Like HDACs,  histone methyltransferases  (HMTs) alter histones, but these enzymes are involved in the transfer of  methyl groups  to the histone's  arginine  and  lysine  residues. Chronic stress has been found to decrease the levels of a number of HMTs, such as  G9a , in the NAc of susceptible mice. [ 13 ]  Conversely, in resilient mice, these HMTs have increased activity. H3K9 and H3K27 have less methylation when depressive behavior is seen. The hippocampus also experiences a number of histone methylation changes: H3K27-trimethylation is hypomethylated in response to stress, while H3K9-trimethylation and H3K4-trimethylation are hypermethylated in response to short term stress. However, H3K9-trimethylation and H3K4-trimethylation can also be hypomethylated in response to chronic, long term stress. In general, stress leading to depression is correlated with a decrease in methylation and a decrease in the activity of HMTs. [ citation needed ]


Brain-derived neurotrophic factor  (BDNF) is a neurotrophic  growth factor  that plays an important role in memory, learning, and higher thinking. It has been found that BDNF  plasma  levels and hippocampal volume are decreased in individuals with depression. [ 7 ]  The expression of BDNF can be affected by different epigenetic modifications, and BDNF  promoters  can be individually regulated by different epigenetic alterations.  MeCP2  can act as a  repressor  and has been shown to regulate BDNF when activated.  Depolarization  of neurons causing an increase in calcium leads to the  phosphorylation  of MeCP2, which results in a decrease in the binding of MeCP2 to BDNF promoter IV. [ 14 ]  Because MeCP2 can no longer bind to the BDNF promoter and repress transcription, BDNF levels increase and neuronal development improves. When there is direct methylation of the BDNF promoter, transcription of BDNF is repressed. Stressful situations have been shown to cause increased methylation of BDNF promoter IV, which causes an increase in MeCP2 binding, and as a result reduction in the activity of BDNF in the hippocampus and depressive behavior. BDNF maintains the survival of neurons in the hippocampus, and decreased levels can cause hippocampal  atrophy . Also, there was found to be increased methylation of BDNF region IV CpGs in the  Wernicke area  of the brain in suicidal individuals. [ 14 ]  The interaction of BDNF and MeCP2 is complex, and there are instances where MeCP2 can cause an increase in BDNF levels instead of repressing. Previous studies have found that in MeCP2 knockout mice, the release and trafficking of BDNF within the neurons are significantly decreased in the hippocampus. [ 15 ]  Another epigenetic modification of BDNF promoters is the neuron-restrictive silencing factor ( REST or NRSF ) which epigenetically regulates the BDNF promoter I and is repressed by MeCP2. Like MeCP2, REST has also been found to inhibit BDNF transcription.


In the  hypothalamic-pituitary-adrenal axis  (HPA axis),  corticotropin-releasing factor  (CRF) is secreted by the hypothalamus in response to stress and other normal body processes. CRH then acts on the  anterior pituitary  and causes it to secrete  adrenocorticotropic hormone  (ACTH). ACTH acts on the  adrenal cortex  to secrete  cortisol , which acts as a  negative feedback  indicator of the pathway. When an individual is exposed to stressful situations, the HPA axis activates the  sympathetic nervous system  and also increases the production of CRF, ACTH, and cortisol, which in turn increases  blood glucose levels  and suppresses the immune system. Increased expression of CRF has been found in the  cerebrospinal fluid  in depressed monkeys and rats, as well as individuals with depression. Increased CRF levels have also been seen in the hypothalamus of depressed individuals. [ 16 ]  It was found that pregnant mice in early gestation stage who were exposed to chronic stress produced offspring with a decreased methylation of the CRF promoter in the hypothalamus area. [ 17 ]  This decreased methylation would cause increased expression of CRF and thus, increased activity of the HPA axis. The higher levels of the HPA axis in response to chronic stress can also cause damage to the hippocampus region of the brain. Increased cortisol levels can lead to a decrease in hippocampal volume which is commonly seen in depressed individuals.


Glial cell-derived neurotrophic factor  (GDNF) is a protein that aids in the survival and differentiation of  dopaminergic  neurons. By looking at expression levels in the nucleus accumbens, it is seen that GDNF expression is decreased in strains of mice susceptible to depression. [ 4 ]  It has also been shown that increased GDNF expression in the  ventral tegmental area  is present in mice that are not susceptible to  social defeat stress  by promoting the survival of neurons. [ 18 ] [ 19 ]  The ventral tegmental area and nucleus accumbens network of the  mesolimbic  dopamine system is thought to be involved in the resistance and susceptibility to chronic stress (which leads to depressed behavior). Thus it is seen that GDNF, by protecting neurons of the mesolimbic pathway, helps to protect against depressive behavior. [ 20 ]  After chronic stress, there are a number of changes that result in the reduction of GDNF levels in the nucleus accumbens. This decrease is associated with decreased H3 acetylation and decreased H3K4-trimethylation, as well as an increased amount of DNA methylation at particular  CpG  sites on the GDNF promoter. [ 20 ]  This DNA methylation is associated with histone deacetylase 2 and  methyl CpG binding protein 2  (MeCP2) recruitment to the GDNF promoter. [ 20 ]  Increased HDAC activity results in a reduction of GDNF expression, since HDAC causes the decreased acetylation at H3. Alternatively, knocking out HDACs (via HDAC  interference ) results in normalization of GDNF levels, and as a result, decreased depression like behavior, even in susceptible strains of mice. [ 21 ]   Cyclic-AMP response element-binding protein  (CREB), which is thought to be involved in GDNF regulation, associates with the aforementioned MeCP2, and complexes to methylated CpG sites on the GDNF promoter. [ 22 ] [ 23 ]  This recruitment of CREB plays a role in the repression of GDNF in the nucleus accumbens. As further evidence that DNA methylation plays a role in depressive behavior, delivery of  DNA methyltransferase inhibitors  results in a reversal of depression-like behaviors.


It is seen that DNA methylation of the GDNF  promoter region  results in the recruitment of MeCP2 and HDACs, resulting in an epigenetic alteration of the histone marks. This correlates to an increase in depression-like behavior.


Glucocorticoid receptors  (GR) are receptors to which cortisol (and other  glucocorticoids ) bind. The bound receptor is involved in the regulation of gene transcription. [ 4 ] [ 24 ]  The GR gene promoter region has a sequence that allows for binding by the transcription factor  nerve growth factor induced protein A  (NGFI-A), which is involved in  neuronal plasticity . In rats, it has been shown that individuals less susceptible to depressive behavior have increased binding of NGFI-A to the promoter region of the GR gene, specifically in the hippocampus. As a result, there is an increased amount of hippocampal GR expression, both in transcription of its  mRNA  and overall protein level.


This is associated with an increase in acetylation of H3K9 in the GR promoter region. Methylation of  CpG islands  in the promoter region of GR leads to a decrease in the ability of NGFI-A to bind to the GR promoter region. It has also been experimentally shown that methylation of CpG sites in the  enhancer region  bound by NGFI-A is detrimental to the ability of NGFI-A to bind to the promoter region. [ 25 ]  Furthermore, the methylation of the promoter region results in a decrease in recruitment of the  CREB-binding protein , which has  histone acetyltransferase  ability. This results in less acetylation of the histones, which has been shown to be a modification that takes place within individuals less susceptible to depression. [ 25 ]


Due to environmental factors, there is a decrease in methylation of the promoter region of the GR gene, which then allows for increased binding of the NGFI-A protein, and as a result, an increase in the expression of the GR gene. This results in decreased depressive behavior.


Through computational methodology, epigenetics has been found to play a critical role in mood disorder susceptibility and development, and has also been shown to mediate treatment response to SSRI medications. SSRI medications including fluoxetine, paroxetine, and escitalopram reduce gene expression and enzymatic activity related to methylation and acetylation pathways in numerous brain regions implicated in patients with major depression. [ 26 ]


Pharmacogenetic research has focused on epigenetic factors related to BDNF, which has been a biomarker for neuropsychiatric diseases. BDNF has been shown to be sensitive to the prolonged effects of stress (a common risk factor of depressive phenotypes), with epigenetic modifications (primarily histone methylation) at BDNF promoters and splice variants. Such variation in gene splicing and repressed hippocampal BDNF expression is associated with major depressive disorder while increased expression in this region is associated with successful antidepressant treatment. [ 26 ]   Patients with major depression and bipolar disorder show increased methylation at BDNF promoters and reduced BDNF mRNA levels in the brain and in blood monocytes while SSRI treatment in patients with depression results in decreased histone methylation and increased BDNF levels. [ 26 ]


In addition to the BDNF gene, micro RNAs (miRNAs) play a role in mood disorders, and transcript levels are suggested in SSRI treatment efficacy. Post-mortem work in patients with major depressive disorder, as well as other psychiatric diseases, show that miRNAs play a critical role in regulating brain structure via synaptic plasticity and neurogenesis. [ 26 ]  Increased hippocampal neural development plays a role in the efficacy of antidepressant treatment, while reductions in such development is related to neuropsychiatric disorders. [ 26 ]  In particular, the miRNA MIR-16 plays a critical role in regulating these processes in individuals with mood disorders. Increased hippocampal MIR-16 inhibits proteins which promote neurogenesis including the serotonin transporter (SERT), which is the target of SSRI therapeutics. [ 26 ]  MIR-16 downregulates SERT expression in humans, which decreases the number of serotonin transporters. [ 26 ]  Inhibition of MIR-16 therefore promotes SERT production and serves as a target for SSRI therapeutics. [ 26 ]  SSRI medications increase neurogenesis in the hippocampus by reductions in MIR-16, thereby restoring hippocampal neuronal activity following treatment in patients with neuropsychiatric disorders. [ 26 ]  In patients with major depressive disorder, treatment with SSRI medications results in differential expression of 30 miRNAs, half of which play a role in modulating neuronal structure and/or are implicated in psychiatric disorders. [ 26 ]


Understanding epigenetic profiles of patients with neuropsychiatric disorders in key brain regions has led to more knowledge of patient outcome following SSRI treatment. Genome wide association studies seek to assess individual polymorphisms in genes which are implicated in depressive phenotypes, and aid in the efficacy of pharmacogenetic studies. [ 27 ]   Single-nucleotide polymorphisms  of the 5-HT(2A) gene correlated with paroxetine discontinuation due to side effects in a group of elderly patients with major depression, but not mirtazapine (a non-SSRI antidepressant) discontinuation. In addition, hypomethylation of the SERT promoter was correlated with poor patient outcomes and treatment success following 6 weeks of escitalopram treatment. [ 26 ]  Such work addressing methylation patterns in the periphery has been shown to be comparable to methylation patterns in brain tissue, and provides information allowing for tailored pharmacogenetic approaches. [ 26 ]


