mediated beneficial effects of HDACi have also been documented in models of  Parkinson disease , suggesting common mechanisms between several  neurodegenerative diseases.


DNA methylation analysis showed that there is significant dysregulation of methylation on  CpG islands  in patients with PD when compared to healthy individuals. Although this was genome-wide, this also occurred on many PD risk genes. [ 32 ]


Mitochondrial DNA  methylation of cytosine has also been shown to fluctuate over time due to age variance, as there is a growing body of literature linking mtDNA methylation to aging and oxidative stress. [ 33 ]  A study from 2015 by Hashizume et al. showed that SHMT2 mRNA levels are significantly reduced in the fibroblasts of old people when compared to younger individuals. The study also further indicated that decreased  GCAT  and  SHMT2  levels of gene expression via  shRNA  and  siRNA , respectively, in the fibroblasts of young patients led to a respiratory chain dysfunction typical for senile individuals-suggesting that an epigenetic mechanism may be the cause for the phenotypic change. As mitochondria plays a role in the development of the PD, [ 34 ]  further research into the area will help uncover any implications that mitochondrial DNA methylation plays in the pathogenesis of PD.


The use of dopaminergic neurons that have been isolated from the PD patients indicated that there were increases in acetylation (at H2A, H3 and H4) when compared to the age-control group. [ 32 ]  Another study involving MPP+ (a compound that can cause a disease state resembling mammals and humans with PD [ 35 ] )-treated cells and (MPP+)-treated mouse brains showed decreased HDAC levels, as well as in midbrain samples from patients with PD. This is seen potentially due to how MPP+ promotes the breakdown of HDAC1 and HDAC2 via  autophagy , a bodily process of cycling out old cells to make room for newer, healthier cells. [ 36 ]  These results point towards the stress of histone modifications in regards to chromatin remodeling and its implication in the  pathogenesis  of PD.


miRNAs are also emerging as relevant contributors to  neurodegeneration  in PD. In particular, the  frontal cortex  PD patients have shown higher levels of LRRK2 and lower levels of miR-205 when compared to healthy individuals. Connecting this to the findings of miR-205's ability to bind to the 3′ UTR of LRRK2 mRNA and suppress expression, as well as miR-205's prevention of defects after introduction to a R1441G LRRK2 mutation, these results point towards miR-205 and its regulatory role in LRRK2 expression-which in turn suggest a regulatory role in the pathogenesis of PD.


In another study in which increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 showed prevention of the association of mutant LRRK2 with microtubules, inhibition of deacetylases HDAC6 and Sirt2 through knockdown processes rescued both axonal transport and locomotor behavior. [ 37 ]  This further connects to the common mechanisms involving HDACi in various neurodegenerative diseases.


Bipolar disorders  are both highly complex and heritable, which makes it an interesting disorder to examine for epigenetic modifications.  DNA methylation , DNA hydroxymethylation, and  histone modifications  are all capable of contributing to the formation of bipolar disorder.


For example, studies of  monozygotic twins  revealed that individuals with bipolar disorder had lower methylation of the peptidylprolyl isomerase E-like (PPIEL) gene, which can be attributed to the dopamine transmission. The studies indicated that hypermethylation of SLC6A4, a serotonin transporter gene, is also involved with bipolar disorder. Greater expression of DNA methyltransferase 1 in cortical GABAergic interneurons may enable hypermethylation. Hypermethylation may prompt hydroxymethylation to occur in order to overcompensate for the repressive effects of hypermethylation. The methylation of CpG regions are relevant to bipolar disorders. Patients with bipolar disorder showed lower methylation levels for the CpG region of the KCNQ3 gene, which is responsible for the voltage-gated K+ channel. Childhood maltreatment contributed to the methylation status of CpG2 III of 5-hydroxytryptamine 3A, which alters how maltreatment affects bipolar disorder.


Moreover, therapeutic interventions such as engineered  transcription factors  could modify chromatin structure to address the epigenetic changes found in those with bipolar disorder.  DNA methyltransferase  (DNMT) inhibitors and  histone deacetylase  (HDAC) inhibitors could possibly reverse epigenetic modifications in order to therapeutically address bipolar disorder. DNMT inhibitors and HDAC often produces antidepressant-like effects.


Epigenetic theories of homosexuality  concern the studies of changes in  gene expression  or cellular  phenotype  caused by mechanisms other than changes in the underlying  DNA sequence , and their role in the development of  homosexuality . [ 1 ] [ 2 ] [ 3 ]   Epigenetics  examines the set of chemical reactions that switch parts of the  genome  on and off at strategic times and locations in the organism's  life cycle . However, epigenetic theories tangle a multiplicity of initiating causes and resulting final effects and will never lead to a single cause or single result. Hence, any interpretation of such theories may not focus on just one isolated reason of a multiplicity of causes or effects. [ 4 ]


Instead of affecting the organism's DNA sequence, non-genetic factors may cause the organism's genes to express themselves differently. DNA in the human body is wrapped around  histones , which are  proteins  that package and order DNA into structural units. DNA and histone are covered with  chemical tags  known as the  epigenome , which shapes the physical structure of the genome. [ 5 ]  It tightly wraps inactive genes on the DNA sequence making those genes unreadable while loosely wrapping active genes making them more expressive. The more tightly wrapped the gene, the less it will be expressed in the organism. These epigenetic tags react to stimuli presented from the outside world. It adjusts specific genes in the genome to respond to humans' rapidly changing environments.
The idea of epigenetics and gene expression has been a theory applied to the origins of homosexuality in humans. One team of researchers examined the effects of  epi-marks  buffering  XX   fetuses  and  XY  fetuses from certain  androgen  exposure and used published data on fetal androgen signaling and  gene regulation  through non-genetic changes in DNA packaging to develop a new model for homosexuality. [ 6 ]  The researchers found that stronger than average epi-marks, epigenomes that are wrapped tightly around the DNA sequence, convert  sexual preference  in individuals without altering  genitalia  or  sexual identity . [ 7 ]  However, a later study found that male homosexuality is not linked to low androgen sensitivity or "sex-reversed" epi-marks. [ 8 ]


Epigenetic marks (epi-marks) are temporary "switches" that control how our genes are expressed during  gestation  and after birth. Moreover, epi-marks  are modifications of histone proteins. [ 9 ]  Epigenetic marks are modifications of the  methyl  and  acetyl  groups that bind to DNA histones thereby changing how the proteins function and as a result, alter gene expression. [ 10 ]  Epi-marks change how the histones function and as a result, influence the way genes are expressed. [ 1 ]  Epigenetic marks promote normal  sexual development  during  fetal development . However, they can be passed on to  offspring  through the process of  meiosis . When they are transferred from one parent to an offspring of the opposite sex, it can contribute to an altered sexual development, thus leading to  masculinization  of female offspring and  feminization  of male offspring. [ 11 ]  However, these epi-marks hold no consistency between individuals in regard to strength and variability. [ citation needed ]


Identical twins  have nearly identical DNA, which leads to the perceived conclusion that all identical twins are either  heterosexual  or homosexual. However, it is evident that this is not the case, consequently leaving a gap in the explanation for homosexuality. Epigenetic modifications act as temporary "switches" that regulate how the genes are expressed. [ 11 ]  Of the pairs of identical twins in which one twin is homosexual, the other twin, despite having the same genome, only has a 20-50% chance of being homosexual as well. [ 12 ]  This leads to the hypothesis that homosexuality is created by something else rather than the genes. Epigenetic transformation allows the on and off switching of certain genes, subsequently shaping how cells respond to androgen signaling, which is critical in sexual development. [ 6 ]
Another example of epigenetic consequences is evident in  multiple sclerosis  in  monozygotic  (identical) twins. There are pairs of twins that are discordant with multiple sclerosis and do not both show the trait. After  gene testing , it was suggested that DNA was identical and that epigenetic differences contributed to the gene difference between identical twins. [ 13 ]


While in the fetal stages, hormonal influences of  androgen , specifically  testosterone , cause feminine qualities in regard to sexual development in females and masculine qualities in males. In typical sexual development, females are exposed to minimal amounts of testosterone, thus feminizing their sexual development, while males are typically exposed to high levels of testosterone, which masculinize their development. Epi-marks play a critical role in this development by acting as a buffer between the fetus and androgen exposure. Moreover, they predominantly protect XY fetuses from androgen underexposure while protecting XX fetuses from androgen overexposure. [ 1 ]  However, when androgen overexposure happens in XX fetuses, research suggests they can show masculinized behavior in comparison to females who undergo normal levels of androgen exposure. The research also suggests that excess androgen exposure in females led to reduced heterosexual interest in adulthood than did females with normal levels of androgen. [ 14 ]


