Some human disorders are associated with genomic imprinting, a phenomenon in mammals where the father and mother contribute different epigenetic patterns for specific genomic loci in their  germ cells . [ 199 ]  The best-known case of imprinting in human disorders is that of  Angelman syndrome  and  Prader–Willi syndrome  – both can be produced by the same genetic mutation,  chromosome 15q partial deletion , and the particular syndrome that will develop depends on whether the mutation is inherited from the child's mother or from their father. [ 200 ]


In the  Överkalix study , paternal (but not maternal) grandsons [ 201 ]  of Swedish men who were exposed during preadolescence to famine in the 19th century were less likely to die of cardiovascular disease. If food was plentiful, then  diabetes  mortality in the grandchildren increased, suggesting that this was a transgenerational epigenetic inheritance. [ 202 ]  The opposite effect was observed for females – the paternal (but not maternal) granddaughters of women who experienced famine while in the womb (and therefore while their eggs were being formed) lived shorter lives on average. [ 203 ]


The use of beta-lactam  antibiotics  can alter glutamate receptor activity and the action of cyclosporine on multiple transcription factors. Additionally,  lithium  can impact autophagy of aberrant proteins, and  opioid  drugs via chronic use can increase the expression of genes associated with addictive phenotypes. [ 204 ]


Parental  nutrition , in utero exposure to stress or  endocrine disrupting chemicals , [ 205 ]  male-induced maternal effects such as the attraction of differential mate quality, and maternal as well as paternal age, and offspring gender could all possibly influence whether a germline epimutation is ultimately expressed in offspring and the degree to which intergenerational inheritance remains stable throughout posterity. [ 206 ]  However, whether and to what extent epigenetic effects can be transmitted across generations remains unclear, particularly in humans. [ 207 ] [ 208 ]


Addiction  is a disorder of the brain's  reward system  which arises through  transcriptional  and neuroepigenetic mechanisms and occurs over time from chronically high levels of exposure to an addictive stimulus (e.g., morphine, cocaine, sexual intercourse, gambling). [ 209 ] [ 210 ] [ 211 ]  Transgenerational epigenetic inheritance of addictive  phenotypes  has been noted to occur in preclinical studies. [ 212 ] [ 213 ]  However, robust evidence in support of the persistence of epigenetic effects across multiple generations has yet to be established in humans; for example, an epigenetic effect of prenatal exposure to smoking that is observed in great-grandchildren who had not been exposed. [ 207 ]


The two forms of heritable information, namely genetic and epigenetic, are collectively called dual inheritance. Members of the APOBEC/AID family of  cytosine deaminases  may concurrently influence genetic and epigenetic inheritance using similar molecular mechanisms, and may be a point of crosstalk between these conceptually compartmentalized processes. [ 214 ]


Fluoroquinolone  antibiotics induce epigenetic changes in  mammalian  cells through iron  chelation . This leads to epigenetic effects through inhibition of α-ketoglutarate-dependent  dioxygenases  that require  iron  as a co-factor. [ 215 ]


Various pharmacological agents are applied for the production of induced pluripotent stem cells (iPSC) or maintain the embryonic stem cell (ESC) phenotypic via epigenetic approach. Adult stem cells like bone marrow stem cells have also shown a potential to differentiate into cardiac competent cells when treated with G9a histone methyltransferase inhibitor BIX01294. [ 216 ] [ 217 ]


Cell plasticity, which is the adaptation of cells to stimuli without changes in their genetic code, requires epigenetic changes. These have been observed in cell plasticity in cancer cells during epithelial-to-mesenchymal transition [ 218 ]  and also in immune cells, such as macrophages. [ 219 ]  Interestingly, metabolic changes underly these adaptations, since various metabolites play crucial roles in the chemistry of epigenetic marks. This includes for instance alpha-ketoglutarate, which is required for histone demethylation, and acetyl-Coenzyme A, which is required for histone acetylation.


