While spontaneous  deamination  of cytosine forms  uracil , which is recognized and removed by DNA repair enzymes, deamination of 5-methylcytosine forms  thymine . This conversion of a DNA base from cytosine (C) to thymine (T) can result in a  transition mutation . [ 11 ]  In addition, active enzymatic deamination of cytosine or 5-methylcytosine by the  APOBEC  family of cytosine deaminases could have beneficial implications on various cellular processes as well as on organismal evolution. [ 12 ]  The implications of deamination on  5-hydroxymethylcytosine , on the other hand, remains less understood.


The NH 2  group can be removed (deamination) from 5-methylcytosine to form  thymine  with use of reagents such as  nitrous acid ; cytosine deaminates to uracil (U) under similar conditions. [ citation needed ]


5-methylcytosine is resistant to deamination by  bisulfite  treatment, which deaminates cytosine residues. This property is often exploited to analyze DNA cytosine methylation patterns with  bisulfite sequencing . [ 13 ]


5mC marks are placed on genomic DNA via  DNA methyltransferases  (DNMTs). There are 5 DNMTs in humans: DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L, and in algae and fungi 3 more are present (DNMT4, DNMT5, and DNMT6). [ 14 ]  DNMT1 contains the replication foci targeting sequence (RFTS) and the CXXC domain which catalyze the addition of 5mC marks. RFTS directs DNMT1 to loci of DNA replication to assist in the maintenance of 5mC on daughter strands during DNA replication, whereas CXXC contains a  zinc finger  domain for  de novo  addition of methylation to the DNA. [ 15 ]  DNMT1 was found to be the predominant DNA methyltransferase in all human tissue. [ 16 ]  Primarily, DNMT3A and DNMT3B are responsible for  de novo  methylation, and DNMT1 maintains the 5mC mark after replication. [ 1 ]  DNMTs can interact with each other to increase methylating capability. For example, 2 DNMT3L can form a complex with 2 DNMT3A to improve interactions with the DNA, facilitating the methylation. [ 17 ]  Changes in the expression of DNMT results in aberrant methylation. Overexpression produces increased methylation, whereas disruption of the enzyme decreased levels of methylation. [ 16 ]


The mechanism of the addition is as follows: first a cysteine residue on the DNMT's PCQ motif creates a nucleophillic attack at carbon 6 on the cytosine nucleotide that is to be methylated.  S-Adenosylmethionine  then donates a methyl group to carbon 5. A base in the DNMT enzyme deprotonates the residual hydrogen on carbon 5 restoring the double bond between carbon 5 and 6 in the ring, producing the 5-methylcytosine base pair. [ 15 ]


After a cytosine is methylated to 5mC, it can be reversed back to its initial state via multiple mechanisms. Passive DNA demethylation by dilution eliminates the mark gradually through replication by a lack of maintenance by DNMT. In active DNA demethylation, a series of oxidations converts it to  5-hydroxymethylcytosine  (5hmC),  5-formylcytosine  (5fC), and 5-carboxylcytosine (5caC), and the latter two are eventually excised by  thymine DNA glycosylase  (TDG), followed by base excision repair (BER) to restore the cytosine. [ 1 ]  TDG knockout produced a 2-fold increase of 5fC without any statistically significant change to levels of 5hmC, indicating 5mC must be iteratively oxidized at least twice before its full demethylation. [ 18 ]  The oxidation occurs through the  TET  (Ten-eleven translocation) family dioxygenases ( TET enzymes ) which can convert 5mC, 5hmC, and 5fC to their oxidized forms. However, the enzyme has the greatest preference for 5mC and the initial reaction rate for 5hmC and 5fC conversions with TET2 are 4.9-7.6 fold slower. [ 19 ]  TET requires Fe(II) as cofactor, and oxygen and  α-ketoglutarate  (α-KG) as substrates, and the latter substrate is generated from isocitrate by the enzyme  isocitrate dehydrogenase  (IDH). [ 20 ]  Cancer however can produce  2-hydroxyglutarate  (2HG) which competes with α-KG, reducing TET activity, and in turn reducing conversion of 5mC to 5hmC. [ 21 ]


In cancer, DNA can become both overly methylated, termed  hypermethylation , and under-methylated, termed hypomethylation. [ 22 ]  CpG islands overlapping gene promoters are  de novo  methylated resulting in aberrant inactivation of genes normally associated with growth inhibition of tumors (an example of hypermethylation). [ 23 ]  Comparing tumor and normal tissue, the former had elevated levels of the methyltransferases DNMT1, DNMT3A, and mostly DNMT3B, all of which are associated with the abnormal levels of 5mC in cancer. [ 16 ]  Repeat sequences in the genome, including satellite DNA, Alu, and long interspersed elements (LINE), are often seen hypomethylated in cancer, resulting in expression of these normally silenced genes, and levels are often significant markers of tumor progression. [ 22 ]  It has been hypothesized that there a connection between the hypermethylation and hypomethylation; over activity of DNA methyltransferases that produce the abnormal  de novo  5mC methylation may be compensated by the removal of methylation, a type of epigenetic repair. However, the removal of methylation is inefficient resulting in an overshoot of genome-wide hypomethylation. The contrary may also be possible; over expression of hypomethylation may be silenced by genome-wide hypermethylation. [ 22 ]   Cancer hallmark capabilities are likely acquired through epigenetic changes that alter the 5mC in both the cancer cells and in surrounding tumor-associated stroma within the tumor microenvironment. [ 24 ]  The anticancer drug  Cisplatin  has been reported to react with 5mC. [ 25 ]


"Epigenetic age" refers to the connection between chronological age and levels of DNA methylation in the genome. [ 26 ]  Coupling the levels of DNA methylation, in specific sets of CpGs called "clock CpGs", with algorithms that regress the typical levels of collective genome-wide methylation at a given chronological age, allow for epigenetic age prediction. During youth (0–20 years old), changes in DNA methylation occur at a faster rate as development and growth progresses, and the changes begin to slow down at older ages. Multiple epigenetic age estimators exist. Horvath's clock measures a multi-tissue set of 353 CpGs, half of which positively correlate with age, and the other half negatively, to estimate the epigenetic age. [ 27 ]  Hannum's clock utilizes adult blood samples to calculate age based on an orthogonal basis of 71 CpGs. [ 28 ]  Levine's clock, known as DNAm PhenoAge, depends on 513 CpGs and surpasses the other age estimators in predicting mortality and lifespan, yet displays bias with non-blood tissues. [ 29 ]  There are reports of age estimators with the methylation state of only one CpG in the gene ELOVL2. [ 30 ]  Estimation of age allows for prediction lifespan through expectations of age related conditions that individuals may be subject to based on their 5mC methylation markers. [ citation needed ]


6- O -Methylguanine  is a derivative of the  nucleobase   guanine  in which a  methyl group  is attached to the oxygen atom. It base-pairs to  thymine  rather than  cytosine , causing a G:C to A:T transition in DNA.


6- O -Methylguanine is formed in DNA by alkylation of the oxygen atom of guanine, most often by N-nitroso compounds (NOC) [ 2 ]  and sometimes due to methylation by other compounds such as  endogenous   S-adenosyl methionine . [ 3 ]   NOC are  alkylating agents  formed by the reaction of nitrite or other nitrogen oxides with secondary  amines  and N-alkylamides, yielding N-alkylnitrosamines and N-alkylnitrosamides. [ 2 ]


NOC are found in some foods (bacon, sausages, cheese) and tobacco smoke, and are formed in the gastrointestinal tract, especially after consumption of red meat. [ 2 ]   In addition, endogenous  nitric oxide  levels were found to be enhanced under chronic inflammatory conditions, and this could favor NOC formation in the large intestine.


Repair of 6- O -methylguanine in DNA is primarily carried out by  O-6-methylguanine-DNA methyltransferase  (MGMT).   Epigenetic  reductions in MGMT expression are one of the most frequent DNA repair defects, associated with carcinogenesis. [ 4 ]  (Also see  MGMT expression in cancer .)


In 1985 Yarosh summarized the early work that established 6- O -methylguanine as the alkylated base in DNA that was the most mutagenic and carcinogenic. [ 5 ]   In 1994 Rasouli-Nia et al. [ 6 ]  showed that about one mutation was induced for every eight unrepaired 6-O-Methylguanines in DNA.


About one third of the time 6- O -methylguanine mispairs  during replication, leading to the incorporation of dTMP rather than dCMP. [ 7 ]  6-O-methylguanine is therefore a mutagenic nucleobase.  However, the mutagenicity of a particular 6-O-methylguanine base depends on the sequence in which it is embedded. [ 8 ]


Unrepaired 6- O -methylguanine can also lead to cell cycle arrest,  sister chromatid exchange , or  apoptosis . [ 9 ]   These effects are due to interaction of the  DNA mismatch repair  pathway with 6- O -methylguanine, and also depend on signaling network activation, led by early ATM, H2AX, CHK1 and p53 phosphorylation. [ 9 ]


EMS mutagenesis


Temozolomide


7-Methylguanine  is a modified  purine   nucleobase .  It is a  methylated  version of  guanine . The 7-methylguanine  nucleoside  is called  7-methylguanosine . However, the free 7-methylguanine base is not involved in the synthesis of nucleotides and not incorporated directly into nucleic acids. [ 1 ] [ 2 ]  7-Methylguanine is a natural inhibitor of  poly (ADP-ribose) polymerase  (PARP) and tRNA guanine transglycosylase (TGT) - and thus may exert anticancer activity. [ 3 ] [ 4 ] [ 5 ]  For example, it was demonstrated that 7-methylguanine could accelerate apoptotic death of BRCA1-deficient breast cancer cells induced by cisplatin and doxorubicin. [ 6 ]


6-Methylisoxanthopterin  ( 6MI ) is a  base analog  for the nucleotide  guanine . It is useful as a fluorescent indicator because unlike most other base analogs,  quenching  does not occur when it is incorporated into a double helix. In fact, it exhibits a 3 to 4-fold increase in  quantum yield  when it is incorporated into a duplex formation. [ 1 ]  This allows 6MI to be used to probe the dynamics of DNA or RNA helices using a technique such as  fluorescence polarization anisotropy .  [ 2 ]


Methylthiouracil  is an  organosulfur compound  that is used antithyroid preparation. It is a  thioamide , closely related to  propylthiouracil . Methylthiouracil is not used clinically in the United States, it has a similar mechanism of action and side effect to that of propylthiouricil. The drug acts to decrease the formation of stored thyroid hormone, as thyroglobulin in the thyroid gland. The clinical effects of the drug to treat the hyperthyroid state can have a lag period of up to two weeks, depending on the stores of thyroglobulin and other factors.


Methylthiouracil is prepared quite simply by condensation of  ethyl acetoacetate  with  thiourea . [ 2 ]


Further work in this series shows that better activity was obtained by incorporation of a lipophilic side chain.