New epi-marks are usually produced with each generation, but these marks sometimes carry over between  generations . Sex-specific epi-marks are produced in early fetal development that protect each sex from the natural disparity in testosterone that occurs during later stages of fetal development. Different epi-marks protect different sex-specific traits from being masculinized or feminized—some affect the genitals, others affect sexual identity, and yet others affect sexual preference. However, when these epi-marks are transmitted across generations from fathers to daughters or mothers to sons, they may cause reversed effects, such as the feminization of some traits in sons and similarly a partial masculinization of daughters. Furthermore, the reversed effects of feminization and masculinization can lead to a reversed sexual preference. For example, sex-specific epi-marks normally prevent female fetuses from being masculinized through exposure of atypically high  testosterone , and vice versa for male fetuses. Sex-specific epi-marks are normally erased and not passed between generations. However, they can sometimes escape erasure and are then transferred from a father's genes to a daughter or from a mother's genes to a son. When this happens, this may lead to an altered sexual preference. [ 1 ]  Epi-marks normally protect parents from variation in sex hormone levels during fetal development, but can carry over across generations and subsequently lead to homosexuality in opposite-sex offspring. This demonstrates that  gene coding  for these epi-marks can spread in the population because they benefit the development and fitness of the parent but only rarely escape erasure, leading to same-sex sexual preference in offspring. [ citation needed ]


Epigenetic explanations for sexual orientation are still purely speculative. W. Rice and colleagues say that they "cannot provide definitive evidence that homosexuality has a epigenetic underpinning". [ 1 ]  Tuck C. Ngun and Eric Vilain published a paper in 2014 in which they evaluated and critiqued the epigenetic model proposed by Rice and colleagues in 2012. Ngun and Vilain agreed with much of Rice's model, but disagreed that "sex-reversing sensitivity to androgen signaling via epigenetic markers will result in homosexuality in both sexes", noting that non-heterosexuality is far more common in women. [ 8 ]
Also, a report of a study of 34 male monozygotic twin pairs discordant for sexual orientation revealed no support for the epigenetic hypothesis. [ 15 ]


Epigenetic therapy  refers to the use of drugs or other interventions to modify gene expression patterns, potentially treating diseases by targeting  epigenetic  mechanisms such as  DNA methylation  and  histone  modifications.


Epigenetics is the study of changes in gene expression that do not arise from alterations in the DNA sequence, resulting in the heritable silencing of genes without changing the coding sequence. Epigenetic therapy involves using drugs or other techniques to influence these epigenetic mechanisms in addressing specific medical conditions. Various diseases, such as  diabetes ,  cancer ,  heart disease , and  mental illnesses , are influenced by epigenetic mechanisms. Emerging areas of epigenetic therapy include its application in heart disease, primarily focusing on  tissue regeneration , and in  schizophrenia , where the focus lies on alleviating symptoms. Overall, epigenetic therapies aim to target the underlying epigenetic molecular pathways responsible for disease manifestation.


Epigenetics refers to the study of changes in gene expressions that do not result from alterations in the DNA sequence'. [ 1 ]  Altered gene expression patterns can result from chemical modifications in DNA and chromatin, to changes in several regulatory mechanisms. Epigenetic markings can be inherited in some cases, and can change in response to environmental stimuli over the course of an organism's life. [ 2 ]


Many diseases are known to have a genetic component, but the epigenetic mechanisms underlying many conditions are still being discovered. A significant number of diseases are known to change the expression of genes within the body, and epigenetic involvement is a plausible hypothesis for how they do this. These changes can be the cause of symptoms to the disease. Several diseases, especially cancer, have been suspected of selectively turning genes on or off, thereby resulting in a capability for the tumorous tissues to escape the host's immune reaction. [ 2 ]


Known epigenetic mechanisms typically cluster into three categories. The first is DNA methylation, where a cytosine residue that is followed by a guanine residue (CpG) is methylated. In general, DNA methylation attracts proteins which fold that section of the chromatin and repress the related genes. [ 3 ]  The second category is histone modifications. Histones are proteins which are involved in the folding and compaction of the chromatin. There are several different types of histones, and they can be chemically modified in a number of ways. Acetylation of histone tails typically leads to weaker interactions between the histones and the DNA, which is associated with gene expression. Histones can be modified in many positions, with many different types of chemical modifications, but the precise details of the  histone code  are currently unknown. [ 4 ]   The final category of epigenetic mechanism is regulatory RNA. MicroRNAs are small, noncoding sequences that are involved in gene expression. Thousands of miRNAs are known, and the extent of their involvement in epigenetic regulation is an area of ongoing research. [ 5 ]  Epigenetic therapies are reversible, unlike  gene therapy . This means that they are  druggable  for targeted therapies. [ 6 ]


Epigenetic alterations can play a major role in the development and progression of cancer. [ 7 ]  This can be seen as a result of the changes made by various epigenetic mechanisms (i.e. DNA methylation, histone modification, non-coding RNAs) which may work to silence or activate cancer-related genes (i.e.  tumor-suppressor genes  or  oncogenes ), therefore invoking changes in gene expression patterns that are responsible for the initiation, progression, and metastasis of cancer. [ 8 ] [ 9 ]  One example includes DNA hypermethylation of  tumor-suppressor genes  which can cause transcriptional inactivation of genes (i.e.  CDKN2A ) encoding for other regulatory proteins (i.e.  p14  and  p16 ) that play a vital role in the cell cycle. [ 10 ]  However, there are numerous avenues in which cancer can arise epigenetically given the various  hallmarks of cancer . Because of this, epigenetic drugs are utilized to target and combat the internal dysregulation caused by these epigenetic alterations.


DNA methyltransferase inhibitors (DNMTis) are used as a treatment option against various forms of cancer. Some of these drugs, including  5-azacytidine ,  Decitabine , and  Zebularine , have been shown to reactivate the cellular anti-tumor systems repressed by  cancer, enabling the body to weaken and kill off the tumor. [ 11 ] [ 12 ] [ 13 ] [ 14 ] [ 9 ]  This can specifically result due to DNMTis ability to block overly active DNMTs, resulting in the hypomethylation of CpG regions which can allow for normal cell growth. [ 10 ]  Other  treatment options including histone deacetylase inhibitors (HDACis), such as  vorinostat , have also shown promising epigenetic therapeutic effects through the reactivation of apoptosis, cell death of undesirable cells, and the inhibition of angiogenesis, the formation of new blood vessels that can be utilized by cancerous cells as an extra source of nutrients. [ 15 ] [ 16 ]  HDACis are an example of a chromatin-modifying drug, which specifically targets the acetylation portion of cancerous cells, and like DMNTis, is able to reactivate the cellular anti-tumor systems within a cell. [ 16 ]  Now because of their wide-ranging effects throughout the entire body, many of these drugs may have major side effects, making dosage regulation important; however, the survival rates are increased significantly when they are used for treatment, whether independently or in congruence with chemotherapy or other cancer-related forms of medication. [ 9 ]


Phytochemical  interventions have also gained traction in serving as an epigenetic treatment option against cancer. Phytochemicals, which are compounds produced by plants that act as a protectant to aid in their survival, can have an impact on the function of DNMTs. [ 17 ]   Polyphenols , a type of phytochemical that can be found in everyday foods and ingredients like tea, apples, and various forms of berries, have been shown to have anticancer effects. [ 17 ]  For example, epigallocatechin gallate (EGCG), the most prevalent polyphenol found in green tea, can work to inhibit cell proliferation by inducing  apoptosis , preventing  angiogenesis , and disrupting regulatory pathways, such as  MAPK  pathways. [ 17 ] [ 18 ] [ 19 ]