Decreased brain-derived neurotrophic factor (BDNF) is known to be associated with depression. Research suggests that increasing BDNF can reverse some symptoms of depression. For instance, increased BDNF signaling can reverse the reduced hippocampal brain signaling observed in animal models of depression. BDNF is involved in depression through its effects on serotonin. BDNF has been shown to promote the development, function, and expression of serotonergic neurons. [ 28 ]  Because more active serotonin results in more positive moods, antidepressants work to increase serotonin levels. Tricyclic antidepressants generally work by blocking  serotonin transporters  in order to keep serotonin in the synaptic cleft where it is still active. Noradrenergic and specific serotonergic antidepressants antagonize serotonin receptors. Noradrenergic and specific serotonergic antidepressants ( NaSSAs )  such as  mirtazapine  and  tricyclic antidepressants  such as  imipramine  both increased BDNF in the cerebral cortices and hippocampi of rats. [ 29 ]  Because BDNF mRNA levels increase with long-term mirtazapine use, increasing BDNF gene expression may be necessary for improvements in depressive behaviors. This also increases the potential for neuronal plasticity. [ 30 ]  Generally, these antidepressants increase peripheral BDNF levels by reducing methylation at BDNF promoters that are known to modulate serotonin. [ 6 ]  As BDNF expression is increased when  H3K27me3  is decreased with antidepressant treatment, BDNF increases its effect on serotonin modulation. [ 31 ]  It modulates serotonin by downregulating the  G protein-coupled receptor ,  5-HT 2A  receptor  protein levels in the hippocampus. [ 32 ]  This increased BDNF increases the inhibition of presynaptic serotonin uptake, which results in fewer symptoms of depression.


Increased NGFI-A binding, and the resulting increase in glucocorticoid receptor (GR) expression, leads to a decrease in depression-like behavior. Antidepressants can work to increase GR levels in affected patients, suppressing depressive symptoms.  Electric shock therapy , is often used to treat patients with depression. It is found that this form of treatment results in an increase in NGFI-A expression levels. [ 33 ]  Electric shock therapy  depolarizes  a number of neurons throughout the brain, resulting in the increased activity of a number of intracellular pathways. This includes the cAMP pathway [ 33 ]  which, through downstream effects, results in expression of NGFI-A. Antidepressant drugs, such as  tranylcypromine  and  imipramine  were found to have a similar effect; treatment with these drugs led to increases in NGFI-A expression and subsequent GR expression. [ 33 ]  These two drugs are thought to alter synaptic levels of 5-HT, which then alters the activity level of the cAMP pathway. It is also known that increased glucocorticoid receptor expression has been shown to modulate the HPA pathway by increasing negative feedback. [ 33 ]  This increase in expression results from decreased methylation, increased acetylation and binding of HGFI-A transcription factor. [ 24 ]  This promotes a more moderate HPA response than seen in those with depression which then decreases levels of hormones associated with stress. [ 25 ]  Another antidepressant,  desipramine  was found to increase GR density and GR mRNA expression in the hippocampus. [ 34 ]  It is thought that this is happening due to an interaction between the response element of GR and the acetyltransferase, CREB Binding Protein. Therefore, this antidepressant, by increasing acetylation, works to lessen the HPA response, and as a result, decrease depressive symptoms.


HDAC inhibitors have been shown to cause antidepressant-like effects in animals. Research shows that antidepressants make epigenetic changes to gene transcription thus altering signaling. These gene expression changes are seen in the BDNF, CRF, GDNF, and GR genes (see above sections). Histone modifications are consistently reported to alter chromatin structure during depression by the removal of acetyl groups, and to reverse this, HDAC inhibitors work by countering the removal of acetyl groups on histones. HDAC inhibitors can decrease gene transcription in the hippocampus and prefrontal cortex that is increased as a characteristic of depression. In animal studies of depression, short-term administration of HDAC inhibitors reduced the fear response in mice, and chronic administration produced antidepressant-like effects. This suggests that long-term treatment of HDAC inhibitors help in the treatment of depression. Some studies show that administration of HDAC inhibitors like  vorinostat  and  romidepsin , hematologic cancer drugs, can augment the effect of other antidepressants. These HDAC inhibitors may become antidepressants in the future, but clinical trials must further assess their efficacy in humans. [ 35 ]


In recent years it has become apparent that the environment and underlying mechanisms affect gene expression and the genome outside of the central dogma of biology. It has been found that many  epigenetic  mechanisms are involved in the regulation and expression of genes such as  DNA methylation  and  chromatin remodeling . These epigenetic mechanisms are believed to be a contributing factor to pathological diseases such as  type 2 diabetes . An understanding of the  epigenome  of diabetes patients may help to elucidate otherwise hidden causes of this disease.


The  PPARGC1A  gene regulates genes involved in energy metabolism. [ 1 ] [ 2 ]  Since  type 2 diabetes  is characterized by chronic  hyperglycaemia  as a result of impaired  pancreatic beta cell  function [ 3 ]  and  insulin resistance  in peripheral tissues, [ 4 ]  it was thought that the gene might be downregulated in type 2 diabetes patients through  DNA methylation .


The defects in pancreatic beta cell function and insulin resistance in peripheral tissues were thought to be the result of impaired  ATP  production from reduced  oxidative phosphorylation . In 2008, it was found that the  mRNA  expression of PPARGC1A was markedly reduced in  pancreatic islets  from type 2 diabetic donors compared with that of non-diabetic donors. Using bisulfite testing, it was also found that there was an approximately twofold increase in DNA methylation of the PPARGC1A  promoter  of human islet cells from diabetics as compared to non-diabetic human islet cells. This means that expression from the PPARGC1A genes were turned down in the diabetic patients. Further testing revealed that the more PPARGC1A was expressed, the more insulin was released from the islets, and as expected, in diabetic patients there was less PPARGC1A expressed and also less insulin secreted. This data supports the idea that PPARGC1A expression is reduced in  animal models  of diabetes and human diabetes and is associated with impaired insulin secretion. [ 5 ]


PGC-1α , the protein encoded by the gene PPARGC1A, can modulate glucose-mediated insulin secretion in human islets, most likely through an effect on ATP production. In human type 2 diabetic islets, reduced PPARGC1A mRNA levels were associated with impaired glucose-mediated insulin secretion. It was suggested that DNA methylation was the mechanism through which the PPARGC1A gene was turned down. [ 5 ]


In a different study where transcriptional changes due to a risk factors for diabetes, were examined, changes in the methylation patterns of the PPARGC1A gene were also found. In the study done on  physical inactivity , where subjects were required to have a sustained bed-rest of 10 days and were then examined, it was also found that there was significant downregulation of the PPARGC1A gene. In addition, it was shown that after the bed rest, there was a marked increase in DNA methylation of the PPARGC1A gene along with a decrease in mRNA expression. [ 6 ]  Another risk factor is  low birth weight  (LBW); in a study, it was found that there was increased DNA methylation in the LBW patients' muscle cells. [ 7 ]


Micro RNAs  (miRNA) are single-stranded transcribed RNAs of 19–25  nucleotides  in length that are generated from endogenous hairpin structured transcripts throughout the genome. Recent studies have shown that miRNAs have pivotal roles in many different gene regulatory pathways. A subset of miRNAs has been shown to be involved in metabolic regulation of glucose homeostasis and in epigenetics of diabetes type 2. [ 8 ] [ 9 ] [ 10 ]


Pancreatic islet-specific  miR-375  inhibits insulin secretion in mouse pancreatic β-cells by inhibiting the expression of the protein  myotrophin . An overexpression of miR-375 can completely suppress glucose-induced insulin secretion, while inhibition of native miR-375 will increase insulin secretion. [ 11 ]  In another study, increasing the level of  miR-9 , a different miRNA, resulted in a severe defect in glucose-stimulated insulin release. This happens because miR-9 down-regulated the transcription factor  Onecut2  (OC2) that controls the expression of  RAB27A  effector  granuphilin , a key factor in controlling insulin release. [ 12 ]  Also miR-192 levels have been shown to be increased in  glomeruli  isolated from diabetic mice when compared to non-diabetic mice, suggesting that it is involved as well. Since miR-192 was shown to regulate extracellular matrix proteins  collagen, type I ,  α 1 and  α2  (Col1α1 and 2) that accumulate during diabetic  nephropathy , [ 13 ]  miR-192 may play a role in  kidney diseases  as well. A correlation between elevated  Notch signaling pathway  gene expression, which is important for cell to  cell communication , and diabetic nephropathy has also been shown. [ 14 ]  MiR-143 has also been experimentally shown to regulate genes that are crucial for  adipocyte  differentiation, (including  GLUT4 ,  hormone-sensitive lipase , the  fatty acid-binding protein, aP2  and  PPAR-γ2 ), demonstrating that miRNAs are also involved in  fat metabolism  and  endocrine  function in humans. [ 15 ] [ 12 ]


Epigenetics may play a role in a wide array of vascular complications and in diabetes. The epigenetic variations involved with diabetes can change  chromatin structure  as well as gene expression. Regardless of whether glycemic control has been achieved through treatment these epigenetic mechanisms are lasting and do not change with the alteration of diet. The most common vascular complication in patients with type 2 diabetes is  retinopathy  which causes many patients to go blind. Studies showed that retinal damage persisted even after the reversal of  hyperglycemia  in dogs. [ 16 ]  Studies with  streptozotocin  injected  type 1 diabetes  rats showed that the re-institution of glycemic control after a short period of hyperglycemia had protective effects in the eyes, including reduction in parameters of  oxidant stress  and inflammation. However, specimens with prolonged diabetes failed to show similar protection. [ 17 ]  It was then seen with  endothelial cells  (which line blood vessels) cultured in high glucose that there was a sustained increase in the expression of key extracellular and pro-fibrotic genes and persistently increased oxidative stress, after subsequent glucose normalization. [ 18 ]  These studies show that the deleterious effects of prior hyperglycemic exposure have long-lasting effects on target organs even after subsequent glycaemic control underscoring the beneficial effects of intensive glycemic control in diabetes. [ 19 ]  The persistence of these symptoms points to epigenesis as an underlying cause.


Studies have shown that the islet dysfunction and development of diabetes in rats is associated with epigenetic silencing via DNA methylation of the gene  Pdx1  promoter, which produces a key transcription factor that regulates  beta-cell  differentiation and insulin gene expression. [ 19 ]   Silencing  at this promoter decreases the amount of beta-cells produced which leads to insulin resistance and the inability of the beta-cells to produce an important peptide that prevents vascular deterioration and neuropathy caused from vascular  inflammatory responses .