Propylthiouracil  ( PTU ) is a medication used to treat  hyperthyroidism . [ 3 ]  This includes hyperthyroidism due to   Graves' disease  and  toxic multinodular goiter . [ 3 ]  In a  thyrotoxic crisis  it is generally more effective than  methimazole . [ 3 ]  Otherwise it is typically only used when methimazole, surgery, and  radioactive iodine  is not possible. [ 3 ]  It is taken  by mouth . [ 3 ]


Common side effects include itchiness, hair loss, parotid swelling, vomiting, muscle pains, numbness, and headache. [ 3 ]  Other severe side effects include  liver problems  and  low blood cell counts . [ 3 ]  Use during pregnancy may harm the baby. [ 3 ]  Propylthiouracil is in the  antithyroid  family of medications. [ 4 ]  It works by decreasing the amount of  thyroid hormone  produced by the  thyroid gland  and blocking the conversion of  thyroxine  (T4) to  triiodothyronine  (T3). [ 3 ]


Propylthiouracil came into medical use in the 1940s. [ 5 ]  It is on the  World Health Organization's List of Essential Medicines . [ 6 ]


Propylthiouracil is generally well tolerated, with side effects occurring in one of every 100 patients. [ citation needed ]  The most common side effects are related to the skin and include rash, itching, hives, abnormal hair loss, and skin pigmentation. [ citation needed ]  Other common side effects are swelling, nausea, vomiting, heartburn, loss of taste, joint or muscle aches, numbness and headache, allergic reactions, and hair whitening. [ citation needed ]


Its notable side effects include a risk of  agranulocytosis  and  aplastic anemia . On 3 June 2009, the  FDA  published an alert "notifying healthcare professionals of the risk of serious liver injury, including liver failure and death, with the use of propylthiouracil." [ 7 ]  As a result, propylthiouracil is no longer recommended in non-pregnant adults and in children as the front line antithyroid medication. [ 8 ]


One possible side effect is  agranulocytosis , [ 9 ]  a decrease of white blood cells in the blood. Symptoms and signs of agranulocytosis include infectious lesions of the throat, the gastrointestinal tract, and skin with an overall feeling of illness and fever. A decrease in blood platelets (thrombocytopenia) also may occur. Since platelets are important for the clotting of blood, thrombocytopenia may lead to problems with excessive bleeding. Side effects are suspected and the drug is sometimes discontinued if the patient complains of recurrent episodes of sore throat.


Another life-threatening side effect is sudden, severe,  fulminant liver failure  resulting in death or the need for a liver transplantation, which occurs in up to 1 in 10,000 people taking propylthiouracil. Unlike agranulocytosis which most commonly occurs in the first three months of therapy, this side effect may occur at any time during treatment. [ 8 ]


Propylthiouracil is classified as  Drug Class D in pregnancy .  Class D signifies there is positive evidence of human fetal risk. The maternal benefit may outweigh fetal risk in life-threatening situations. [ 10 ]  PTU is preferred over  methimazole  (which is also a class D) only in the first trimester of pregnancy and in women who may become pregnant because of the increased risk of teratogenicity of methimazole during critical organogenesis. In the second and third trimester, this risk is diminished and  methimazole  is preferred to avoid the risk of liver complications from PTU in the mother. [ 8 ]


The primary effect on the fetus from transplacental passage of PTU is the production of a mild hypothyroidism when the drug is used close to term.  This usually resolves within a few days without treatment.  The hypothyroid state may be observed as a goiter in the newborn, and is the result of increased levels of fetal pituitary thyrotropin. [ 11 ]   The incidence of fetal goiter after PTU treatment in reported cases is approximately 12%.


PTU inhibits the enzyme  thyroperoxidase , which normally acts in thyroid hormone synthesis by oxidizing the anion  iodide  (I − ) to  iodine  (I 0 ), facilitating iodine's addition to tyrosine residues on the hormone precursor  thyroglobulin .  This is one of the essential steps in the formation of  thyroxine  (T4). [ 12 ]


PTU does not inhibit the action of the  sodium-dependent iodide transporter  located on follicular cells' basolateral membranes. Inhibition of this step requires competitive inhibitors, such as  perchlorate  and  thiocyanate .


PTU also acts by inhibiting the enzyme 5'-deiodinase ( tetraiodothyronine 5' deiodinase ), which converts T 4  to the more active form T 3 . (This is in contrast to  methimazole , which shares propylthiouracil's central mechanism, but not its peripheral one.)


It is important to recognize that these enzymes only work on the conjugated tyrosine molecules of T3 and T4: a completely different enzyme family is responsible for the deiodinase activity of iodized single tyrosine molecules within the thyroid follicular cells. For information on that enzyme family, see  Iodotyrosine deiodinase .


The administration is oral, with peak serum concentrations occurring in one hour, and actively concentrated to the thyroid gland. Depending on several patient variables, however, euthyroid status may not be achieved until 2–4 months after treatment initiation. Of note, the drug is approximately 70% protein-bound and significantly ionized at normal physiologic pH, while the antithyroid agent  methimazole  is substantially less protein bound. However, both are equally transferred across the placenta. [ 13 ]


The plasma half-life is one hour and is not altered appreciably by the thyroid status of the patient. Due to the concentration in the thyroid, however, dosing intervals may last 8 hours or longer. Less than 10% of the drug is excreted unchanged, with the remaining fraction undergoing extensive hepatic metabolism via  glucuronidation .


Propylthiouracil can be prepared from  ethyl 3-oxohexanoate  and  thiourea . [ 14 ]


Propylthiouracil, together with  phenylthiocarbamide  (PTC), are known to have bitter taste. [ 15 ]  However, it seems the  propensity for tasting  these compounds is genetically based and the bitter taste is likely to be engendered by the  thiocyanate  moiety, also present in PTC. [ 15 ]


It was approved by the United States  Food and Drug Administration  in 1947.


Queuine  ( / k j uː iː n / ) ( Q ) is a hypermodified  nucleobase  found in the first (or  wobble ) position of the  anticodon  of  tRNAs  specific for Asn, Asp, His, and Tyr, in most  eukaryotes  and  prokaryotes . [ 1 ]  Because it is utilized by all eukaryotes but produced exclusively by bacteria, it is a putative vitamin. [ 2 ]


The  nucleoside  of queuine is  queuosine .  Queuine is not found in the tRNA of archaea; however, a related 7-deazaguanine derivative, the nucleoside of which is  archaeosine , occurs in different tRNA position, the dihydrouridine loop, and in tRNAs with more specificities.


In 1967, it was discovered that the four above-mentioned tRNAs contained an as-yet unknown nucleoside, which was designated "Nucleoside Q". This name remained in use throughout much of the work to characterize the compound, after which it was proposed that its common name should be based on the sound of the letter Q—thus producing "queuine" by analogy to  guanine  and other nucleobases, and "queuosine" by analogy to  guanosine  and other nucleosides. [ 3 ]


The presence of queuine in certain tRNA is a nearly ubiquitous feature of eukaryotic life, meaning it is found in every healthy cell of the human body. It is also found in all other animals, plants, and fungi. The only known exception is brewer's yeast,  Saccharomyces cerevisiae . However, queuosine can be produced only by bacteria; higher organisms must obtain queuine from the diet, or salvage it from symbiotic microbes: a process for which dedicated enzymatic machinery exists. [ 4 ]  Because queuine is necessary for healthy cellular function in animals, but is produced exclusively by microbes, it can be considered a vitamin, akin to the  B vitamins —many which are also produced primarily or exclusively by bacteria. [ 5 ]


The biosynthesis pathway for queuine shares a common enzymatic starting step with  folate . Because queuosine in dietary or gut-bacterial RNA can be salvaged and converted to queuine by the human body, queuosine could be considered a  vitamer  of queuine. As of 2019, human queuine requirements are not well understood, and the prevalence of queuine deficiency in humans is unknown. [ 6 ]  Plants obtain queuine from the tRNA of symbiotic bacteria in and around their roots.


Once salvaged, queuine replaces a guanine base in the anticodon of certain tRNAs, where it appears to play a role in ensuring rapid and accurate recognition of the corresponding  mRNAs ' codons. In the absence of queuosine modification, translation at Q-decoded codons slows down to the point that many proteins cannot fold properly. [ 7 ]


In animal experiments using "germ-free" mice, even the total absence of queuine in the diet is not lethal in the presence of an adequate supply of the dietary amino acid  tyrosine . Withdrawal of tyrosine from the diet, however, causes rapid physical deterioration and death over a period of two weeks. Tyrosine is not typically an essential nutrient for animals provided dietary phenylalanine, suggesting that queuine depletion impairs the activity of  phenylalanine hydroxylase .


BH 4  is a cofactor for the  biopterin-dependent aromatic amino acid hydroxylase  enzymes, which catalyze the conversion of  phenylalanine  to  tyrosine , tyrosine to  L-DOPA , and  tryptophan  to  5-HTP , oxidizing BH 4  to  dihydrobiopterin  (BH 2 ) in the process. BH 2  must then be converted back to BH 4  by the enzyme  dihydropteridine reductase  before it can be used again. Queuine depletion appears to impair this "recycling" process, resulting in a deficit of BH 4  and an excess of BH 2 , which in turn impairs the activity of the aromatic amino acid hydroxylase enzymes. [ 8 ]


Because the aromatic amino acid hydroxylase enzymes are the rate-limiting steps in the body's biosynthesis of  serotonin  and  dopamine  (and subsequent metabolites including  melatonin ,  norepinephrine , and  adrenaline ), queuine deficiency is under investigation as a potential cause of human diseases linked to a deficit of these neurotransmitters. [ 9 ]


4-Thiouracil  is a heterocyclic organic compound having a  pyrimidine  skeleton. It is a derivative of the nucleobase  uracil  with a sulfur instead of oxygen in position 4. It is found naturally in the  4-thiouridine  nucleoside.


This article about a  heterocyclic compound  is a  stub . You can help Wikipedia by  expanding it .


2-Thiouracil  is a chemical derivative of  uracil  in which the oxygen atom in the 2-position of the ring is  substituted  by sulfur.


The substance is a historically relevant anti-thyroid preparation.  Edwin Astwood  used it in 1943 as therapy for  Graves' disease  for the first time. [ 1 ] [ 2 ] [ 3 ]


Thiouracil inhibits thyroid activity by blocking the enzyme  thyroid peroxidase . [ 4 ]  Its use in recent times has been replaced by advent of more potent and safer antithyroid drugs. It occurs in seeds of Brassica and Crucifera species. Thiouracil has been used as antithyroid, coronary vasodilator, and in congestive heart failure although its use has been largely supplanted by other drugs. [ 5 ]


Thymine  ( / ˈ θ aɪ m ɪ n / ) ( symbol   T  or  Thy ) is one of the four  nucleotide bases  in the  nucleic acid  of  DNA  that are represented by the letters G–C–A–T. The others are  adenine ,  guanine , and  cytosine . Thymine is also known as  5-methyluracil,  a  pyrimidine  nucleobase. In  RNA , thymine is replaced by the nucleobase  uracil .  Thymine was first isolated in 1893 by  Albrecht Kossel  and Albert Neumann from calf  thymus  glands, hence its name. [ 2 ]


As its alternate name (5-methyluracil) suggests, thymine may be derived by  methylation  of uracil at the 5th carbon. In  RNA , thymine is replaced with  uracil  in most cases.  In DNA, thymine (T) binds to  adenine  (A) via two hydrogen bonds, thereby stabilizing the nucleic acid structures.