Although various mechanisms have been utilized to mitigate the negative effects of cancer development and progression, drug resistance does still occur.  Drug resistance  can emerge as a result of continued and repetitive usage of a drug, giving pathogens time to develop defenses against drugs designed to kill them. [ 20 ]  In cancer, overexpression, for instance in DNA methylation and histone deactylase, can cause deleterious effects which can enhance drug resistance behaviors such as drug efflux. [ 21 ]  Drug efflux is regulatory process in which toxic compounds (i.e. drugs for cancer) get removed from a cell to reduce its cytotoxic effects. [ 22 ]  However, research suggests there are ways to overcome this type of drug resistance, with one being through the use of epigenetic factor inhibitors. [ 23 ]


Cardiovascular disease is one of the leading cause of death. Factors such as age, smoking, obesity, diabetes, and hypertension increase the likelihood of developing cardiovascular diseases. As of now, initial investigations have established links between DNA methylation, histone modifications, RNA-based mechanisms, and the onset of  cardiovascular  diseases such as atherosclerosis, cardiac hypertrophy, myocardial infarction, and heart failure. [ 24 ]  Epigenetic alterations like DNA methylation enzymes can reverse abnormal DNA methylation, histone modifications, and non-coding RNA regulation play critical roles in cardiovascular disease.  A number of cardiac dysfunctions have been linked to cytosine methylation patterns. DNA methylation is an important epigenetic mechanism, it is able to transfer to offspring DNA through the regulation of DNMT. [ 25 ]  DNA demethylation can happen through either an active or passive process. Passive demethylation refers to the failure of maintenance DNA methyltransferases (DNMTs) to methylate the newly synthesized DNA strand during mitosis. [ 25 ]  DNMT deficient mice show upregulation of inflammatory mediators, which cause increased atherosclerosis and inflammation.  Atherosclerotic tissue has increased methylation in the promoter region for the estrogen gene, although any connection between the two is unknown. Hypermethylation of the  HSD11B2  gene, which catalyzes conversions between cortisone and cortisol, and is therefore influential in the stress response in mammals, has been correlated with hypertension. Decreased  LINE-1  methylation is a strong predictive indicator of ischemic heart disease and stroke, although the mechanism is unknown. Various impairments in lipid metabolism, leading to clogging of arteries, has been associated with the hypermethylation of  GNASAS ,  IL-10 ,  MEG3 ,  ABCA1 , and the hypomethylation of  INSIGF  and  IGF2 . Additionally, upregulation of a number of miRNAs has been shown to be associated with acute myocardial infarction, coronary artery disease, and heart failure. Strong research efforts into this area are very recent, with all of the aforementioned discoveries being made since 2009. Mechanisms are entirely speculative at this point, and an area of future research. [ 26 ]  It has been demonstrated that miRNAs regulate gene expression by suppressing messenger RNA (mRNA) translation and increasing mRNA degradation. Studies have shown that miRNAs can influence myocardial angiogenesis and the survival and proliferation of cardiomyocytes by regulating target gene expression. [ 27 ]


Epigenetic treatment methods for cardiac dysfunction are still highly speculative. SiRNA therapy targeting the miRNAs mentioned above is being investigated. The primary area of research in this field is on using epigenetic methods to increase the regeneration of cardiac tissues damaged by various diseases. [ 26 ]  By comprehending the roles these mechanisms play in disease pathology, researchers can devise precise treatments to regulate gene expression, potentially halting or alleviating the progression of cardiovascular diseases.


Diabetes is a disease where an affected individual is unable to convert food into energy. When left untreated, the condition can lead to other, more severe complications. [ 28 ]  A common sign of diabetes is the degradation of blood vessels in various tissues throughout the body. Retinopathy refers to damage from this process in the retina, the part of the eye that senses light. [ 29 ] [ 30 ]  Diabetic retinopathy is known to be associated with a number of epigenetic markers, including methylation of the Sod2 and MMP-9 genes, an increase in transcription of  LSD1 , a H3K4 and H3K9 demethylase, and various DNA Methyl-Transferases (DNMTs), and increased presence of miRNAs for transcription factors and  VEGF . [ 30 ]


It is believed that much of the retinal vascular degeneration characteristic of diabetic retinopathy is due to impaired mitochondrial activity in the retina.  Sod 2 codes for a superoxide disputes enzyme, which scavenges free radicals and prevents oxidative damage to cells. LSD1 may play a major role in diabetic retinopathy through the downregulation of Sod2 in retinal vascular tissue, leading to oxidative damage in those cells.  MMP-9  is believed to be involved in cellular apoptosis, and is similarly downregulated, which may help to propagate the effects of diabetic retinopathy. [ 30 ]


Several avenues to epigenetic treatment of diabetic retinopathy have been studied. One approach is to inhibit the methylation of the Sod2 and MMP-9. The DNMT inhibitors  5-azacytidine  and  5-aza-20-deoxycytidine  have both been approved by the FDA for the treatment of other conditions, and studies have examined the effects of those compounds on diabetic retinopathy, where they seem to inhibit these methylation patterns with some success at reducing symptoms. The DNA methylation inhibitor  Zebularine  has also been studied, although results are currently inconclusive. A second approach is to attempt to reduce the miRNAs observed at elevated levels in retinopathic patients, although the exact role of those miRNAs is still unclear. The Histone Acetyltransferase (HAT) inhibitors  Epigallocatechin-3-gallate ,  Vorinostat , and  Romidepsin  have also been the subject of experimentation for this purpose, with some limited success. [ 30 ]   The possibility of using Small Interfering RNAs, or siRNAs, to target the miRNAs mentioned above has been discussed, but there are currently no known methods to do so. This method is somewhat hindered by the difficulty involved in delivering the siRNAs to the affected tissues. [ 30 ]


Type 2 diabetes mellitus (T2DM) has many variations and factors that influence how it affects the body. DNA methylation is a process by which methyl groups attach to DNA structure causing the gene to not be expressed. This is thought to be an epigenetic cause of T2DM by causing the body to develop an insulin resistance and inhibit the production of beta cells in the pancreas. [ 31 ]  Because of the repressed genes the body does not regulate blood sugar transport to cells, causing a high concentration of glucose in the blood stream.


Another variation of T2DM is mitochondrial reactive oxygen species (ROS) which causes a lack of antioxidants in the blood. This leads to oxidation stress of cells leading to the release of free radicals inhibiting blood glucose regulation and hyperglycemic conditions. This leads to persistent vascular complications that can inhibit blood flow to limbs and the eyes. This persistent hyperglycemic environment leads to DNA methylation as well because the chemistry within chromatin in the nucleus is affected. [ 32 ]


Current medicine used by T2DM sufferers includes Metformin hydrochloride which stimulates production in the pancreas and promotes insulin sensitivity. A number of preclinical studies have suggested that adding a treatment to metformin that would inhibit acetylation and methylation of DNA and histone complexes. [ 32 ]  DNA methylation occurs throughout the human genome and is believed to be a natural method of suppressing genes during development. Treatments targeting specific genes with methylation and acetylation inhibitors is being studied and debated. [ 33 ]


Autism or autism spectrum disorder (ASD) is a neurodevelopmental disorder discovered in 1943. [ 34 ]  It is part of a growing group of disorders called pervasive developmental disorders (PDDs), which are becoming increasingly common up to 1 in 110 in the United States and 1 in 64 in the United Kingdom. [ 34 ]


While there are many causes for Autism spectrum disorder to manifest (viral infection, encephalitis, or an auto immune reaction, [ 34 ]  but the primary reasons are genetic or epigenetic. in the 1970s karyotyping allowed for the understanding of chromosomes, over time techniques such as fluorescence  in situ  hybridization (FISH))  we began to be able to analyze chromosomes with more resolution. Chromosome microarray and single nucleotide genotyping, and even whole genotype sequencing allow resolution at the gene level. One issue that possibly leads to autism is copy number variation in a gene the most studied of which is the 15q multiplication from the maternal chromosome. [ 35 ]  A database of genes (AutDB) has over 800 potential targets for study as a cause for autism. [ 36 ]  While not an exhaustive list a few examples are  NLGN4X, PAH ,  PEX7 ,and  SYNE1 . [ 36 ]


The heterogeneity of autism causes and symptoms has led to research in epigenetic factors. Maternal health during pregnancy, including taking folic acid, has been shown to affect the chances of not having ASD through epigenetic means. DNA methylation and histone modification are the two leading causes for epigenetic causes for autism. Targets for research for DNA methylation in relation to autism are  oxytocin receptor, SHANK3, and BCL-2. [ 37 ]  Oxytocin is a hormone that partially controls social interactions, this has been epigenetically linked to autism. Methylation controls SHANK3 levels, which in turn activate CpG- island genes, which can lead to autism. BCL-2 is involved in cell death, and an event that misfires could contribute to brain development in that region. [ 37 ]  Histone modification also potentially leads to autism. Histone modification contributes to brain development. Modification of lysine residues on the H3 histone effect brain development. [ 38 ]  histone deacetylase inhibitors sodium butyrate and trichostatin A are regulators of oxytocin and vasopressin which are linked to autism symptoms. [ 37 ]


Epigenetic therapies are a new emerging medical field. There are a number of potential therapies to treat ASD, using various methods. While the research into potential therapies is still in its infancy one of the positives of epigenetic therapy is it is reversible, this leads to many less potential side effects. While there are no current therapeutics approved for epigenetic use in autism there are a number of potential categories, histone deacetylase inhibitors (HDACis), [ 39 ]  and DNA methyltransferases. While there is great hope for epigenetic therapy for autism and some powerful research, there is currently no available drugs in clinical trials.