A subsequent study shows that under high glucose conditions, islet-specific transcription factor Pdx1 was shown to stimulate insulin expression by recruiting co-activators  p300  and the  histone methyl transferase   SETD7/9 , which increased histone acetylation and  H3K4me2 , respectively, and the formation of open  chromatin  at the insulin promoter. In contrast, under low-glucose conditions, Pdx1 could recruit co-repressors  HDAC1 / 2 , which led to inhibition of insulin gene expression. Furthermore, Pdx1 also mediated β-cell-specific expression of SET7/9, which may regulate genes involved in glucose-induced insulin secretion. [ 19 ]


Nephropathy  is another common symptom of diabetes patients and is caused by  angiopathy  of the  capillaries  in the  kidneys . A gene known as  UNC13B  shows hypermethylation in diabetes patients genomes and is linked to diabetic nephropathy. [ 19 ]  The  National Center for Biotechnology Information  claims that hyperglycemia leads to an upregulation of this gene due to the increase in methylation at important  CpG sites  within the gene.  UNC13B  produces a protein with a  diacylglycerol  (DAG) binding domain. Hyperglycemia increases DAG levels in the blood which causes  apoptosis  in cells upregulating this gene and renal complications when DAG binds to the product of the UNC13B gene.


The production of fat hinders the ability for muscles and other cells in the body to respond properly to glucose and insulin, furthering the complications involved with diabetes. Increased levels of fat in the body and blood raises blood pressure, increases cholesterol, and causes arteriosclerosis; all of which are severely dangerous vascular complications for patients with diabetes and can lead to death. The epigenetic marks  H3K27me3 ,  H3K4me3 , and the  polycomb group of proteins  such as  Bmi-1 , the H3K27me3 transferase  Ezh2 , its demethylase  JMJD3 , and the H3K4me3 transferase  MLL  were shown to be regulators in the expression of tumour suppressor  p16INK4a  in β-cell proliferation and regeneration. Post-translational modifications of histones (H3K4me2 and  H3K9me2 ), H3K4  demethylase   lysine-specific demethylase 1  (LSD1), and an H3K9me2 methyltransferase  SET domain  bifurcated 1 ( SETDB1 ) were also implicated in diabetes related  adipogenesis . [ 19 ]


The  inflammatory response  arises from vascular tissues and specialized  white blood cells , and a persistent state of inflammation under diabetic stress leads to  clots  and vascular deterioration. Patients experience edema, aneurysms, and injuries that cannot heal properly because the  vascular system  is unable to respond properly when under epigenetic influences. Diabetes and the associated hyperglycemia can lead to production of pro-inflammatory mediators such as  cytokines  and  growth factors . Together, they activate multiple signal transduction pathways including oxidant stress,  tyrosine kinases ,  PKC , and  MAPKs  leading to activation of transcription factors such as  NF-κB , and dysregulation of epigenetic mechanisms including HKme, histone lysine acetylation, and DNA methylation via the action of corresponding methyltransferases, demethylases, acetylases, and deacetylases. This leads to higher accessibility of pathological gene expression products and activation of pathological genes. [ 19 ]  Being in this state of diabetic stress leads to long term metabolic memory and altered epigenome with adverse side effects on the  cardiovascular system .


Oxidized  low-density lipoprotein -induced  chemokine  expression was associated with H3KAc and phosphorylation, and recruitment of HATs along with NF-κB in  endothelial cells , and these were reversed by pre-treatment with  statins . Studies show alterations in histone modification patterns, along with changes in expression of the corresponding histone methyltransferases, in vascular smooth muscle cells and endothelial cells from aortas of adult mice exposed to  hypercholesterolaemia . Studies in  monocytes , white blood cells that travel along vascular surfaces, showed that H3K9/14Ac and HATs CBP/p300, H3R17me and its methyltransferase  CARM1 , play key roles in inflammatory gene expression.  HDACs  also played key roles in  lipopolysaccharide  (LPS)-induced inflammatory gene expression in monocytes and  macrophages . Prolonged inflammatory responses in the cardiovascular system lead to  atherosclerosis  and  high blood pressure  which contribute to the many heart attacks and strokes caused by diabetes each year. [ 19 ]


In humans it has been shown that inflammatory gene expression induced by diabetic stimuli like high glucose and a  RAGE  ligand  S100B  was associated with increased H3K9/14Ac genome wide along with increased recruitment of  NF-κB  and HATs  CBP /p300 at inflammatory gene promoters in  THP1 cell line  monocytes. In vivo, increased histone lysine acetylation at these promoters in monocytes obtained from  diabetes mellitus type 1  and  diabetes type 2  patients was seen. Acetylation at these promoters increases gene expression and increases the inflammatory response as a result. [ 20 ]  Genome-wide location studies using  chromatin immunoprecipitation  ( ChIP ) coupled with  DNA microarray  analysis revealed significant changes in H3K4me2 and H3K9me2 patterns at key gene regions in high glucose-treated THP-1 monocytes, with relevant changes being observed in primary monocytes from diabetes patients. [ 19 ]


A possible treatment for vascular complications of diabetes exists with the  SIRT1  gene, a member of  Sirtuin  family of genes. The SIRT1 enzyme has  HDAC  activity, and has been shown to modulate energy metabolism and inflammation. SIRT1 overexpression or activation by  resveratrol  could improve  insulin resistance  and SIRT1 activators are being developed for diabetes treatment. The role of other HDACs and the potential use of HDAC inhibitors in diabetes is not very clear. [ 19 ]  Other treatments look towards an anti-inflammatory agent and HAT inhibitor,  curcumin , which in trials ameliorated high glucose-induced inflammatory gene expression and histone acetylation at their promoters as well as changes in HAT and HDAC activities in human monocytes. [ 19 ]


Metabolic memory  is the phenomenon of diabetic vascular stresses persisting after glucose normalization in diabetic patients because of early an glycemic environment. Hyperglycemia appears to be remembered in organs such as the kidney, eyes, and heart. [ 21 ]  Evidence of this is found in patients who were always in intensive therapy when they are compared to patients who began in conventional therapy and then switched to intensive therapy. The first group had delayed progression of  nephropathy ,  retinopathy , and  peripheral neuropathy . Metabolic memory may be regulated by epigenetics.


Recent studies have shown that diabetic patients have decreased H3K9me3 and an increase in the  Histone methyltransferase  called  SUV39H1 , [ 22 ]  and all of these changes serve to repress chromatin. Normal patients treated with high glucose exhibited the same changes in DNA methylation, implying that the persistent changes in these marks could be due to the prior hyperglycemia. Oxidant stress may also play an important role in preserving this metabolic memory by modifying or damaging essential lipids, proteins, and/or DNA. [ 22 ]


Histone acetyltransferases  (HATs) and  HDACs  have been found to play key roles in genes linked to diabetes. One example is the SIRT family of HDACs, specifically  SIRT1  has been found to regulate several factors involved in metabolism, adipogenesis, and insulin secretion. It has also been seen that histone acetylation promotes some gene expression related to diabetic conditions. This was seen in an experiment when a high glucose treatment of cultured monocytes increased recruitment of the HATs  CREB-binding protein  (CPB) and  PCAF , leading to increased histone lysine acetylation at the  cyclooxygenase-2  (COX-2) and  TNF -inflammatory gene promoters. This led to a corresponding increase in gene expression, which was similar to the increased histone lysine acetylation at these gene promoters in type 1 diabetes and type 2 diabetes patients compared to healthy control volunteers. [ 22 ]


There are several factors that put people at higher risk of contracting type 2 diabetes. Among these are  obesity ,  lack of exercise , and  aging . But, not everyone that becomes diabetic falls under these categories. However, what has become clear is that there are multiple loci which increase a person's susceptibility to Type 2 diabetes. One study [ citation needed ]  examined a multitude of papers, creating a detailed list of epigenetic modifications and loci associated with type 2 diabetes. Prominent among these, was DNA methylation, or lack thereof. After examining type 2 diabetes patients, it was found that levels of  homocysteine  were exceptionally high when compared to levels in individuals without the disease. Homocysteine is an intermediate that is responsible for maintaining methylation reactions in critical metabolic processes. It can be re-methylated to form  methionine , be taken into the  cysteine biosynthetic pathway , or be freed into the  extracellular medium . [ 23 ]  When a person lacks sulfur in their diet, it prompts the body to use  methionine  and form cysteine. This in turn increases the risk of a person contracting type 2 diabetes later in life. The reason behind this turns out to be rather simple. Methionine directly affects  S-adenosyl methionine  (SAM) levels. SAM is the substance that provides the methyl groups for DNA methylation. A shortage of SAM leads to an inability to develop proper methylation patterns, and is thought to be an indicator of an increased risk of contracting type 2 diabetes. [ 24 ]


There are a number of genes involved in chromatin methylation. One of these genes encodes the enzyme  methylenetetrahydrofolate reductase  (MTHFR). MTHFR is involved in reducing  5,10-methylenetetrahydrofolate  to  5-methyltetrahydrofolate . [ 25 ]  This reaction is a critical step in the conversion of homocysteine to methionine. The resulting product is a methyl donor that is required for CpG and histone methylation. Mutations in this gene can lead to reduced methylation at  CpG sites , and these changes in methylation patterns may increase susceptibility for type 2 diabetes. The most common at the gene encoding MTHFR is the  C677t  mutation. This is not a spontaneous mutation; it is actually hereditary. While the mutation does not inactivate the gene, it greatly reduces the efficiency, thus impairing the formation of methionine. The lack of this amino acid prevents methylation from occurring, and as said above, hypomethylation leads to increased susceptibility to type 2 diabetes. [ 26 ]


Type 2 diabetes associated  sequence polymorphisms  have been identified [ citation needed ]  in 30  linkage disequilibrium  (LD) blocks across the human genome, but their effects only explain a minor fraction of the observed  phenotypic diversity  among individuals. Previously, in other studies it has been demonstrated that connections between small methylation differences at isolated CpG sites have large differences in gene-expression levels On a genome wide scale studies have proven that hypomethylation in genes known to be associated with type 2 diabetes has been associated with an increase in susceptibility for the disease. CpG sites in particular display a small but significant case of hypomethylation consistently. The odds of belonging to the type 2 diabetes group increased by 6.1% for every 1% decrease in methylation based on microarray-based assay. These observed methylation differences are capable of producing or indicating real expression differences, which lead to the observed enhanced disease risk. Based on this study further investigation proved that if hypomethylation was induced, then the individual was far more likely to develop type 2 diabetes than those who led a  healthy lifestyle . This proven presence of low methylation at certain sites could be used to diagnose diabetes 2 in individuals earlier in future treatment. [ 27 ]