Epigenetic regulation of gene expression that could be altered or used in  epigenome editing  are or include  mRNA/lncRNA modification ,  DNA methylation  modification and  histone modification . [ 220 ] [ 221 ] [ 222 ]


Methylation is a widely characterized mechanism of genetic regulation that can determine biological traits. However, strong experimental evidences correlate methylation patterns in SNPs as an important additional feature for the classical activation/inhibition epigenetic dogma. Molecular interaction data, supported by colocalization analyses, identify multiple nuclear regulatory pathways, linking sequence variation to disturbances in DNA methylation and molecular and phenotypic variation. [ 223 ]


UBASH3B  encodes a protein with tyrosine phosphatase activity, which has been previously linked to advanced neoplasia. [ 224 ]  SNP rs7115089 was identified as influencing DNA methylation and expression of this locus, as well as and Body Mass Index (BMI). [ 223 ]  In fact, SNP rs7115089 is strongly associated with BMI [ 225 ]  and with genetic variants linked to other cardiovascular and metabolic traits in GWASs. [ 226 ] [ 227 ] [ 228 ]  New studies suggesting  UBASH3B  as a potential mediator of adiposity and cardiometabolic disease. [ 223 ]  In addition, animal models demonstrated that  UBASH3B  expression is an indicator of caloric restriction that may drive programmed susceptibility to obesity and it is associated with other measures of adiposity in human peripherical blood. [ 229 ]


SNP rs730775 is located in the first intron of  NFKBIE  and is a  cis  eQTL for  NFKBIE  in whole blood. [ 223 ]  Nuclear factor (NF)-κB inhibitor ε (NFKBIE) directly inhibits NF-κB1 activity and is significantly co-expressed with NF-κB1, also, it is associated with rheumatoid arthritis. [ 230 ]  Colocalization analysis supports that variants for the majority of the CpG sites in SNP rs730775 cause genetic variation at the  NFKBIE  locus which is suggestible linked to rheumatoid arthritis through  trans  acting regulation of DNA methylation by NF-κB. [ 223 ]


Fatty acid desaturase 1 (FADS1) is a key enzyme in the metabolism of fatty acids. [ 231 ]  Moreover, rs174548 in the  FADS1  gene shows increased correlation with DNA methylation in people with high abundance of CD8+ T cells. [ 223 ]  SNP rs174548 is strongly associated with concentrations of arachidonic acid and other metabolites in fatty acid metabolism, [ 232 ] [ 233 ]  blood eosinophil counts. [ 234 ]  and inflammatory diseases such as asthma. [ 235 ]  Interaction results indicated a correlation between rs174548 and asthma, providing new insights about fatty acid metabolism in CD8+ T cells with immune phenotypes. [ 223 ]


As epigenetics is in the early stages of development as a science and is surrounded by  sensationalism  in the public media,  David Gorski  and geneticist  Adam Rutherford  have advised caution against the proliferation of false and  pseudoscientific  conclusions by  new age  authors making unfounded suggestions that a person's genes and health can be manipulated by  mind control . Misuse of the scientific term by  quack authors  has produced misinformation among the general public. [ 2 ] [ 236 ]


Beck–Fahrner syndrome , also known as  BEFAHRS  and  TET3 deficiency , is a  rare   genetic disorder  caused by mutations of the  TET3  gene. The clinical presentation varies among individuals, but typically includes  global developmental delay ,  slow progress in mental and physical activities ,  autism ,  decreased muscle tone ,  epilepsy  and  dysmorphic features .


Mutations in the TET3 gene disrupt  DNA demethylation  during  embryogenesis , an essential  epigenetic  process contributing to the early  development of the nervous system . They can occur from  new genetic mutations  or be  inherited  in an  autosomal dominant  manner.  Diagnosis involves  molecular  and  genetic testing  in the context of typical symptoms. Management is  supportive , aimed at improving  quality of life . As of 2023, approximately 50 individuals have been diagnosed with Beck–Fahrner syndrome.


