Genome-wide levels of DNA methylation vary widely between plant species, and  Arabidopsis  cytosines tend to be less densely methylated than those in other plants. For example, ~92.5% of CpG cytosines are methylated in  Beta vulgaris . [ 79 ]  The patterns of methylation also differ between cytosine sequence contexts; universally, CpG methylation is higher than CHG and CHH methylation, and CpG methylation can be found in both active genes and transposable elements, while CHG and CHH are usually relegated to silenced transposable elements. [ 80 ] [ 76 ]


It is not clear how the cell determines the locations of  de novo  DNA methylation, but evidence suggests that for many (though not all) locations,  RNA-directed DNA methylation  (RdDM) is involved. In RdDM, specific RNA transcripts are produced from a genomic DNA template, and this RNA forms secondary structures called double-stranded RNA molecules. [ 81 ]  The double-stranded RNAs, through either the small interfering RNA ( siRNA ) or microRNA ( miRNA ) pathways direct de-novo DNA methylation of the original genomic location that produced the RNA. [ 81 ]  This sort of mechanism is thought to be important in cellular defense against  RNA viruses  and/or  transposons , both of which often form a double-stranded RNA that can be mutagenic to the host genome. By methylating their genomic locations, through an as yet poorly understood mechanism, they are shut off and are no longer active in the cell, protecting the genome from their mutagenic effect. Recently, it was described that methylation of the DNA is the main determinant of embryogenic cultures formation from explants in woody plants and is regarded the main mechanism that explains the poor response of mature explants to somatic embryogenesis in the plants (Isah 2016). [ citation needed ]


Diverse orders of insects show varied patterns of DNA methylation, from almost undetectable levels in  flies  to low levels in  butterflies  and higher in  true bugs  and some cockroaches (up to 14% of all CG sites in  Blattella asahinai ). [ 82 ]


Functional DNA methylation has been discovered in Honey Bees. [ 83 ] [ 84 ]  DNA methylation marks are mainly on the gene body, and current opinions on the function of DNA methylation is gene regulation via alternative splicing [ 85 ]


DNA methylation levels in  Drosophila melanogaster  are nearly undetectable. [ 86 ]  Sensitive methods applied to Drosophila DNA Suggest levels in the range of 0.1–0.3% of total cytosine. [ 87 ]  A 2014 study of found that the low level of methylation in fruit fruit flies appeared "at specific short motifs and is independent of DNMT2 activity." [ 88 ]  Further, highly sensitive mass spectrometry approaches, [ 89 ]  have now demonstrated the presence of low (0.07%) but significant levels of adenine methylation during the earliest stages of Drosophila embryogenesis.


Many  fungi  have low levels (0.1 to 0.5%) of cytosine methylation, whereas other fungi have as much as 5% of the genome methylated. [ 90 ]  This value seems to vary both among species and among isolates of the same species. [ 91 ]  There is also evidence that DNA methylation may be involved in state-specific control of  gene expression  in fungi. [ citation needed ]  However, at a detection limit of 250 attomoles by using ultra-high sensitive  mass spectrometry  DNA methylation was not confirmed in single cellular yeast species such as  Saccharomyces cerevisiae  or  Schizosaccharomyces pombe , indicating that yeasts do not possess this DNA modification. [ 9 ] : abstract


Although brewers' yeast ( Saccharomyces ), fission yeast ( Schizosaccharomyces ), and  Aspergillus flavus [ 92 ]  have no detectable DNA methylation, the model filamentous fungus  Neurospora crassa  has a well-characterized methylation system. [ 93 ]  Several genes control methylation in  Neurospora  and mutation of the DNA methyl transferase,  dim-2 , eliminates all DNA methylation but does not affect growth or sexual reproduction. While the  Neurospora  genome has very little repeated DNA, half of the methylation occurs in repeated DNA including  transposon  relics and centromeric DNA. The ability to evaluate other important phenomena in a DNA methylase-deficient genetic background makes  Neurospora  an important system in which to study DNA methylation. [ citation needed ]


DNA methylation is largely absent from  Dictyostelium discoideum [ 94 ]  where it appears to occur at about 0.006% of cytosines. [ 6 ]  In contrast, DNA methylation is widely distributed in  Physarum polycephalum [ 95 ]  where 5-methylcytosine makes up as much as 8% of total cytosine [ 4 ]


Adenine  or  cytosine  methylation are mediated by  restriction modification systems  of many  bacteria , in which specific DNA sequences are methylated periodically throughout the genome. [ 97 ]  A  methylase  is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNAs (which are not methylated in this manner) that are introduced into the cell are degraded by sequence-specific  restriction enzymes  and cleaved. Bacterial genomic DNA is not recognized by these restriction enzymes. The methylation of native DNA acts as a sort of primitive immune system, allowing the bacteria to protect themselves from infection by  bacteriophage . [ citation needed ]


E. coli   DNA adenine methyltransferase  (Dam) is an enzyme of ~32 kDa that does not belong to a restriction/modification system. The target recognition sequence for  E. coli  Dam is GATC, as the methylation occurs at the N6 position of the adenine in this sequence (G meATC). The three base pairs flanking each side of this site also influence DNA–Dam binding. Dam plays several key roles in bacterial processes, including mismatch repair, the timing of DNA replication, and gene expression. As a result of DNA replication, the status of GATC sites in the  E. coli  genome changes from fully methylated to hemimethylated. This is because adenine introduced into the new DNA strand is unmethylated. Re-methylation occurs within two to four seconds, during which time replication errors in the new strand are repaired. Methylation, or its absence, is the marker that allows the repair apparatus of the cell to differentiate between the template and nascent strands. It has been shown that altering Dam activity in bacteria results in an increased spontaneous mutation rate. Bacterial viability is compromised in dam mutants that also lack certain other DNA repair enzymes, providing further evidence for the role of Dam in DNA repair.


One region of the DNA that keeps its hemimethylated status for longer is the  origin of replication , which has an abundance of GATC sites. This is central to the bacterial mechanism for timing DNA replication. SeqA binds to the origin of replication, sequestering it and thus preventing methylation. Because hemimethylated origins of replication are inactive, this mechanism limits DNA replication to once per cell cycle.


Expression of certain genes, for example, those coding for  pilus  expression in  E. coli , is regulated by the methylation of GATC sites in the promoter region of the gene operon. The cells' environmental conditions just after DNA replication determine whether Dam is blocked from methylating a region proximal to or distal from the promoter region. Once the pattern of methylation has been created, the pilus gene transcription is locked in the on or off position until the DNA is again replicated. In  E. coli , these pili  operons  have important roles in virulence in urinary tract infections. It has been proposed [ by whom? ]  that inhibitors of Dam may function as antibiotics.


On the other hand, DNA cytosine methylase targets CCAGG and CCTGG sites to methylate cytosine at the C5 position (C meC(A/T) GG). The other methylase enzyme, EcoKI, causes methylation of adenines in the sequences AAC(N 6 )GTGC and GCAC(N 6 )GTT.


In  Clostridioides difficile , DNA methylation at the target motif CAAAAA was shown to impact  sporulation , a key step in disease transmission, as well as cell length, biofilm formation and host colonization. [ 98 ]


Most strains used by molecular biologists are derivatives of  E. coli  K-12, and possess both Dam and Dcm, but there are commercially available strains that are dam-/dcm- (lack of activity of either methylase). In fact, it is possible to unmethylate the DNA extracted from dam+/dcm+ strains by transforming it into dam-/dcm- strains. This would help digest sequences that are not being recognized by methylation-sensitive restriction enzymes. [ 99 ] [ 100 ]


The  restriction enzyme   DpnI  can recognize 5'-GmeATC-3' sites and digest the methylated DNA. Being such a short motif, it occurs frequently in sequences by chance, and as such its primary use for researchers is to degrade template DNA following  PCRs  (PCR products lack methylation, as no methylases are present in the reaction). Similarly, some commercially available restriction enzymes are sensitive to methylation at their cognate restriction sites and must as mentioned previously be used on DNA passed through a dam-/dcm- strain to allow cutting. [ citation needed ]


DNA methylation can be detected by the following assays currently used in scientific research: [ 101 ]


Differentially methylated regions , which are genomic regions with different methylation statuses among multiple samples (tissues, cells, individuals or others), are regarded as possible functional regions involved in gene transcriptional regulation. The identification of DMRs among multiple tissues (T-DMRs) provides a comprehensive survey of epigenetic differences among human tissues. [ 113 ]  For example, these methylated regions that are unique to a particular tissue allow individuals to differentiate between tissue type, such as semen and vaginal fluid. Current research conducted by Lee et al., showed DACT1 and USP49 positively identified semen by examining T-DMRs. [ 114 ]  The use of T-DMRs has proven useful in the identification of various body fluids found at crime scenes. Researchers in the forensic field are currently seeking novel T-DMRs in genes to use as markers in forensic DNA analysis. DMRs between cancer and normal samples (C-DMRs) demonstrate the aberrant methylation in cancers. [ 115 ]  It is well known that DNA methylation is associated with cell differentiation and proliferation. [ 116 ]  Many DMRs have been found in the development stages (D-DMRs) [ 117 ]  and in the reprogrammed progress (R-DMRs). [ 118 ]  In addition, there are intra-individual DMRs (Intra-DMRs) with longitudinal changes in global DNA methylation along with the increase of age in a given individual. [ 119 ]  There are also inter-individual DMRs (Inter-DMRs) with different methylation patterns among multiple individuals. [ 120 ]


QDMR (Quantitative Differentially Methylated Regions) is a quantitative approach to quantify methylation difference and identify DMRs from genome-wide methylation profiles by adapting Shannon entropy. [ 121 ]  The platform-free and species-free nature of QDMR makes it potentially applicable to various methylation data. This approach provides an effective tool for the high-throughput identification of the functional regions involved in epigenetic regulation. QDMR can be used as an effective tool for the quantification of methylation difference and identification of DMRs across multiple samples. [ 122 ]


Gene-set analysis (a.k.a. pathway analysis; usually performed tools such as DAVID, GoSeq or GSEA) has been shown to be severely biased when applied to high-throughput methylation data (e.g. MeDIP-seq, MeDIP-ChIP, HELP-seq etc.), and a wide range of studies have thus mistakenly reported hyper-methylation of genes related to development and differentiation; it has been suggested that this can be corrected using sample label permutations or using a statistical model to control for differences in the numbers of CpG probes / CpG sites that target each gene. [ 123 ]