Studies in 2011 have shown that  insulin resistance  (IR), the hallmark of type 2 diabetes, may also involve epigenetic control as a contributing factor. Promoter methylation of the  mitochondrial transcription factor A  gene, an important gene essential for  mitochondrial DNA  maintenance, was associated with IR in adolescents. A possible link between DNA methylation and insulin resistance was shown through  monozygotic twin  comparisons and  bisulfite pyrosequencing  to measure the global DNA methylation of  Alu repeats  in peripheral blood  leukocytes . Alu elements are known to create genomic instability and affect gene expression, and have been implicated in numerous human diseases. Epigenetic alterations arising during the lifetime of monozygotic twins in Alu repeats resulted in an increase in genomic s instability, and consequently cause insulin resistance and type 2 diabetes and show that methylation levels at all four CpG sites displayed an increase in Alu methylation. This study provides the first evidence that alteration in global DNA hypermethylation is associated with increased risk of IR independent of established risk factors. Because epigenetic modifications are possibly reversible, this research suggests the potential for lifestyle or therapeutic interventions for insulin resistance. [ 28 ]


As shown by various studies, there are a number of genes that affect a person's risk of contracting type 2 diabetes. The same applies for  obesity , which has several loci in common with the disease. Both are  polygenic , but it is possible to identify at least part of the regions via  DNA assays . Among these regions is the  fat mass  and obesity associated  FTO gene , which has shown to increase susceptibility to both obesity and type 2 diabetes. [ 29 ]  When looked at further, it was shown to have increased methylation levels in a  haplotype  associated with type 2 diabetes. To find exactly which portion of the gene led to the higher levels of risk, one group of researchers performed a  sliding window analysis . Using the information, they narrowed the search down to the 46 kb  linkage disequilibrium  block, and within that region found a 7.7 kb region in which methylation levels were abnormally high.  Pyrosequencing  found that this difference was due to  single nucleotide polymorphisms  (SNPs) that created CpGs across the haplotype. [ 30 ]


Within this 7.7 kb, haplotype-specific methylation area, a highly  conserved non-coding element  (HCNE) was found. Anja Ragvin, a researcher at the  University of Bergen , has proven that this HCNE directly effects  IRX3  expression. First, HCNE-driven expression patterns were identified via  protein imaging . Next, they were compared to IRX3 expression patterns. When compared, it was found that the two patterns matched. Researchers used this information to conclude that the linkage disequilibrium block of the FTO gene acts upon IRX3. These results were also supported by the presence of the  H3K4me1  enhancer signature left behind during both HCNE-driven expression and IRX3 expression. [ 29 ] [ 31 ]


In summary, persons at high risk for type 2 diabetes and obesity have a highly methylated 7.7 kb region in the linkage disequilibrium block of the FTO gene. This methylation leads to the deactivation of the HCNE located within it, and decreased IRX3 expression. When IRX3 was knocked out, there was a drop in the number of  β-cells  that produce  insulin  and the  α-cells  that produce  glucagon . This sudden drop indicates a direct relationship between the FTO gene, IRX3, and type 2 diabetes. [ 31 ]


Epigenetics of human development  is the study of how  epigenetics  (hertiable characteristics that do not involve changes in DNA sequence) effects  human development .


Development before birth, including  gametogenesis ,  embryogenesis , and  fetal development , is the process of body development from the  gametes  are formed to eventually combine into a  zygote  to when the fully developed organism exits the  uterus . Epigenetic processes are vital to fetal development due to the need to differentiate from a single cell to a variety of cell types that are arranged in such a way to produce cohesive tissues, organs, and systems.


Epigenetic  modifications such as  methylation  of CpGs (a dinucleotide composed of a 2'-deoxycytosine and a 2' deoxyguanosine) and  histone  tail modifications allow  activation or repression  of certain  genes  within a  cell , in order to create cell memory either in favor of using a gene or not using a gene. These modifications can either originate from the parental  DNA , or can be added to the gene by various  proteins  and can contribute to  differentiation . Processes that alter the epigenetic profile of a gene include production of activating or repressing  protein complexes , usage of  non-coding RNAs  to guide proteins capable of modification, and the proliferation of a signal by having protein complexes attract either another protein complex or more DNA in order to modify other locations in the gene.


Gene expression  refers to the  transcription  of a gene but the  RNA  produced does not necessarily have to encode a protein product.  Transcription may produce so called noncoding RNA products such as  tRNA  and  regulatory RNA .  Repression may refer to the decrease in transcription of a gene or inhibition of a protein.  Proteins are often inhibited by binding the active site or causing a  conformational change  so that the active site can no longer bind. By making these alterations, proteins, like  transcription factors , may bind DNA less or some protein may be inhibited so that it becomes a block in a  signaling cascade  and certain genes will then not be induced to be expressed. Repression can occur pre- or post-transcriptionally.  Methylating  the DNA or modifying the histones that the DNA wraps around is one example that commonly leads to repression.  Pre-transcriptional repression can also occur by altering the proteins that allow transcription to occur, namely the  polymerase  complex.  Proteins can sit on the DNA strand and serve as a kind of block to polymerase proteins, halting them from transcribing.  Post-transcriptional repression generally refers to the degradation of the RNA product or binding the RNA with proteins so that it cannot be translated or carry out its function.


DNA methylation  in humans and most other mammals refers to the methylation of a CpG. Methylation of these  cytosines  are common in DNA, and in sufficient numbers can prevent proteins from attaching to the DNA by obscuring the domain binding site's matching DNA to the protein. Regions in which cytosines prior to  guanines  are clustered and highly unmethylated are called  CpG islands , and often serve as  promoters , or transcription start sites.


Histone modifications are modifications made to the  amino acid  residues in the tails of the histones that either restrict the histone's ability to bind to DNA or boost the histone's ability to bind to DNA. Histone modifications also act as sites for proteins to attach, which then further alter the gene's expression. Two common histone modifications are  acetylation  and methylation. Acetylation is when a protein adds an  acetyl group  to a  lysine  in a histone tail in order to restrict the ability of the histone to bind to DNA. This acetylation is commonly found on lysine 9 of histone 3, notated as H3K9ac. This results in the DNA being more open to transcription, due to the decreased binding to the histone. Methylation, meanwhile, is when a protein adds a methyl group to a lysine in a histone tail, although more than one methyl group can be added at a time. Two sites for histone methylation are common in current studies: trimethylation of lysine 4 on histone 3 ( H3K4me3 ), which causes activation, and trimethylation of lysine 27 on histone 3 ( H3K27me3 ), which causes repression.


Cis acting elements refer to mechanisms that act on the same  chromosome  they come from, usually either in the same region from which they were produced or a region very close to this origin region. For example, a long non-coding RNA that is produced at one location silences the same or a different location on the same chromosome. Trans acting elements, however, are gene products from one location that act on a different chromosome, either the other in a chromosomal pair, or on a different chromosome from a separate chromosome pair. An example of this is a long non coding RNA from Hox gene C silences Hox gene D on a different chromosome, from a different chromosomal pair.


Hox genes  are genes in humans that regulate body plan development. Humans have four sets of Hox genes, numbering 39 genes altogether, all of which aid in the differentiation of cells by location. Hox genes are activated early in the  development of the embryo , in order to plan the development of the differing structures of the body. They also show colinearity with the body plan, meaning that the order of the Hox genes is similar to the expression levels of the Hox genes on the anterior-posterior axis. This colinearity allows for a spatial and temporal activation of genes in order to produce a proper body structure. [ 1 ] [ 2 ]


Hox genes are regulated using a variety of epigenetic mechanisms, including the use of  lncRNAs  such as  HOTAIR , the  Trithorax  (TrxG) group of proteins, and the  Polycomb  (PcG) group of proteins.


In Hox genes, long non-coding RNAs allow for communication between different Hox genes and different sets of Hox genes in order to coordinate body plan in the cell. One example of a long non-coding RNA that coordinates between Hox gene sets is HOTAIR, which is an RNA transcript produced in the HoxC cassette that represses transcription of a large number of genes in the HoxD cassette. Thus, HOTAIR regulates the HoxD genes from the HoxC genes in order to coordinate transcription of the Hox genes. [ 1 ]


The PcG and TrxG genes that produce protein complexes responsible for continuing the activation and the repression patterns in the Hox genes initially formed by the maternal factors. PcG genes are responsible for repressing  chromatin  in Hox clusters meant to be inactivated in the differentiated cell. PcG proteins repress genes by forming polycomb repressive complexes, such as  PRC1  and  PRC2 . PRC2 complexes repress by trimethylating histone 3 at lysine 27 through histone  methyltransferases   Ezh2  and  Ezh1 . PRC2 is recruited by many elements, including CpG islands. [ 1 ]  PRC1, meanwhile,  ubiquitinates  H2AK119 using Ring1A/B's  E3 ligase  activity, causing stalling of  RNA polymerase II . Furthermore, Ring1B, a member of the PRC1 complex, also represses Hox genes with Me118, Mph2, and RYBP by compacting the chromatin into higher-order structures. [ 3 ]  TrxG genes, meanwhile, are responsible for activating genes by trimethylating lysine 4 of the histone H3 tail. Genes with similar transcriptional marks tend to cluster together in distinct structures. In bivalent domains, both of these marks are present, indicating genes that are silenced but can be rapidly activated when necessary. [ 1 ] [ 4 ]


231 ncRNAs are present in the four Hox  gene cassettes . Similarly to the Hox protein-coding genes, the ncRNAs show differential expression according to the cell's location on the anterior-posterior and proximal-distal axes. These lncRNAs can act either on the set of genes which they are present in, or can act on a separate gene set within the Hox genes. [ 5 ] [ 6 ] [ 7 ]


HOTTIP  is a long non-coding RNA that assists in regulating the HoxA genes. It is produced from the 5' end of the HoxA gene cassette, and activates HoxA genes. Loops within the chromosome bring HOTTIP closer to its targets; this allows HOTTIP to bind to  WDR5 /MLL protein complexes to aid in trimethylation of lysine 4 of histone 3. [ 5 ]


HOTAIR is a long non-coding RNA that assists in regulating the HoxD genes. It is produced in the HoxC cassette, near the divide between expressed and unexpressed genes, and represses HoxD genes. HOTAIR acts by attaching to  Suz12  in the PRC2 complex, and then guides this complex to the genes to be repressed. PRC2 then trimethylates the lysine 27 of histone 3, repressing the gene of interest. [ 1 ]


In female humans,  Barr bodies  are defined as the condensed and inactivated  X-chromosome  that is found in every cell of the adult.  Because females have two nearly identical X chromosomes, one of them must be silenced so that the expression levels of the genes on the X-chromosome are of the proper dosage. Thus, males and females have the same level of X-chromosome expression, despite being born with one X for males and two for females. This is also why individuals with  Klinefelter syndrome , a disease in which more than two sex chromosomes are present in the body, have fewer symptoms than individuals with other types of  aneuploidy , which are often fatal before birth. [ 8 ]