Thymine combined with  deoxyribose  creates the  nucleoside   deoxythymidine , which is synonymous with the term  thymidine . Thymidine can be phosphorylated with up to three phosphoric acid groups, producing  dTMP  ( d eoxy t hymidine  m ono p hosphate),  dTDP , or  dTTP  (for the  d i- and  t ri- phosphates, respectively).


One of the common mutations of DNA involves two adjacent thymines or cytosine, which, in presence of  ultraviolet light , may form  thymine dimers , causing "kinks" in the DNA molecule that inhibit normal function.


Thymine could also be a target for actions of 5- fluorouracil  (5-FU) in  cancer  treatment. 5-FU can be a metabolic analog of thymine (in DNA synthesis) or uracil (in RNA synthesis).  Substitution of this analog inhibits  DNA synthesis  in actively dividing cells.


Thymine bases are frequently oxidized to  hydantoins  over time after the death of an organism. [ 3 ]


During growth of  bacteriophage T4 , an imbalance of thymine availability, either a deficiency or an excess of thymine, causes increased  mutation . [ 4 ]   The mutations caused by thymine deficiency appear to occur only at AT  base pair  sites in DNA and are often AT to GC  transition mutations . [ 5 ]    In the bacterium  Escherichia coli , thymine deficiency was also found to be mutagenic and cause AT to GC transitions. [ 6 ]


In March 2015, NASA scientists reported that, for the first time, complex  DNA  and  RNA   organic compounds  of  life , including  uracil ,  cytosine  and thymine, have been formed in the laboratory under  outer space  conditions, using starting chemicals, such as  pyrimidine , found in  meteorites . Pyrimidine, like  polycyclic aromatic hydrocarbons  (PAHs), another carbon-rich compound, may have been formed in  red giants  or in  interstellar dust  and gas clouds, according to the scientists. [ 7 ]  Thymine has not been found in meteorites, which suggests the first strands of DNA had to look elsewhere to obtain this building block. Thymine likely formed within some meteorite parent bodies, but may not have persisted within these bodies due to an  oxidation  reaction with  hydrogen peroxide . [ 8 ]


Thymine was first prepared by hydrolysis of the corresponding  nucleoside  obtained from natural sources. Interest in its direct chemical synthesis began in the early 1900s:  Emil Fischer  published a method starting from  urea  but a more practical synthesis used methylisothiourea in a  condensation reaction  with ethyl formyl propionate, followed by hydrolysis of the pyrimidine intermediate: [ 9 ]


Many other preparative methods have been developed, including optimised conditions so that urea can be used directly in the reaction shown above, preferably with methyl formyl propionate. [ 10 ]


Tioguanine , also known as  thioguanine  or  6-thioguanine  ( 6-TG ) or  tabloid   is a medication used to treat  acute myeloid leukemia  (AML),  acute lymphocytic leukemia  (ALL), and  chronic myeloid leukemia  (CML). [ 2 ]  Long-term use is not recommended. [ 2 ]  It is given  by mouth . [ 2 ]


Common side effects include  bone marrow suppression ,  liver problems  and  inflammation of the mouth . [ 2 ] [ 3 ]  It is recommended that  liver enzymes  be checked weekly when on the medication. [ 2 ]  People with a genetic deficiency in  thiopurine S-methyltransferase  are at higher risk of side effects. [ 3 ]  Avoiding  pregnancy  when on the medication is recommended. [ 2 ]  Tioguanine is in the  antimetabolite  family of medications. [ 3 ]  It is a  purine analogue  of  guanine  and works by disrupting  DNA  and  RNA . [ 4 ]


Tioguanine was developed between 1949 and 1951. [ 5 ] [ 6 ]  It is on the  World Health Organization's List of Essential Medicines . [ 7 ]


The major concern that has inhibited the use of thioguanine has been veno-occlusive disease (VOD) and its histological precursor nodular regenerative hyperplasia (NRH). The incidence of NRH with thioguanine was reported  as between 33 and 76%. [ 10 ]  The risk of ensuing VOD is serious and frequently irreversible so this side effect has been a major concern.
However, recent evidence using an animal model for thioguanine-induced NRH/VOD has shown that, contrary to previous assumptions, NRH/VOD is dose dependent and the mechanism for this has been demonstrated. [ 11 ]  This has been confirmed in human trials, where thioguanine has proven to be safe but efficacious for coeliac disease when used at doses below those commonly prescribed. [ 12 ]  This has led to a revival of interest in thioguanine because of its higher efficacy and faster action compared to other thiopurines and immunosuppressants such as mycophenylate. [ 13 ]


Cancers that do not respond to treatment with mercaptopurine do not respond to thioguanine. On the other hand, some cases of IBD that are resistant to mercaptopurine (or its pro-drug azathioprine) may be responsive to thioguanine.


The enzyme  thiopurine S-methyltransferase  (TPMT) is responsible for the direct inactivation of thioguanine to its methylthioguanine base – this  methylation  prevents thioguanine from further conversion into active,  cytotoxic  thioguanine nucleotide (TGN) metabolites. [ 15 ] [ 16 ] [ 17 ]  Certain  genetic variations  within the TPMT gene can lead to decreased or absent TPMT enzyme activity, and individuals who are  homozygous  or  heterozygous  for these types of  genetic variations  may have increased levels of TGN metabolites and an increased risk of severe bone marrow suppression ( myelosuppression ) when receiving thioguanine. [ 15 ]  In many ethnicities,  TPMT  polymorphisms that result in decreased or absent TPMT activity occur with a frequency of approximately 5%, meaning that about 0.25% of patients are  homozygous  for these variants. [ 15 ] [ 18 ]  However, an assay of TPMT activity in  red blood cells  or a TPMT  genetic test  can identify patients with reduced TPMT activity, allowing for the adjustment of thiopurine dose or avoidance of the drug entirely. [ 15 ] [ 19 ]  The FDA-approved drug label for thioguanine notes that patients who are TPMT-deficient may be prone to developing  myelosuppression  and that laboratories offer testing for TPMT deficiency. [ 20 ]  Indeed, testing for TPMT activity is currently one of the few examples of  pharmacogenetics  being translated into routine clinical care. [ 21 ]


A single oral dose of thioguanine has incomplete metabolism, absorption and high interindividual variability. The bioavailability of thioguanine has an average of 30% (range 14-46%). The maximum concentration in plasma after a single oral dose is attained after 8 hours.


Thioguanine, like other thiopurines, is cytotoxic to white cells; as a result it is immunosuppressive at lower doses and anti-leukemic/anti-neoplastic at higher doses. Thioguanine is incorporated into human bone marrow cells, but like other thiopurines, it is not known to cross the blood-brain barrier. Thioguanine cannot be demonstrated in  cerebrospinal fluid , similar to the closely related compound 6-mercaptopurine which also cannot penetrate to the brain.


The plasma half-life of thioguanine is short, due to the rapid uptake into liver and blood cells and conversion to 6-TGN. The median plasma half-life of 80-minutes with a range of 25–240 minutes. Thioguanine is excreted primarily through the kidneys in urine, but mainly as a metabolite, 2-amino-6-methylthiopurine.  However, the intra-cellular thio-nucleotide metabolites of thioguanine (6-TGN) have longer half-lives and can therefore be measured after thioguanine is eliminated from the plasma.


Thioguanine is catabolized (broken down) via two pathways. [ 22 ]  One route is through the deamination by the enzyme  guanine deaminase  to 6-thioxanthine, which has minimal anti-neoplastic activity, then by oxidation by  xanthine oxidase  of the thioxanthine to  thiouric acid . This metabolic pathway is not dependent on the efficacy of xanthine oxidase, so that the inhibitor of xanthine oxidase, the drug allopurinol, does not block the breakdown of thioguanine, in contrast to its inhibition of the breakdown of the related thiopurine 6-mercaptopurine. The second pathway is the methylation of thioguanine to 2-amino-6-methylthiopurine, which is minimally effective as an anti-neoplastic and significantly less toxic than thioguanine. This pathway also is independent of the enzyme activity of xanthine oxidase.


6-Thioguanine is a thio analogue of the naturally occurring purine base guanine. 6-thioguanine utilises the enzyme  hypoxanthine-guanine phosphoribosyltransferase  (HGPRTase) to be converted to 6-thioguanosine monophosphate (TGMP). High concentrations of TGMP may accumulate intracellularly and hamper the synthesis of guanine nucleotides via the enzyme Inosine monophosphate dehydrogenase ( IMP dehydrogenase ), leading to DNA mutations. [ 23 ]


TGMP is converted by phosphorylation to thioguanosine diphosphate (TGDP) and thioguanosine triphosphate (TGTP). Simultaneously deoxyribosyl analogs are formed, via the enzyme  ribonucleotide reductase . The TGMP, TGDP and TGTP are collectively named 6-thioguanine nucleotides (6-TGN). 6-TGN are cytotoxic to cells by: (1) incorporation into DNA during the synthesis phase (S-phase) of the cell; and (2) through inhibition of the GTP-binding protein ( G protein )  Rac1 , which regulates the Rac/Vav pathway. [ 24 ]


It is a pale yellow, odorless, crystalline powder.


Tioguanine ( INN ,  BAN ,  AAN ), or thioguanine ( USAN ).


Thioguanine is administered by mouth (as a tablet – 'Lanvis').


Uracil  ( / ˈ j ʊər ə s ɪ l / ) ( symbol   U  or  Ura ) is one of the four  nucleotide bases  in the  nucleic acid   RNA . The others are  adenine  (A),  cytosine  (C), and  guanine  (G). In RNA, uracil binds to  adenine  via two  hydrogen bonds . In  DNA , the uracil nucleobase is replaced by  thymine  (T). Uracil is a  demethylated  form of  thymine .


Uracil is a common and naturally occurring  pyrimidine  derivative. [ 2 ]  The name "uracil" was coined in 1885 by the German chemist  Robert Behrend , who was attempting to synthesize derivatives of  uric acid . [ 3 ]  Originally discovered in 1900 by  Alberto Ascoli , it was isolated by  hydrolysis  of  yeast   nuclein ; [ 4 ]  it was also found in  bovine   thymus  and  spleen ,  herring   sperm , and  wheat   germ . [ 5 ]  It is a planar, unsaturated compound that has the ability to absorb light. [ 6 ]


Uracil that was formed extraterrestrially has been detected in the  Murchison meteorite , [ 7 ]  in  near-Earth asteroid   Ryugu , [ 8 ]  and possibly on the surface of the moon  Titan . [ 9 ]  It has been synthesized under cold laboratory conditions similar to outer space, from pyrimidine embedded in water ice and exposed to ultraviolet light. [ 10 ]


In RNA, uracil  base-pairs  with adenine and replaces thymine during DNA transcription.  Methylation  of uracil produces thymine. [ 11 ]  In DNA, the evolutionary substitution of thymine for uracil may have increased DNA stability and improved the efficiency of  DNA replication  (discussed below). Uracil pairs with adenine through  hydrogen bonding . When  base pairing  with adenine, uracil acts as both a  hydrogen bond  acceptor and a hydrogen bond donor. In RNA, uracil binds with a  ribose  sugar to form the  ribonucleoside   uridine . When a  phosphate  attaches to uridine, uridine 5′-monophosphate is produced. [ 6 ]


Uracil undergoes amide-imidic acid tautomeric shifts because any nuclear instability the molecule may have from the lack of formal  aromaticity  is compensated by the cyclic-amidic stability. [ 5 ]  The amide  tautomer  is referred to as the  lactam  structure, while the imidic acid tautomer is referred to as the  lactim  structure. These tautomeric forms are predominant at  pH  7. The lactam structure is the most common form of uracil.