DNA methylation marks  – genomic regions with specific methylation patterns in a specific biological state such as tissue, cell type, individual – are regarded as possible functional regions involved in gene transcriptional regulation. Although various human cell types may have the same genome, these cells have different methylomes. The systematic identification and characterization of methylation marks across cell types are crucial to understanding the complex regulatory network for cell fate determination. Hongbo Liu et al. proposed an entropy-based framework termed SMART to integrate the whole genome bisulfite sequencing methylomes across 42 human tissues/cells and identified 757,887 genome segments. [ 124 ]  Nearly 75% of the segments showed uniform methylation across all cell types. From the remaining 25% of the segments, they identified cell type-specific hypo/hypermethylation marks that were specifically hypo/hypermethylated in a minority of cell types using a statistical approach and presented an atlas of the human methylation marks. Further analysis revealed that the cell type-specific hypomethylation marks were enriched through  H3K27ac  and transcription factor binding sites in a cell type-specific manner. In particular, they observed that the cell type-specific hypomethylation marks are associated with the cell type-specific super-enhancers that drive the expression of cell identity genes. This framework provides a complementary, functional annotation of the human genome and helps to elucidate the critical features and functions of cell type-specific hypomethylation. [ citation needed ]


The entropy-based Specific Methylation Analysis and Report Tool, termed "SMART", which focuses on integrating a large number of DNA methylomes for the de novo identification of cell type-specific methylation marks. The latest version of SMART is focused on three main functions including de novo identification of differentially methylated regions (DMRs) by genome segmentation, identification of DMRs from predefined regions of interest, and identification of differentially methylated CpG sites. [ 125 ]


DNA methylation allows for several tissues to be analyzed in one assay as well as for small amounts of body fluid to be identified with the use of extracted DNA. Usually, the two approaches of DNA methylation are either methylated-sensitive restriction enzymes or treatment with sodium bisulphite. [ 126 ]  Methylated sensitive restriction enzymes work by cleaving specific CpG, cytosine and guanine separated by only one phosphate group, recognition sites when the CpG is methylated. In contrast, unmethylated cytosines are transformed to uracil and in the process, methylated cytosines remain methylated. In particular, methylation profiles can provide insight on when or how body fluids were left at crime scenes, identify the kind of body fluid, and approximate age, gender, and phenotypic characteristics of perpetrators. [ 127 ]  Research indicates various markers that can be used for DNA methylation. Deciding which marker to use for an assay is one of the first steps of the identification of body fluids. In general, markers are selected by examining prior research conducted. Identification markers that are chosen should give a positive result for one type of cell. One portion of the chromosome that is an area of focus when conducting DNA methylation are tissue-specific differentially methylated regions, T-DMRs. The degree of methylation for the T-DMRs ranges depending on the body fluid. [ 127 ]  A research team developed a marker system that is two-fold. The first marker is methylated only in the target fluid while the second is methylated in the rest of the fluids. [ 110 ]  For instance, if venous blood marker A is un-methylated and venous blood marker B is methylated in a fluid, it indicates the presence of only venous blood. In contrast, if venous blood marker A is methylated and venous blood marker B is un-methylated in some fluid, then that indicates venous blood is in a mixture of fluids. Some examples for DNA methylation markers are Mens1(menstrual blood), Spei1(saliva), and Sperm2(seminal fluid).


DNA methylation provides a relatively good means of sensitivity when identifying and detecting body fluids. In one study, only ten nanograms of a sample was necessary to ascertain successful results. [ 128 ]  DNA methylation provides a good discernment of mixed samples since it involves markers that give "on or off" signals. DNA methylation is not impervious to external conditions. Even under degraded conditions using the DNA methylation techniques, the markers are stable enough that there are still noticeable differences between degraded samples and control samples. Specifically, in one study, it was found that there were not any noticeable changes in methylation patterns over an extensive period of time. [ 127 ]


The detection of DNA methylation in cell-free DNA and other body fluids has recently become one of the main approaches to  Liquid biopsy . [ 129 ]  In particular, the identification of tissue-specific and disease-specific patterns allows for non-invasive detection and monitoring of diseases such as cancer. [ 130 ]  If compared to strictly genomic approaches to liquid biopsy, DNA methylation profiling offers a larger number of differentially methylated CpG sites and differentially methylated regions (DMRSs), potentially enhancing its sensitivity. Signal deconvolution algorithms based on DNA methylation have been successfully applied to cell-free DNA and can nominate the tissue of origin of cancers of unknown primary, allograft rejection, and resistance to hormone therapy. [ 131 ]


DNA methylation can also be detected by computational models through sophisticated algorithms and methods. Computational models can facilitate the global profiling of DNA methylation across chromosomes, and often such models are faster and cheaper to perform than biological assays. Such up-to-date computational models include Bhasin,  et al. , [ 132 ]  Bock,  et al ., [ 133 ]  and Zheng,  et al . [ 134 ] [ 135 ]  Together with biological assay, these methods greatly facilitate the DNA methylation analysis.


DNA methylation in cancer  plays a variety of roles, helping to change the healthy cells by  regulation of gene expression  to a cancer cells or a diseased cells  disease pattern . One of the most widely studied DNA methylation dysregulation is the promoter hypermethylation where the CPGs islands in the promoter regions are methylated contributing or causing genes to be silenced. [ 1 ]


All mammalian cells descended from a fertilized egg (a  zygote ) share a common DNA sequence (except for new mutations in some lineages). However, during development and formation of different tissues  epigenetic  factors change.  The changes include  histone  modifications,  CpG island  methylations and chromatin reorganizations which can cause the stable silencing or activation of particular genes. [ 2 ]   Once differentiated tissues are formed, CpG island methylation is generally stably inherited from one cell division to the next through the DNA methylation maintenance machinery. [ 2 ]


In cancer, a number of mutational changes are found in protein coding genes.  Colorectal cancers typically have 3 to 6  driver  mutations and 33 to 66  hitchhiker  or passenger mutations that silence protein expression in the genes affected. [ 3 ]    However, transcriptional silencing may be more important than mutation in causing gene silencing in progression to cancer. [ 4 ]   In colorectal cancers about 600 to 800 genes are transcriptionally silenced, compared to adjacent normal-appearing tissues, by CpG island methylation. Such CpG island methylation has also been described in  glioblastoma [ 5 ]  and  mesothelioma . [ 6 ]  Transcriptional repression in cancer can also occur by other  epigenetic  mechanisms, such as altered expression of  microRNAs . [ 7 ]


CpG islands are commonly 200 to 2000 base pairs long, have a C:G  base pair  content >50%, and have frequent 5' → 3' CpG sequences.  About 70% of human  promoters  located near the  transcription start site  of a gene contain a  CpG island . [ 8 ] [ 9 ]


Promoters located at a distance from the  transcription  start site of a gene  also frequently contain CpG islands.  The promoter of the DNA repair gene  ERCC1 , for instance,  was identified and located about 5,400 nucleotides upstream of its coding region. [ 10 ]   CpG islands also occur frequently in promoters for  functional noncoding RNAs  such as  microRNAs  and  Long non-coding RNAs  (lncRNAs).


Genes can be silenced by multiple methylation of CpG sites in the CpG islands of their promoters.[11] Even if silencing of a gene is initiated by another mechanism, this often is followed by methylation of CpG sites in the promoter CpG island to stabilize the silencing of the gene.[11] On the other hand, hypomethylation of CpG islands in promoters can result in gene over-expression.


Causes of DNA hypermethylation   are:
- Mediation of mutated K-ras induced jun protein (Serra RW. et al. 2014; Leppä S. et al. 1998)
- the inhibitory effect of lnRNA on miRNAs causing demethylation - their "absorption" in the sponge effect or direct repression of demethylation factors TET1 and TGD (Thakur S. Brenner C. 2017; Ratti M. et al. 2020; Morita S. et al. 2013)
- Activation of DNA methylases (Kwon JJ. et al. 2018)
- Changes in isocitrate dehydrogenase (Christensen BC. et al. 2011)
- Effects of viruses (Wang X. et al. )


Causes of DNA hypomethylation:
- The effect of mutated K-ras on long non-coding RNAs, which, when acting, a) directly inhibits the activity or translation of genes encoding DNA methylases (Sarkar D. et al. 2015) b) rather, "sponges" absorb miRNAs (Ratti M. et al. 2020 ), which should ensure the functioning of DNA methylases
- The effect of mutated K-Ras through the activation of the myc-ODC axis, the mTor complex, with the consequence of the synthesis of  polyamines , the activation of which, figuratively speaking, "pumps out" single-carbon fragments from the Methionine cycle and creates a lack of substrate for DNA methylation, leading to a hypomethylated state of DNA (Урба К. 1991 )
- Changes in the activity of methylases DNMT1/3A/3B, their relocalization (Hoffmann MJ, Schulz WA. 2005; Nishiyama A. et al. 2021)
- Changes in TET performance (Nishiyama A. et al. 2021)
- Changes in the synthesis of SAM from methionine due to changes in the enzymes MAT (Frau M. et al. 2013)
- Changes in serine catabolism (Snell K., Weber G. 1986), causing more intensive removal of homocysteine from the methionine cycle, when serine binds to homocysteine (Урба К. 1991)
- Other, unspecified reasons for supplying the Met cycle with single-carbon fragments, causing e.g. "methyl trap" phenomenon (Shane B. Stokstad EL. 1985; Zheng Y, Cantley LC. 2019), sietin and with disorders of vitamin B12 metabolism, disruption of the spare methionine resynthesis pathway (Ouyang Y. et al. 2020; Ozyerli-Goknar E, Bagci-Onder T. 2021; Barekatain, Yasaman et al. 2021) or other monocarbon fragment metabolism disorders (Urba K. 1991).


In cancers, loss of expression of genes occurs about 10 times more frequently by hypermethylation of promoter CpG islands than by mutations.  For instance, in colon tumors compared to adjacent normal-appearing colonic mucosa, about 600 to 800 heavily methylated CpG islands occur in promoters of genes in the tumors while these CpG islands are not methylated in the adjacent mucosa. [ 11 ] [ 12 ] [ 13 ]   In contrast, as Vogelstein et al. [ 3 ]  point out, in a colorectal cancer there are typically only about 3 to 6  driver  mutations and 33 to 66  hitchhiker  or passenger mutations.