Beck–Fahrner syndrome, also referred to as "BEFAHRS", is characterized by a mnemonic encompassing its prominent features: behavioral differences, epilepsy, facial features, autistic features, hypotonia, retardation of psychomotor development, and size differences. [ 1 ]


The most common  neurodevelopmental  symptoms associated with Beck–Fahrner syndrome include a  delay in global development ,  slow progress in mental and physical activities ,  delayed speech , and difficulties with  fine  and  gross motor skills . [ 2 ]   Intellectual  and  learning disabilities  are commonly present, and more than two-thirds of affected individuals have  autism  or  social communication disorder . [ 3 ]  Additionally,  attention deficit hyperactivity disorder ,  obsessive–compulsive tendencies ,  anxiety ,  depression  and  psychosis  have been observed. [ 2 ]


Decreased tone of muscles  is most noticeable in early life, causing feeding difficulties and constipation in infants, and further impeding the development of speech and motor skills in children.  Epilepsy  and seizure disorders affect over one-third of individuals, while some individuals encounter  motor  and  movement disorders . Eye involvement can lead to  vision ,  movement  and  alignment  issues, and ear involvement may result in  hearing loss . Affected individuals may at times demonstrate  benign  brain  malformations  in  neuroimaging  studies. [ 3 ]


Most individuals affected by Beck–Fahrner syndrome exhibit similar  facial features , including a long face with a broad forehead, an open mouth appearance,  protruding ears , arched eyebrows and a  high-arched palate .  Musculoskeletal abnormalities  encompass  kyphosis ,  scoliosis ,  hyperflexible joints ,  hip misalignment  and  flat feet . [ 3 ]  Overgrowth may manifest in some individuals, presenting with characteristics such as a  larger head size  and  tall stature ; [ 2 ]  rarely this may be correlated with  enlarged kidneys  and  heart . Conversely, undergrowth has also been reported, associated with a  smaller head size  and  short stature .  Congenital heart defects ,  pyloric stenosis ,  inguinal hernia ,  hypospadias  and  undescended testis  have been observed less frequently. [ 3 ]


The TET3 gene on  chromosome 2  (2p13.1)  encodes  the  tet methylcytosine dioxygenase 3  (TET3) enzyme. [ 4 ] [ A ]  TET3 enzyme facilitates conversion of  5-methylcytosine  (5mC) to  5-hydroxymethylcytosine  (5hmC), initiating active  DNA demethylation , an  epigenetic  mechanism. [ 6 ]  TET3 enzyme is produced in  embryonic stem cells  during  embryogenesis , where it contributes to the  development of the nervous system . It sustains  neural stem cells  and subsequently promotes the  maturation  of  neurons . [ 7 ]


Beck–Fahrner syndrome is caused by specific mutations in the TET3 gene. These mutations can be heterozygous (one normal copy and one mutated copy),  compound heterozygous  (two different mutated copies), or  homozygous  (two identical mutated copies). [ 8 ]  The mutations—which can be of various types like  nonsense ,  missense  or  frameshift —disrupt the normal DNA demethylation process during early embryonic development and the formation of the nervous system. This disruption leads to an elevation in methylated  CpG sites , causing DNA hypermethylation. [ 2 ]


The mutations can either occur due to   new genetic mutations  during embryogenesis or be  inherited  in a way where one copy of the mutated gene is sufficient to cause the condition –  autosomal dominant . The signs and symptoms may vary among individuals due to differences in  gene expression  and  partial loss of gene function . [ 3 ]


Beck–Fahrner syndrome shares clinical findings with several genetic disorders. These include  Bainbridge–Ropers syndrome ,  Fragile X syndrome ,  Heyn–Sproul–Jackson syndrome ,  Kabuki syndrome ,  Luscan-Lumish syndrome ,  Malan syndrome ,  Sotos syndrome  and  Tatton-Brown–Rahman syndrome . There is no consensus on diagnostic criteria for Beck–Fahrner syndrome. Diagnosis involves confirming the presence of a  pathogenic  or likely pathogenic TET3 gene mutation in conjunction with identification of characteristic signs and symptoms. [ 3 ]


Various  molecular  and  genetic testing  methods are employed to identify mutations associated with Beck–Fahrner syndrome. These may include multigene panels incorporating the TET3 gene,  whole genome sequencing ,  exome sequencing ,  sequence analysis , and single-gene testing followed by targeted gene deletion or duplication analysis.  GeneReviews  recommends exome sequencing as the preferred diagnostic test due to the recent identification of the condition and the limited availability of TET3 gene analysis on most multigene panels. [ 3 ]