Uracil also recycles itself to form nucleotides by undergoing a series of phosphoribosyltransferase reactions. [ 2 ]  Degradation of uracil produces the substrates  β-alanine ,  carbon dioxide , and  ammonia . [ 2 ]


Oxidative degradation of uracil produces urea and maleic acid in the presence of  H 2 O 2  and  Fe 2+  or in the presence of diatomic  oxygen  and Fe 2+ .


Uracil is a  weak acid . The first site of  ionization  of uracil is not known. [ 12 ]  The negative charge is placed on the oxygen anion and produces a  p K a  of less than or equal to 12. The basic p K a  = −3.4, while the acidic p K a  = 9.38 9 . In the gas phase, uracil has four sites that are more acidic than water. [ 13 ]


Uracil is rarely found in DNA, and this may have been an evolutionary change to increase genetic stability. This is because cytosine can deaminate spontaneously to produce uracil through hydrolytic deamination. Therefore, if there were an organism that used uracil in its DNA, the deamination of cytosine (which undergoes base pairing with guanine) would lead to formation of uracil (which would base pair with adenine) during DNA synthesis.
 Uracil-DNA glycosylase  excises uracil bases from double-stranded DNA. This enzyme would therefore recognize and cut out both types of uracil – the one incorporated naturally, and the one formed due to cytosine deamination, which would trigger unnecessary and inappropriate repair processes. [ 14 ]


This problem is believed to have been solved in terms of evolution, that is by "tagging" (methylating) uracil. Methylated uracil is identical to thymine. Hence the hypothesis that, over time, thymine became standard in DNA instead of uracil. So cells continue to use uracil in RNA, and not in DNA, because RNA is shorter-lived than DNA, and any potential uracil-related errors do not lead to lasting damage. Apparently, either there was no evolutionary pressure to replace uracil in RNA with the more complex thymine, or uracil has some chemical property that is useful in RNA, which thymine lacks. Uracil-containing DNA still exists, for example in:


Organisms synthesize uracil, in the form of  uridine monophosphate  (UMP), by decarboxylating  orotidine 5'-monophosphate  (orotidylic acid). In humans this decarboxylation is achieved by the enzyme  UMP synthase . In contrast to the purine nucleotides, the pyrimidine ring (orotidylic acid) that leads uracil is synthesized first and then linked to  ribose phosphate , forming UMP. [ 16 ]


There are many laboratory  synthesis  of uracil available. The first reaction is the simplest of the syntheses, by adding water to  cytosine  to produce uracil and  ammonia : [ 2 ]


The most common way to synthesize uracil is by the  condensation  of  malic acid  with urea in  fuming sulfuric acid : [ 5 ]


Uracil can also be synthesized by a double decomposition of  thiouracil  in aqueous  chloroacetic acid . [ 5 ]


Photodehydrogenation  of 5,6-diuracil, which is synthesized by beta- alanine  reacting with  urea , produces uracil. [ 17 ]


In 2009,  NASA  scientists reported having produced uracil from  pyrimidine  and water ice by exposing it to  ultraviolet light  under space-like conditions. [ 10 ]  This suggests a possible natural original source for uracil. [ 18 ]  In 2014, NASA scientists reported that additional complex  DNA  and  RNA   organic compounds  of  life , including uracil,  cytosine  and  thymine , have been formed in the laboratory under  outer space  conditions, starting with ice,  pyrimidine , ammonia, and methanol, which are compounds found in astrophysical environments. [ 19 ]  Pyrimidine, like  polycyclic aromatic hydrocarbons  (PAHs), a carbon-rich chemical found in the  Universe , may have been formed in  red giants  or in  interstellar dust  and gas clouds. [ 20 ]


Based on  12 C/ 13 C  isotopic ratios  of  organic compounds  found in the  Murchison meteorite , it is believed that uracil,  xanthine , and related molecules can also be formed extraterrestrially. [ 7 ]   Data from the  Cassini mission , orbiting in the  Saturn  system, suggests that uracil is present in the surface of the moon  Titan . [ 9 ]   In 2023, uracil was observed in a sample from  162173 Ryugu , a  near-Earth asteroid , with no exposure to Earth's biosphere, giving further evidence for synthesis in space. [ 8 ]


Uracil readily undergoes regular reactions including  oxidation ,  nitration , and  alkylation . While in the presence of  phenol  (PhOH) and  sodium hypochlorite  (NaOCl), uracil can be visualized in  ultraviolet light . [ 5 ]  Uracil also has the capability to react with elemental  halogens  because of the presence of more than one strongly electron donating group. [ 5 ]


Uracil readily undergoes addition to  ribose   sugars  and  phosphates  to partake in synthesis and further reactions in the body.  Uracil becomes  uridine ,  uridine monophosphate  (UMP),  uridine diphosphate  (UDP),  uridine triphosphate  (UTP), and  uridine diphosphate glucose  (UDP-glucose). Each one of these molecules is synthesized in the body and has specific functions.


When uracil reacts with anhydrous  hydrazine , a first-order kinetic reaction occurs and the uracil ring opens up. [ 21 ]  If the  pH  of the reaction increases to > 10.5, the uracil anion forms, making the reaction go much more slowly. The same slowing of the reaction occurs if the pH decreases, because of the protonation of the hydrazine. [ 21 ]  The reactivity of uracil remains unchanged, even if the temperature changes. [ 21 ]


Uracil's use in the body is to help carry out the synthesis of many enzymes necessary for cell function through bonding with riboses and phosphates. [ 2 ]  Uracil serves as  allosteric  regulator and  coenzyme  for reactions in animals and in plants. [ 22 ]  UMP controls the activity of  carbamoyl phosphate synthetase  and  aspartate transcarbamoylase  in plants, while UDP and UTP regulate  CPSase II  activity in  animals . UDP-glucose regulates the conversion of  glucose  to  galactose  in the  liver  and other tissues in the process of  carbohydrate metabolism . [ 22 ]  Uracil is also involved in the  biosynthesis  of  polysaccharides  and the transportation of sugars containing  aldehydes . [ 22 ]  Uracil is important for the detoxification of many  carcinogens , for instance those found in tobacco smoke. [ 23 ]  Uracil is also required to detoxify many drugs such as cannabinoids (THC) [ 24 ]  and morphine (opioids). [ 25 ]  It can also slightly increase the risk for cancer in unusual cases in which the body is extremely deficient in  folate . [ 26 ]  The deficiency in folate leads to increased ratio of  deoxyuridine monophosphates  (dUMP)/ deoxythymidine monophosphates  (dTMP) and uracil misincorporation into DNA and eventually low production of DNA. [ 26 ]


Uracil can be used for  drug delivery  and as a  pharmaceutical . When elemental  fluorine  reacts with uracil, they produce  5-fluorouracil . 5-Fluorouracil is an anticancer drug ( antimetabolite ) used to masquerade as uracil during the nucleic acid replication process. [ 2 ]  Because 5-fluorouracil is similar in shape to, but does not undergo the same chemistry as, uracil, the drug inhibits  RNA  transcription enzymes, thereby blocking RNA synthesis and stopping the growth of cancerous cells. [ 2 ]  Uracil can also be used in the synthesis of caffeine. [ 27 ]  Uracil has also shown potential as a HIV viral capsid inhibitor. [ 28 ]  Uracil derivatives have antiviral, anti-tubercular and anti-leishmanial activity. [ 29 ] [ 30 ] [ 31 ]


Uracil can be used to determine  microbial  contamination of  tomatoes . The presence of uracil indicates  lactic acid   bacteria  contamination of the fruit. [ 32 ]  Uracil derivatives containing a  diazine  ring are used in  pesticides . [ 33 ]  Uracil derivatives are more often used as  antiphotosynthetic   herbicides , destroying weeds in  cotton ,  sugar beet ,  turnips ,  soya ,  peas ,  sunflower  crops,  vineyards ,  berry  plantations, and  orchards . [ 33 ]  Uracil derivatives can enhance the activity of antimicrobial  polysaccharides  such as  chitosan . [ 34 ]


In  yeast , uracil concentrations are inversely proportional to uracil permease. [ 35 ]


Mixtures containing uracil are also commonly used to test  reversed-phase   HPLC  columns. As uracil is essentially unretained by the non-polar stationary phase, this can be used to determine the dwell time (and subsequently dwell volume, given a known flow rate) of the system.


In  biology ,  epigenetics  is the study of  heritable traits , or a stable change of cell function, that happen without changes to the  DNA sequence . [ 1 ]  The  Greek  prefix  epi-  ( ἐπι-  "over, outside of, around") in  epigenetics  implies features that are "on top of" or "in addition to" the traditional (DNA sequence based)  genetic  mechanism of inheritance. [ 2 ]  Epigenetics usually involves a change that is not erased by cell division, and affects the  regulation of gene expression . [ 3 ]  Such effects on  cellular  and  physiological   phenotypic traits  may result from  environmental  factors, or be part of normal development. Epigenetic factors can also lead to cancer. [ 4 ]


The term also refers to the mechanism of changes: functionally relevant alterations to the  genome  that do not involve mutation of the  nucleotide sequence . Examples of mechanisms that produce such changes are  DNA methylation  and  histone modification , each of which alters how genes are expressed without altering the underlying  DNA  sequence. [ 5 ]  Further, non-coding RNA sequences have been shown to play a key role in the regulation of gene expression. [ 6 ]  Gene expression can be controlled through the action of  repressor proteins  that attach to  silencer  regions of the DNA. These epigenetic changes may last through  cell divisions  for the duration of the cell's life, and may also last for multiple generations, even though they do not involve changes in the underlying DNA sequence of the organism; [ 7 ]  instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently. [ 8 ]


One example of an epigenetic change in  eukaryotic  biology is the process of  cellular differentiation . During  morphogenesis ,  totipotent   stem cells  become the various  pluripotent   cell lines  of the  embryo , which in turn become fully differentiated cells. In other words, as a single fertilized  egg cell  – the  zygote  – continues to  divide , the resulting daughter cells change into all the different cell types in an organism, including  neurons ,  muscle cells ,  epithelium ,  endothelium  of  blood vessels , etc., by activating some genes while inhibiting the expression of others. [ 9 ]


The term  epigenesis  has a generic meaning of "extra growth" that has been used in English since the 17th century. [ 10 ]  In scientific publications, the term  epigenetics  started to appear in the 1930s (see Fig. on the right). However, its contemporary meaning emerged only in the 1990s. [ 11 ]


A definition of the concept of  epigenetic trait  as a "stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence" was formulated at a  Cold Spring Harbor  meeting in 2008, [ 12 ]  although alternate definitions that include non-heritable traits are still being used widely. [ 13 ]


The hypothesis of epigenetic changes affecting the expression of  chromosomes  was put forth by the Russian biologist  Nikolai Koltsov . [ 14 ]  From the generic meaning, and the associated adjective  epigenetic , British embryologist  C. H. Waddington  coined the term  epigenetics  in 1942 as pertaining to  epigenesis , in parallel to  Valentin Haecker 's 'phenogenetics' ( Phänogenetik ). [ 15 ]   Epigenesis  in the context of the biology of that period referred to the  differentiation  of cells from their initial  totipotent  state during  embryonic development . [ 16 ]