Inactivation  of one of the X chromosomes is initiated by a long non coding RNA called  Xist . This lncRNA is expressed on the same chromosome it represses, known as working in cis.  Recent research has shown that a repeat element in the RNA of Xist causes PRC2 to bind to the RNA. Another part of the RNA binds to the X-chromosome positioning PRC2 such that it can methylate various regions on the X-chromosome. This methylation causes other factors like  histone deacetylases  (HDACs) to bind to the chromosome and propagate  heterochromatin  formation, even into active gene regions. This heterochromatin greatly reduces, if not completely silences gene expression of the Barr body. Xist will be continuously created to maintain a condensed and silenced Barr body. [ 9 ] [ 10 ] [ 11 ]


In human cells with more than one X chromosome, two long non-coding RNAs are produced:  Tsix  is produced by one X chromosome, and Xist is produced by all of the other X chromosomes. Tsix is a long non-coding RNA that prevents repression of an X chromosome, while Xist is a long non-coding RNA that acts to repress and condense an entire X chromosome. The actions of Xist serve to create a Barr body in the cell. [ 9 ]


In embryonic development, when the zygote is still composed of just a few cells, each cell of the zygote will randomly choose an X-chromosome to condense and silence. From then on, the  daughter cells  of that cell will always silence the same X-chromosome as the parent cell it propagated from. This creates what is known as the “mosaic effect,” in which differential X-chromosome expression creates differing  genotypes  throughout a single organism. This may or may not be evident in females, depending on how the genes of the X-chromosomes affect  phenotype . If the  alleles  for a gene are identical on both X-chromosomes, then you will see no difference between the cells that chose one X over the other. If the alleles are different for, say, fur color, then you may see patches of one color and patches of the other color. In  calico cats  the mosaic pattern of X inactivation is easily seen because a gene affecting coat color is carried on the X, resulting in patches of color on the coat. The mosaic pattern of X inactivation may also determine how penetrant a disease is, if the disease allele is present on one X-chromosome and not the other. The organism may have few cells in which the diseased allele has not been condensed, leading to little expression of the disease allele. This is referred to as skewed X-chromosome inactivation.


Imprinting  is defined as the differential expression of paternal and maternal alleles of a gene, due to epigenetic marks introduced onto the chromosome during the production of  egg  and  sperm . These marks usually lead to differential expression of the specific sets of genes from the maternal and paternal chromosomes. Imprinting is carried out through many epigenetic mechanisms like methylation, histone modifications, rearrangement of higher order chromatin structure, non-coding RNAs, and interfering RNAs. [ 12 ] [ 13 ]


A single evolutionary purpose of imprinting is still unknown, since the mechanisms and effects seem to be so diverse. One hypothesis states that imprinting occurs in order to carry out the evolutionary goal of the parent, that being the differential partition of resources. The male seeks to provide maximum resources for his offspring so that his genes may be passed on successfully to the next generation, whereas the female must partition resources between all her offspring, and so must limit resources given. [ 14 ] [ 15 ]


Another hypothesis states that imprinting may help protect the female from ovarian  trophoblastic disease  and  parthenogenesis . Trophoblastic disease occurs when a sperm fertilizes an egg with no nucleus and a cancer-like mass forms in the  placenta . [ 16 ]  Parthenogenesis occurs when an unfertilized egg develops into a fully functional organism that is genetically identical to the parent, who is female in the case of animals or both sexes, in the case of plants. [ 17 ]   This does not occur naturally in mammals.  In most animals, especially mammals, uniparental inheritance of chromosomes is often lethal or results in developmental abnormalities, sometimes physically but often cognitively. Other hypotheses point to the function of imprinting as a way of establishing the proper amount of expression or functional  haploidy , much like silencing the extra X-chromosome in females (see section on Barr bodies). Imprinting may help in the differentiation of cells by silencing  pluripotency  genes or other developmental genes. Supporting this hypothesis, imprinted genes have been shown to differ in their expression between tissue types in the same organism, pointing to divergent outcomes as a result of developmental events during embryogenesis. Regardless of whether there is a single purpose for imprinting, numerous studies have shown that a normal and functional organism cannot be made without the various imprinting mechanisms.


In mammals, imprinted genes are often clustered in the genome, probably because they share transcriptional regulators or regulatory regions that impact the expression of multiple genes. It is easier for a lncRNA to silence multiple genes if they are closer together, making silencing more efficient. In some cases, when a gene is transcribed it overlaps another region nearby or opposite (antisense) to it, often silencing it. In the case of the Ifg2 and  H19  genes,  CTCF , a transcriptional repressor protein, is involved. CTCF binds to the unmethylated maternal ICR region but not the methylated paternal ICR region. ICR is a shared control region of Ifg2 and H19 that, when deleted, results in the loss of imprinting of these genes. CTCF then binds another region of the chromosome, creating a loop where Igf2 is blocked from transcription, but H19 is not, resulting in the maternal chromosome expressing H19 but not Igf2. CTCF has been shown to directly interact with Suz12, a subunit of PRC2, in order to silence the Ifg2 promoter region through hypermethylation. Conversely, the paternal H19 promoter is highly methylated during embryogenesis so that Ifg2 will not be silenced. Should CTCF fail to bind, H19 on the maternal chromosome has reduced expression and Igf2 is not silenced properly, resulting in biallelic expression. Mice have homologues of these genes, but silence them in a different way, where biallelic expression occurs and then  antisense RNA  is used to silence one of the genes. [ 18 ] [ 19 ] [ 20 ]


Airn  is an lncRNA used to silence Igf2r and other surrounding genes. In the mechanism to silence Igf2r, the transcription of the lncRNA Airn silences the expression of Igf2r, as opposed to an active repression mechanism. Airn is the antisense gene of Ifg2r, so if Airn is being transcribed, the transcriptional machinery may cover a part of or the entire promoter region of Igf2r, so RNA polymerase cannot bind to the promoter region of Igf2r in order to initiate transcription.  This mechanism is very efficient in that Igf2r is silenced by transcription of Airn, while the RNA product silences other genes near Igf2r.  The imprinting mechanisms described above work on the chromosome that the Airn lncRNA is produced, but there are many other imprinted genes that work to silence genes on other chromosomes or to silence the similar allele(s) on the opposing chromosome of the same pair. Some imprinted genes code for regulatory RNA elements such as lncRNA,  small nucleolar RNA , and  micro RNA , so the expression of these genes results in the silencing of some other gene. [ 21 ]


From these examples, researchers have seen similar patterns in  developmental genetics . It is imperative that many genes are silenced at the right time so that cells can maintain their identity and expressional integrity. Failure to do so often leads to symptoms such as cognitive abnormalities, if not fatality. [ 22 ]


The lncRNA Airn is an lncRNA that regulates Igf2r expression. Igf2r is a gene which expresses a receptor for  insulin-like growth factor 2 , and assists in  lysosomal  enzyme transport, activation of  growth factors , and degradation of insulin-like growth factor 2. [ 23 ]  This lncRNA is an RNA modified by imprinting, leading to Airn expression in the paternal allele, but not in the maternal allele. Airn acts by cis-acting silencing of the Igf2r region through overlapping the Igf2r gene through the antisense transcript encoded by Airn. Airn is silenced in the maternal allele through Igf2r transcription. In the brain, however, Igf2r alleles are both expressed due to Airn mediation being repressed in neuronal cells. [ 24 ]


PRC2 (Polycomb repressive complex 2) is a complex of proteins that repress chromatin by histone methylation and by working to recruit other proteins that help further the repression of chromatin. The structure of this complex and group of mechanisms used by this complex are highly conserved across various  eukaryotic  species. Very few species have duplicates of these complexes in the genome beyond PRC1 and PRC2. [ 25 ] [ 26 ]


Long non-coding RNAs, or lncRNAs, are RNA transcripts produced by RNA polymerase II that are not translated but participate in the regulation of gene expression. Long non-coding RNAs are used in various epigenetic processes in development, including the regulation of Hox genes, as well as in the creation of Barr bodies. [ 27 ] [ 28 ]


Although PRC2 seems to have a very simple mechanism and works on many genes and chromosomes across the genome, it often has very specific binding regions and has been observed to localize to specific genes to cause their repression. Recent research shows that it probably does this through the binding of long non coding RNAs (lncRNAs). Xist and Hox genes have both been studied extensively and display this mechanism very well. The lncRNA that the complex binds does not necessarily need to hybridize to the target region in order to silence it, as evidenced by the PRC2-lncRNA complex working on regions other than the region from which this complex was produced. However, the three-dimensional configuration of the RNA often gives the complex specific localization to regions where the RNA is created to bind.


PRC2 is a multi-protein complex composed of four major subunits (E2H1/2, SUZ12, EED, and RbAp46/48) and three variable subunits ( AEBP2 ,  JARID2 , and PCLs).  The three variable subunits are used for catalysis of enzymatic reactions or binding to specific regions, not for repression of genes or chromatin. Similar to a  zinc finger , AEBP2 docks onto the major grooves of DNA to assist in binding. [ 29 ]  PRC2 is usually recruited by other proteins or lncRNa and then catalyzes the trimethylation of lysine 27 of histone 3 tails (H3K27me3). This methylation is thought to cause repression by steric hindrance of RNA polymerase II. Even though the polymerase is not prevented from binding, the polymerase, after beginning transcription, will pause at H3K27me3 marks. The short transcript produced by the pausing of the polymerase often recruits regulatory complexes, like PRC2. Thus, PRC2 represses by two mechanisms: by directly altering the structure of the chromatin through methylation or by binding of transcripts. [ 18 ] [ 30 ]


PRC2 has been shown in many experiments to be necessary for the proper  formation of organs , starting with the maintenance of cellular differentiation and silencing of pluripotency genes.  The exact mechanism in early embryogenesis that induces cells to differentiate is still unclear, but this mechanism has been closely linked to  protein kinase A  (PKA). Since the PRC2 complex has sites able to be phosphorylated and has differentiated behavior based on the level of phosphorylation, a logical hypothesis can be made that PKA affects PRC2 behavior and may phosphorylate PRC2, activating the protein and starting the methylation cascade that silences genes. [ 31 ]


Experimentally, PRC2 has been shown to be highly enriched at the Hox genes and near developmental gene regulators, resulting in their methylation. Some time after the second or third cleavage event, PRC2 begins to bind to these developmental genes, even though they have the markers for highly active genes like H3K9me3. This has been described as the “leaking” of PRC2 binding. Variable binding will cause some genes to be silenced before others, causing differentiation, but this is likely regulated by the organism. What causes the specificity of cell differentiation is still unknown but some hypotheses say it largely has to do with the cell environment and the “awareness” of the cells to each other, considering all cells in this stage contain identical genomes at this point. The maintained cell lines after this differentiation event are largely dependent on PRC2. Without it, pluripotency genes will still be active, causing the cells to be unstable and reversion back to a  stem cell -like stage where the cell would have to undergo differentiation again in order to return to its normal state. Properly differentiated cells have silenced pluripotency genes. [ 32 ]


PRC2 is also highly associated with  intergenic regions , subtelomeric regions, and long-terminal repeat  transposons . PRC2 acts to create heterochromatin in these regions through similar mechanisms to the mechanism used to repress genes. Heterochromatin formation is imperative in these regions in order to regulate gene expression, maintain chromatin shape, prevent degradation of the chromosome, and reduce the event of transposon “hopping” or spontaneous recombination. [ 32 ]


Thus, PRC2 is not only essential to the initiation of differentiation in development, but also for maintaining heterochromatin in all cell stages and for silencing genes and chromosome regions that would undo the cell differentiation that had already occurred or negatively affect the survival of the cell or the organism as a whole.