Levy et al. (2021) discovered a distinct DNA methylation pattern or  epigenetic signature  (episignature)—demonstrating DNA hypermethylation—unique to pathogenic and likely pathogenic TET3 gene mutations. This episignature can be assessed through  whole blood  genome-wide DNA methylation analysis, [ 1 ]  and may serve as a tool to confirm the  pathogenicity  of a TET3  variant of uncertain significance . [ 9 ]


The management approach for Beck–Fahrner syndrome is primarily  supportive , with a focus on improving the  quality of life . The care is coordinated by  medical genetics  and  pediatrics , involving a multidisciplinary team of specialists. Depending on specific symptoms, various medical specialists may be involved, including  neurology  for seizures and  cardiology  for heart defects. Early interventions, such as  autism therapies  and participation in  special education  programs like  behavior therapy  and  speech therapy , can help manage developmental and cognitive issues.  Physical  and  occupational therapy  can support in addressing physical symptoms, and assistive technology such as  mobility aid  and  standing frame  can be utilized when necessary. [ 3 ]


Genetic counseling  plays a role in educating patients and their families about the condition, assessing the risk of other family members having the disorder, offering guidance on  family planning , and providing information on  prenatal testing . Furthermore,  social work  assists patients and families in exploring  palliative ,  respite  and  nursing home  care options when necessary. [ 3 ]


In 2018, a research paper suggested that mutations in the TET3 gene could cause intellectual disability. [ 10 ]  Beck–Fahrner syndrome, initially referred to as "TET3 deficiency", [ 2 ]  was first described in 2020. [ 11 ]  It was the first human disorder of DNA demethylation to be delineated. [ 12 ]  As of 2023, approximately 50 individuals have been diagnosed with this condition. [ 13 ]


Bisulfite [ 1 ]   sequencing  (also known as  bisulphite sequencing ) is the use of  bisulfite  treatment of  DNA  before routine  sequencing  to determine the pattern of  methylation .  DNA methylation  was the first discovered  epigenetic  mark, and remains the most studied. In animals it predominantly involves the addition of a  methyl group  to the carbon-5 position of  cytosine  residues of the dinucleotide  CpG , and is implicated in repression of  transcriptional activity .


Treatment of DNA with bisulfite converts cytosine residues to  uracil , but leaves  5-methylcytosine  residues unaffected. Therefore, DNA that has been treated with bisulfite retains only methylated cytosines. Thus, bisulfite treatment introduces specific changes in the  DNA sequence  that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA. Various analyses can be performed on the altered sequence to retrieve this information.  The objective of this analysis is therefore reduced to differentiating between  single nucleotide polymorphisms  (cytosines and  thymidine ) resulting from bisulfite conversion (Figure 1).


Bisulfite sequencing applies routine sequencing methods on bisulfite-treated genomic DNA to determine methylation status at CpG dinucleotides.  Other non-sequencing strategies are also employed to interrogate the methylation at specific loci or at a  genome -wide level.  All strategies assume that bisulfite-induced conversion of unmethylated cytosines to uracil is complete, and this serves as the basis of all subsequent techniques.  Ideally, the method used would determine the methylation status separately for each  allele .  Alternative methods to bisulfite sequencing include  Combined Bisulphite Restriction Analysis  and  methylated DNA immunoprecipitation  (MeDIP).


Methodologies to analyze bisulfite-treated DNA are continuously being developed.  To summarize these rapidly evolving methodologies, numerous review articles have been written. [ 2 ] [ 3 ] [ 4 ] [ 5 ]


The methodologies can be generally divided into strategies based on methylation-specific PCR (MSP) (Figure 4), and strategies employing  polymerase chain reaction  (PCR) performed under non-methylation-specific conditions (Figure 3).  Microarray-based methods use PCR based on non-methylation-specific conditions also.