In sporadic cancers, a DNA repair deficiency is occasionally found to be due to a mutation in a DNA repair gene.  However, much more frequently, reduced or absent expression of a DNA repair gene in cancer is due to methylation of its promoter.  For example, of 113 colorectal cancers examined, only four had a  missense mutation  in the DNA repair gene  MGMT , while the majority had reduced  MGMT  expression due to methylation of the  MGMT  promoter region. [ 14 ]   Similarly, among 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene  PMS2  expression, 6 had a mutation in the  PMS2  gene, while for 103 PMS2 was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1). [ 15 ]   In the remaining 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1. [ 16 ]


Twenty-two DNA repair genes with hypermethylated promoters, and reduced or absent expression, were found to occur among 17 types of cancer, as listed in two review articles. [ 17 ]   Promoter hypermethylation of  MGMT  occurs frequently in a number of cancers including 93% of bladder cancers, 88% of stomach cancers, 74% of thyroid cancers, 40%-90% of colorectal cancers and 50% of brain cancers. [ citation needed ]   That review also indicated promoter hypermethylation of  LIG4 ,  NEIL1 ,  ATM ,  MLH1  or  FANCB  occurs at frequencies between 33% and 82% in one or more of  head and neck cancers ,  non-small-cell lung cancers  or  non-small-cell lung cancer  squamous cell carcinomas.  The article  Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer  indicates the DNA repair gene  WRN  has a promoter that is frequently hypermethylated in a number of cancers, with hypermethylation occurring in 11% to 38% of  colorectal ,  head and neck ,  stomach ,  prostate ,  breast ,  thyroid ,  non-Hodgkin lymphoma ,  chondrosarcoma  and  osteosarcoma  cancers (see  WRN ).


As discussed by Jin and Roberston in their review, [ 17 ]  silencing of a DNA repair gene by hypermethylation may be a very early step in progression to cancer.  Such silencing is proposed to act similarly to a germ-line mutation in a DNA repair gene, and predisposes the cell and its descendants to progression to cancer.  Another review [ 18 ]  also indicated an early role for hypermethylation of DNA repair genes in cancer.  If a gene necessary for DNA repair is hypermethylated, resulting in deficient DNA repair, DNA damages will accumulate.  Increased DNA damage tends to cause increased errors during DNA synthesis, leading to mutations that can give rise to cancer.


If hypermethylation of a DNA repair gene is an early step in carcinogenesis, then it may also occur in the normal-appearing tissues surrounding the cancer from which the cancer arose (the  field defect ).  See the table below.


While DNA damages may give rise to mutations through error prone  translesion synthesis , DNA damages can also give rise to epigenetic alterations during faulty DNA repair processes. [ 28 ] [ 29 ] [ 30 ] [ 31 ]   The DNA damages that accumulate due to hypermethylation of the promoters of DNA repair genes can be a source of the increased epigenetic alterations found in many genes in cancers.


In an early study, looking at a limited set of transcriptional promoters, Fernandez et al. [ 32 ]  examined the DNA methylation profiles of 855 primary tumors.  Comparing each tumor type with its corresponding normal tissue, 729 CpG island sites (55% of the 1322 CpG island sites evaluated) showed differential DNA methylation.  Of these sites, 496 were hypermethylated (repressed) and 233 were hypomethylated (activated). Thus, there is a  high level of promoter methylation alterations in tumors.  Some of these alterations may contribute to cancer progression.


In mammals,  microRNAs  (miRNAs) regulate the  transcriptional  activity of about 60% of protein-encoding genes. [ 33 ]   Individual miRNAs can each target, and repress transcription of, on average, roughly 200 messenger RNAs of protein coding genes. [ 34 ]   The promoters of about one third of the 167 miRNAs evaluated by Vrba et al. [ 35 ]  in normal breast tissues were differentially hyper/hypo-methylated in breast cancers.  A more recent study pointed out that the 167 miRNAs evaluated by Vrba et al. were only 10% of the miRNAs found expressed in breast tissues. [ 36 ]   This later study found that 58% of the miRNAs in breast tissue had  differentially methylated regions  in their promoters in breast cancers, including 278 hypermethylated miRNAs and 802 hypomethylated miRNAs.


One miRNA that is over-expressed about 100-fold in breast cancers is miR-182. [ 37 ]   MiR-182 targets the BRCA1 messenger RNA and may be a major cause of reduced BRCA1 protein expression in many breast cancers [ 38 ]  (also see  BRCA1 ).


Some miRNAs target the messenger RNAs for  DNA methyltransferase  genes  DNMT1 ,  DNMT3A  and  DNMT3B , whose gene products are needed for initiating and stabilizing promoter methylations.  As summarized in three reviews, [ 39 ] [ 40 ] [ 41 ]  miRNAs miR-29a, miR-29b and miR-29c target DNMT3A and DNMT3B; miR-148a and miR-148b target DNMT3B; and miR-152 and miR-301 target DNMT1.  In addition, miR-34b targets DNMT1 and the promoter of miR-34b itself is hypermethylated and under-expressed in the majority of prostate cancers. [ 42 ]   When expression of these microRNAs is altered, they may also be a source of the hyper/hypo-methylation of the promoters of protein-coding genes in cancers.


Ruben Agrelo,* Wen-Hsing Cheng,† Fernando Setien,* Santiago Ropero,* Jesus Espada,* Mario F. Fraga,* Michel Herranz,* Maria F. Paz,* Montserrat Sanchez-Cespedes,* Maria Jesus Artiga,* David Guerrero,‡ Antoni Castells,§ Cayetano von Kobbe,* Vilhelm A. Bohr,† and Manel Esteller*¶Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer.Proc Natl Acad Sci U S A. 2006; 103(23): 8822–8827.


Environmental epigenetics  is a branch of  epigenetics  that studies the influence of external environmental factors on the  gene expression  of a developing  embryo . [ 1 ]  The way that  genes  are expressed may be passed down from parent to offspring through epigenetic modifications, although environmental influences do not alter the  genome  itself.


During embryonic development, epigenetic modifications determine which genes are expressed, which in turn determines the embryo's  phenotype . When the offspring is still developing, genes can be turned on and off depending on exposure to certain environmental factors. While certain  genes  being turned on or off can increase the risk of developmental diseases or abnormal phenotypes, there is also the possibility that the phenotype will be non-functional. [ 2 ]  Environmental influence on epigenetics is highly variable, but certain environmental factors can greatly increase the risk of detrimental diseases being expressed at both early and adult life stages. [ 3 ]


The way that genes are expressed is influenced by the environment that the genome is in. These environmental influences are referred to as triggers and can involve anything that influences normal gene expression. How the genome is expressed depends on the environmental factors present during  gestation . It is possible for the environmental effects of epigenetics to be deleterious or to be a natural part of the development pathway. [ 4 ]  When these environmental factors are detrimental it causes the deactivation of some DNA sequences, which can lead to atypical phenotypes. Some of the most common triggers include diet, temperature, exposure to harmful substances, and lifestyle. These triggers can cause low birth weight, neurological disorders, cancers, autoimmune diseases, and many other malformations.


These epigenetic triggers can change the way that an  organism  develops and have lifelong effects. Epigenetic changes can be passed down through  offspring , present in multiple generations, and continue to change throughout a lifetime. Each sequence of affected DNA is not expressed at the same time. There are specific stages in which these expressions happen during development. [ 5 ]  The combined epigenetic mechanisms of DNA methylation and histone modification are responsible for how the genome is altered. For example, the suppression of  oncogenes  is regulated by DNA methylation and whether or not methylation is activated. This activation depends on the environment. [ 4 ]  When these activations happen, they can be passed down to the next generation through germ-cell differentiation.


Offspring can experience phenotypic changes depending on their access to nutrients. When nutrients are limited during pregnancy, the offspring's phenotypic expression can be disrupted. Nutrient intake is also important during  lactation  for the purpose of transferring nutrients to the offspring. [ 6 ]


When the maternal figure is exposed to stressors it can lead to a greater likelihood of expressing or stunting DNA expression. If the maternal figure experiences high levels of  depression  or stress it can lead to small litter sizes with lower birth rates. Decreased  hormone  production is the suspected cause of this. [ 7 ]


Changes in temperature can have varied effects on an organism. DNA methylation can be impacted by temperature when the temperature deviates from its normal value, preventing regular processes from taking place. Temperature can be considered a stressor in environmental epigenetics since it has the potential to change how offspring respond and react to their environment. [ 2 ]   Monarch butterflies  are an example of how temperature can impact the survival and  fitness  of an organism. [ 8 ]  If exposed to stressors such as varying temperatures, these butterflies may express coloring that deviates from their normal color.


Epigenetic marks  can result from a number of exposures and choices made by an individual in their lifetime. Exposure to environmental pollutants, psychological stress, dietary choices or restrictions, working habits, and consumption of drugs or alcohol all influence the epigenetics of an individual and what may be passed down to future offspring.  [ 9 ]  Such exposures can affect important processes of epigenetics such as DNA methylation and histone acetylation, influencing the risk for noncommunicable diseases such as  obesity .


Exposure to certain materials or chemicals can cause an epigenetic reaction. The epigenetic causing substances cause issues like altered DNA methylation, CpG islands, chromatin, along with other transcription factors. [ 10 ]  Environmental epigenetics aims to relate such environmental triggers or substances to phenotypic variation. [ 11 ]  Numerrous studies have demonstrated how exposure to environmental pollutants, such as heavy metals, pesticides, and air pollutants, can induce epigenetic changes in various organisms. [ 12 ]  For example, research has shown that exposure to pollutants like biphenol A (BPA) and polycyclic acromatic hydrocarbons (PAHs) can lead to DNA methylation changes and histone modifications in plants, animals, and humans. [ 13 ]


Epigentic mechanisms play a role in the adaptation of species of changing environmental conditions, including climate change. [ 14 ]  Studies have shown that organisms can exhibit phenotypic plasticity through epigenetic modifications in response to environmental stressors such as temperature fluctuation, drought, and habitat loss. [ 15 ]


Environmental epigenetics has revealed the potential for transgenerational effects, where environmental exposures experienced by one generation can influence the phenotypes and health outcomes of subsequent generations through epigenetic inheritance mechanisms. [ 16 ]  Studies in various organisms, including plants, insects, and mammals, have shown transgenerational epigenetic effects resulting from parental exposure to stressors such as toxins, dietary changes, and environmental contaminants. [ 17 ]  Epigenetic modifications can influence gene expression and phenotypic traits in oragamisms across different trophic levels, with implications for ecosystem stability. [ 18 ]


An example of exposure causing environmental epigenetic can be seen in  lemon sharks ,  Negaprion brevirostris . Due to a dredging event, lemon sharks in the  Bahamas  experienced an epigenetic change. Dredging is done with a machine that clears out all mud and debris found at the bottom of a body of water. Dredging is extremely harmful to the physical environment and the organisms living there. This dredging caused exposure to different toxic metals like Manganese along with other trace amount of heavy metals, which then affected  DNA methylation  in juvenile lemon sharks. This exposure caused the lemon sharks’ DNA to methylate at abnormal rates which caused gene expression to be altered. Scientists hypothesized that this aberrant DNA methylation could be caused by the stress that the dredging caused. Exposure to a stressful event is also an example of an environmental epigenetic factor. [ 19 ]


Plants need some types of metals to help with their development, but when exposed to high amounts, the metals can become toxic to plants. Since plants can process metals that are important for their growth, they also process metals that are not needed to help them grow and develop. Once the levels of the metals get too high, they start to affect plants directly and indirectly because metals cannot be broken down. Direct toxic effects that occur due to high levels of metal are inhibition of cytoplasmic and damage to the structure of the cell because of oxidative stress. Oxidative stress is like a bad storm that messes up the plant by damaging it so it cannot get the nutrients it needs to be healthy because things that are not supposed to be there are taking up space where the essential nutrients should go. Indirect toxic effects are the proxy nutrients at the plant's cation exchange. The cation exchange site is where the plant picks up the nutrients it needs, though if something comes in and starts taking all the valuable nutrients, such as heavy metal, there is nothing there to help the plant grow. An example of a heavy metal that is not required for plant growth is mercury. The relationship between Hg in the soil and how much plants take in is complex. It relies on many other factors, such as the pH of the soil, the type of plant species, etc.