Histone acetlytion is a standard way to measure gene activity, HDACS remove histones thereby lowering the gene activity at that site. HDACi drugs are being tested and used as cancer theraputics, but are showing potential for neurodevelopmental disorders such as ASD. Valproic acid, commonly called Vorinostat, is an HDACi that helps with mood stabilization, this shows that it has potential for other neuro applications. MGCD0103 is a drug that is in preclinical trials as a cancer drug. Is mechanism is to affect HDAC1. HDAC1 has a large impact on many functions in the body, including many neuro regions, this makes it a good candidate for a potential Autism therapy. [ 39 ]


DNA Methyltransferases (DNMT) have the potential to silence specific genes. As we learn more about genes related to ASD such as PAH (see above) these compounds become more relevant as therapies.  There are three types of DNMT's. DNMT1 is a maintenance DNMT whereas DNMT3a and DNMT 3b are a de novo factor.  There are currently two therapeutics being tested for this process, azacitidine and decitabine. [ 37 ]


Traumatic experiences can lead to several mental illnesses including  post-traumatic stress disorder . It was previously thought that PTSD could be treated with advances in  cognitive behavioral therapy  methods like  Exposure therapy . In exposure therapy, patients are exposed to stimuli that provoke fear and anxiety. In theory, repeated exposure can lead to a decreased connection between the stimuli and the anxiety. While exposure therapy helps many patients, many patients do not experience improvement in their symptoms while others may experience more symptoms. [ 40 ]


The biochemical mechanisms underlying these systems are not completely understood. However, brain-derived neurotrophic factor ( BDNF ) and the N-methyl-D-aspartate receptors ( NMDA ) have been identified as crucial in the exposure therapy process. Successful exposure therapy is associated with increased acetylation of these two genes.  Acetylation  is a biochemical modification that affects how tightly DNA is wound around histone proteins, thereby influencing gene expression. When the genes encoding BDNF and NMDA receptors experience increased acetylation, they become more "accessible" for transcription, the first step in protein production. So, enhanced expression of BDNF and NMDA receptors seems to bolster neural plasticity, facilitating the brain's capacity to form new connections and adjust its responses to anxiety-provoking stimuli.  For these reasons, increasing the acetylation of these two genes has been a major area of recent research into the treatment of  anxiety disorders . [ 41 ]


N-methyl-D-aspartate receptors (NMDA receptors) play a crucial role in synaptic plasticity and learning, including fear extinction, which is a core mechanism targeted in exposure therapy. These receptors are involved in the consolidation of new memories and the extinction of fear responses by regulating the strength of synaptic connections. Exposure therapy aims to reduce the fear response to specific stimuli by repeated exposure to those stimuli in a safe environment. During exposure therapy, the process of fear extinction occurs, where individuals learn that the feared stimuli no longer predict harm. NMDA receptors are implicated in this fear extinction learning process through their involvement in synaptic plasticity mechanisms, such as long-term potentiation (LTP) and long-term depression (LTD), which underlie the formation and extinction of fear memories. Research has shown that pharmacological manipulation of NMDA receptors can influence fear extinction learning and enhance the efficacy of exposure therapy. For example, drugs that enhance NMDA receptor function, such as D-cycloserine, have been used as adjuncts to exposure therapy to facilitate fear extinction in individuals with anxiety disorders. The article "Effects of D-cycloserine on extinction: translation from preclinical to clinical work" (Davis et al., 2006) [ 42 ]  discusses the preclinical and clinical studies investigating the effects of D-cycloserine, a partial agonist at the glycine site of the NMDA receptor, on fear extinction and its translation to clinical applications in exposure therapy for anxiety disorders.


In relation to epigenetic therapy, modulating NMDA receptor-related pathways and enhancing fear extinction learning. By targeting epigenetic modifications of NMDA receptor genes, such as promoting DNA demethylation or histone acetylation, it may be possible to enhance NMDA receptor function and facilitate fear extinction processes. This could involve adjunctive treatments with epigenetic-modifying agents, tailored to individual genetic and epigenetic profiles, to optimize exposure therapy outcomes in individuals with anxiety disorders.


Going deeper into BDNF as well, utilizing BDNF as more than a mere marker in exposure therapy and integrating it into epigenetic therapy to target BDNF-related pathways could serve as a valuable addition to complement exposure therapy. The relationship between BDNF, exposure therapy, and epigenetic therapy can be illuminated by considering the role of BDNF in fear extinction learning, which is a core mechanism targeted in exposure therapy, and the potential for epigenetic mechanisms to modulate BDNF expression and function.


BDNF plays a pivotal role in fear extinction learning, crucial for exposure therapy, by facilitating synaptic plasticity and consolidating safety memories. [ 43 ]  Individuals with BDNF dysfunction, like those with the Val66Met SNP (The Val66Met  single nucleotide polymorphism  (SNP) of BDNF), may exhibit impaired fear extinction, potentially leading to treatment resistance in exposure therapy. [ 44 ]  Consequently, strategies aimed at enhancing BDNF function or expression could enhance the efficacy of exposure therapy interventions. Moreover, epigenetic mechanisms, such as DNA methylation and histone modifications, dynamically regulate BDNF expression and function. Environmental factors and genetic variants like the Val66Met SNP can influence BDNF epigenetic marks, impacting its transcriptional activity. Targeting epigenetic modifications of BDNF presents a novel approach to modulating fear extinction processes, potentially improving exposure therapy outcomes. [ 45 ]  Epigenetic therapy could complement exposure therapy by targeting BDNF-related pathways, such as demethylating the BDNF gene promoter or altering histone acetylation patterns to facilitate BDNF transcription. Integrating genetic information, such as BDNF polymorphisms, with epigenetic profiles and clinical data allows for personalized treatment approaches. Identifying individuals likely to benefit from targeted epigenetic interventions could enhance fear extinction learning and improve treatment outcomes in exposure therapy. This interplay between BDNF, exposure therapy, and epigenetic regulation holds promise for optimizing treatment outcomes and advancing personalized medicine approaches for anxiety disorders.


HDAC inhibitors are also a topic that can be discussed in this area. Exposure therapy's effectiveness in rodents is increased by the administration of Vorinostat, Entinostat, TSA, sodium butyrate, and VPA, all known histone deacetylase inhibitors. Several studies in the past two years have shown that in humans, Vorinostat and Entinostat increase the clinical effectiveness of exposure therapy as well, and human trials using the drugs successfully in rodents are planned. [ 41 ]  In addition to research on the effectiveness of HDAC inhibitors, some researchers have suggested that histone acetyltransferase activators might have a similar effect, although not enough research has been completed to draw any conclusions. However, none of these drugs are likely to be able to replace exposure therapy or other cognitive behavioral therapy methods.  Rodent studies  have indicated that administration of HDAC inhibitors without successful exposure therapy worsens anxiety disorders significantly, [ 46 ]  although the mechanism for this trend is unknown. [ 41 ]  The most likely explanation is that exposure therapy works by a learning process, and can be enhanced by processes that increase neural plasticity and learning. However, if a subject is exposed to a stimulus that causes anxiety in such a way that their fear does not decrease, compounds that increase learning may also increase re-consolidation, ultimately strengthening the memory.