The first reported method of methylation analysis using bisulfite-treated DNA utilized PCR and standard dideoxynucleotide  DNA sequencing  to directly determine the nucleotides resistant to bisulfite conversion. [ 6 ]   Primers are designed to be strand-specific as well as bisulfite-specific (i.e., primers containing non-CpG cytosines such that they are not complementary to non-bisulfite-treated DNA), flanking (but not involving) the methylation site of interest.  Therefore, it will amplify both methylated and unmethylated sequences, in contrast to methylation-specific PCR.  All sites of unmethylated cytosines are displayed as  thymines  in the resulting amplified sequence of the sense strand, and as  adenines  in the amplified  antisense  strand.  By incorporating high throughput sequencing adaptors into the PCR primers, PCR products can be sequenced with massively parallel sequencing. Alternatively, and labour-intensively, PCR product can be cloned and sequenced.  Nested PCR  methods can be used to enhance the product for  sequencing .


All subsequent DNA methylation analysis techniques using bisulfite-treated DNA is based on this report by Frommer et al. (Figure 2). [ 6 ]  Although most other modalities are not true sequencing-based techniques, the term "bisulfite sequencing" is often used to describe bisulfite-conversion DNA methylation analysis techniques in general.


Pyrosequencing  has also been used to analyze bisulfite-treated DNA without using methylation-specific PCR. [ 7 ] [ 8 ]   Following PCR amplification of the region of interest, pyrosequencing is used to determine the bisulfite-converted sequence of specific  CpG sites  in the region.  The ratio of C-to-T at individual sites can be determined quantitatively based on the amount of C and T incorporation during the sequence extension.  The main limitation of this method is the cost of the technology.  However, Pyrosequencing does well allow for extension to  high-throughput screening  methods.


A variant of this technique, described by Wong  et al. , uses allele-specific primers that incorporate  single-nucleotide polymorphisms  into the sequence of the sequencing primer, thus allowing for separate analysis of maternal and paternal  alleles . [ 9 ]  This technique is of particular usefulness for  genomic imprinting  analysis.


This method is based on the  single-strand conformation polymorphism  analysis (SSCA) method developed for  single-nucleotide polymorphism  (SNP) analysis. [ 10 ]  SSCA differentiates between single-stranded DNA fragments of identical size but distinct sequence based on differential migration in non-denaturating  electrophoresis .  In MS-SSCA, this is used to distinguish between bisulfite-treated, PCR-amplified regions containing the CpG sites of interest.  Although SSCA lacks sensitivity when only a single  nucleotide  difference is present, bisulfite treatment frequently makes a number of C-to-T conversions in most regions of interest, and the resulting sensitivity approaches 100%.  MS-SSCA also provides semi-quantitative analysis of the degree of DNA methylation based on the ratio of band intensities.  However, this method is designed to assess all  CpG sites  as a whole in the region of interest rather than individual methylation sites.


A further method to differentiate converted from unconverted bisulfite-treated DNA is using high-resolution melting analysis (HRM), a  quantitative PCR -based technique initially designed to distinguish SNPs. [ 11 ]   The PCR  amplicons  are analyzed directly by temperature ramping and resulting liberation of an intercalating  fluorescent dye  during melting.  The degree of methylation, as represented by the C-to-T content in the  amplicon , determines the rapidity of melting and consequent release of the dye.  This method allows direct quantitation in a single-tube assay, but assesses methylation in the amplified region as a whole rather than at specific  CpG sites .


MS-SnuPE employs the primer extension method initially designed for analyzing  single-nucleotide polymorphisms . [ 12 ]   DNA is bisulfite-converted, and bisulfite-specific primers are annealed to the sequence up to the base pair immediately before the CpG of interest. The primer is allowed to extend one base pair into the C (or T) using  DNA polymerase  terminating  dideoxynucleotides , and the ratio of C to T is determined quantitatively.


A number of methods can be used to determine this C:T ratio. At the beginning, MS-SnuPE relied on radioactive  ddNTPs  as the reporter of the primer extension.  Fluorescence-based methods or  Pyrosequencing  can also be used. [ 13 ]  However, matrix-assisted laser desorption ionization/time-of-flight ( MALDI-TOF )  mass spectrometry  analysis to differentiate between the two polymorphic primer extension products can be used, in essence, based on the GOOD assay designed for  SNP genotyping .  Ion pair reverse-phase  high-performance liquid chromatography  (IP-RP- HPLC ) has also been used to distinguish primer extension products. [ 14 ]