Many types of heavy metals are toxic to plants, such as  lead . Typically, land plants absorb lead(Pb) from the soil, most retaining it in their roots with some evidence of foliage uptakes and potential distribution to other plant parts. Calcium and phosphorus can reduce the uptake of lead, a common and toxic soil element that impacts the plant, growth structure, and photosynthesis of the plant. Lead, in particular, inhibits the process by which a plant grows from a seed into a seedling, known as seed germination in various species, by interfering with crucial enzymes. Studies have shown that lead acetate reduces protease and amylase activity in rice endosperm considerably. This interferes with early seeding growth across plant species such as soybean, rice, tomato, barley, maize, and some legumes.


Furthermore, lead delays root and steam elongation and leaf expansion, with the extent of root elongation inhibition varying based on the lead concentration, the medium's ionic composition, and pH. [ 20 ]  Soil levels that have high levels of lead can also cause irregular root thickening, cell wall modifications in peas, growth reduction in sugar beets, oxidative stress due to increased reactive oxygen species (ROS) production, biomass, and protein content in maize, along with diminished lead count and area, plant height in Portia trees, and enzyme activity affecting CO2 fixation in oats.


Manganese  (Mn), is crucial for plants and involves in photosynthesis and other physiological processes. Deficiency commonly affects sandy, organic, or tropical soils with a high pH above six and heavily weathered tropical soils. Mn can move easily from roots to shoots, though it is not efficiently redistributed from leaves to other parts of the plant. The signs of Mn toxicity are necrotic brown spots on leaves, petioles, and steams that start on the lower leaves and move upward, leading to death. [ 21 ] [ 22 ]  When damage to young leaves and stems, coupled with chlorosis and browning, called a "crinkle leaf." In some species, toxicity can begin with chlorosis in older leaves, advancing to younger ones, and can inhibit chlorophyll synthesis by interfering with iron-related processes. [ 23 ]  Mn toxicity is more present in soils with a pH level lower than six. In the broad bean plant, Mn affects shoot and root length [ 24 ]  The spearmint plant, lowers chlorophyll and carotenoid levels and increases root Mn accumulation. [ 25 ]  Pea plant, lowers chlorophyll a and b, growth rate, and photosynthesis. [ 26 ]  In the tomato plant, it slows growth and decreases chlorophyll concentration. [ 27 ] [ 28 ]


Humans have displayed evidence of epigenetic changes such as DNA methylation, differentiation in expression, and histone modification due to environmental exposures. Carcinogen development in humans has been studied in correlation to environmental inducements such as chemical and physical exposures and their transformative abilities on epigenetics. Chemical and physical environmental factors are contributors to epigenetic statuses amongst humans. [ 11 ]


Firstly, a study was performed on drinking water populations in China involving three generations: the F1 generation consisting of grandparents exposed to arsenic in adulthood, the F2 generation including the parents exposed to arsenic in utero and early childhood, and the F3 generation which were the grandchildren exposed to arsenic from germ cells. [ 29 ]  This area in China was historically known for its dangerously high levels of arsenic, therefore, there was opportunity to examine the timeline As exposures across the three generations. The study was conducted to discover the linkage between the timeline effects of As exposure and DNA methylations. The population and environment for which the study was conducted were reportedly not exposed to other environmental exposures besides arsenic. [ 29 ]


The results concluded from this experiment were that 744 CpG sites [ 30 ]  had been differentially methylated. The 744 sites were found across all three generations in the group exposed to arsenic. The concluding argument based on the results of this study is that the DNA methylation changes were more prevalent in those that developed arsenic-induced diseases. [ 29 ]


Exposures to environmental factors during human lifetimes and their potential effect on phenotypes is a highly question topic involving epigenetics and disease development. [ 11 ]  In the case of humans, "unhealthy" phenotypes have been identified to carry such evidence that environmental epigenetics could be a leading cause in gene regulation, disease development, cell development and differentiation, aging, and carcinogenic effect. [ 11 ]  Although the way that environmental factors and the human genome work together is not completely understood, their influence has been identified and is continuing to drive explanations for human genome modifications and their outcomes. Driving evidence for adverse effects implemented by extrinsic factors from the environment, comes from studies done on nutrition and exposures to toxins. [ 11 ]


Besides arsenic exposures, other metals have been identified to cause such hypermethylations. Concentrations of Cd, Cr, Cu, Pb and Zn metals were identified in fishermen's blood and resulted in an increase in the expression of the IGF2 gene. [ 31 ]  The IGF2 gene is responsible for making the insulin-like growth factor 2. [ 32 ]  The insulin-like growth factor is involved in growth and can result in disorders where cell growth and overgrowth are abnormal. [ 33 ]  Such disorders include breast and lung cancers and Silver–Russell and Beckwith–Wiedemann syndromes  [ 33 ] [ 34 ]  The significance of IGF2 gene expression is found in its relationships to human health. There is remaining uncertainty between the long-term environmental exposures and epigenetic changes, but conducted research has provided that heavy metal exposures cause DNA methylation changes. [ 31 ]


Exposures to certain triggers such as alcohol or drugs can disrupt the normal expression of the offspring's phenotype. Antipsychotic drugs can lead to abnormal or stunted development during the  fetal  or embryonic stages. [ 35 ]


Organisms respond to the habitat around them in many different ways, one way is by changing its gene expression to one that is most suitable for their surroundings. More often than not, this has a direct correlation to  phenotypic plasticity . Phenotypic plasticity is when a species develops new physical features in response to the environment they’re in. Passing down epigenetics that are in relation to mitotic cell divisions allows for the belief of a possibility that this is also passed down from parents to offspring. Parents could be responsible for the development of new phenotypes in these cases. [ 36 ]


Epigenetic inheritance refers to passing down or transferring epigenetic information between the parent and offspring. Some believe that these occurrences can be passed down for many generations. For example, the language a given species utilizes develops a specific phenotype that will be passed down from generation to generation. [ 36 ]


Cultural inheritance is a behavioral factor that is passed down from generation to generation, similar to inheritance. For example, in rats, mothers that lick and groom their pups pass down a specific behavior to their offspring causing them to do the same to the subsequent generation. Epigenetic inheritance is involved in this, but they are separate things that work together. [ 36 ]


DNA replication  is a highly conserved process involving the copying of genetic information from parent one generation to the next. Within this complex process,  chromatin  disassembles and reassembles in a precise and regulated manner in order to compact large amounts of genetic material into the nucleus, while also maintaining the integrity of epigenetic information carried by  histone proteins  bound to  DNA  in the process of  cell division . Half of the histones present during replication are from chromatin found in the parent DNA and thus carry the parent's epigenetic information. [ 37 ]  These epigenetic marks play a critical role in determining chromatin structure and thus gene expression in the newly synthesized DNA. The other half of the histones present in replication are newly synthesized.


A major formative mechanism of epigenetic modification is  DNA methylation . DNA methylation is the process of adding a  methyl group  to a  cytosine  base in the  DNA  strand, via  covalent bond . This process is carried out by specific  enzymes . [ 38 ]  These methyl additions can be reversed in a process known as  demethylation . The presence or absence of methyl groups can attract proteins involved in gene repression, or inhibit the binding of certain transcription factors, thus preventing methylated genes from being transcribed, ultimately affecting phenotypic expression. [ 39 ]


Acetylation  is a reaction that introduces an  acetyl group  into an organic chemical compound, typically by substituting an acetyl group for a hydrogen atom. Deacetylation is the removal of an acetyl group from an organic chemical compound.  Histone acetylation and deacetylation  affect the three-dimensional structure of  chromatin . A more relaxed chromatin structure leads to greater rates of genetic transcription, whereas a tighter structure inhibits transcription.


Transcriptional regulation  is a complex process involving the binding of  transcriptional machinery  to  regulatory proteins —specifically chromatin remodeling or modifying proteins-directly onto a specific target. This may sometimes be facilitated by the contribution of  accessory complexes  that function primarily to repress and activate transcription in a cell. Transcriptional regulation additionally focuses on the epigenetic regulation of a target locus, as the epigenetic status of the locus determines either the facilitation of or prohibition of transcription. Epigenetic regulation is necessary for the precise deployment of transcriptional programs. [ 40 ]


An  epigenetic clock  is a  biochemical test  that can be used to measure age. The test is based on modifications that change over time and regulate how genes are expressed. Typically they can use  DNA methylation  levels, measuring the accumulation of methyl groups to one's DNA molecules, or more recently, based on the  histone code .


The strong effects of age on  DNA methylation  levels have been known since the late 1960s. [ 1 ]  A vast literature describes sets of  CpGs  whose DNA methylation levels correlate with age. [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ]  The first robust demonstration that DNA methylation levels in saliva could generate age predictors with an average accuracy of 5.2 years was published by a  UCLA  team including Sven Bocklandt,  Steve Horvath , and  Eric Vilain  in 2011 (Bocklandt et al. 2011). [ 7 ] [ 8 ]  The laboratories of  Trey Ideker  and  Kang Zhang  at the  University of California, San Diego  published the Hannum epigenetic clock (Hannum 2013), [ 9 ]  which consisted of 71 markers that accurately estimate age based on blood methylation levels. The first multi-tissue epigenetic clock, Horvath's epigenetic clock, was developed by Steve Horvath, a professor of  human genetics   and  biostatistics  at  UCLA  (Horvath 2013). [ 10 ] [ 11 ]  Horvath spent over 4 years collecting publicly available  Illumina  DNA methylation data and identifying suitable statistical methods. [ 12 ]


The personal story behind the discovery was featured in  Nature . [ 13 ]  The age estimator was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. The major innovation of Horvath's epigenetic clock lies in its wide applicability: the same set of 353 CpGs and the same prediction algorithm is used irrespective of the DNA source within the organism, i.e. it does not require any adjustments or offsets. [ 10 ]  This property allows one to compare the ages of different areas of the human body using the same aging clock. Shortly afterwards, a derivation of Horvath's clock, the IEAA (Intrinsic Epigenetic Age Acceleration), an estimator based on the cellular composition of the blood, was developed.