Neat1 is an lncRNA which assists in forming the structure of nuclear structures known as  paraspeckles : nuclear bodies which contain RNA-binding proteins. [ 33 ]  They control gene expression in the nucleus by retaining RNA in the  nucleus  that would otherwise alter gene expression. [ 34 ]  Paraspeckles form a significant portion of the  corpus luteum  of the  ovary ; in  Neat1  impaired mice, corpus luteum formation is highly dysfunctional, causing ovarian defects and lowered  progesterone  levels resulting in a lack of pregnancy in Neat1 deficient mice. Neat1 assists in regulation of luteal genes by preventing the protein  Sfpq  from inhibiting  Nr5a1  and Sp1, allowing luteal genes to be regularly transcribed. Neat1 is regulated by histone deacetylases. [ 33 ] [ 35 ] [ 36 ]


Evf2  is a lncRNA that acts in  forebrain  neuronal differentiation during embryonic development.  Evf2 is transcribed from an ultraconserved region, or a region that is very highly conserved among most  vertebrate  species, within the region from  Dlx5  to  Dlx6 . This region is a target for SHH, a highly important regulator of  central nervous system  development. Evf2, when transcribed, recruits Dlx and  Mecp2  through cis and trans-acting mechanisms to the Dlx5/6 region in the ventral forebrain, causing  GABAergic  interneurons in the  hippocampus  to be formed. [ 37 ]  Evf2 acts by forming a complex with  Dlx4  that increases Dlx4 transcription activation ability and stability. [ 38 ]


Malat1, another neurological lncRNA, causes increased synaptic function and greater amounts of dendrite development. Increases of Malat1 increase neuronal density, while decreases of Malat1 decrease neuronal density. Malat1 acts by regulating the expression levels of Nlgn1 and SynCAM1 which are important genes in synapse formation. [ 38 ]


Bromodomain protein 4, or  BRD4 , is a protein which binds to acetylated tails of histones H3 and H4 to aid active gene transcription by decompaction using the  bromodomain  with the assistance of the acetylated K5 on H4. BRD4 is a member of the BET protein family, which includes other bromodomain-containing proteins and their homologues in other species. BRD4 is a protein which functions in both gene activation and repression in  cell cycle  control and  DNA replication . BRD4 functions by binding to the acetylated tails and then attaching to other proteins, allowing those proteins to
either activate or repress the histones next to BRD4. [ 39 ]


BRD4 aids in early cell development by activating pluripotent genes through interacting with  Oct4  and recruiting  P-TEFb  (positive transcription elongation factor). By occupying pluripotent genes and X-chromosome inactivation lncRNAs in their regulatory regions, BRD4 enhances activation of these DNA regions. BRD4 enhances this activation by recruiting P-TEFb; if either BRD4 or P-TEFb is not functional, pluripotent gene transcription is blocked, and the cell differentiates into a neuroectodermal cell. [ 40 ]


BRD4 can act as epigenetic bookmarking throughout the cell cycle, including after transcription, due to its association with P-TEFb, allowing BRD4 to enhance RNAPII. [ 40 ]


BRD4 also assists in the hyperacetylation of histones in the  sperm nucleus . Histone hyperacetylation, the addition of acetyl groups to lysines on the amino tails of histones in an amount much larger than normal, is believed to assist in histone removal from the sperm nucleus. [ 41 ] [ 42 ]


Examples of diseases caused by epigenetic dysfunction in development include:


Human herpes viruses , also known as  HHV s, are part of a family of  DNA viruses  that cause several diseases in humans. One of the most notable functions of this virus family is their ability to enter a latent phase and lay dormant within animals for extended periods of time. [ 1 ]  The mechanism that controls this is very complex because expression of viral proteins during  latency  is decreased a great deal, meaning that the virus must have transcription of its  genes repressed . There are many factors and mechanisms that control this process and  epigenetics  is one way this is accomplished.  Epigenetics  refers to persistent changes in expression patterns that are not caused by changes to the  DNA sequence . This happens through mechanisms such as  methylation  and  acetylation  of  histones ,  DNA methylation , and  non-coding RNAs  (ncRNA).  Altering the acetylation of histones  creates changes in expression by changing the binding affinity of histones to DNA, making it harder or easier for  transcription  machinery to access the DNA.  Methyl  and  acetyl groups  can also act as binding sites for  transcription factors  and  enzymes  that further modify histones or alter the DNA itself. [ 2 ]


Human herpesviruses are all  double stranded DNA viruses  that enter the  nucleus  of the host cell. HHVs replicate in the nucleus of the infected cells and can enter both a lytic and latent phase. In the lytic phase, the viral genes are being actively transcribed and many times ultimately lead to cell death. In the lytic phase the virus is able to spread to other hosts due to open sores and other such mechanisms. In the latent phase the amount of viral transcript is heavily reduced. In this phase the host will show little to no signs of virus infection. This state can be maintained for very long periods of time and infection with HHVs can last a lifetime. [ 3 ]


Herpes simplex virus 1 and 2  (HSV 1 and HSV 2) are part of the  alpha subfamily  of herpesviruses. The lytic phase of infection occurs within mucoepithelial cells while the latent infection of these cells occurs in neurons. These two viruses are the cause of  oral  and  genital herpes . [ 4 ]


Latency is maintained in a variety of ways, one of which is the  latency-associated transcript , or LAT. This long non-coding RNA accumulates in the latent phase and has been found to suppress the lytic genes. It is also possible that the LAT transcript plays a role in determining which neurons become infected and maintain the lytic phase. LAT manages this suppression of the viral genome through  miRNAs  that block viral  transcription  factors such as  ICP0  and  ICP4  that play a very important role in the lytic phase of infection. Another mechanism that is used to maintain the latent phase is  chromatin remodeling . During the latent phase, researchers have found more methylated chromatin in the lytic genes and less acetylated chromatin, whereas in the LAT region there is less methylation and more acetylation found during latency. This was reversed during the lytic phase, Suggesting that this change of chromatin structure is used to control the [ clarification needed ]  and maintain the lytic and latent phases. [ 5 ]


Varicella zoster virus  (VZV) is a member of the alpha subfamily of herpesviruses and is responsible for the human diseases of the  chicken pox  and  shingles . These viruses have their lytic phase in mucoepithelial cells and are latent in neuronal cells. [ 6 ]


Unlike HSV 1 and 2, there has not been any LAT found in the VZV latent cells. Instead, it appears that the main way that expression of viral transcripts is maintained at a lower level during latency is through  chromatin  structure. There are 5 transcripts identified during latent infection of VZV.  Open reading frames  (ORFs) 21, 29, 62, 63, and 66 are all expressed with 62 and 63 being the most abundant. The  promoters  of these two genes,  ORFs  62 and 63, contain large amounts of acetylated H3K9, which makes these genes  euchromatic . This contrasts other genes normally found in lytic infection that are not associated with acetylated H3K9 histones during latency. This means that these genes are controlled through a mechanism affecting the chromatin structure that allows them to be expressed in the latent phase. The mechanism of how this happens is not clearly understood and research is currently being done in this area. [ 7 ] [ 8 ]


Epstein–Barr virus  (EBV) is a member of the  gammaherpesvirus  subfamily. EBV is responsible for infectious mononucleosis and is linked to several forms of cancer. EBV infects both  B cells  and  epithelial cells  and is able to undergo latency in both of these cells. A remarkable fact concerning the latency of EBV is that different patterns of expression exist while it is latent, known as latency programs. [ 9 ]


One of the ways that latency is achieved and maintained is through histone modification. It is known that  methylation  of histones often serve as marks that cause the formation of heterochromatin, an inactive form of chromatin, while acetylation often marks euchromatin, an active form of  chromatin . In EBV, The amount and location of the histone modifications is different for each latency program. There are several promoter regions which are the key targets of this histone modification in each of these programs.  There are the Cp and  Qp promoter  regions. The Cp promoter promotes the transcription of all the EBNA genes from a  polycistronic mRNA . The Q promoter (Qp) promotes the transcription of  EBNA1 . In type I latency, the Qp is  acetylated  and active while the Cp region is methylated and inactive. The opposite is true in type III latency where the Cp region is active and the Qp region is inactive. In both of these types of latency, H3K27me is slightly enriched in the lytic immediate early promoters. This suggests that facultative heterochromatin interacts with the  polycomb repressive complex  in order to limit lytic reactivation. [ 10 ]


Cytomegalovirus  (CMV) is a member of the  betaherpesvirinae  subfamily. CMV is responsible for a range of diseases, but mainly affects the immunocompromised. The primary targets for infection include  monocytes , lymphocytes, and epithelial cells with the known sites of latency including monocytes and  lymphocytes . [ 11 ]


One epigenetic mechanism employed in the control of latency for CMV is heterochromatization of the viral genome. Studies have shown that during latency the IE promoter is  heterochromatic  while the LUNA promoter is euchromatic. It has also been found that latently infected cells are more enriched for histones relative to the cellular genes. This keeps the transcriptional machinery from being able to access the viral transcripts. [ 12 ]


This control over the chromatin is brought about and maintained by recruited transcriptional repressors. These complexes, which are composed of enzymes such as  histone  deacetylases (HDACs), bind to the major immediate-early promoter (MIEP) of CMV and condense the DNA and prevent any remodeling. HDACs appear to be important for the continued maintenance of latency as HDAC inhibitors induce a lytic infection. It is also of not to mention that while chromatin methylation is utilized in this control mechanism, methylation of the DNA has not been observed. This is important due to the fact that HDACs do not function on their own but instead are part of larger co-repressor complexes that are recruited by methylated DNA or transcription factors which bind to specific sequences on DNA. Since there is no methylated DNA, it is likely that transcription factors are used to recruit these co-repressor complexes. [ 13 ]