A recently described method by Ehrich et al. further takes advantage of bisulfite-conversions by adding a base-specific cleavage step to enhance the information gained from the nucleotide changes. [ 15 ]   By first using in vitro  transcription  of the region of interest into  RNA  (by adding an  RNA polymerase   promoter  site to the PCR primer in the initial amplification),  RNase A  can be used to cleave the  RNA   transcript  at base-specific sites.  As  RNase A  cleaves  RNA  specifically at cytosine and uracil  ribonucleotides , base-specificity is achieved by adding incorporating cleavage-resistant  dTTP  when cytosine-specific (C-specific) cleavage is desired, and incorporating dCTP when uracil-specific (U-specific) cleavage is desired.  The cleaved fragments can then be analyzed by  MALDI-TOF .  Bisulfite treatment results in either introduction/removal of cleavage sites by C-to-U conversions or shift in fragment mass by G-to-A conversions in the amplified reverse strand.  C-specific cleavage will cut specifically at all methylated  CpG sites .  By analyzing the sizes of the resulting fragments, it is possible to determine the specific pattern of DNA methylation of  CpG sites  within the region, rather than determining the extent of methylation of the region as a whole.  This method demonstrated efficacy for  high-throughput screening , allowing for interrogation of numerous  CpG sites  in multiple tissues in a cost-efficient manner.


This alternative method of methylation analysis also uses bisulfite-treated DNA but avoids the need to sequence the area of interest. [ 16 ]  Instead, primer pairs are designed themselves to be "methylated-specific" by including sequences complementing only unconverted  5-methylcytosines , or, on the converse, "unmethylated-specific", complementing  thymines  converted from unmethylated cytosines.  Methylation is determined by the ability of the specific primer to achieve amplification. This method is particularly useful to interrogate  CpG islands  with possibly high methylation density, as increased numbers of CpG pairs in the primer increase the specificity of the assay.  Placing the CpG pair at the 3'-end of the primer also improves the sensitivity.  The initial report using MSP described sufficient sensitivity to detect methylation of 0.1% of  alleles .  In general, MSP and its related protocols are considered to be the most sensitive when interrogating the methylation status at a specific  locus .


The MethyLight method is based on MSP, but provides a quantitative analysis using  quantitative PCR . [ 17 ]   Methylated-specific primers are used, and a methylated-specific fluorescence reporter probe is also used that anneals to the amplified region.  In alternative fashion, the primers or probe can be designed without methylation specificity if discrimination is needed between the CpG pairs within the involved sequences. Quantitation is made in reference to a methylated reference DNA.  A modification to this protocol to increase the specificity of the PCR for successfully bisulfite-converted DNA (ConLight-MSP) uses an additional probe to bisulfite-unconverted DNA to quantify this non-specific amplification. [ 18 ]


Further methodology using MSP-amplified DNA analyzes the products using  melting curve analysis  (Mc-MSP). [ 19 ]   This method amplifies bisulfite-converted DNA with both methylated-specific and unmethylated-specific primers, and determines the quantitative ratio of the two products by comparing the differential peaks generated in a melting curve analysis. A high-resolution melting analysis method that uses both  quantitative PCR  and melting analysis has been introduced, in particular, for sensitive detection of low-level methylation [ 20 ]


Microarray -based methods are a logical extension of the technologies available to analyze bisulfite-treated DNA to allow for genome-wide analysis of methylation. [ 21 ]   Oligonucleotide microarrays  are designed using pairs of  oligonucleotide   hybridization probes  targeting  CpG sites  of interest.  One is complementary to the unaltered methylated sequence, and the other is complementary to the C-to-U-converted unmethylated sequence.  The probes are also bisulfite-specific to prevent binding to DNA incompletely converted by bisulfite. The  Illumina Methylation Assay  is one such assay that applies the bisulfite sequencing technology on a microarray level to generate genome-wide methylation data.


Bisulfite sequencing is used widely across mammalian genomes, however complications have arisen with the discovery of a new mammalian DNA modification  5-hydroxymethylcytosine . [ 22 ] [ 23 ]  5-Hydroxymethylcytosine converts to cytosine-5-methylsulfonate upon bisulfite treatment, which then reads as a C when sequenced. [ 24 ]  Therefore, bisulfite sequencing cannot discriminate between 5-methylcytosine and 5-hydroxymethylcytosine. This means that the output from bisulfite sequencing can no longer be defined as solely DNA methylation, as it is the composite of 5-methylcytosine and 5-hydroxymethylcytosine.