A second generation of epigenetic clocks emerged a few years later and improved on the first in age estimation. This was thanks to the incorporation not only of epigenetic variants such as  DNA methylation  but also environmental variants such as smoking or chronological age. Among these clocks, the PhenoAge and GrimAge clocks stand out. PhenoAge is an  epigenetic  clock that takes chronological age into account, and GrimAge uses the mortality risks of age together with the smoking variant among others as a risk factor. Taking into account environmental variants allows GrimAge to outperform any other epigenetic clock in "predicting death".


Third-generation epigenetic clocks are designed to be applicable across multiple species simultaneously. Specifically, pan-mammalian epigenetic clocks determine the age of tissues from all mammalian species by analyzing cytosine methylation in DNA regions that are highly conserved. [ 14 ]


More recently in 2025, age-related changes in histone marks have been leveraged to build a new class of epigenetic clocks that do not rely on DNA methylation. [ 15 ]  These new predictors show promise as an alternative to clocks that use cytosine methylation.


New age estimation tools have been developed continuously, which also facilitate the prognosis of certain diseases.


E longation  O f  V ery  L ong  C hain  F atty  A cids- L ike  2  is a gene that codes for a transmembrane protein that plays a role in the synthesis of  VLCFAs . [ 16 ]  The inhibition of its expression has been associated with increased aging of the retina in mice while its upregulation resulted in a slower aging of the retina. [ 17 ]  Methylation sites in the promoter region of this gene have consistently been part of the top most age correlated in different studies. [ 18 ] [ 19 ] [ 20 ]  The methylation in those sites increases with age which reduce its expression. [ 21 ]


F our-and-a- H alf  L IM domain protein  2  is a gene implicated in  signal transduction . Increase in its expression has been associated with obesity. [ 22 ]  The methylation in its promoter is also strongly correlated with age in numerous studies. [ 23 ] [ 18 ] [ 24 ]  In this case the methylation, which increases with age, [ 25 ]  is associated with an increase in  FHL2  expression [ 26 ]  but surprisingly also with a decreased expression in some tissues. [ 23 ]


It is not yet known what exactly is measured by DNA methylation age. Horvath hypothesized that DNA methylation age measures the cumulative effect of an epigenetic maintenance system but details are unknown. The fact that DNA methylation age of blood predicts all-cause mortality in later life [ 27 ] [ 28 ] [ 29 ] [ 30 ]  has been used to argue that it relates to a process that causes aging. [ 27 ]  However, if a particular CpG played a direct causal role in the aging process, the mortality it created would make it less likely to be observed in older individuals, making the site less likely to have been chosen as a predictor; the 353 clock CpGs therefore likely have no causal effect whatsoever. [ 31 ]  Rather, the epigenetic clock captures an  emergent property  of the epigenome.


In 2010, a new unifying model of aging and the development of complex diseases was proposed, incorporating classical aging theories and epigenetics. [ 32 ] [ 33 ]  Horvath and Raj [ 34 ]  extended this theory, proposing an epigenetic clock theory of aging with the following tenets:


In general, biological aging clocks and  biomarkers of aging   are expected to find many uses in biological research since age is a fundamental characteristic of most  organisms . Accurate measures of biological age (biological aging clocks) could be useful for


Overall, biological clocks are expected to be useful for studying what causes aging and what can be done against it. However, they can only capture the effects of interventions that affect the rate of future aging, i.e. the slope of the  Gompertz curve  by which mortality increases with age, and not that of interventions that act at one moment in time, e.g. to lower mortality across all ages, i.e. the intercept of the Gompertz curve. [ 31 ]


The clock is defined as an age estimation method based on 353  epigenetic  markers on the DNA. The 353 markers measure  DNA methylation  of  CpG dinucleotides . Estimated age ("predicted age" in mathematical usage), also referred to as DNA methylation age, has the following properties: first, it is close to zero for embryonic and  induced pluripotent stem cells ; second, it correlates with cell  passage number ; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues (which are used as human analogs for biological testing purposes). Organismal growth (and concomitant cell division) leads to a high ticking rate of the epigenetic clock that slows down to a constant ticking rate (linear dependence) after adulthood (age 20). [ 10 ]  The fact that DNA methylation age of blood predicts all-cause mortality in later life even after adjusting for known risk factors [ 27 ] [ 28 ]  is compatible with a variety of causal relationships, e.g. a common cause for both. Similarly, markers of physical and mental fitness are associated with the epigenetic clock (lower abilities associated with age acceleration). [ 35 ]  It systematically underestimates age from older individuals. [ 36 ]


Salient features of Horvath's epigenetic clock include its applicability to a broad spectrum of tissues and cell types. Since it allows one to contrast the ages of different tissues from the same subject, it can be used to identify tissues that show evidence of accelerated age due to disease.


The Horvath clock, specifically the IEAA variant, is associated with several ageing-related genes:14


The basic approach is to form a weighted average of the 353 clock CpGs, which is then transformed to DNAm age using a calibration function. The calibration function reveals that the epigenetic clock has a high ticking rate until adulthood, after which it slows to a constant ticking rate. Using the training data sets, Horvath used a penalized regression model ( Elastic net regularization ) to regress a calibrated version of chronological age on 21,369 CpG probes that were present both on the Illumina 450K and 27K platform and had fewer than 10 missing values. DNAm age is defined as estimated ("predicted") age. The elastic net predictor automatically selected 353 CpGs. 193 of the 353 CpGs correlate positively with age while the remaining 160 CpGs correlate negatively with age. R software and a freely available web-based tool can be found  at the following webpage. [ 38 ]


The median error of estimated age is 3.6 years across a wide spectrum of tissues and cell types, [ 10 ]  although this increases for older individuals [ 36 ]  The epigenetic clock performs well in heterogeneous tissues (for example, whole blood, peripheral blood mononuclear cells, cerebellar samples, occipital cortex, buccal epithelium, colon, adipose, kidney, liver, lung, saliva, uterine cervix, epidermis, muscle) as well as in individual cell types such as CD4 T cells, CD14 monocytes, glial cells, neurons, immortalized B cells, mesenchymal stromal cells. [ 10 ]  However, accuracy depends to some extent on the source of the DNA.


The epigenetic clock leads to a chronological age prediction that has a  Pearson correlation coefficient  of r = 0.96 with chronological age (Figure 2 in [ 10 ] ). Thus the age correlation is close to its maximum possible correlation value of 1.  Other biological clocks are based on a)  telomere  length, b)  p16INK4a  expression levels (also known as INK4a/ARF locus), [ 39 ]  and c)  microsatellite  mutations. [ 40 ]  The correlation between chronological age and  telomere  length is r = −0.51 in women and r = −0.55 in men. [ 41 ]  The correlation between chronological age and expression levels of  p16INK4a  in T cells is r = 0.56. [ 42 ]


By contrasting DNA methylation age (estimated age) with chronological age, one can define measures of age acceleration. Age acceleration can be defined as the difference between DNA methylation age and chronological age. Alternatively, it can be defined as the residual that results from regressing DNAm age on chronological age. The latter measure is attractive because it does not correlate with chronological age.
A positive/negative value of epigenetic age acceleration suggests that the underlying tissue ages faster/slower than expected.


The broad sense heritability (defined via  Falconer's formula ) of age acceleration of blood from older subjects is around 40% but it appears to be much higher in newborns. [ 10 ]  Similarly, the age acceleration of brain tissue (prefrontal cortex) was found to be 41% in older subjects. [ 43 ]  Genome-wide association studies (GWAS) of epigenetic age acceleration in postmortem brain samples have identified several  SNPs  at a genomewide significance level. [ 44 ] [ 45 ]  GWAS of age acceleration in blood have identified several genome-wide significant genetic loci including the telomerase reverse transcriptase gene ( TERT ) locus. [ 46 ]  Genetic variants associated with longer leukocyte telomere length in TERT gene paradoxically confer higher epigenetic age acceleration in blood. [ 46 ]


In general, lifestyle factors have only weak associations with epigenetic age acceleration in blood. [ 47 ] [ 48 ] [ 49 ]  Cross sectional studies of extrinsic epigenetic aging rates in blood show reduced epigenetic aging correlates with higher education, eating a high plant diet with lean meats, moderate alcohol consumption, and physical activity [ 48 ]  and the risks associated with  metabolic syndrome . However, studies suggest that high levels of alcohol consumption are associated with accelerated aging of certain epigenetic clocks. [ 49 ]


The epigenetic clock was used to study the relationship between high  body mass index  (BMI) and the DNA methylation ages of human blood, liver, muscle and adipose tissue. [ 50 ]  A significant correlation (r = 0.42) between BMI and epigenetic age acceleration could be observed for the liver. A much larger sample size (n = 4200 blood samples) revealed a weak but statistically significant correlation (r = 0.09) between BMI and intrinsic age acceleration of blood. [ 47 ]  The same large study found that various biomarkers of  metabolic syndrome  (glucose-, insulin-, triglyceride levels, C-reactive protein,  waist-to-hip ratio ) were associated with epigenetic age acceleration in blood. [ 47 ]  Conversely, high levels of  HDL cholesterol  were associated with a lower epigenetic aging rate of blood. [ 47 ]  Other research suggests very strong associations between higher  body mass index ,  waist-to-hip ratio , and  waist circumference  and accelerated epigenetic clocks, with evidence that  physical activity  may lessen these effects. [ 48 ]


DNAm age is higher than chronological age in female breast tissue that is adjacent to breast cancer tissue. [ 10 ]  Since normal tissue which is adjacent to other cancer types does not exhibit a similar age acceleration effect, this finding suggests that normal female breast tissue ages faster than other parts of the body. [ 10 ]  Similarly, normal breast tissue samples from women without cancer have been found to be substantially older than blood samples collected from the same women at the same time. [ 51 ]


In a study of three epigenetic clocks and breast cancer risk, DNAm age was found to be accelerated in blood samples of cancer-free women, years before diagnosis. [ 52 ]