Kaposi's sarcoma-associated herpesvirus  (KSHV) is  a part of the gammaherpesvirus subfamily. It undergoes lytic infection in lymphocytes and latency in B cells. It causes  Kaposi's sarcoma  and  primary effusion lymphoma . KSHV is one of eight known human cancer-causing viruses. The control of KSHV latency is most notably controlled by a set of viral miRNAs. During latency there are only a few transcripts that are produced. These transcripts include latency-associated nuclear antigen (LANA), viral cyclin (vCyclin), vFLIP (ORF71), kaposin (K12), and 12 miRNAs. [ 14 ]


One mechanism for this control is that the miRNAs down regulate the lytic activator protein known as Rta or orf50. This down regulation is accomplished through increasing the amount of  methyltransferases  and  CpG  methylation. The main miRNA responsible for this is miR-K12-5 which interacts with orf50 through its  3′ untranslated region  (UTR). These miRNAs also change the methylation of the histones in the viral genome. In the latent phase there is a lot of  H3K9 trimethylation  near the terminal repeats, which is an indication of heterochromatin in these areas. Another of these miRNAs, miR-K12-4-5p, targets  retinoblastoma-like protein 2  (Rbl2). Rbl2 regulates  DNA methyltransferases  (DNMTs), which in turn regulates the amount of DNA methylation. miR-K12-4-5p has been shown to upregulate the amount of DNMTs which increases DNA methylation and allows the latent phase of the infection to continue. [ medical citation needed ]


Neurodegenerative diseases  are a heterogeneous group of complex disorders linked by the degeneration of  neurons  in either the  peripheral nervous system  or the  central nervous system . Their underlying causes are extremely variable and complicated by various genetic and/or environmental factors. These diseases cause progressive deterioration of the neuron resulting in decreased  signal transduction  and in some cases even neuronal death. Peripheral nervous system diseases may be further categorized by the type of nerve cell ( motor ,  sensory , or both) affected by the disorder. Effective treatment of these diseases is often prevented by lack of understanding of the underlying molecular and genetic pathology.  Epigenetic therapy  is being investigated as a method of correcting the expression levels of misregulated genes in neurodegenerative diseases.


Neurodengenerative diseases of motor neurons  can cause degeneration of motor neurons involved in voluntary muscle control such as muscle contraction and relaxation.  This article will cover the epigenetics and treatment of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). See the Motor Neuron Fact Sheet [ 1 ]  for details regarding other motor neuron diseases.  Neurodegenerative diseases of the central nervous system  can affect the brain and  spinal cord . This article will cover the epigenetics and treatment of  Alzheimer's disease  (AD),  Huntington's disease  (HD), and  Parkinson's disease  (PD).  These diseases are characterized by chronic and progressive neuronal dysfunction, sometimes leading to behavioral abnormalities (as with PD), and, ultimately, neuronal death, resulting in  dementia .


Neurodegenerative diseases of sensory neurons can cause degeneration of sensory neurons involved in transmitting sensory information such as  hearing  and  seeing . The main group of sensory neuron diseases are hereditary sensory and autonomic neuropathies (HSAN) such as  HSAN I ,  HSAN II , and  Charcot-Marie-Tooth  Type 2B (CMT2B). [ 2 ] [ 3 ]  Though some sensory neuron diseases are recognized as neurodegenerative, epigenetic factors have not yet been clarified in the molecular pathology.


The term  epigenetics  refers to three levels of gene regulation: (1)  DNA methylation , (2)  histone modifications , and (3)  non-coding RNA  (ncRNA) function.  Briefly, histone-mediated transcriptional control occurs by the wrapping of DNA around a  histone  core.  This DNA-histone structure is called a  nucleosome ; the more tightly the DNA is bound by the nucleosome, and the more tightly a string of nucleosomes are compressed among each other, the greater the repressive effect on  transcription  of genes in the DNA sequences near or wrapped around the histones, and vice versa (i.e. looser DNA binding and relaxed compaction leads to a comparatively derepressed state, resulting in facultative  heterochromatin  or, even further derepressed,  euchromatin ).  At its most repressive state, involving many folds into itself and other scaffolding proteins, DNA-histone structures form constitutive heterochromatin.  This chromatin structure is mediated by these three levels of gene regulation. The most relevant epigenetic modifications to treatment of neurodegenerative diseases are DNA methylation and histone protein modifications via methylation or acetylation. [ 4 ] [ 5 ]


Epigenetic drugs target the proteins responsible for modifications on DNA or histone. Current epigenetic drugs include but are not limited to:  HDAC inhibitors  (HDACi), HAT modulators, DNA methyltransferase inhibitors, and histone demethylase inhibitors. [ 7 ] [ 8 ]  The majority of epigenetic drugs tested for use against neurodegenerative diseases are HDAC inhibitors; however, some DNMT inhibitors have been tested as well. While the majority of epigenetic drug treatments have been conducted in mouse models, some experiments have been performed on human cells as well as in human drug trials (see table below). There are inherent risks in using epigenetic drugs as therapies for neurodegenerative disorders as some epigenetic drugs (e.g. HDACis such as  sodium butyrate ) are non-specific in their targets, which leaves potential for off-target epigenetic marks causing unwanted epigenetic modifications.


D (y)  16, 18 ;  H (ny)


H (v)  26, 27, 28, 29


Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a motor neuron disease that involves neurogeneration. All skeletal muscles in the body are controlled by motor neurons that communicate signals from the brain to the muscle through a  neuromuscular junction . When the motor neurons degenerate, the muscles no longer receive signals from the brain and begin to waste away. ALS is characterized by stiff muscles, muscle twitching, and progressive muscle weakness from muscle wasting. The parts of the body affected by early symptoms of ALS depend on which motor neurons in the body are damaged first, usually the limbs. As the disease progresses most patients are unable to walk or use their arms and eventually develop difficulty speaking, swallowing and breathing. Most patients retain cognitive function and sensory neurons are generally unaffected. Patients are often diagnosed after the age of 40 and the median survival time from onset to death is around 3–4 years. In the final stages, patients can lose voluntary control of eye muscles and often die of  respiratory failure  or  pneumonia  as a result of degeneration of the motor neurons and muscles required for breathing. Currently there is no cure for ALS, only treatments that may prolong life.


To date, multiple genes and proteins have been implicated in ALS. One of the common themes between many of these genes and their causative mutations is the presence of  protein aggregates  in motor neurons. [ 43 ]  Other common molecular features in ALS patients are altered RNA metabolism [ 44 ]  and general histone hypoacetylation. [ 45 ]


ALS patients and mouse models show general histone hypoacetylation that can ultimately trigger  apoptosis  of cells. [ 54 ]  In experiments with mice, HDAC inhibitors counteract this hypoacetylation, reactivate aberrantly down-regulated genes, and counteract apoptosis initiation. [ 13 ] [ 55 ]  Furthermore, HDAC inhibitors are known to prevent SOD1 protein aggregates in vitro. [ 56 ]


Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by mutations in the  SMN1  gene. [ 59 ]  Symptoms vary greatly with each subset of SMA and the stage of the disease. General symptoms include overall muscle weakness and poor muscle tone including extremities and respiratory muscles leading to difficulty walking, breathing, and feeding. Depending on the type of SMA, the disease can present itself from infancy through adulthood. As SMN protein generally promotes the survival of motor neurons, mutations in SMN1 results in slow degeneration motor neurons leading to progressive system-wide muscle wasting. Specifically, over time, decreased levels of SMN protein results in gradual death of the  alpha motor neurons  in the  anterior horn of the spinal cord  and brain. Muscles depend on connections to motor neurons and the central nervous system to stimulate muscle maintenance and therefore degeneration of motor neurons and subsequent denervation of muscles lead to loss of muscle control and muscle atrophy. The muscles of the lower extremities are often affected first followed by upper extremities and sometimes the muscles of respiration and mastication. In general, proximal muscle is always affected more than distal muscle.


Spinal muscular atrophy is linked to genetic mutations in the SMN1 (Survival of Motor Neuron 1) gene. The SMN protein is widely expressed in neurons and serves many functions within neurons including  spliceosome  construction, mRNA axon transport,  neurite  outgrowth during development, and  neuromuscular junction  formation. The causal function loss in SMA is currently unknown.


SMN1 is located in a  telomeric  region of  human chromosome 5  and also contains SMN2 in a  centromeric  region. SMN1 and  SMN2  are nearly identical except for a single  nucleotide change  in SMN2 resulting in an alternative splicing site where intron 6 meets exon 8. This single base pair change leads to only 10–20% of SMN2 transcripts resulting in fully functional SMN protein and 80–90% of transcripts leading to a truncated protein that is rapidly degraded. Most SMA patients have 2 or more copies of the SMN2 gene with more copies resulting in a decrease in disease severity. [ 60 ]  Most SMA patients have either  point mutations  or a deletion in exon 7 often leading to a protein product similar to the truncated and degraded version of the SMN2 protein. In SMA patients this small amount of functional SMN2 protein product allows for some neurons to survive.


Although SMA is not caused by an epigenetic mechanism, therapeutic drugs that target epigenetic marks may provide SMA patients with some relief, halting or even reversing the progression of the disease. As SMA patients with higher copy numbers of the SMN2 gene have less severe symptoms, researchers predicted that epigenetic drugs that increased SMN2 mRNA expression would increase the amount of functional SMN protein in neurons leading to a reduction in SMA symptoms. Histone deacetylase (HDAC) inhibitors are the main compounds that have been tested to increase SMN2 mRNA expression. Inhibiting HDACs would allow for hyperacetylation of the SMN2 gene loci theoretically resulting in an increase in SMN2 expression. [ 41 ]  Many of these HDAC inhibitors (HDACi) are first tested in mouse models of SMA created through a variety of mutations in the mouse SMN1 gene. If the mice show improvement and the drug does not cause very many side effects or toxicity, the drug may be used in human clinical trials. Human trials with all of the below HDAC inhibitors are extremely variable and often impacted by the patient's exact SMA subtype.


Myasthenia gravis is an autoimmune disease affecting synapses at the neuromuscular junction, whereby antibodies produced primarily in the thymus gland by B-cells associate with postsynaptic nicotinic acetylcholine receptors (AChR), along with other NMJ post-synaptic receptors (MuSK-R and low-density lipoprotein receptor). These antibodies include acetylcholine receptor antibodies, MuSK antibodies, and low-density lipoprotein receptor related protein 4 antibodies (LRP4-Ab). [ 62 ]  Antibody binding to their respective receptors causes the destruction of those receptors, leading to a reduction in the number of postsynaptic acetylcholinergic receptors and a reduction in overall acetylcholine transport. Disease symptoms include muscular weakness that fatigues due to overuse, but improves with rest. Hallmark symptoms due to muscular weakness include ptosis, double vision, dysphagia, as well as aberrant speech. [ 63 ]


Myasthenia gravis is a relatively rare disease, occurring in about 3–30 individuals per 100,000, but has been rising over the past couple decades. There exists two variations of myasthenia gravis with respect to age and gender demographics: early-onset myasthenia gravis, which has a higher incidence among females, and late-onset myasthenia gravis, which has a higher incidence among males. [ 63 ]


There has been extensive research on the genetic basis of myasthenia gravis, however evidence does not suggest that it is an inherited disease. [ 64 ]  There has also been extensive research on the epigenetic contribution to myasthenia gravis. [ 65 ]  DNA methylation and noncoding RNA, such as miRNA (micro RNA) and long noncoding RNA (lncRNA), are epigenetic factors that play a significant role in increasing the likelihood of acquiring myasthenia gravis. In addition, the thymus is a key organ in the immune response that is often negatively affected by abnormal miRNA expression and DNA methylation.