Bisulfite sequencing relies on the conversion of every single unmethylated cytosine residue to uracil. If conversion is incomplete, the subsequent analysis will incorrectly interpret the unconverted unmethylated cytosines as methylated cytosines, resulting in  false positive  results for methylation. Only cytosines in single-stranded DNA are susceptible to attack by bisulfite, therefore  denaturation  of the DNA undergoing analysis is critical. [ 2 ]  It is important to ensure that reaction parameters such as temperature and salt concentration are suitable to maintain the DNA in a single-stranded conformation and allow for complete conversion. Embedding the DNA in  agarose  gel has been reported to improve the rate of conversion by keeping strands of DNA physically separate. [ 25 ]  Incomplete conversion rates can be estimated and adjusted-for after sequencing by including an internal control in the sequencing library, such as  lambda phage  DNA (which is known to be unmethylated) or by aligning bisulfite sequencing reads to a known unmethylated region in the organism, such as the chloroplast genome. [ 26 ]


A major challenge in bisulfite sequencing is the degradation of DNA that takes place concurrently with the conversion. The conditions necessary for complete conversion, such as long incubation times, elevated temperature, and high bisulfite concentration, can lead to the degradation of about 90% of the incubated DNA. [ 27 ]   Given that the starting amount of DNA is often limited, such extensive degradation can be problematic. The degradation occurs as  depurinations  resulting in random strand breaks. [ 28 ]  Therefore, the longer the desired  PCR   amplicon , the more limited the number of intact template molecules will likely be. This could lead to the failure of the PCR amplification, or the loss of quantitatively accurate information on methylation levels resulting from the limited  sampling  of template molecules. Thus, it is important to assess the amount of DNA degradation resulting from the reaction conditions employed, and consider how this will affect the desired  amplicon . Techniques can also be used to minimize DNA degradation, such as cycling the incubation temperature. [ 28 ]


In 2020,  New England Biolabs  developed NEBNext Enzymatic Methyl-seq, an alternative enzymatic approach to minimize DNA damage. Instead of bisulfite,  APOBEC  is used to convert C into U. Distinction between C, 5mC, and 5hmC is granted by the further modifications that "protect" the modified bases from APOBEC. [ 29 ] [ 30 ]


A potentially significant problem following bisulfite treatment is incomplete  desulfonation  of  pyrimidine  residues due to inadequate alkalization of the solution. This may inhibit some  DNA polymerases , rendering subsequent PCR difficult. However, this situation can be avoided by monitoring the  pH  of the solution to ensure that desulfonation will be complete. [ 2 ]


A final concern is that bisulfite treatment greatly reduces the level of complexity in the sample, which can be problematic if multiple PCR reactions are to be performed (2006). [ 5 ]   Primer  design is more difficult, and inappropriate cross-hybridization is more frequent.


The advances in bisulfite sequencing have led to the possibility of applying them at a  genome -wide scale, where, previously, global measure of DNA methylation was feasible only using other techniques, such as  Restriction landmark genomic scanning . The mapping of the human  epigenome  is seen by many scientists as the logical follow-up to the completion of the  Human Genome Project . [ 31 ] [ 32 ]  This epigenomic information will be important in understanding how the function of the genetic sequence is implemented and regulated. Since the epigenome is less stable than the genome, it is thought to be important in  gene-environment interactions . [ 33 ]


Epigenomic mapping is inherently more complex than  genome sequencing , however, since the epigenome is much more variable than the  genome . One's epigenome varies with age, differs between tissues, is altered by environmental factors, and shows aberrations in diseases. Such rich epigenomic mapping, however, representing different ages, tissue types, and disease states, would yield valuable information on the normal function of  epigenetic  marks as well as the mechanisms leading to aging and disease.