Cancer tissues show both positive and negative age acceleration effects. For most tumor types, no significant relationship can be observed between age acceleration and tumor morphology (grade/stage). [ 10 ] [ 53 ]  On average, cancer tissues with mutated  TP53  have a lower age acceleration than those without it. [ 10 ]  Further, cancer tissues  with high age acceleration tend to have fewer somatic mutations than those with low age acceleration. [ 10 ] [ 53 ]  Age acceleration is highly related to various genomic aberrations in cancer tissues. Somatic mutations in  estrogen receptors  or  progesterone receptors  are associated with accelerated DNAm age in breast cancer. [ 10 ]  Colorectal cancer samples with a  BRAF  (V600E) mutation or promoter hypermethylation of the mismatch repair gene  MLH1  are associated with an increased age acceleration. [ 10 ]  Age acceleration in  glioblastoma multiforme  samples is highly significantly associated with certain mutations in  H3F3A . [ 10 ]  One study suggests that the epigenetic age of blood tissue may be prognostic of lung cancer incidence. [ 54 ]


Down syndrome  entails an increased risk of many chronic diseases that are typically associated with older age. The clinical manifestations of accelerated aging suggest that trisomy 21 increases the biological age of tissues, but molecular evidence for this hypothesis has been sparse. According to the epigenetic clock, trisomy 21 significantly increases the age of blood and brain tissue (on average by 6.6 years). [ 55 ]


Epigenetic age acceleration of the human prefrontal cortex was found to be correlated with several neuropathological measurements that play a role in Alzheimer's disease [ 43 ]   Further, it was found to be associated with a decline in global cognitive functioning, and memory functioning among individuals with Alzheimer's disease. [ 43 ]  The epigenetic age of blood relates to cognitive functioning in the elderly. [ 35 ]  Overall, these results strongly suggest that the epigenetic clock lends itself for measuring the biological age of the brain.


It has been difficult to identify tissues that seem to evade aging due to the lack of biomarkers of tissue age that allow one to contrast compare the ages of different tissues. An application of epigenetic clock to 30 anatomic sites from six centenarians and younger subjects revealed that the  cerebellum  ages slowly: it is about 15 years younger than expected in a centenarian. [ 56 ]  This finding might explain why the cerebellum exhibits fewer neuropathological hallmarks of age related dementias compared to other brain regions. In younger subjects (e.g. younger than 70), brain regions and brain cells appear to have roughly the same age. [ 10 ] [ 56 ]  Several SNPs and genes have been identified that relate to the epigenetic age of the cerebellum. [ 44 ]


Huntington's disease  has been found to increase the epigenetic aging rates of several human brain regions. [ 57 ]


The offspring of semi-supercentenarians (subjects who reached an age of 105–109 years) have a lower epigenetic age than age-matched controls (age difference = 5.1 years in blood) and centenarians are younger (8.6 years) than expected based on their chronological age. [ 30 ]


Infection with the Human Immunodeficiency Virus-1 ( HIV ) is associated with clinical symptoms of accelerated aging, as evidenced by increased incidence and diversity of age-related illnesses at relatively young ages. But it has been difficult to detect an accelerated aging effect on a molecular level. An epigenetic clock analysis of human DNA from HIV+ subjects and controls detected a significant age acceleration effect in brain (7.4 years) and blood (5.2 years) tissue due to HIV-1 infection. [ 58 ]  These results are consistent with an independent study that also found an age advancement of 5 years in blood of HIV patients and a strong effect of the HLA locus. [ 59 ]


A large-scale study suggests that the blood of Parkinson's disease subjects, in particular, their granulocyte ratio, exhibits (relatively weak) accelerated aging effects. [ 60 ]


Children with a very rare disorder known as  syndrome X  maintain the façade of persistent toddler-like features while aging from birth to adulthood. Since the physical development of these children is dramatically delayed, these children appear to be a toddler or at best a preschooler. According to an epigenetic clock analysis, blood tissue from syndrome X cases is not younger than expected. [ 61 ]


The following results strongly suggest that the loss of female hormones resulting from menopause accelerates the epigenetic aging rate of blood and possibly that of other tissues. [ 62 ]  First, early  menopause  has been found to be associated with an increased epigenetic age acceleration of blood. [ 62 ]  Second, surgical menopause (due to bilateral  oophorectomy ) is associated with epigenetic age acceleration in blood and saliva. Third,  menopausal hormone therapy , which mitigates hormonal loss, is associated with a negative age acceleration of buccal cells (but not of blood cells). [ 62 ]  Fourth, genetic markers that are associated with early menopause are also associated with increased epigenetic age acceleration in blood. [ 62 ]


A confounding aspect of biological aging is the nature and role of senescent cells. It is unclear whether the three major types of cellular senescence, namely replicative senescence, oncogene-induced senescence and DNA damage-induced senescence are descriptions of the same phenomenon instigated by different sources, or if each of these is distinct, and how they are associated with epigenetic aging.
Induction of replicative senescence (RS) and oncogene-induced senescence (OIS) were found to be accompanied by epigenetic aging of primary cells but senescence induced by DNA damage was not, even though RS and OIS activate the cellular DNA damage response pathway. [ 63 ]  These results highlight the independence of cellular senescence from epigenetic aging. Consistent with this, telomerase-immortalised cells continued to age (according to the epigenetic clock) without having been treated with any senescence inducers or DNA-damaging agents, re-affirming the independence of the process of epigenetic ageing from telomeres, cellular senescence, and the DNA damage response pathway.
Although the uncoupling of senescence from cellular aging appears at first sight to be inconsistent with the fact that senescent cells contribute to the physical manifestation of organism ageing, as demonstrated by Baker et al., where removal of senescent cells slowed down aging. [ 64 ]


The epigenetic clock analysis of senescence, however, suggests that cellular senescence is a state that cells are forced into as a result of external pressures such as DNA damage, ectopic oncogene expression and exhaustive proliferation of cells to replenish those eliminated by external/environmental factors. [ 63 ]  These senescent cells, in sufficient numbers, will probably cause the deterioration of tissues, which is interpreted as organism ageing. However, at the cellular level, aging, as measured by the epigenetic clock, is distinct from senescence. It is an intrinsic mechanism that exists from the birth of the cell and continues. This implies that if cells are not shunted into senescence by the external pressures described above, they would still continue to age. This is consistent with the fact that mice with naturally long telomeres still age and eventually die even though their telomere lengths are far longer than the critical limit, and they age prematurely when their telomeres are forcibly shortened, due to replicative senescence. Therefore, cellular senescence is a route by which cells exit prematurely from the natural course of cellular aging. [ 63 ]


Men age faster than women according to epigenetic age acceleration in blood, brain, saliva, but it depends on the structure being researched and the lifestyle. [ 65 ]
The epigenetic clock method applies to all examined racial/ethnic groups in the sense that DNAm age is highly correlated with chronological age. But ethnicity can be associated with epigenetic age acceleration. [ 65 ]  For example, the blood of Hispanics and the  Tsimané  ages more slowly than that of other populations which might explain the  Hispanic mortality paradox . [ 65 ]


Hematopoietic stem cell transplantation , which transplants these cells from a young donor to an older recipient, rejuvenates the epigenetic age of blood to that of the donor. However,  graft-versus-host disease  is associated with increased DNA methylation age. [ 66 ]


Adult progeria also known as  Werner syndrome  is associated with epigenetic age acceleration in blood. [ 67 ]
Fibroblast samples from children with Hutchinson-Gilford  Progeria  exhibit accelerated epigenetic aging effects according to the "skin & blood" epigenetic clock but not according to the original pan tissue clock from Horvath. [ 68 ]


Horvath hypothesized that his clock arises from a methylation footprint left by an epigenomic maintenance system. [ 10 ]


Endogenous DNA damages occur frequently including about 50 double-strand DNA breaks per cell cycle [ 69 ]  and about 10,000 oxidative damages per day (see  DNA damage (naturally occurring) ).  During repair of double-strand breaks many epigenetic alterations are introduced, and in a percentage of cases epigenetic alterations remain after repair is completed, including increased methylation of CpG island promoters. [ 70 ] [ 71 ] [ 72 ]   Similar, but usually transient epigenetic alterations were recently found during repair of oxidative damages caused by H 2 O 2 , and it was suggested that occasionally these epigenetic alterations may also remain after repair. [ 73 ]   These accumulated epigenetic alterations may contribute to the epigenetic clock.  Accumulation of epigenetic alterations may parallel the accumulation of un-repaired DNA damages that are proposed to cause aging (see  DNA damage theory of aging ). In line with stochastic DNA damage accumulation, age-related alterations in DNA methylation have been observed to predominantly undergo stochastic changes as individuals age. [ 74 ]  This accumulation of stochastic variation has demonstrated sufficient capacity to build aging clocks, further supporting the notion that epigenetic changes may be driven by the gradual accrual of unprogrammed stochastic damage. [ 75 ]


The  epigenetic code  is hypothesised to be a defining code in every  eukaryotic   cell  consisting of the specific  epigenetic  modification in each cell. It consists of histone modifications defined by the  histone code  and additional epigenetic modifications such as  DNA methylation . The base for the epigenetic code is a system above the  genetic code  of a single cell. While in one individual the genetic code in each cell is the same, the epigenetic code is tissue and cell specific. [ 1 ]  The epigenetic code can be multidimensional in nature. It could include any of the three major cellular macromolecucles; namely, DNA (code independent), RNA, and/or protein. In some ciliates potential structural codes have also been suggested. [ 2 ]


Epigenetic controls in ciliates  is about the unique characteristic of  Ciliates , which is that they possess two kinds of nuclei (this phenomenon is called  nuclear dimorphism ): a  micronucleus  used for inheritance, and a  macronucleus , which controls the  metabolism . The micronucleus contains the entirety of the genome whereas the macronucleus only contains the DNA necessary for vegetative growth. The macronucleus divides via  amitosis , whereas the micronucleus undergoes typical mitosis. During sexual development a new macronucleus is formed from the meiosis of the micronucleus, where the removal of  transposons  occurs. [ 1 ]  On the division or reproduction of ciliates, the two nuclei are under several epigenetic controls.


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Epigenetic effects of smoking  concerns how  epigenetics  (hertiable characteristics that do not involve changes in DNA sequence) contributes to the deleterious effects of  smoking . Cigarette smoking has been found to affect global epigenetic regulation of transcription across tissue types. Studies have shown differences in epigenetic markers like  DNA methylation ,  histone modifications  and  miRNA  expression between smokers and non-smokers. Similar differences exist in children whose mothers smoked during pregnancy. These epigenetic effects are thought to be linked to many of negative health effects associated with smoking.