Micro RNA (miRNA) are single-stranded non-coding RNAs that bind their target mRNAs. From there, they can regulate gene expression by inhibiting translation or degrading the mRNA strand, oftentimes in B-cells and T-cells of the immunological process. With respect to myasthenia gravis, abnormal miRNA function is associated with immunoregulatory pathogenesis, and each miRNA has its own unique downstream effects.


The thymus is an important endocrine organ implicated in myasthenia gravis. In normal, healthy development, the thymus shrinks in size over time. In those with thymus-associated myasthenia gravis there are correlations with thymomas in late-onset myasthenia gravis as well as thymic hyperplasia with germinal centers in early-onset myasthenia gravis, and each of these conditions can be attributed partly to irregular miRNA function. [ 66 ]  In late-onset myasthenia gravis subjects, it was shown that miRNA-12a-5p expression was increased in thymoma-associated myasthenia gravis. MiRNA-12a-5p inhibits expression of the gene FoxP3, a gene known to be associated with normal thymus development and whose alteration is attributed to thymomas. [ 67 ]  Additionally, an association between thymoma-associated myasthenia gravis and decreased miR-376a/miR-376c expression was found. Autoimmune regulation is known to be downregulated in thymoma-associated myasthenia gravis, and in thymus cells with downregulated autoimmune regulation there was simultaneous downregulation in miR-376a, miR-376c, and miRNA-12a-5p expression. [ 67 ]  In early-onset myasthenia gravis patients, 61 miRNA's were found to be either significantly downregulated or upregulated. The most downregulated miRNA was found to be miR-7-5p, whose target gene is CCL21. CCL21 is known to aberrantly recruit B-cells in the thymus of early-onset myasthenia gravis patients, and was found to be highly expressed in early-onset myasthenia gravis patients, potentially explaining the abnormally large amounts of B cells found in thymic hyperplasia. [ 68 ]


Aside from miRNA's corresponding to altered thymus function, there are other several key miRNA's that are correlated with myasthenia gravis. MiR-15 cluster (miR-15a, miR-15b, and miR-15c) was shown to be associated with autoimmunity, in that its downregulation increased CXCL10 expression, a target gene involved in T-cell signaling. CXCL10 expression was also shown to be increased in the thymus of myasthenia gravis patients. [ 69 ]  Additionally, miR-146 was found to be upregulated in myasthenia gravis patients. In these patients with upregulated miR-146, there was a concurrent increase in proteins that correspond to a wide array of immune responses, specifically TLR4, CD40, and CD80. [ 70 ]


DNA methylation is the epigenetic process by which methyl groups are added to either adenine or cytosine bases, which results in the repression of that sequence when cytosine methylation occurs. [ 71 ]  DNA methylation was found to be a factor in increasing the likelihood of acquiring myasthenia gravis, albeit this topic has not been widely researched. Research in China has identified the gene CTLA-4 (cytotoxic T lymphocyte antigen-4) as being highly methylated in myasthenia gravis patients compared to control groups throughout the entire span of the disease. The CTLA-4 gene produces an antigen of the same name that is presented on killer T-cells and allows for the suppression of the immune response. Methylation of this gene represses production of the antigen CTLA-4—a pattern seen in a significant majority of myasthenia gravis patients—and can explain the elevated immune response seen in myasthenia gravis. [ 72 ]  Furthermore, myasthenia gravis patients with thymic abnormalities (approximately 10–20% of all myasthenia gravis patients) [ 73 ]  had even higher levels of CTLA-4 methylation than other myasthenia gravis patients. It is not extensively researched why certain genes are hypermethylated in these cases, but information on myasthenia gravis largely points to upregulation of the DNA methyltransferase genes DNMT1, DNMT3A, and DNMT3B in patients with myasthenia gravis. [ 74 ]


In addition to CTLA-4 methylation, hypermethylation of the growth hormone secretagogue receptor gene was seen in patients with thymoma-associated late-onset myasthenia gravis. [ 75 ]  Growth hormone secretagogue receptor hypermethylation is detected in a wide variety of cancers, however only recently has been correlated with the development of thymoma-associated myasthenia gravis. Although it is seen in approximately 1/4 of thymoma-associated myasthenia gravis subjects, it is not a reliable biomarker for the disease, and its relevance to disease progression is not well known.


Long ncRNA (lncRNA) are a second type of non-coding RNA that are key post-transcriptional modifiers of protein-coding gene expression. These also play a significant role in myasthenia gravis. Their aberrant regulation can cause differential expression in downstream genes. For instance, the differential expression of lncRNA interferon gamma antisense RNA negatively regulates the expression of HLA-DRB and HLA-DOB, [ 75 ]  two genes implicated in the body's autoimmune response by differentiating endogenous and foreign proteins. [ 76 ]  As seen in myasthenia gravis patients with downregulated lethal (let)-7 lncRNA, it was also found that the level of let-7 lncRNA was negatively correlated with levels of interleukin (IL)-10, a gene responsible for inhibiting cytokine secretion/activation, antigen presentation, and macrophage activity, [ 77 ]  but also for exhibiting anti-tumor effects. [ 78 ]  Therefore, the negative correlation between let-7 lncRNA and IL-10 levels and its specific effects on myasthenia gravis development are ambiguous.


In addition to aberrant regulation of downstream target genes, lncRNA also affect expression by acting as competing endogenous RNA (ceRNA). The competing endogenous RNA theory states that transcripts sharing common miRNA binding sites can compete to bind these identical miRNAs, and in this way lncRNAs can bind miRNAs, regulating their downstream binding activity and affecting their function. In the case of myasthenia gravis, the lncRNA small nucleolar RNA host gene (SNHG) 16 regulates the expression of IL-10 by adsorbing let-7c-5p, a miRNA that commonly associates with IL-10, as a competing endogenous RNA.


Diagnosis of myasthenia gravis, individual prognosis, and the level of treatment needed can be determined by detecting the amounts of circulating miRNA.


Immunosuppressants represent a large category in clinical studies for myasthenia gravis treatment, as they reduce the hyperactive immunological response in T-cells presenting acetylcholine receptor-binding antigens. [ 66 ]  By overexpressing miR-146, studies show that patients with early-onset myasthenia gravis can have antigen-specific suppressive effects. This has implications in reducing the immune response of myasthenia gravis patients and improving prognosis. Likewise, miR-155 is proven to be correlated with myasthenia gravis-associated thymic inflammation and immune response. Research is being conducted whereas repression of miR-155 could reduce these aberrant effects. [ 66 ]  Lastly, the miRNA's miR-150-5p and miR-21-5p are consistently shown to be elevated in myasthenia gravis patients with acetylcholinergic receptor antibodies (in contrast to the MuSK-binding variant of myasthenia gravis), therefore these two miRNA's are reliable biomarkers in detecting this variant of myasthenia gravis. [ 79 ]


Alzheimer's disease (AD) is the most prevalent form of dementia among the elderly.  The disease is characterized behaviorally by chronic and progressive decline in cognitive function, beginning with short-term memory loss, and neurologically by buildup of misfolded  tau protein  and associated  neurofibrillary tangles , and by amyloid-beta senile plaques  amyloid-beta senile plaques .  Several genetic factors have been identified as contributing to AD, including mutations to the  amyloid precursor protein  ( APP )  and  presenilins 1 and 2  genes, and familial inheritance of  apolipoprotein E  allele epsilon 4.  In addition to these common factors, there are a number of other genes that have shown altered expression in Alzheimer's disease, some of which are associated with epigenetic factors.


Treatment for prevention or management of Alzheimer's disease has proven troublesome since the disease is chronic and progressive, and many epigenetic drugs act globally and not in a gene-specific manner.  As with other potential treatments to  prevent  or  ameliorate  symptoms of AD, these therapies do not work to cure, but only ameliorate symptoms of the disease temporarily, underscoring the chronic, progressive nature of AD, and the variability of methylation in AD brains.


Huntington's disease (HD) is an inherited disorder that causes progressive degeneration of neurons within the  cerebral cortex  and  striatum  of the brain [ 98 ]  resulting in loss of motor functions (involuntary muscle contractions), decline in cognitive ability (eventually resulting in dementia),  and changes in behavior. [ 7 ]


Huntington's is caused by an autosomal dominant mutation expanding the number of glutamine codon repeats (CAG) within the  Huntingtin  gene (Htt). [ 98 ]  The Htt gene encodes for the huntingtin protein which plays a role in normal development but its exact function remains unknown. [ 99 ]  The length of this CAG repeat correlates with the age-of-onset of the disease. The average person without Huntington's has less than 36 CAG repeats present within the Htt gene. When this repeat length exceeds 36, the onset of neuronal degradation and the physical symptoms of Huntington's can range from as early as 5 years of age (CAG repeat > 70) to as late as 80 years of age (CAG repeat < 39). [ 100 ]


This CAG expansion results in mRNA downregulation of specific genes, decreased histone acetylation, and increased histone methylation. [ 101 ] [ 102 ]  The exact mechanism of how this repeat causes gene dysregulation is unknown, but epigenome modification may play a role. For early-onset Huntington's (ages 5–15), both transgenic mice and mouse striatal cell lines show brain specific histone H3 hypoacetylation and decreased histone association at specific downregulated genes within the striatum (namely Bdnf, Cnr1, Drd2 – dopamine 2 receptor, and Penk1 – preproenkephalin). [ 103 ]  For both late- and early-onset Huntington's, the H3 and H4 core histones associated with these downregulated genes in Htt mutants have hypoacetylation (decreased acetylation) compared to wild-type Htt. [ 102 ] [ 103 ]  This hypoacetylation is sufficient to cause tighter chromatin packing and mRNA downregulation. [ 102 ]


Along with H3 hypoacetylation, both human patients and mice with the mutant Htt have increased levels of histone H3 lysine 9 trimethylation. [ 101 ]   This increase in H3-K9 trimethylation is linked to an increased expression of the methyltransferase ESET/SETDB1 (ERG-associated protein with SET domain (ESET)), which targets and trimethylates H3-K9 residues. [ 101 ]  It is proposed that this hypermethylation may account for the onset of specific gene repression in Htt mutants. [ 101 ]