When Waddington coined the term, the physical nature of  genes  and their role in heredity was not known. He used it instead as a conceptual model of how genetic components might interact with their surroundings to produce a  phenotype ; he used the phrase " epigenetic landscape " as a metaphor for  biological development . Waddington held that cell fates were established during development in a process he called  canalisation  much as a marble rolls down to the point of  lowest local elevation . [ 17 ]  Waddington suggested visualising increasing irreversibility of cell type differentiation as ridges rising between the valleys where the marbles (analogous to cells) are travelling. [ 18 ]


In recent times, Waddington's notion of the epigenetic landscape has been rigorously formalized in the context of the  systems dynamics  state approach to the study of cell-fate. [ 19 ] [ 20 ]  Cell-fate determination is predicted to exhibit certain dynamics, such as attractor-convergence (the attractor can be an equilibrium point, limit cycle or  strange attractor ) or oscillatory. [ 20 ]


Robin Holliday  defined in 1990 epigenetics as "the study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms." [ 21 ]


More recent usage of the word in biology follows stricter definitions. As defined by  Arthur Riggs  and colleagues, it is "the study of  mitotically  and/or  meiotically  heritable changes in gene function that cannot be explained by changes in DNA sequence." [ 22 ]


The term has also been used, however, to describe processes which have not been demonstrated to be heritable, such as some forms of histone modification. Consequently, there are attempts to redefine "epigenetics" in broader terms that would avoid the constraints of requiring  heritability . For example,  Adrian Bird  defined epigenetics as "the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states." [ 7 ]  This definition would be inclusive of transient modifications associated with  DNA repair  or  cell-cycle  phases as well as stable changes maintained across multiple cell generations, but exclude others such as templating of membrane architecture and  prions  unless they impinge on chromosome function. Such redefinitions however are not universally accepted and are still subject to debate. [ 23 ]  The  NIH  "Roadmap Epigenomics Project", which ran from 2008 to 2017, uses the following definition: "For purposes of this program, epigenetics refers to both heritable changes in gene activity and  expression  (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable." [ 24 ]  In 2008, a consensus definition of the epigenetic trait, a "stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence," was made at a  Cold Spring Harbor  meeting. [ 12 ]


The similarity of the word to "genetics" has generated many parallel usages. The " epigenome " is a parallel to the word " genome ", referring to the overall epigenetic state of a cell, and  epigenomics  refers to global analyses of epigenetic changes across the entire genome. [ 13 ]  The phrase " genetic code " has also been adapted – the " epigenetic code " has been used to describe the set of epigenetic features that create different phenotypes in different cells from the same underlying DNA sequence. Taken to its extreme, the "epigenetic code" could represent the total state of the cell, with the position of each molecule accounted for in an  epigenomic map , a diagrammatic representation of the gene expression, DNA methylation and histone modification status of a particular genomic region. More typically, the term is used in reference to systematic efforts to measure specific, relevant forms of epigenetic information such as the  histone code  or  DNA methylation  patterns. [ citation needed ]


Covalent  modification of either DNA (e.g. cytosine methylation and hydroxymethylation) or of histone proteins (e.g. lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation) play central roles in many types of epigenetic inheritance. Therefore, the word "epigenetics" is sometimes used as a synonym for these processes. However, this can be misleading. Chromatin remodeling is not always inherited, and not all epigenetic inheritance involves chromatin remodeling. [ 25 ]  In 2019, a further lysine modification appeared in the scientific literature linking epigenetics modification to cell metabolism, i.e. lactylation [ 26 ]


Because the  phenotype  of a cell or individual is affected by which of its genes are transcribed, heritable  transcription states  can give rise to epigenetic effects. There are several layers of regulation of  gene expression . One way that genes are regulated is through the remodeling of chromatin. Chromatin is the complex of DNA and the  histone  proteins with which it associates. If the way that DNA is wrapped around the histones changes, gene expression can change as well. Chromatin remodeling is accomplished through two main mechanisms:


There is frequently a reciprocal relationship between DNA methylation and histone lysine methylation. [ 32 ]  For instance, the  methyl binding domain protein MBD1 , attracted to and associating with  methylated cytosine  in a DNA  CpG site , can also associate with H3K9  methyltransferase  activity to methylate histone 3 at lysine 9. On the other hand, DNA maintenance methylation by  DNMT1  appears to partly rely on recognition of histone methylation on the nucleosome present at the DNA site to carry out cytosine methylation on newly synthesized DNA. [ 32 ]  There is further crosstalk between DNA methylation carried out by  DNMT3A  and  DNMT3B  and histone methylation so that there is a correlation between the genome-wide distribution of DNA methylation and histone methylation. [ 33 ]


Mechanisms of heritability of histone state are not well understood; however, much is known about the mechanism of heritability of DNA methylation state during cell division and differentiation. Heritability of methylation state depends on certain enzymes (such as  DNMT1 ) that have a higher affinity for 5-methylcytosine than for cytosine. If this enzyme reaches a "hemimethylated" portion of DNA (where 5-methylcytosine is in only one of the two DNA strands) the enzyme will methylate the other half. However, it is now known that DNMT1 physically interacts with the protein  UHRF1 . UHRF1 has been recently recognized as essential for DNMT1-mediated maintenance of DNA methylation. UHRF1 is the protein that specifically recognizes hemi-methylated DNA, therefore bringing DNMT1 to its substrate to maintain DNA methylation. [ 33 ]


Although histone modifications occur throughout the entire sequence, the unstructured N-termini of histones (called histone tails) are particularly highly modified. These modifications include  acetylation ,  methylation ,  ubiquitylation ,  phosphorylation ,  sumoylation , ribosylation and citrullination. Acetylation is the most highly studied of these modifications. For example, acetylation of the K14 and K9  lysines  of the tail of histone H3 by histone acetyltransferase enzymes (HATs) is generally related to transcriptional competence [ 35 ]  (see Figure).


One mode of thinking is that this tendency of acetylation to be associated with "active" transcription is biophysical in nature. Because it normally has a positively charged nitrogen at its end, lysine can bind the negatively charged phosphates of the DNA backbone. The acetylation event converts the positively charged amine group on the side chain into a neutral amide linkage. This removes the positive charge, thus loosening the DNA from the histone. When this occurs, complexes like  SWI/SNF  and other transcriptional factors can bind to the DNA and allow transcription to occur. This is the "cis" model of the epigenetic function. In other words, changes to the histone tails have a direct effect on the DNA itself. [ 36 ]


Another model of epigenetic function is the "trans" model. In this model, changes to the histone tails act indirectly on the DNA. For example, lysine acetylation may create a binding site for chromatin-modifying enzymes (or transcription machinery as well). This chromatin remodeler can then cause changes to the state of the chromatin. Indeed, a bromodomain – a protein domain that specifically binds acetyl-lysine – is found in many enzymes that help activate transcription, including the  SWI/SNF  complex. It may be that acetylation acts in this and the previous way to aid in transcriptional activation.


The idea that modifications act as docking modules for related factors is borne out by  histone methylation  as well. Methylation of lysine 9 of histone H3 has long been associated with constitutively transcriptionally silent chromatin (constitutive  heterochromatin ) (see bottom Figure). It has been determined that a chromodomain (a domain that specifically binds methyl-lysine) in the transcriptionally repressive protein  HP1  recruits HP1 to K9 methylated regions. One example that seems to refute this biophysical model for methylation is that tri-methylation of histone H3 at lysine 4 is strongly associated with (and required for full) transcriptional activation (see top Figure). Tri-methylation, in this case, would introduce a fixed positive charge on the tail.


It has been shown that the histone lysine methyltransferase (KMT) is responsible for this methylation activity in the pattern of histones H3 & H4. This enzyme utilizes a catalytically active site called the SET domain (Suppressor of variegation, Enhancer of Zeste, Trithorax). The SET domain is a 130-amino acid sequence involved in modulating gene activities. This domain has been demonstrated to bind to the histone tail and causes the methylation of the histone. [ 37 ]


Differing histone modifications are likely to function in differing ways; acetylation at one position is likely to function differently from acetylation at another position. Also, multiple modifications may occur at the same time, and these modifications may work together to change the behavior of the  nucleosome . The idea that multiple dynamic modifications regulate gene transcription in a systematic and reproducible way is called the  histone code , although the idea that histone state can be read linearly as a digital information carrier has been largely debunked. One of the best-understood systems that orchestrate chromatin-based silencing is the  SIR protein  based silencing of the yeast hidden mating-type loci HML and HMR.


DNA methylation  frequently occurs in repeated sequences, and helps to suppress the expression and mobility of ' transposable elements ': [ 38 ]  Because  5-methylcytosine  can be spontaneously deaminated (replacing nitrogen by oxygen) to  thymidine , CpG sites are frequently mutated and become rare in the genome, except at  CpG islands  where they remain unmethylated. Epigenetic changes of this type thus have the potential to direct increased frequencies of permanent genetic mutation. DNA methylation patterns are known to be established and modified in response to environmental factors by a complex interplay of at least three independent  DNA methyltransferases , DNMT1, DNMT3A, and DNMT3B, the loss of any of which is lethal in mice. [ 39 ]  DNMT1 is the most abundant methyltransferase in somatic cells, [ 40 ]  localizes to replication foci, [ 41 ]  has a 10–40-fold preference for hemimethylated DNA and interacts with the  proliferating cell nuclear antigen  (PCNA). [ 42 ]


By preferentially modifying hemimethylated DNA, DNMT1 transfers patterns of methylation to a newly synthesized strand after  DNA replication , and therefore is often referred to as the 'maintenance' methyltransferase. [ 43 ]  DNMT1 is essential for proper embryonic development, imprinting and X-inactivation. [ 39 ] [ 44 ]  To emphasize the difference of this molecular mechanism of inheritance from the canonical Watson-Crick base-pairing mechanism of transmission of genetic information, the term 'Epigenetic templating' was introduced. [ 45 ]  Furthermore, in addition to the maintenance and transmission of methylated DNA states, the same principle could work in the maintenance and transmission of histone modifications and even cytoplasmic ( structural ) heritable states. [ 46 ]


RNA methylation of N6-methyladenosine (m6A) as the most abundant eukaryotic RNA modification has recently been recognized as an important gene regulatory mechanism. [ 47 ]


Histones H3 and H4 can also be manipulated through demethylation using  histone lysine demethylase  (KDM). This recently identified enzyme has a catalytically active site called the Jumonji domain (JmjC). The demethylation occurs when JmjC utilizes multiple cofactors to hydroxylate the methyl group, thereby removing it. JmjC is capable of demethylating mono-, di-, and tri-methylated substrates. [ 48 ]


Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Epigenetic control is often associated with alternative  covalent modifications  of histones. [ 49 ]  The stability and heritability of states of larger chromosomal regions are suggested to involve positive feedback where modified  nucleosomes  recruit enzymes that similarly modify nearby nucleosomes. [ 50 ]  A simplified stochastic model for this type of epigenetics is found here. [ 51 ] [ 52 ]