It has been well established that smoking cigarettes has a number of negative health effects, including increased risk for  cancer ,  cardiovascular disease , and  chronic obstructive pulmonary disease  (COPD). [ 1 ]  Children exposed prenatally to cigarette smoke demonstrate increased risk for  fetal growth restriction ,  sudden infant death syndrome , and  addictive behaviors  later in life, as well as a host of other secondary health effects. It is thought that epigenetic changes that arise from smoking cigarettes or exposure to cigarette smoke play a role in the development of these conditions.


Epigenetic modifications to the genome, including  histone modifications ,  DNA methylation , and the modulation of  RNAi , are major epigenetic events used to modulate gene expression. When the  epigenome  of an organism is altered by an environmental cue like smoking, gene expression changes accordingly. Changes in the regulation of critical genes can have disastrous consequences on health and quality of life. Irregular gene expression is one of the hallmarks of  cancer , but is also found in a number of diseases and disorders.


One of the most prominent and well studied epigenetic consequences of cigarette smoke is altered DNA methylation. Cigarette smoke acts through a number of mechanisms to effect this, chief among these being smoke-induced damage to the DNA and altered expression levels of proteins involved in DNA methylation and transcriptional regulation.


Chemicals in smoke can damage DNA, which subsequently leads to changes in DNA methylation during the repair process. Damage typically comes in the form of double-strand breaks that are linked to carcinogens like  arsenic ,  chromium ,  formaldehyde ,  polycyclic aromatic hydrocarbons , and  nitrosamines  which are found in cigarette smoke.  DNMT1  is the enzyme involved in the maintenance of DNA methylation marks.  DNMT1  is recruited to DNA during its replication, or during DNA repair. As a new DNA strand is synthesized, unmethylated cytosines are incorporated into the sequence. This leads to hemimethylated DNA, where an older methylated strand is bound to a younger unmethylated one.


DNMT1  is an enzyme which recognizes and corrects hemimethylation by transferring the appropriate methyl groups to the newly synthesized strand. Like all biological processes, DNMT1 based hemimethylation correction is not perfect. Mistakes in hemimethylation correction can occur, and are more likely to appear the more a DNA sequence is replicated or repaired. This is compounded by the fact that cigarette smoke compromises the expression
and activity of  DNMT1 . The end result is a decrease in the body's ability to maintain proper methylation patterns, leading to misimpression of gene.


Exposure to cigarette smoke impacts proteins involved in  DNA methylation . These effects come from either hypoxia induced by the cigarette smoke, or the chemical consequences of  nicotine .
Inhaling cigarette smoke increases blood levels of  carbon monoxide  which negatively affects oxygenation throughout the body leading to hypoxia. [ 1 ]  One response to hypoxia is the upregulation in synthesis of the major methyl donor  S-adenosylmethionine . Upregulation of this methyl donor through heightened expression of methionine adenosyltransferase 2A leads to increased DNA methylation, which can lead to the down-regulation of target genes.


Nicotine  found in cigarette smoke binds to  nicotinic acetylcholine receptors . [ 1 ]
This binding leads to an increase in calcium levels which in turn can activate the  cAMP response element-binding protein  (CREB) transcription factor. The most striking downstream effect of the upregulation of this transcription factor is the downregulation of the  DNMT1  gene, which has a cAMP response element in its promoter. This down-regulation of  DNMT1  can have serious consequences on DNA methylation, namely a failure to maintain normal methylation patterns during DNA replication and repair. The upregulation of  DNMT3b  has also been shown to occur as a result of cigarette exposure. [ 2 ]


DNMT3b  is thought to be critical to de novo methylation, or the production of new methylation marks on DNA. This increased expression of  DNMT3b  and methionine adenosyltransferase 2A, taken together with the down-regulation of  DNMT1 , results in myriad unintended epigenetic consequences.


Sp1 , a transcription factor that plays a crucial role in early development, was shown to be expressed at higher rates in lung epithelial cells in the presence of cigarette-smoke condensate. [ 1 ]  This is relevant because Sp1 binds to GC-rich promoter regions which prevent the methylation of these regions during embryonic development. Increased Sp1 expression can lead to a global reduction in DNA methylation, leading to a number of downstream health effects in developing embryos exposed to maternal cigarette smoke.


Regardless of mechanism, several known differences in DNA methylation have been observed between smokers and non-smokers. An overall average decrease in DNA methylation is observed, leading to an increase in the expression of a number of genes. Several genes known to be affected by differential methylation are the  CYP1A1  xenobiotic response element,  AHRR , and  F2RL3 . CYP1A1 is critical to the detoxification of carcinogens, and is found to be hypomethylated in frequent smokers.  AHRR  and  F2RL3  are similarly hypomethylated in smokers. [ 1 ]


AHRR  is known to inhibit the aryl hydrocarbon receptor, which is important to metabolizing harmful chemicals. The resultant increase in  AHRR  expression could lead to a decrease in the body's ability to break down carcinogens, increasing the risk of cancer.  F2RL3  is known to be involved in blood clotting and the inflammation response. [ 1 ]  Effects on the regulation of  F2RL3  in particular could be a link between epigenetic changes from smoking and increased risk of heart disease. Time specific changes in methylation of D4Z4 and NBL2 repeats, which are known factors in carcinogenesis, have also been observed. [ 2 ]


Though smoking leads to an overall decrease in DNA methylation, several critical genes become hypermethylated. Two of the most noteworthy of these genes are  p16  and  p53 . These genes are critical to cell cycle regulation and were shown to have higher levels of methylation in smokers than in non smokers. [ 3 ]  The subsequent loss of function of these genes could potentially lead to dysregulation of the cell cycle, wherein cells are able to bypass normal growth impeding signals. Ultimately, uncontrolled cellular divisions and failure to properly regulate the cell cycle leads to cancer.


Fetuses exposed in utero to cigarette smoke are also known to have some distinct epigenetic differences from smoke-free cohorts. CYP1A1 was found to be hypomethylated in the placentas of fetuses prenatally exposed to cigarette smoke, along with the transposable element AluYB8. [ 4 ]  Methylation of transposable elements is one of the primary ways they are prevented from replicating or moving within the genome. Similarly observed hypomethylation in a number of  Alu elements  results in a general decrease in genomic stability and an increase in the risk of cancer from mutation resulting from the random insertion of transposable elements. [ 2 ]


Strikingly,  BDNF  appears to be hypermethylated in children who were exposed to smoke prenatally. BDNF is critical to long term memory formation and the upkeep of neurons. Downregulation of BDNF has also been linked to clinical depression. [ 4 ]  Taken as a whole, the epigentic consequences of prenatal exposure to cigarette smoke result in increased metabolic stress, decreased genomic stability, heightened risk of mutation and altered brain development.


Histone modifications  are another epigenetic phenomenon known to be affected by smoking. Cigarette smoke has been observed to globally alter  histone modifications  near the promoter regions of pro-inflammatory genes, mainly through an overall increase in acetylation. [ 3 ] [ 5 ]  Cigarette smoke can alter histone acetylation through a number of pathways. Chief among these is the degradation of  HDAC2 , thereby preventing the removal of acetylation marks in affected cells. The degradation of this enzyme results from phosphorylation and subsequent ubiquitination induced by cigarette smoke. [ 3 ]


In rat and mouse models, cigarette smoke was observed to increase acetylation of lysine 9 on histone H3 (H3K9), lysine 12 on histone H4 (H4K12) and phosphorylation of serine 10 on histone H3 (H3S10). These marks are associated with an increase in gene expression and prevent the accumulation of repressive  histone modifications . Mechanistically, the increased frequency of these marks, especially the modifications on histone H3, are linked to the activation of  IKK-α  which directly phosphorylates histone H3 as a consequence of exposure to cigarette smoke. It is thought that this increase in acetylation of histones H3 and H4 in  macrophages  in the  alveolus  could potentially lead to the development of COPD.


MicroRNAs , or miRNAs, are known to be major epigenetic regulators of gene expression in humans. These RNAs are short molecules which bind to  mRNA  through complementary base pairing. This binding impacts the expression of proteins encoded by those  mRNAs  by either inducing the cleavage of the  mRNA , destabilizing the molecule, or limiting the efficiency of its translation. Unlike differences in DNA methylation, changes in miRNA activity induced by cigarette smoke are largely unknown. [ 1 ]  Primary research data suggests that cigarette smoke promotes the dysregulation of a number of miRNA's. One such study showed that cigarette smoke downregulates miR-16, miR-21, and  miR-146a  in the placenta. [ 6 ]


A downregulation of miR-16 is predicted to inhibit apoptosis via the subsequent upregulation of  BCL2L2  and  EDA , both of which contribute to anti-apoptotic signaling. Downregulaton of miR-146a is predicted to influence the expression of  TRAF6 , which has a number of downstream effects, including regulation of  inflammatory responses and anti-apoptotic signaling. Taken as a whole, dysregulation of these miRNA's could lead to dysregulation of normal cell death, cell cycling and immune responses, all of which have negative health impacts and could potentially lead to cancerous growths and improper placental formation.


Epigenetic priming  (also known as  gene priming ) is the modification to a cell's  epigenome  whereby specific  chromatin domains  within a  cell  are  converted  from a closed state to an open state, usually as the result of an external biological trigger or  pathway , allowing for  DNA  access by  transcription factors  or other modification mechanisms. The action of epigenetic priming for a certain region of  DNA  dictates how other  gene regulation mechanisms  will be able to act on the DNA later in the cell’s life. Epigenetic priming has been chiefly investigated in  neuroscience  and  cancer research , as it has been found to play a key role in  memory formation  within  neurons [ 1 ]  and  tumor-suppressor gene  activation in  cancer treatment [ 2 ]  respectively.


Epigenetic priming refers to a latent  epigenetic state  triggered by  stimuli , such as a  drug  or environmental changes. The epigenetically primed state is characterized by  chromatin loosening , which is the change of chromatin state from  heterochromatin  (tightly bound and inaccessible) to  euchromatin  (loosely bound and fully accessible), which leads to an increased  transcription  of certain  genes  as a result of the easier access and binding of  transcription factors . [ 1 ]  The triggering signal is effectuated by various epigenetic mechanisms, the most prominent of which are  histone acetylation  and  histone methylation . Most of the epigenetic agents involved in histone modifications, such as  histone deacetylase  (HDAC) variants, are non-targeted, meaning the loosening and tightening of the chromatin is unspecific within the  cell . [ 3 ]  Therefore, epigenetic priming and resultant  gene transcription  occurs throughout the cell and affects a large variety of  chromatin  sites.


Chromatin remodeling processes such as histone acetylation and methylation are reversible, and  euchromatin  sites resulting from epigenetic priming are eventually converted back to  heterochromatin  by reversal agents such as  histone deacetylase . Thus, priming may be artificially controlled by  inhibiting  these reversal agents within the cell so that the  chromatin  remains open. Among these approaches, the most well studied is  HDAC inhibition .