Psychotherapy can be linked to alterations in epigenetic markers. An example of the growing evidence that indicates that successful psychotherapy can be linked to alterations in epigenetic markers, particularly DNA methylation, and could serve as a potential indicator of treatment efficacy is a research paper titled "Epigenetics of traumatic stress: The association of NR3C1 methylation and posttraumatic stress disorder symptom changes in response to narrative exposure therapy". [ 47 ]  This study explored the relationship between DNA methylation at the glucocorticoid receptor gene (NR3C1) and the success of psychotherapy in treating Posttraumatic Stress Disorder (PTSD) among conflict survivors in Northern Uganda. The researchers used Narrative Exposure Therapy (NET) on a sample of 153 individuals with PTSD and conducted diagnostic interviews and saliva sampling before treatment and at 4 and 10 months after treatment completion. They found that changes in methylation at a specific CpG site (cg25535999) were associated with PTSD symptom development. Treatment responders showed an increase in methylation at this site after therapy, while lower methylation levels before treatment predicted greater symptom improvement. These findings suggest that epigenetic changes at NR3C1 may play a role in the success of trauma-focused therapy, highlighting the importance of glucocorticoid signaling in PTSD treatment.


Research findings have demonstrated that schizophrenia is linked to numerous epigenetic alterations, including DNA methylation and histone modifications. [ 48 ]  For example, the therapeutic efficacy of schizophrenic drugs such as antipsychotics are limited by epigenetic alterations [ 49 ]  and future studies are looking into the related biochemical mechanisms to improve the efficacy of such therapies. Even if epigenetic therapy wouldn't allow to fully reverse the disease, it can significantly improve the quality of life. [ 50 ]


Melanoma  is a rare but aggressive  malignant cancer  that originates from  melanocytes . These melanocytes are cells found in the  basal layer  of the  epidermis  that produce  melanin  under the control of  melanocyte-stimulating hormone . Despite the fact that melanoma represents only a small number of all skin cancers, it is the cause of more than 50% of cancer-related deaths. The high metastatic qualities and death rate, and also its prevalence among people of younger ages have caused melanoma to become a highly researched malignant cancer.  Epigenetic modifications  are suspected to influence the emergence of many types of cancer-related diseases, and are also suspected to have a role in the development of melanoma.


In the last few years, chemical alterations in the  genome  have become more evident, and these alterations can be critical in the development of malignancy. This alteration process is referred to as epigenetics (Patino et al. 2008).  Epigenetics  is the term used to refer to stable changes in DNA that affect  gene expression  but do not involve changes in the underlying nucleotide sequence of the organism (Patino et al. 2008). The mechanisms by which epigenetics occur involve hypo- and hypermethylation of DNA,  histone modifications  by  acetylation ,  methylation , and  phosphorylation , and  posttranslational modifications  which include  RNA silencing . These modifications can cause different expression patterns to occur, which can result in alterations to cells. Some of these alterations could result in the formation of cancerous cells or various other dangerous changes in cell function, among many other outcomes when paired together. Cancerous cells are not formed from just one change.


In epigenetic alterations in cancer,  DNA methylation  is the most studied, although it is not the only alteration that can occur. DNA methylation is a covalent modification of DNA where a methyl group is added to the C-5 position of cytosine by DNA-methyltransferases. This occurs mostly at the cytosine-phosphate-guanine dinucleotide rich regions, known as  CpG islands , and are located particularly in the promoter regions of genes in the  human genome  (Patino et al. 2008). These promoter regions are methylated in certain ways or can be completely unmethylated. However, in an altered methylation of the CpG islands (generally where the methylation pattern is inverted),  transcription  can be altered which can lead to cancer. This is due to chromosomes being highly condensed, preventing RNA polymerase and other transcription factors from recognizing and binding to the DNA. This can result in  gene silencing . This silencing of genes can be dangerous to cells, especially when the genes silenced are active in maintaining the cell cycle. The table below shows some of the important genes targeted by promoter hypermethylation in melanoma.


TABLE 1.  Various genes targeted by promoter hypermethylation in malignant melanoma


Some of the genes affected by cytosine methylation in melanoma formation


INK4A, also known as  p16 , is a  tumor suppressor gene  and is found to have hypermethylated promoter regions in 10- 20% of melanoma cells and is involved in 40- 87% of gene alterations in melanoma cases (Gonzalgo et al., 1997). This means that 10- 20% of the time there is an epigenetic change in the INK4A gene, and 40- 87% of the time there is a nucleotide mutation in the gene. INK4A is one of the genes that is both epigenetically and genomically altered. As a regularly functioning gene, INK4A is a tumor suppressor that functions to repress the formation of tumors. The hypermethylation of this gene can cause it to become inactivated (Straume et al., 2002). When INK4 is inactivated through hypermethylation, it causes an interruption of the  CDK4  and  CDK6  genes, which normally stop cell growth in the G1 phase of cell division. When this happens, there is no regulation in the cell and it grows quickly and becomes cancerous.


SYK


SYK  is another gene that is affected by cytosine methylation during cancer progression. It is a gene that, when hypermethylated, loses function (Muthusamy et al., 2006). This gene is found in 30- 89% of melanoma cases (Dahl et al., 2007), and causes cells to grow quickly. This fast growth is important in the quick metastasis of melanoma cells, and when hypermethylated, the growth and spread of cells slow considerably (Hoeller et al., 2005). This is a controversial finding with inconclusive results, though.  Some findings show that normally functioning SYK genes aids in tumor suppression, while other studies find that it is a transforming factor that facilitates cancer formation.  Normally functioning, the SYK gene produces a non- receptor protein kinase enzyme that aids in differentiation, proliferation, and phagocytosis among many other important processes. SYK is found expressed in a variety of cells including fibroblasts, epithelial cells (where it controls cell division and acts as a tumor suppressor), hepatocytes, and neuronal cells (TORCIS Bioscience, 2014). Although SYK does not have any reported DNA modifications, the epigenetic changes still cause sufficient damage to cells. When SYK silencing is coupled with other alterations, both genetically and epigenetically, cancer can form.


Histone modifications play a large role in epigenetics because of their widely varied methods of occurrence and affect. Modifications to histones come about in many forms, with many still not being clearly understood. Methylation, acetylation, phosphorylation, and ubiquitination are the main categories of histone modification, with combinations of changes resulting in a range of genetic expression.


Histone methylation  generally causes genes to turn off. Methylation of histones is also important for maintaining  heterochromatin  boundaries and a misregulation can lead to over expression or under expression of genes. This over expression or under expression can lead to the formation of cancer cells within an organism. On the other end of the spectrum, demethylation causes genes to turn on. This activation can be just as detrimental to cells, as their processes are disrupted by unnecessary gene products. Most histone methylation occurs in the promoter region, and is similar to cytosine methylation in process and function. [ 1 ]


Histone acetylation  is generally associated with transcription activation, which can cause certain proteins to be coded for and expressed when they shouldn't be. This can potentially lead to cancer. Conversely, deacetylation of genes causes transcription to be inhibited, shutting down important biological processes within the cell. In melanoma,  Ku70  and  Ku86  genes involved in DNA repair, were found to be inactivated when a  histone deacetylase , or HDAC, was exposed to the gene (Munshi et al., 2005).


Histone phosphorylation is the process by which a phosphate group is added to histone tails. This process drastically changes histone structure and shape. It has been discovered that phosphorylation causes post translational changes, with binding domains having been found, in addition to regulations in DNA repair, transcription regulation, and chromatin condensation. One of the major phosphorylations in DNA repair is the H2AX location. When H2AX is phosphorylated, it spreads throughout the DNA break and is thought to recruit acetyltransferases to relax the DNA and allow repair proteins to access the damaged portion (Rossetto et al., 2012). Phosphorylation of histones begins the process of DNA repair, and it is important for signaling other processes to begin, including cell division . H2AX is an indicator of melanoma because of its high correlation to chromosomal instability (Warters et al., 2005). When high levels of H2AX are present, more phosphorylation occurs and chromosomes are more susceptible to damage. Histone phosphorylation of H3S10 has been shown to associate with H3 acetylation, which is integral in transcription activation (Rossetto et al., 2012). If transcription is altered, cells are generally negatively impacted and they react in adverse ways that may harm the organism.


Scientists have wondered how melanoma cells travel from major  tumors  found on the surface of the skin to the liver, brain, lungs and other organs where they become very destructive, resistant to treatments and even cause death. Research being conducted tries to answer the question of how and why melanoma cells become defective and cause harm to people. Many studies have found that some classes of short strands of RNA, called  microRNAs , are linked to these harmful properties of melanoma cells.