It has been suggested that chromatin-based transcriptional regulation could be mediated by the effect of small RNAs.  Small interfering RNAs  can modulate transcriptional gene expression via epigenetic modulation of targeted  promoters . [ 53 ]


Sometimes a gene, after being turned on, transcribes a product that (directly or indirectly) maintains the activity of that gene. For example,  Hnf4  and  MyoD  enhance the transcription of many liver-specific and muscle-specific genes, respectively, including their own, through the  transcription factor  activity of the  proteins  they encode. RNA signalling includes differential recruitment of a hierarchy of generic chromatin modifying complexes and DNA methyltransferases to specific loci by RNAs during differentiation and development. [ 54 ]  Other epigenetic changes are mediated by the production of  different splice forms  of  RNA , or by formation of double-stranded RNA ( RNAi ). Descendants of the cell in which the gene was turned on will inherit this activity, even if the original stimulus for gene-activation is no longer present. These genes are often turned on or off by  signal transduction , although in some systems where  syncytia  or  gap junctions  are important, RNA may spread directly to other cells or nuclei by  diffusion . A large amount of RNA and protein is contributed to the  zygote  by the mother during  oogenesis  or via  nurse cells , resulting in  maternal effect  phenotypes. A smaller quantity of sperm RNA is transmitted from the father, but there is recent evidence that this epigenetic information can lead to visible changes in several generations of offspring. [ 55 ]


MicroRNAs  (miRNAs) are members of  non-coding RNAs  that range in size from 17 to 25 nucleotides. miRNAs regulate a large variety of biological functions in plants and animals. [ 56 ]  So far, in 2013, about 2000 miRNAs have been discovered in humans and these can be found online in a miRNA database. [ 57 ]  Each miRNA expressed in a cell may target about 100 to 200 messenger RNAs(mRNAs) that it downregulates. [ 58 ]  Most of the downregulation of mRNAs occurs by causing the decay of the targeted mRNA, while some downregulation occurs at the level of translation into protein. [ 59 ]


It appears that about 60% of human protein coding genes are regulated by miRNAs. [ 60 ]  Many miRNAs are epigenetically regulated. About 50% of miRNA genes are associated with  CpG islands , [ 56 ]  that may be repressed by epigenetic methylation. Transcription from methylated CpG islands is strongly and heritably repressed. [ 61 ]  Other miRNAs are epigenetically regulated by either histone modifications or by combined DNA methylation and histone modification. [ 56 ]


In 2011, it was demonstrated that the  methylation  of  mRNA  plays a critical role in human  energy homeostasis . The obesity-associated  FTO gene  is shown to be able to  demethylate   N6-methyladenosine  in RNA. [ 62 ] [ 63 ]


sRNAs  are small (50–250 nucleotides), highly structured, non-coding RNA fragments found in bacteria. They control gene expression including  virulence  genes in pathogens and are viewed as new targets in the fight against drug-resistant bacteria. [ 64 ]  They play an important role in many biological processes, binding to mRNA and protein targets in prokaryotes. Their phylogenetic analyses, for example through sRNA–mRNA target interactions or protein  binding properties , are used to build comprehensive databases. [ 65 ]  sRNA- gene maps  based on their targets in microbial genomes are also constructed. [ 66 ]


Numerous investigations have demonstrated the pivotal involvement of long non-coding RNAs (lncRNAs) in the regulation of gene expression and chromosomal modifications, thereby exerting significant control over cellular differentiation. These long non-coding RNAs also contribute to genomic imprinting and the inactivation of the X chromosome. [ 67 ]
In invertebrates such as social insects of honey bees, long non-coding RNAs are detected as a possible epigenetic mechanism via allele-specific genes underlying aggression via reciprocal crosses. [ 68 ]


Prions  are  infectious  forms of  proteins . In general, proteins fold into discrete units that perform distinct cellular functions, but some proteins are also capable of forming an infectious conformational state known as a prion. Although often viewed in the context of  infectious disease , prions are more loosely defined by their ability to catalytically convert other native state versions of the same protein to an infectious conformational state. It is in this latter sense that they can be viewed as epigenetic agents capable of inducing a phenotypic change without a modification of the genome. [ 69 ]


Fungal prions  are considered by some to be epigenetic because the infectious phenotype caused by the prion can be inherited without modification of the genome.  PSI+  and URE3, discovered in  yeast  in 1965 and 1971, are the two best studied of this type of prion. [ 70 ] [ 71 ]  Prions can have a phenotypic effect through the sequestration of protein in aggregates, thereby reducing that protein's activity. In PSI+ cells, the loss of the Sup35 protein (which is involved in termination of translation) causes ribosomes to have a higher rate of read-through of stop  codons , an effect that results in suppression of  nonsense mutations  in other genes. [ 72 ]  The ability of Sup35 to form prions may be a conserved trait. It could confer an adaptive advantage by giving cells the ability to  switch into a PSI+ state  and express dormant genetic features normally terminated by stop codon mutations. [ 73 ] [ 74 ] [ 75 ] [ 76 ]


Prion-based epigenetics has also been observed in  Saccharomyces cerevisiae . [ 77 ]


Epigenetic changes modify the activation of certain genes, but not the genetic code sequence of DNA. [ 78 ]  The microstructure (not code) of DNA itself or the associated  chromatin  proteins may be modified, causing activation or silencing. This mechanism enables differentiated cells in a multicellular organism to express only the genes that are necessary for their own activity. Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism's lifetime; however, these epigenetic changes can be transmitted to the organism's offspring through a process called  transgenerational epigenetic inheritance . Moreover, if gene inactivation occurs in a sperm or egg cell that results in fertilization, this epigenetic modification may also be transferred to the next generation. [ 79 ]


Specific epigenetic processes include  paramutation ,  bookmarking ,  imprinting ,  gene silencing ,  X chromosome inactivation ,  position effect ,  DNA methylation reprogramming ,  transvection ,  maternal effects , the progress of  carcinogenesis , many effects of  teratogens , regulation of  histone  modifications and  heterochromatin , and technical limitations affecting  parthenogenesis  and  cloning . [ 80 ] [ 81 ] [ 82 ]


DNA damage can also cause epigenetic changes. [ 83 ] [ 84 ] [ 85 ]  DNA damage is very frequent, occurring on average about 60,000 times a day per cell of the human body (see  DNA damage (naturally occurring) ). These damages are largely repaired, however, epigenetic changes can still remain at the site of DNA repair. [ 86 ]  In particular, a double strand break in DNA can initiate unprogrammed epigenetic gene silencing both by causing DNA methylation as well as by promoting silencing types of histone modifications (chromatin remodeling - see next section). [ 87 ]  In addition, the enzyme  Parp1 (poly(ADP)-ribose polymerase)  and its product poly(ADP)-ribose (PAR) accumulate at sites of DNA damage as part of the repair process. [ 88 ]  This accumulation, in turn, directs recruitment and activation of the chromatin remodeling protein, ALC1, that can cause  nucleosome  remodeling. [ 89 ]  Nucleosome remodeling has been found to cause, for instance, epigenetic silencing of DNA repair gene MLH1. [ 22 ] [ 90 ]  DNA damaging chemicals, such as  benzene ,  hydroquinone ,  styrene ,  carbon tetrachloride  and  trichloroethylene , cause considerable hypomethylation of DNA, some through the activation of oxidative stress pathways. [ 91 ]


Foods are known to alter the epigenetics of rats on different diets. [ 92 ]  Some food components epigenetically increase the levels of DNA repair enzymes such as  MGMT  and  MLH1 [ 93 ]  and  p53 . [ 94 ]  Other food components can reduce DNA damage, such as soy  isoflavones . In one study, markers for oxidative stress, such as modified nucleotides that can result from DNA damage, were decreased by a 3-week diet supplemented with soy. [ 95 ]  A decrease in oxidative DNA damage was also observed 2 h after consumption of  anthocyanin -rich  bilberry  ( Vaccinium myrtillius  L.)  pomace  extract. [ 96 ]


Damage to DNA is very common and is constantly being repaired. Epigenetic alterations can accompany DNA repair of oxidative damage or double-strand breaks. In human cells, oxidative DNA damage occurs about 10,000 times a day and DNA double-strand breaks occur about 10 to 50 times a cell cycle in somatic replicating cells (see  DNA damage (naturally occurring) ). The selective advantage of DNA repair is to allow the cell to survive in the face of DNA damage. The selective advantage of epigenetic alterations that occur with DNA repair is not clear. [ citation needed ]


In the steady state (with endogenous damages occurring and being repaired), there are about 2,400 oxidatively damaged guanines that form  8-oxo-2'-deoxyguanosine  (8-OHdG) in the average mammalian cell DNA. [ 97 ]  8-OHdG constitutes about 5% of the oxidative damages commonly present in DNA. [ 98 ]  The oxidized guanines do not occur randomly among all guanines in DNA. There is a sequence preference for the guanine at a  methylated   CpG site  (a cytosine followed by guanine along its  5' → 3' direction  and where the cytosine is methylated (5-mCpG)). [ 99 ]  A 5-mCpG site has the lowest ionization potential for guanine oxidation. [ citation needed ]


Oxidized guanine has mispairing potential and is mutagenic. [ 101 ]   Oxoguanine glycosylase  (OGG1) is the primary enzyme responsible for the excision of the oxidized guanine during DNA repair. OGG1 finds and binds to an 8-OHdG within a few seconds. [ 102 ]  However, OGG1 does not immediately excise 8-OHdG. In HeLa cells half maximum removal of 8-OHdG occurs in 30 minutes, [ 103 ]  and in irradiated mice, the 8-OHdGs induced in the mouse liver are removed with a half-life of 11 minutes. [ 98 ]


When OGG1 is present at an oxidized guanine within a methylated  CpG site  it recruits  TET1  to the 8-OHdG lesion (see Figure). This allows TET1 to demethylate an adjacent methylated cytosine. Demethylation of cytosine is an epigenetic alteration. [ citation needed ]


As an example, when human mammary epithelial cells were treated with H 2 O 2  for six hours, 8-OHdG increased about 3.5-fold in DNA and this caused about 80% demethylation of the 5-methylcytosines in the genome. [ 100 ]  Demethylation of CpGs in a gene promoter by  TET enzyme  activity increases transcription of the gene into messenger RNA. [ 104 ]  In cells treated with H 2 O 2 , one particular gene was examined,  BACE1 . [ 100 ]  The methylation level of the  BACE1   CpG island  was reduced (an epigenetic alteration) and this allowed about 6.5 fold increase of expression of  BACE1  messenger RNA. [ citation needed ]


While six-hour incubation with H 2 O 2  causes considerable demethylation of 5-mCpG sites, shorter times of H 2 O 2  incubation appear to promote other epigenetic alterations. Treatment of cells with H 2 O 2  for 30 minutes causes the mismatch repair protein heterodimer MSH2-MSH6 to recruit DNA methyltransferase 1 ( DNMT1 ) to sites of some kinds of oxidative DNA damage. [ 105 ]  This could cause increased methylation of cytosines (epigenetic alterations) at these locations.