In order to maintain the  epigenome  plasticity,  enzymes  that add (writers) and remove (erasers) the different epigenetic marks are needed. As exemplified by  histone acetylation  in the  Epigenetic Priming Model  figure above, there is an interplay between these writers and erasers that allows the genome to be responsive to external or internal stimuli. In the case of acetylation,  histone acetyltransferases  add acetyl groups to the  histones  and  histone deacetylases  (HDAC) remove them. Both are present within a  cell  at a given time, meaning that an acetylated (open) region of  chromatin  might be reverted to closed form.  HDAC inhibition  ensures that chromatin is left in an open state by prohibiting the open to closed transition, leading to lasting  gene expression  and other epigenetic activity.


Epigenetic priming was first described in  cancer research  when  epigenetic alterations  on  tumor-suppressor genes  (TSG) were found to be drivers of  carcinogenesis . [ 2 ]   Epigenetic alterations  (e.g.  DNA methylation ) resulting in TSG inactivation as a common means of tumor formation. Contrary to regular DNA mutations common in cancer, methylation is reversible, provided that the chromatin is adequately open to allow  hypomethylating agents  to access the DNA and prevent methylation. Therefore, priming was investigated as a ‘pre-treatment’ to sensitize the  tumerogenic cells  to hypomethylating chemotheraputics such as  decitabine . [ 2 ]  Many types of cancer (e.g. gastric) are known for having aberrant epigenetic changes, particularly in DNA methylation. In contrast to DNA mutations which cannot be easily changed through treatment, these aberrant epigenetic changes allow for a reversible treatment avenue.


Epigenetic agents have proved to increase expression of aberrantly silenced genes (i.e.  Runx3 ,  Tnf ,  Pycard ,  Fas ) in mice models after 5-aza-CR treatment . [ 4 ]  Thus, helping overcome cancer-induced cell dysfunction. Additionally, epigenetic priming has been shown to enhance  cytotoxicity  of cancer drugs (i.e.  SN38  and  CDDP ), showing promising results in  lung  and  ovarian cancer . [ 5 ]  Due to their proven effectivity, the  FDA  approved  5-azacytidine ,  romidepsin  and other DMNT inhibitors (i.e. 5-azacytidine, hydralazine, 5-Aza-2’-deoxycytidine) and HDAC inhibitors (i.e. romidepsin, belinostat, panobinostat) for clinical use . [ 4 ]   [ 6 ]


Several clinical trials have been performed to assess the  safety  and effectivity of epigenetic therapy as a pretreatment in cancer therapy. Preclinical usage of epigenetic agents like  5-azacytidine  ( DNMT  inhibitor) and  romidepsin  (HDAC inhibitor) sensitizes cancer cells for further treatment. Some examples of clinical trials performed are listed below.


Epigenetic treatment with  5-azacytidine  (5-AZA) and  romidepsin  before  pembrolizumab  administration was tested for safety in a  clinical trial  from 2016 - 2018. Drug administration ( 5-AZA ,  romidepsin ,  5-AZA  +  romidepsin ) was followed for 14 days in 24 patients between 40–69 years old. Side effects in groups included  diarrhea ,  nausea  and  fatigue . Moreover,  lack of appetite ,  anemia  and  thrombocytopenia  were independent of the drug combination received by the patient. After this study, epigenetic agent 'pretherapy' with  5-AZA  and  romidepsin  followed by  pembrolizumab  treatment was deemed feasible and overall safe for patients. [ 7 ]


Gastric cancer is heavily influenced by epigenetic aberrations. Analysis showed that DNA methylation changes have a higher influence on gastric cancer than point mutations. A phase I study on  gastric cancer  5-AZA pretreatment in combination with  epirubicin ,  oxaliplatin  and  capecitabine  was successful. [ 8 ]  The epigenetic intervention was fruitful in demethylating loci (i.e. CDKN2A, ESR1, HPP1, MGMT, TIMP3) abnormally methylated in gastric carcinomas.


A phase I study explored the feasibility of epigenetic priming with  decitabine  in patients with Acute Myelogenous Leukemia (AML) followed by  cytarabine  and  daunorubicin  treatment. Patients were treated two weeks before the immunotherapy either with 1 hour infusion (group A) or continuous infusion for 3, or 7 days (group B). Group B showed higher levels of hypomethylation after treatment than group A, but neither showed toxicity by the epigenetic agent. Finally, no significant side effects were encountered. [ 9 ]


Older patients with  AML  diagnosis have poor  prognosis , lower rates of  complete remission  and worsening of overall survival. A phase 2 study was performed evaluating the efficacy and safety of epigenetic priming through  decitabine  in elderly patients with  AML . In 2015, 46 patients who were not candidates for intensive  chemotherapy  enrolled in the study. Treatment consisted of continuous  IV  administration of  decitabine , followed by 5 days of  cytarabine   immunotherapy . Patients continued with a second cycle if evidence of disease was found on 15 day  bone marrow biopsy , otherwise, they proceeded with  decitabine  maintenance. The study showed that pretreatment with  decitabine  followed by  cytarabine  promoted a higher number of  complete remissions  (70%) in older patients with  AML . [ 10 ]


It is believed that  epigenetic modifications , and in particular those which perform epigenetic priming, are fundamentally responsible for the encoding of  memory  within neurons. This idea is supported by various pieces of evidence. Firstly, despite  inhibition  of  protein synthesis  during  memory formation , memories may be retrieved later on. [ 11 ]  This, along with the discovery that  long-term memory  can be restored after  synapse  deterioration, [ 12 ]  suggests  synaptic structuring  (which requires  protein synthesis  during memory formation) is not the fundamental source of  engram  encoding within a cell. [ 13 ]  Furthermore, in mice it has been found that proper  histone acetytransferase  function is required for memory formation [ 14 ] [ 15 ]  and that  HDAC inhibition  in  neurons  can improve learning behavior and  long-term memory . [ 16 ]  An explanation is that, when present in combination with memory-associated neural activity, HDAC inhibitors (HDACi) allow chromatin to remain open and increase  transcription of genes  that remodel  synapses , resulting in increased  plasticity  and improved memory formation. [ 13 ] [ 17 ]  As a result of these observations, it has been proposed that epigenetic priming is the initial phase of memory formation. [ 13 ]


It has been found that different forms of  long-term memory  are associated with different types of  histone acetylation , such as acetylation of  H3  versus  H4 . [ 15 ]  This suggests that epigenetic priming in neurons which result in different memory profile expressions may be encoded by different  histone acetyltransferases . Thus, although the mechanisms that  loosen chromatin  are unspecific in their target, likely have specificity depending on which ones are activated. In a similar vein, it is believed that the action of different of priming agents, such as the varieties of histone acetyltransferases, may combine to create a stacking effect on neuron chromatin, resulting in significantly increased expression of the associated genes. [ citation needed ]


Epidemiological and experimental studies have shown that  environmental factors  during early development, such as maternal nutrition and body composition, can influence the metabolic phenotype of the offspring. [ 18 ]  Epigenetic priming is thought to mediate the persistent changes in  gene expression  that could eventually lead to  metabolic syndrome . [ 19 ]  Potentially, these induced metabolic disruptions benefit progeny developing in a low resources environment to increased success later in life. [ 18 ]  The  Agouti  mouse exemplifies a variation of the aforementioned effect of early environmental exposures on offspring’s  fitness . [ 20 ]


Epigenetic regulation of neurogenesis  is the role that  epigenetics  (hertitable characteristics that do not involve changes in DNA sequence) plays in the regulation of  neurogenesis  (the production of  neurons  from neural  stem cells ).


Epigenetics is the study of heritable changes in  gene expression  which do not result from modifications to the  sequence of DNA .  Neurogenesis  is the mechanism for  neuron  proliferation and  differentiation . It entails many different complex processes which are all time and order dependent. [ 1 ]


Processes such as neuron proliferation, fate specification, differentiation, maturation, and functional integration of newborn cells into existing  neuronal networks  are all interconnected. [ 2 ]   In the past decade [ when? ]  many  epigenetic  regulatory mechanisms have been shown to play a large role in the timing and determination of neural  stem cell  lineages. [ 1 ]


Three important methods of epigenetic regulation include  histone modification ,  DNA methylation  and  demethylation , and microRNA ( miRNA ) expression.  Histones  keep the DNA of the  eukaryotic cell  tightly packaged through charge interactions between the positive charge on the histone tail and the negative charge of the DNA, as well as between histone tails of nearby  nucleosomes . While there are many different types of histone modifications, in neural epigenetics there are two primary mechanisms which have been explored:  histone methylation  and  histone acetylation . [ 1 ] [ 3 ]
In the former,  methyl groups  are either added or removed to the histone altering its structure and exposing  chromatin  and leading to  gene activation  or deactivation. In the latter, histone acetylation causes the histone to hold the DNA more loosely, allowing for more gene activation. DNA methylation, in which methyl groups are added to cytosine or adenosine residues on the DNA, is a more lasting method of gene inactivation than histone modification, though is still reversible in some cases. [ 1 ] [ 3 ]  MicroRNAs are a small form of  non-coding RNA  (ncRNA) which often act as "fine-tuning" mechanisms for gene expression by repressing or inducing messenger RNA (mRNA) in neural cells but can also act directly with  transcription factors  to guide neurogenesis. [ 1 ] [ 3 ] [ 4 ] [ 5 ]


Neural stem cells are involved in the development of the cortex in a precise "inside out" manner with carefully controlled timing mechanisms. Early born neurons form  deep layers  in the cortex while newer born neurons form the upper layers. This timing program is seen in vitro as well as in vivo. [ 1 ] [ 3 ]  Mutant analysis has shown that histone methylation modulates the production of deep layer and upper layer neurons through epigenetic regulation. Specifically, deletion of a portion of the  PRC2  complex,  Ezh2 , encoding histone  methyltransferase , led to a twofold reduction of  POU3F2 /BRN2-expressing and  SATB2 -expressing upper layer neurons without affecting the number of neurons in layers V and VI . In mouse  embryonic stem cell -derived neural progenitors, increased histone acetylation induced by the  histone deacetylase  (HDAC) inhibitor  valproic acid  not only induced neuronal differentiation, but also selectively enriched the upper layer neuronal population. Therefore, it has been proposed that HDAC inhibition promotes the progression of neuronal differentiation, leading to a fate-switch from deep-layer producing progenitors into upper-layer progenitors. However, the reasons behind this selective differentiation and timing control as a result of HDAC inhibition are not yet fully understood. [ 3 ]