MicroRNAs (miRNAs)  are non-coding RNAs of roughly 20-22 nucleotides in length. They function in post-transcriptional gene regulation, preventing a particular protein from being produced. They can do this in many ways, such as binding to and destroying the messenger RNA that otherwise would have produced the protein. MiRNAs have also been shown to bind directly to messenger RNA and silence them before they are able to be translated into proteins which would play important roles in transcription (O'Donnell et al., 2005). There are roughly 1000 miRNAs in the human genome, and about 33% of all human  messenger RNAs  are under their control (Liu et al. 2012).


MiRNAs function in several areas of biological processes, including differentiation, proliferation and apoptosis. They have also been found to play important roles in the development of cancers. Some of these non-coding RNAs, including miRNA-205, have been linked, recently, to the over or under expression of several genes associated with cancer and other life-threatening diseases such as heart disease, and alterations in their expressions have been shown to impact cancer cell growth, survival and the ability to spread (Liu et al. 2012). For instance, the loss of the miRNA-205 expression is associated with the metastasis of melanoma (Liu et al. 2012). An overproduction of MiRNAs can cause the epigenetic silencing of many important genes such as MITF, FOXO3, TFAP2C, CCND1, ITGA3 and c-KIT, needed in cell cycle regulation, and this can cause a cell to become cancerous (He et al., 2005).


MicroRNAs and genes involved in melanoma


A particular miRNA, MiR-182, has been found to impact  MITF  and  FOXO3 , which are tumor suppressor genes. MiR-182 is a member of a miRNA cluster in a chromosomal locus that is most often amplified in melanoma (Segura  et al.  2009). It is frequently up-regulated in human melanoma tissue samples and cell lines, and this up-regulation causes its ectopic expression, which stimulates migration of melanoma cells, both in vitro and in vivo. The down-regulation of this miRNA triggers apoptosis. Over-expression of miR-182 in malignancy has been shown to negatively impact these tumor suppressor genes by directly repressing them. The repression of these genes allows miRNA to promote the spread and migration of melanoma cells, not taking into account the properties necessary for metastasis to occur.


In a fairly recent experiment done by Penna  et al.  on miRNA-214, it was shown that the miRNA regulates the expression of a panel of 11 target genes, including  TFAP2C  and  ITGA3 , and contributes to melanoma tumor progression by suppressing those genes. In this experiment, these genes, known to contain one or more binding sites of the miRNA, were obtained and their three prime untranslated regions (3'UTRs) cloned into a vector.  Luciferase  assays were then performed in MA-2 and/or MC-1 cells transfected with the miRNA. It was reported that luciferase expression, which was driven by the  3'UTRs  of  integrin  alpha 3 (ITGA3) or  transcription factor  AP-2 gamma (TFAP2C), was repressed significantly. The team then tried to assess whether the luciferase expression regulation seen in the ITGA3 and TFAP2C genes depended on the binding between the miRNA and the complementary sequence that was present on the 3'UTRs of either gene. A four nucleotide deletion or three point mutations were respectively inserted in the ITGA3 or TFAP2C 3'UTRs, and the 3'UTR alterations entirely abrogated the effect of the miRNA over-expression on luciferase expression in the MA-2 and MC-1 cells. This observation indicated the direct regulation of miRNA-214 on the 3'UTR binding sites of the ITGA3 and TFAP2C genes.


Further experiment on protein expression was also conducted on these two particular genes, TFAP2C and ITGA3, and it was shown that miRNA-214 over-expression led to a 30-90% protein decrease in ITGA3 and a 40-80% decrease in TFAP2C (Penna et al., 2011). Consistently, proteins were upregulated 20% in the ITGA3 and 40% in the TFAP2C when miRNA-214 was silenced in the MC-1 cell. This suggests that miRNA-214 importantly regulates the expression of the ITGA3 and TFAP2C genes and promotes melanoma tumor progression when overly expressed.


TABLE 2.  Various genes altered by general mutations or amplification in malignant melanoma


Many genes are altered genetically and epigenetically in cancers, which is one of the reasons cancer is so hard to combat. INK4A and PTEN are two genes that are in both Tables 1 and 2 above, as they are both epigenetically and genetically mutated in melanoma cases. This is not uncommon, as cells need multiple changes to the genome to initiate a change as large as cancer. The epigenetic side of cancer research is growing and uncovering many overlaps like in the cases of INK4A and PTEN, giving a larger, more accurate image of cancer and melanoma. The complexity and variability of cancer epigenetics makes this a growing and important field. Since epigenetic modifications are able to be reversed, this makes them a target for therapeutics and part of the future of cancer combat, and specifically melanoma, due to its deadliness and difficulty to catch. The genes listed above are only the beginning of a long list of available treatment areas that could potentially reverse or prevent melanoma if detected early.


Epigenetics in forensic science  is the application of  epigenetics  to solving crimes. [ 1 ] [ 2 ]


Forensic science  has been using DNA as evidence since 1984, however this does not give information about any changes in the individual since birth and will not be useful in distinguishing identical siblings.  The focus of epigenetics in the forensic field is on non-heritable changes such as aging and diseases. [ 2 ]


Epigenetics involves any changes to the DNA that does not affect the sequence, but instead affects the activity of the DNA, such as the level of transcription of a particular gene. These changes can be passed down transgenerationally through the germline or arise after birth from environmental factors. [ 3 ] [ 4 ]  In humans and other mammals,  CpG dinucleotides  are the main sequence that develops methylation, and because of this most studies on try and find unique methylation sites. There are a few methylation sites that have been determined as a cause of environmental influences from age, lifestyle, or certain diseases.


DNA methylation  is a common epigenetic mark being studied as potential evidence in forensic science. [ 5 ] [ 6 ]  Unlike DNA, realistic DNA methylation is less likely be planted at crime scenes.> [ 6 ]  Current methods to fabricate DNA usually exclude important methylation marks found in biological tissues making this a way to confirm the identity of an individual when evidence is being assessed. [ 2 ]


Many different tissues can be used to analyze methylation.


The effect of  cryopreservation  on epigenetic marks in tissues is a new area of study. The primary focus of this research is on oocytes and sperm for the purpose of  assisted reproductive technology , however it can be useful in forensics for the preservation of evidence. [ 7 ]  Methylation can be analyzed in fresh tissue that is cryo-preserved within 24 hours of death and it can then be analyzed in this tissue for up to 1 year. [ 8 ]  If the tissue is formalin-fixed or putrefied, methylation analysis is much more difficult.


Although blood is the primary sample used in studies, most tissues consistently show that methylation increases early in life and slowly decreases, globally, throughout late adulthood. [ 9 ]  This process is referred to as epigenetic drift.


The  epigenetic clock  refers to methylation sites that are highly associated with aging. [ 10 ]  These sites consistently change across individuals and can therefore be used as age markers for an individual. There are some models that have been developed to predict ages for specific samples, such as saliva and buccal epithelial cells, blood, or semen, but others have been made to age any tissue. In 2011, three significant, hypermethylated CpG sites related to aging across all samples were found in the  KCNQ1DN ,  NPTX2 , and  GRIA2  genes. [ 9 ]  The age guess for over 700 samples had a mean absolute deviation from chronological age (MAD) of 11.4 years. Two years later, almost 8,000 samples were used in an  elastic net regularized regression  to create a new age predictive model. [ 9 ]  This resulted in 353 CpG sites being chosen for the age prediction, and the model had a MAD of 3.6 years.


There is evidence for specific methylation sites to be associated with the circadian clock, meaning a sample could have a time of day associated with their death through methylation marks. In whole blood from humans, plasma homocysteine and global DNA methylation change in levels throughout the day. [ 11 ]  Homocysteine levels peak in the evening and are at their lowest overnight while DNA methylation follows an inverse pattern. Other studies with rats found that expression of  DNMT3B  and other methylation enzymes oscillate with the circadian clock and may be regulated by the circadian clock. [ 11 ]  Another methylation associated factor, MECP2, is phosphorylated by the  superchiasmatic nucleus  in response to light signaling. In a group of subjects that died from a variety of causes, there was partial methylation at the PER2, PER3, CRY1, and TIM promoters which are important genes in controlling the circadian clock. [ 8 ]  The methylation of CRY1 varied within an individual's tissues and between two individuals, however the difference between individuals may have been due to methamphetamine exposure.