Jiang et al. [ 106 ]  treated  HEK 293 cells  with agents causing oxidative DNA damage, ( potassium bromate  (KBrO3) or  potassium chromate  (K2CrO4)).  Base excision repair  (BER) of oxidative damage occurred with the DNA repair enzyme  polymerase beta  localizing to oxidized guanines. Polymerase beta is the main human polymerase in short-patch BER of oxidative DNA damage. Jiang et al. [ 106 ]  also found that polymerase beta recruited the  DNA methyltransferase  protein DNMT3b to BER repair sites. They then evaluated the methylation pattern at the single nucleotide level in a small region of DNA including the  promoter  region and the early transcription region of the  BRCA1  gene. Oxidative DNA damage from bromate modulated the DNA methylation pattern (caused epigenetic alterations) at CpG sites within the region of DNA studied. In untreated cells, CpGs located at −189, −134, −29, −19, +16, and +19 of the BRCA1 gene had methylated cytosines (where numbering is from the  messenger RNA  transcription start site, and negative numbers indicate nucleotides in the upstream  promoter  region). Bromate treatment-induced oxidation resulted in the loss of cytosine methylation at −189, −134, +16 and +19 while also leading to the formation of new methylation at the CpGs located at −80, −55, −21 and +8 after DNA repair was allowed.


At least four articles report the recruitment of  DNA methyltransferase 1 (DNMT1)  to sites of DNA double-strand breaks. [ 107 ] [ 108 ] [ 109 ] [ 110 ]  During  homologous recombinational repair (HR)  of the double-strand break, the involvement of DNMT1 causes the two repaired strands of DNA to have different levels of methylated cytosines. One strand becomes frequently methylated at about 21  CpG sites  downstream of the repaired double-strand break. The other DNA strand loses methylation at about six CpG sites that were previously methylated downstream of the double-strand break, as well as losing methylation at about five CpG sites that were previously methylated upstream of the double-strand break. When the chromosome is replicated, this gives rise to one daughter chromosome that is heavily methylated downstream of the previous break site and one that is unmethylated in the region both upstream and downstream of the previous break site. With respect to the gene that was broken by the double-strand break, half of the progeny cells express that gene at a high level and in the other half of the progeny cells expression of that gene is repressed. When clones of these cells were maintained for three years, the new methylation patterns were maintained over that time period. [ 111 ]


In mice with a CRISPR-mediated homology-directed recombination insertion in their genome there were a large number of increased methylations of CpG sites within the double-strand break-associated insertion. [ 112 ]


Non-homologous end joining  (NHEJ) repair of a double-strand break can cause a small number of demethylations of pre-existing cytosine DNA methylations downstream of the repaired double-strand break. [ 108 ]  Further work by Allen et al. [ 113 ]  showed that NHEJ of a DNA double-strand break in a cell could give rise to some progeny cells having repressed expression of the gene harboring the initial double-strand break and some progeny having high expression of that gene due to epigenetic alterations associated with NHEJ repair. The frequency of epigenetic alterations causing repression of a gene after an NHEJ repair of a DNA double-strand break in that gene may be about 0.9%. [ 109 ]


Epigenetic research uses a wide range of  molecular biological  techniques to further understanding of epigenetic phenomena. These techniques include  chromatin immunoprecipitation  (together with its large-scale variants  ChIP-on-chip  and  ChIP-Seq ),  fluorescent in situ hybridization , methylation-sensitive  restriction enzymes , DNA adenine methyltransferase identification ( DamID ) and  bisulfite sequencing . [ 114 ]  Furthermore, the use of  bioinformatics  methods has a role in  computational epigenetics . [ 114 ]


Chromatin Immunoprecipitation (ChIP) has helped bridge the gap between DNA and epigenetic interactions. [ 115 ]  With the use of ChIP, researchers are able to make findings in regards to gene regulation, transcription mechanisms, and chromatin structure. [ 115 ]


Fluorescent  in situ  hybridization (FISH) is very important to understand epigenetic mechanisms. [ 116 ]  FISH can be used to find the location of genes on chromosomes, as well as finding noncoding RNAs. [ 116 ] [ 117 ]  FISH is predominantly used for detecting chromosomal abnormalities in humans. [ 117 ]


Methylation sensitive restriction enzymes paired with PCR is a way to evaluate methylation in DNA - specifically the CpG sites. [ 118 ]  If DNA is methylated, the restriction enzymes will not cleave the strand. [ 118 ]  Contrarily, if the DNA is not methylated, the enzymes will cleave the strand and it will be amplified by PCR. [ 118 ]


Bisulfite sequencing is another way to evaluate DNA methylation. Cytosine will be changed to uracil from being treated with sodium bisulfite, whereas methylated cytosines will not be affected. [ 118 ] [ 119 ] [ 120 ]


Certain sequencing methods, such as  nanopore sequencing , allow sequencing of native DNA. Native (=unamplified) DNA retains the epigenetic modifications which would otherwise be lost during the amplification step. Nanopore basecaller models can distinguish between the signals obtained for epigenetically modified bases and unaltered based and provide an epigenetic profile in addition to the sequencing result. [ 121 ]


In  ciliates  such as  Tetrahymena  and  Paramecium , genetically identical cells show heritable differences in the patterns of ciliary rows on their cell surface. Experimentally altered patterns can be transmitted to daughter cells. It seems existing structures act as templates for new structures. The mechanisms of such inheritance are unclear, but reasons exist to assume that multicellular organisms also use existing cell structures to assemble new ones. [ 122 ] [ 123 ] [ 124 ]


Eukaryotic genomes have numerous  nucleosomes . Nucleosome position is not random, and determine the accessibility of DNA to regulatory proteins. Promoters active in different tissues have been shown to have different nucleosome positioning features. [ 125 ]  This determines differences in gene expression and cell differentiation. It has been shown that at least some nucleosomes are retained in sperm cells (where most but not all histones are replaced by  protamines ). Thus nucleosome positioning is to some degree inheritable. Recent studies have uncovered connections between nucleosome positioning and other epigenetic factors, such as DNA methylation and hydroxymethylation. [ 126 ]


Different  histone variants  are incorporated into specific regions of the genome non-randomly. Their differential biochemical characteristics can affect genome functions via their roles in gene regulation, [ 127 ]  and maintenance of chromosome structures. [ 128 ]


The three-dimensional configuration of the genome (the 3D genome) is complex, dynamic and crucial for regulating genomic function and nuclear processes such as DNA replication, transcription and DNA-damage repair. [ 129 ]


Memory formation  and maintenance are due to epigenetic alterations that cause the required dynamic changes in  gene transcription  that create and renew memory in neurons. [ 31 ]


An event can set off a chain of reactions that result in altered methylations of a large set of genes in neurons, which give a representation of the event, a memory. [ 31 ]


Areas of the brain important in the formation of memories include the hippocampus, medial prefrontal cortex (mPFC), anterior cingulate cortex and amygdala, as shown in the diagram of the human brain in this section. [ 130 ]


When a strong memory is created, as in a rat subjected to  contextual fear conditioning  (CFC), one of the earliest events to occur is that more than 100 DNA double-strand breaks are formed by  topoisomerase IIB  in neurons of the hippocampus and the medial prefrontal cortex (mPFC). [ 131 ]  These double-strand breaks are at specific locations that allow activation of transcription of  immediate early genes  (IEGs) that are important in memory formation, allowing their expression in  mRNA , with peak mRNA transcription at seven to ten minutes after CFC. [ 131 ] [ 132 ]


Two important IEGs in memory formation are  EGR1 [ 133 ]  and  the alternative promoter variant of  DNMT3A ,  DNMT3A2 . [ 134 ]  EGR1 protein binds to DNA at its binding motifs, 5′-GCGTGGGCG-3′ or 5′-GCGGGGGCGG-3', and there are about 12,000 genome locations at which EGR1 protein can bind. [ 135 ]  EGR1 protein binds to DNA in gene  promoter  and  enhancer  regions. EGR1 recruits the demethylating enzyme  TET1  to an association, and brings TET1 to about 600 locations on the genome where TET1 can then demethylate and activate the associated genes. [ 135 ]


The DNA methyltransferases DNMT3A1, DNMT3A2 and DNMT3B can all methylate cytosines (see image this section) at  CpG sites  in or near the promoters of genes. As shown by Manzo et al., [ 136 ]  these three DNA methyltransferases differ in their genomic binding locations and DNA methylation activity at different regulatory sites. Manzo et al. located 3,970 genome regions exclusively enriched for DNMT3A1, 3,838 regions for DNMT3A2 and 3,432 regions for DNMT3B. When DNMT3A2 is newly induced as an IEG (when neurons are activated), many new cytosine methylations occur, presumably in the target regions of DNMT3A2. Oliviera et al. [ 134 ]  found that the neuronal activity-inducible IEG levels of Dnmt3a2 in the hippocampus determined the ability to form long-term memories.


Rats form long-term associative memories after  contextual fear conditioning (CFC) . [ 137 ]  Duke et al. [ 30 ]  found that 24 hours after CFC in rats, in hippocampus neurons, 2,097 genes (9.17% of the genes in the rat genome) had altered methylation. When newly methylated cytosines are present in  CpG sites  in the promoter regions of genes, the genes are often repressed, and when newly demethylated cytosines are present the genes may be activated. [ 138 ]  After CFC, there were 1,048 genes with reduced mRNA expression and 564 genes with upregulated mRNA expression. Similarly, when mice undergo CFC, one hour later in the hippocampus region of the mouse brain there are 675 demethylated genes and 613 hypermethylated genes. [ 139 ]  However, memories do not remain in the hippocampus, but after four or five weeks the memories are stored in the anterior cingulate cortex. [ 140 ]  In the studies on mice after CFC, Halder et al. [ 139 ]  showed that four weeks after CFC there were at least 1,000 differentially methylated genes and more than 1,000 differentially expressed genes in the anterior cingulate cortex, while at the same time the altered methylations in the hippocampus were reversed.


The epigenetic alteration of methylation after a new memory is established creates a different pool of nuclear mRNAs. As reviewed by Bernstein, [ 31 ]  the epigenetically determined new mix of nuclear  mRNAs  are often packaged into neuronal granules, or  messenger RNP , consisting of mRNA,  small and large ribosomal subunits , translation initiation factors and RNA-binding proteins that regulate mRNA function. These neuronal granules are transported from the neuron nucleus and are directed, according to 3′ untranslated regions of the mRNA in the granules (their "zip codes"), to neuronal  dendrites . Roughly 2,500 mRNAs may be localized to the dendrites of hippocampal pyramidal neurons and perhaps 450 transcripts are in excitatory presynaptic nerve terminals (dendritic spines). The altered assortments of transcripts (dependent on epigenetic alterations in the neuron nucleus) have different sensitivities in response to signals, which is the basis of altered synaptic plasticity. Altered synaptic plasticity is often considered the neurochemical foundation of learning and memory.


Epigenetics play a major role in  brain aging  and age-related cognitive decline, with relevance to  life extension . [ 141 ] [ 142 ] [ 143 ] [ 144 ] [ 145 ]


In adulthood, changes in the  epigenome  are important for various higher cognitive functions. Dysregulation of epigenetic mechanisms is implicated in  neurodegenerative disorders  and diseases. Epigenetic modifications in  neurons  are dynamic and reversible. [ 146 ]  Epigenetic regulation impacts neuronal action, affecting learning, memory, and other  cognitive  processes. [ 147 ]


Early events, including during  embryonic development , can influence development, cognition, and health outcomes through  epigenetic mechanisms . [ 148 ]


Epigenetic mechanisms have been proposed as "a potential molecular mechanism for effects of endogenous  hormones  on the organization of developing brain circuits". [ 149 ]


Nutrients  could interact with the epigenome to "protect or boost cognitive processes across the lifespan". [ 150 ] [ 151 ]


A review suggests  neurobiological effects of physical exercise  via  epigenetics  seem "central to building an 'epigenetic memory' to influence long-term brain function and behavior" and may even be heritable. [ 152 ]


With the axo-ciliary  synapse , there is communication between  serotonergic   axons  and antenna-like  primary cilia  of  CA1   pyramidal   neurons  that alters the neuron's  epigenetic  state in the  nucleus  via the signalling distinct from that at the  plasma membrane  (and longer-term). [ 153 ] [ 154 ]


Epigenetics also play a major role in the  brain evolution in and to humans . [ 155 ]


Developmental epigenetics can be divided into predetermined and probabilistic epigenesis. Predetermined epigenesis is a unidirectional movement from structural development in DNA to the functional maturation of the protein. "Predetermined" here means that development is scripted and predictable. Probabilistic epigenesis on the other hand is a bidirectional structure-function development with experiences and external molding development. [ 156 ]


Somatic epigenetic inheritance, particularly through DNA and histone covalent modifications and  nucleosome  repositioning, is very important in the development of multicellular eukaryotic organisms. [ 126 ]  The genome sequence is static (with some notable exceptions), but cells differentiate into many different types, which perform different functions, and respond differently to the environment and intercellular signaling. Thus, as individuals develop,  morphogens  activate or silence genes in an epigenetically heritable fashion, giving cells a memory. In mammals, most cells terminally differentiate, with only  stem cells  retaining the ability to differentiate into several cell types ("totipotency" and "multipotency"). In  mammals , some stem cells continue producing newly differentiated cells throughout life, such as in  neurogenesis , but mammals are not able to respond to loss of some tissues, for example, the inability to regenerate limbs, which some other animals are capable of. Epigenetic modifications regulate the transition from neural stem cells to glial progenitor cells (for example, differentiation into oligodendrocytes is regulated by the deacetylation and methylation of histones). [ 157 ]  Unlike animals, plant cells do not terminally differentiate, remaining totipotent with the ability to give rise to a new individual plant. While plants do utilize many of the same epigenetic mechanisms as animals, such as  chromatin remodeling , it has been hypothesized that some kinds of plant cells do not use or require "cellular memories", resetting their gene expression patterns using positional information from the environment and surrounding cells to determine their fate. [ 158 ]


Epigenetic changes can occur in response to environmental exposure – for example, maternal dietary supplementation with  genistein  (250 mg/kg) have epigenetic changes affecting expression of the  agouti gene , which affects their fur color, weight, and propensity to develop cancer. [ 159 ] [ 160 ] [ 161 ]  Ongoing research is focused on exploring the impact of other known  teratogens , such as  diabetic embryopathy , on  methylation  signatures. [ 162 ]


Controversial results from one study suggested that traumatic experiences might produce an epigenetic signal that is capable of being passed to future generations. Mice were trained, using foot shocks, to fear a cherry blossom odor. The investigators reported that the mouse offspring had an increased aversion to this specific odor. [ 163 ] [ 164 ]  They suggested epigenetic changes that increase gene expression, rather than in DNA itself, in a gene, M71, that governs the functioning of an odor receptor in the nose that responds specifically to this cherry blossom smell. There were physical changes that correlated with olfactory (smell) function in the brains of the trained mice and their descendants. Several criticisms were reported, including the study's low statistical power as evidence of some irregularity such as bias in reporting results. [ 165 ]  Due to limits of sample size, there is a probability that an effect will not be demonstrated to within statistical significance even if it exists. The criticism suggested that the probability that all the experiments reported would show positive results if an identical protocol was followed, assuming the claimed effects exist, is merely 0.4%. The authors also did not indicate which mice were siblings, and treated all of the mice as statistically independent. [ 166 ]  The original researchers pointed out negative results in the paper's appendix that the criticism omitted in its calculations, and undertook to track which mice were siblings in the future. [ 167 ]


Epigenetic mechanisms were a necessary part of the evolutionary origin of  cell differentiation . [ 168 ] [ need quotation to verify ]  Although epigenetics in multicellular organisms is generally thought to be a mechanism involved in differentiation, with epigenetic patterns "reset" when organisms reproduce, there have been some observations of transgenerational epigenetic inheritance (e.g., the phenomenon of  paramutation  observed in  maize ). Although most of these multigenerational epigenetic traits are gradually lost over several generations, the possibility remains that multigenerational epigenetics could be another aspect to  evolution  and adaptation.
As mentioned above, some define epigenetics as heritable.


A sequestered germ line or  Weismann barrier  is specific to animals, and epigenetic inheritance is more common in plants and microbes.  Eva Jablonka ,  Marion J. Lamb  and Étienne Danchin have argued that these effects may require enhancements to the standard conceptual framework of the  modern synthesis  and have called for an  extended evolutionary synthesis . [ 169 ] [ 170 ] [ 171 ]  Other evolutionary biologists, such as  John Maynard Smith , have incorporated epigenetic inheritance into  population-genetics  models [ 172 ]  or are openly skeptical of the extended evolutionary synthesis ( Michael Lynch ). [ 173 ]  Thomas Dickins and Qazi Rahman state that epigenetic mechanisms such as DNA methylation and histone modification are genetically inherited under the control of  natural selection  and therefore fit under the earlier  "modern synthesis" . [ 174 ]


Two important ways in which epigenetic inheritance can differ from traditional genetic inheritance, with important consequences for evolution, are:


In plants, heritable DNA methylation mutations are 100,000 times more likely to occur compared to DNA mutations. [ 177 ]  An epigenetically inherited element such as the  PSI+  system can act as a "stop-gap", good enough for short-term adaptation that allows the lineage to survive for long enough for mutation and/or recombination to  genetically assimilate  the adaptive phenotypic change. [ 178 ]  The existence of this possibility increases the  evolvability  of a species.


More than 100 cases of  transgenerational epigenetic inheritance  phenomena have been reported in a wide range of organisms, including prokaryotes, plants, and animals. [ 179 ]  For instance,  mourning-cloak butterflies  will change color through hormone changes in response to experimentation of varying temperatures. [ 180 ]


The filamentous fungus  Neurospora crassa  is a prominent model system for understanding the control and function of cytosine methylation. In this organism, DNA methylation is associated with relics of a genome-defense system called RIP (repeat-induced point mutation) and silences gene expression by inhibiting transcription elongation. [ 181 ]


The  yeast prion  PSI is generated by a conformational change of a translation termination factor, which is then inherited by daughter cells. This can provide a survival advantage under adverse conditions, exemplifying epigenetic regulation which enables unicellular organisms to respond rapidly to environmental stress. Prions can be viewed as epigenetic agents capable of inducing a phenotypic change without modification of the genome. [ 182 ]


Direct detection of epigenetic marks in microorganisms is possible with  single molecule real time sequencing , in which polymerase sensitivity allows for measuring methylation and other modifications as a DNA molecule is being sequenced. [ 183 ]  Several projects have demonstrated the ability to collect genome-wide epigenetic data in bacteria. [ 184 ] [ 185 ] [ 186 ] [ 187 ]


While epigenetics is of fundamental importance in  eukaryotes , especially  metazoans , it plays a different role in bacteria. [ 188 ]  Most importantly, eukaryotes use epigenetic mechanisms primarily to regulate gene expression which bacteria rarely do. However, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Bacteria also use DNA  adenine  methylation (rather than DNA  cytosine  methylation) as an epigenetic signal. DNA adenine methylation is important in bacteria virulence in organisms such as  Escherichia coli ,  Salmonella ,  Vibrio ,  Yersinia ,  Haemophilus , and  Brucella . In  Alphaproteobacteria , methylation of adenine regulates the cell cycle and couples gene transcription to DNA replication. In  Gammaproteobacteria , adenine methylation provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage, transposase activity and regulation of gene expression. [ 182 ] [ 189 ]  There exists a genetic switch controlling  Streptococcus pneumoniae  (the pneumococcus) that allows the bacterium to randomly change its characteristics into six alternative states that could pave the way to improved vaccines. Each form is randomly generated by a phase variable methylation system. The ability of the pneumococcus to cause deadly infections is different in each of these six states. Similar systems exist in other bacterial genera. [ 190 ]  In  Bacillota  such as  Clostridioides difficile ,  adenine methylation regulates  sporulation ,  biofilm  formation and host-adaptation. [ 191 ]


Epigenetics has many and varied potential medical applications. [ 192 ]


Direct comparisons of identical twins constitute an optimal model for interrogating  environmental epigenetics . In the case of humans with different environmental exposures, monozygotic (identical) twins were epigenetically indistinguishable during their early years, while older twins had remarkable differences in the overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation. [ 11 ]  The twin pairs who had spent less of their lifetime together and/or had greater differences in their medical histories were those who showed the largest differences in their levels of  5-methylcytosine  DNA and  acetylation  of  histones  H3 and H4. [ 193 ]


Dizygotic (fraternal) and monozygotic (identical) twins show evidence of epigenetic influence in humans. [ 193 ] [ 194 ] [ 195 ]  DNA sequence differences that would be abundant in a singleton-based study do not interfere with the analysis. Environmental differences can produce long-term epigenetic effects, and different developmental monozygotic twin subtypes may be different with respect to their susceptibility to be discordant from an epigenetic point of view. [ 196 ]


A  high-throughput  study, which denotes technology that looks at extensive genetic markers, focused on epigenetic differences between monozygotic twins to compare global and locus-specific changes in  DNA methylation  and histone modifications in a sample of 40 monozygotic twin pairs. [ 193 ]  In this case, only healthy twin pairs were studied, but a wide range of ages was represented, between 3 and 74 years. One of the major conclusions from this study was that there is an age-dependent accumulation of epigenetic differences between the two siblings of twin pairs. This accumulation suggests the existence of epigenetic "drift".  Epigenetic drift  is the term given to epigenetic modifications as they occur as a direct function with age. While age is a known risk factor for many diseases, age-related methylation has been found to occur differentially at specific sites along the genome. Over time, this can result in measurable differences between biological and chronological age. Epigenetic changes have been found to be reflective of  lifestyle  and may act as functional  biomarkers  of disease before clinical  threshold  is reached. [ 197 ]


A more recent study, where 114 monozygotic twins and 80 dizygotic twins were analyzed for the DNA methylation status of around 6000 unique genomic regions, concluded that epigenetic similarity at the time of blastocyst splitting may also contribute to phenotypic similarities in monozygotic co-twins. This supports the notion that  microenvironment  at early stages of embryonic development can be quite important for the establishment of epigenetic marks. [ 194 ]  Congenital genetic disease is well understood and it is clear that epigenetics can play a role, for example, in the case of  Angelman syndrome  and  Prader–Willi syndrome . These are normal genetic diseases caused by gene deletions or inactivation of the genes but are unusually common because individuals are essentially  hemizygous  because of  genomic imprinting , and therefore a single gene knock out is sufficient to cause the disease, where most cases would require both copies to be knocked out. [ 198 ]