An age model using dentin from teeth is currently being studied. [ 12 ]  Over 300 genes have been found that are a part of odontogenesis and quite a few affect the epigenome. For example, JMJD3 is a histone demethylase that modifies the methylation of homeobox and bone morphogenetic proteins. [ 13 ]  More studies are being done to differentiate genetic, epigenetic, and environmental factors on methylation in teeth so that aging algorithms are more accurate.


Previously, measuring differences in between sets of teeth was done with calipers, but 2D and 3D imaging has become more available and allows for better accuracy of measurements. New programs are being developed to analyze these images of teeth. [ 14 ]  Mono-zygotic twin studies reveal 8-29% of changes between the twins' teeth is from the environment. Several studies of mono-zygotic twins have shown that when they have a tooth defect, such as congenitally missing or supernumerary teeth, the twins can share the same number or position of the defective tooth, but sometimes not both of these factors. [ 15 ]


Mono zygotic  twins provide information on epigenetic differences that are not from genetic factors. Epigenetic markers differ the most in monozygotic twins who spend time apart or have a very different medical history. As twins age, their methylation and acetylation of histone H3 and H4 increasingly vary. [ 16 ]  These marks are specific to the environmental changes between the twins and not changes in methylation from general aging. The rate of disease discordance between monozygotic twins is usually over 50%, including heritable diseases. [ 17 ]  This does not correlate to the disease prevalence rate.


There are more phenotypic methylation differences in twins discordant for bipolar, schizophrenia, or systemic lupus erythematosus than in unrelated cases. [ 17 ]  There is no difference between twins discordant for rheumatoid arthritis or dermatomyositis. A limitation to the current studies on twin disease discordance is the lack of a baseline epigenetic profile of the twins before they develop the disease. [ 17 ]  This baseline will be used to distinguish the environmental changes between the twins to narrow down the methylation sites related to the disease. Several studies are obtaining newborn epigenetic profiles for long-term research.


Epigenetics in insects  is the role that  epigenetics  (hertiable characteristics that do not involve changes in DNA sequence) plays in  insects .


Epigenetic mechanisms are regulatory mechanisms, which change  expression levels of genes . Several mechanisms are considered  epigenetic , including  DNA methylation ,  histone modifications  and  non-coding RNAs . Epigenetic mechanisms play a role in processes like  development ,  learning and memory formation ,  aging ,  diseases ,  cell differentiation  and genome defence.


DNA methylation is an epigenetic mechanism. It is a chemical modification of the DNA where a  methyl group  is attached to  cytosine . This modification is set by  DNA methyltransferases  (Dnmts). There are three known types of DNA methyltransferases in  mammals . Those DNA methyltransferases are present in  insects  as well, although it varies between different species which specific Dnmt types are present. It still is a matter of discussion what the specific role of DNA methylation in insects is, as some insects such as  Drosophila melanogaster  just have traces of DNA methylation in their  genome  and in general insect genomes are much less methylated compared to mammalian genomes (0.034% vs. 7.6% in  Mus musculus ). [ 1 ]  In a comparison of different insect species and their respective methylation levels, there was a clear relationship between cell turn over and DNA methylation, but not between genome size or the number of repetitive sequences and DNA methylation.


Honeybees ( Apis mellifera ) possess  homologs  for all three DNA methyltransferases known in mammals. [ 2 ] [ 3 ]  But unlike mammals they possess two DNA methyltransferases 1 and just one DNA methyltransferase 3. DNA methylation predominantly occurs in  coding regions  in honeybees. [ 4 ]  The function of the DNA methylation in honey bees is to regulate gene alternative splicing  [ 5 ]


DNA methylation plays a major role in honeybee caste and subcaste development. In honeybees there are two different castes which are workers and queens. They are genetically the same, but show morphological, physiological and behavioral differences. Among the worker caste there are two subcastes, which are nurses and foragers. Which subcaste a worker bee belongs to depends on its age. The DNA methylation pattern in queens and workers, [ 4 ] [ 6 ] [ 7 ]  and between nurses and foragers is different. [ 8 ] [ 9 ]  DNA methylation also increases in worker larvae with age, especially in coding regions and  CpG islands . If DNA methyltransferase 3 is silenced in honeybee larvae they develop into queens, whereas they otherwise would develop into workers. [ 10 ]


Using the DNA methyltransferase inhibitor  zebularine , the role of DNA methyltransferases during  learning  and  memory  formation has been studied. If DNA methyltransferases are inhibited during an  associative learning  paradigm, in which the bee is trained to associate an odour with a food reward, the odour specific associative  long-term memory  of bees is impaired, [ 11 ]  as well as their  extinction  memory. [ 12 ]   Short-term memory  formation and  acquisition  are not affected by DNA methyltransferase inhibition.


The fruitfly  D. melanogaster  possess just one DNA methyltransferase, which is Dnmt 2-like. Dnmt 2 is not known to methylate DNA in mammals. In Drosophila however a knock down of Dnmt 2-like protein is sufficient to deplete DNA methylation completely and an overexpression of Dnmt 2 causes hypermethylation of the DNA. [ 13 ]


While the cellular and molecular mechanisms of  learning  and  memory  have long been a central focus of  neuroscience , it is only in recent years that attention has turned to the  epigenetic mechanisms  behind the dynamic changes in  gene transcription  responsible for  memory formation  and maintenance.  Epigenetic gene regulation  often involves the physical marking (chemical modification) of  DNA  or associated proteins to cause or allow long-lasting changes in gene activity. Epigenetic mechanisms such as  DNA methylation  and  histone  modifications ( methylation ,  acetylation , and  deacetylation ) have been shown to play an important role in learning and memory. [ 1 ]


DNA methylation  involves the addition of a  methyl group  to a 5'  cytosine  residue. This usually occurs at cytosines that form part of a cytosine-guanine dinucleotide ( CpG sites ). Methylation can lead to activation or repression of  gene transcription  and is mediated through the activity of  DNA methyltransferases  (DNMTs).  DNMT3A  and  DNMT3B  regulate  de novo  methylation of CpG sites, while  DNMT1  maintains established methylation patterns. [ 2 ]  S-adenosyl methionine acts as the methyl donor. [ 3 ]


One current hypothesis for how DNA methylation contributes to the storage of memories is that dynamic DNA methylation changes occur temporally to activate transcription of genes that encode for proteins whose role is to stabilize memory. Another hypothesis is that changes in DNA methylation that occur even early in life can persist through adulthood, affecting how genes are able to be activated in response to different environmental cues.


The first demonstration about the role of epigenetics in learning in memory was the landmark work of Szyf and Meaney (PMID 15220929) where they showed that licking and grooming by mother rats (maternal care) prevented methylation of the glucocorticoid receptor gene. When these pups become adults, they respond better to stressors than rats who, as pups, were not licked and groomed by their mothers and instead had a buildup of methylation in the glucocorticoid receptor gene.


Miller and Sweatt demonstrated that rats trained in a contextual  fear conditioning  paradigm had elevated levels of  mRNA  for  DNMT3a  and  DNMT3b  in the  hippocampus . [ 4 ]  Fear conditioning is an associative memory task where a context, like a room, is paired with an  aversive stimulus , like a foot shock; animals who have learned the association show higher levels of freezing behavior when exposed to the context even in the absence of the aversive stimulation. However, when rats were treated with the  DNMT inhibitors   zebularine  or  5-aza-2′-deoxycytidine  immediately after fear-conditioning, they demonstrated reduced learning (freezing behavior). When treated rats were re-trained 24 hours later, they performed as well as non-treated rats. Furthermore, it was shown that when these DNMT inhibitors were given 6 hours after training, and the rats were tested 24 hours later, the rats displayed normal fear memory, indicating that DNMTs are involved specifically in memory consolidation. [ 4 ]  These findings reveal the importance of dynamic changes in methylation status in memory formation.


Feng et al. created double conditional  knock out  (DKO) mice for the genes DNMT3a and DNMT1. These mice were shown to have significantly weakened  long-term potentiation  (LTP) and much more easily stimulated  long-term depression  (LTD) in the hippocampus. When tested in the  Morris water navigation task , which is used to study hippocampus-dependent  spatial memory , the DNMT3a/DNMT1 DKO mice took longer to find the platform than control mice. Single knock-out mice (SKO) for either DNMT3a or DNMT1 performed normally. [ 5 ]  DKO mice were also unable to  consolidate memory  after fear-conditioning. Since SKO mice did not exhibit the same learning and memory defects as the DKO mice, it was concluded that DNMT3a and DNMT1 play redundant roles in regulating learning and memory.


When DNMTs are inhibited in the  prefrontal cortex , recall of existing memories is impaired, but not the formation of new ones. This indicates that DNA methylation may be circuit-specific when it comes to regulating the formation and maintenance of memories. [ 6 ]


The memory suppressor gene,  protein phosphatase 1  ( PP1 ), was shown to have increased  CpG island  methylation after contextual-fear conditioning. This corresponded to decreased levels of PP1 mRNA in the hippocampus of the trained rats. When DNMTs were inhibited, increased methylation at the  PP1  gene was no longer observed. [ 4 ]  These data suggest that during memory consolidation in associative learning tasks, CpG methylation is used to inhibit the expression of  PP1 , a gene that negatively inhibits memory formation.


While DNA methylation is necessary to inhibit genes involved in  memory suppression ,  DNA demethylation  is important in activating genes whose expression is positively correlated with memory formation. Sweatt and Miller also showed that the gene  reelin , which is involved in long term potentiation induction, had a reduced methylation profile and increased reelin mRNA in fear-conditioned versus control rats.  Brain-derived neurotrophic factor  ( BDNF ), another important gene in neural plasticity, has also been shown to have reduced methylation and increased transcription in animals that have undergone learning. [ 7 ]  While these studies have been linked to the  hippocampus , recent evidence has also shown increased demethylation of  reelin  and  BDNF  in the  medial prefrontal cortex  (mPFC), an area involved in cognition and emotion. [ 8 ]


The mechanism behind this experience-dependent demethylation response was previously not fully understood, with some evidence showing that DNMTs may be involved in demethylation. [ 7 ]  It was also suggested that members of the DNA damage repair  GADD45  family may contribute to this demethylation process. [ 2 ] [ 3 ]   However, more recently, the pathways illustrated in the Figure below, titled "Demethylation of 5-Methylcytosine (5mC) in neuron DNA," especially the  TET  dependent pathway, have been confirmed as pathways of DNA demethylation. [ 9 ]   A role for GADD45 has also recently been indicated, since GADD45 physically interacts with  thymine-DNA glycosylase  (TDG) and GADD45 may promote the activity of TDG in its role(s) during conversion of 5mC to cytosine. [ 9 ]


Mice that have genetic disruptions for  CpG binding protein 2  (MeCP2) have been shown to have significant problems in  hippocampus#Role in memory -dependent memory and have impaired hippocampal LTP. [ 2 ]


Changes in expression of genes associated with  post-traumatic stress disorder  (PTSD), which is characterized by an impaired extinction of traumatic memory, may be mediated by DNA methylation. [ 10 ]
In people with  schizophrenia , it has been shown that  reelin  is down-regulated through increased DNA methylation at  promoter  regions in  GABAergic  interneurons.  DNMT1  has also been shown to be upregulated in these cells. [ 10 ]


Methylation of  histones  may either increase or decrease gene transcription depending on which histone is modified, the amino acid that is modified, and the number of methyl groups added. [ 11 ]  In the case of  lysine   methylation , three types of modifications exist: monomethylated, dimethylated, or trimethylated lysines. The di- or trimethylation of  histone H3  at lysine 9 (H3K9) has been associated with transcriptionally silent regions, while the di- or trimethylation of histone H3 at lysine 4 (H3K4) is associated with transcriptionally active genes. [ 12 ]


The hippocampus is an important brain region in memory formation. H3K4 trimethylation is associated with active transcription. In contextual fear conditioning experiments in rats, it was found that levels of H3K4 trimethylation increases in the  hippocampus  after fear conditioning. [ 13 ]  In these experiments by Gupta et al., a connection was made between changes in histone methylation and active gene expression during the consolidation of associative memories. [ 13 ]  In this same study, it was also found that these histone methylations were reversible, as the levels of trimethylation of H3K4 returned to basal levels after a period of 24 hours. This indicated that active demethylation was occurring following  memory consolidation . To further explore the role of  methyltransferases  in long-term memory formation, this study applied the same fear conditioning tests on rats deficient in  Mll , a H3K4-specific methyltransferase. The rats with a heterozygous mutant Mll+/− gene showed a significant reduction in their ability to form  long-term memories  compared to normal rats with an intact Mll gene. Therefore, H3K4 methyltransferases, such as Mll, must have an essential role in long-term memory formation in the hippocampus. [ 13 ]


The change in methylation state of histones at the location of specific gene promoters, as opposed to just genome-wide, is also involved in memory formation. [ 13 ]   Zif268  and  BDNF  genes are critical for memory consolidation. [ 14 ]  H3K4 trimethylation increases around both of the Zif268 and BDNF promoters following contextual fear conditioning, when these genes are transcriptionally active. This demonstrates that at the time of memory consolidation, the transcription of memory formation genes such as Zif268 and bdnf is regulated by histone methylation. [ 13 ]


Histone H3 lysine 9 dimethylation is associated with  transcriptional silencing . [ 12 ]  The  G9a / G9a-like protein  (GLP) complex is a methyltransferase specific for producing this modification. [ 15 ]  One study examined the role of G9a/GLP-mediated transcriptional silencing in the hippocampus and  entorhinal cortex  (EC) during memory consolidation. It was found that the inhibition of G9a/GLP in the EC, but not in the hippocampus, results in the enhancement of long-term memory formation. [ 16 ]  In addition, G9a/GLP inhibition in the entorhinal cortex altered histone H3 lysine 9 dimethylation in the  Cornu Ammonis area 1  of the hippocampus, suggesting the importance of this complex in mediating connectivity between these two brain regions. Therefore, the G9a/GLP complex plays an important role in histone methylation and long-term memory formation in the hippocampus and the EC. [ 16 ]


Histone methylation marks are also correlated with other epigenetic modifications, such as  histone deacetylation  and DNA methylation, in the context of learning and memory. Reduced histone deacetylation is correlated with an increase in H3K9 dimethylation, a modification associated with transcriptional silencing. [ 13 ]  Therefore, histone deacetylase inhibitors may be applied to increase histone acetylation and suppress H3K9 dimethylation, thereby increasing gene transcription. In the case of DNA methylation, it was found that increases in H3K4 trimethylation correlate with altered DNA methylation of  CpG sites  at the promoter of  Zif268 , a gene involved in memory formation, after fear conditioning. Gupta et al. showed that DNA methylation at the Zif268 promoter increased after fear conditioning, correlating with an increase in Zif268 gene expression. [ 13 ]  This finding was surprising, since it was previously thought that DNA methylation resulted in transcriptional silencing. [ 13 ]


Acetylation  involves the replacement of a hydrogen with an  acetyl group . In a biological context, acetylation is most often associated with the modification of proteins, specifically  histones . The acetylation reaction is most often catalyzed by enzymes that contain  histone acetyltransferase  (HAT) activity.


HATs are enzymes responsible for the acetylation of amino acids. HATs acetylate by converting the  lysine  side group of  amino acids  with the addition of an acetyl group from an  acetyl CoA  molecule, creating  acetyl lysine . HAT enzymes are most often associated with histone proteins and work to regulate the interaction between histones and the DNA that is wrapped around them. HATs are not only restricted to the acetylation of histone but can also acetylate many other proteins implicated in the manipulation of gene expression like that of transcription factors and receptor proteins.


Acetylation is one of the main mechanisms implicated in the process of  chromatin remodeling . Chromatin remodeling affects the regulation of gene expression by altering the relationship between  nucleosomes  and DNA. Acetylation of histones removes positive charge, which reduces the level of interaction between the formerly positively charged histone and the negatively charged phosphate groups of the DNA wrapped around the nucleosome complex. This alteration in charges causes a relaxation of DNA from the nucleosome, this relaxed section is seen to have higher levels of gene expression than non acetylated regions.


Patterns of histone acetylation have been useful as a source of epigenetic information due to their ability to reflect changes in transcription rates and the maintenance of gene expression patterns. This acetylation code can then be read and provide generous information for the study of inheritance patterns of epigenetic changes like that of learning, memory and disease states.