Direct benefits of epigenomic mapping include probable advances in  cloning  technology. It is believed that failures to produce cloned animals with normal viability and lifespan result from inappropriate patterns of epigenetic marks. Also, aberrant methylation patterns are well characterized in many  cancers . Global hypomethylation results in decreased genomic stability, while local hypermethylation of  tumour suppressor gene   promoters  often accounts for their  loss of function . Specific patterns of methylation are indicative of specific cancer types, have  prognostic  value, and can help to guide the best course of treatment. [ 32 ]


Large-scale epigenome mapping efforts are under way around the world and have been organized under the  Human Epigenome Project . [ 33 ]  This is based on a multi-tiered strategy, whereby bisulfite sequencing is used to obtain high-resolution methylation profiles for a limited number of reference epigenomes, while less thorough analysis is performed on a wider spectrum of samples. This approach is intended to maximize the insight gained from a given amount of resources, as high-resolution genome-wide mapping remains a costly undertaking.


Gene-set analysis (for example using tools like DAVID and GoSeq) has been shown to be severely biased when applied to high-throughput methylation data (e.g. genome-wide bisulfite sequencing); it has been suggested that this can be corrected using sample label permutations or using a statistical model to control for differences in the numberes of CpG probes / CpG sites that target each gene. [ 34 ]


5-Methylcytosine and 5-hydroxymethylcytosine both read as a C in bisulfite sequencing. [ 24 ]  In oxidative bisulfite sequencing (oxBS), Tet is used to convert 5-hydroxymethylcytosine to 5-formylcytosine, which subsequently converts to uracil during bisulfite treatment. [ 35 ]  The only base that then reads as a C is 5‑methylcytosine, giving a map of the true methylation status in the DNA sample. Levels of 5‑hydroxymethylcytosine can also be quantified by measuring the difference between bisulfite and oxidative bisulfite sequencing.


Another method, Tet-assisted oxidative bisulfite sequencing (TAB-Seq) by  Chuan He  at the University of Chicago, converts the bases differently: 5hmC reads as C, while 5mC and C both read as T. To achieve this, 5hmC bases are first "protected" by conversion to β-glucosyl-5-hydroxymethylcytosine (5gmC). The Tet enzyme is introduced to convert all 5mC to 5caC. Bisulfite then converts both C and 5caC into uracil. 5gmC will be read out like C in PCR amplification. [ 36 ]


Bookmarking  (also " gene bookmarking " or " mitotic bookmarking ") refers to a potential mechanism of transmission of gene expression programs through cell division.


During  mitosis ,  gene transcription  is silenced and most  transcription factors  are removed from  chromatin . [ 1 ] [ 2 ]  The term bookmarking compares transcription to reading from a book. The pause in transcription during mitosis is like closing the book. "Molecular bookmarks" are the factors that allow transcription to resume in an orderly fashion in newborn cells following mitosis (when the book is re-opened).


Bookmarks fulfill the following criteria:


The term bookmarking was originally coined to describe the non-compaction of some  gene promoters  during  mitosis . [ 3 ]  More recently genome accessibility during interphase and mitosis has been directly compared on a genome-wide scale. [ 4 ]  While gene promoters tend to be better preserved than distal regulatory elements, substantial variation exists at individual sites.


Some patterns of  histone  modification and the presence of some histone variants on DNA remain unchanged during mitosis and have the potential to act as bookmarks. [ 5 ] [ 6 ] [ 7 ]  Patterns of  DNA methylation  are generally unchanged and also have the potential to function as bookmarks.


Notable transcription factors implicated in mitotic bookmarking include:


Cancer epigenetics  is the study of  epigenetic  modifications to the DNA of  cancer cells  that do not involve a change in the nucleotide sequence, but instead involve a change in the way the genetic code is expressed. Epigenetic mechanisms are necessary to maintain normal sequences of tissue specific gene expression and are crucial for normal development. [ 1 ]  They may be just as important, if not even more important, than  genetic mutations  in a cell's transformation to cancer. The disturbance of epigenetic processes in cancers, can lead to a loss of  expression of genes  that occurs about 10 times more frequently by transcription silencing (caused by epigenetic promoter hypermethylation of  CpG islands ) than by mutations. As Vogelstein et al. points out, in a colorectal cancer there are usually about 3 to 6 driver mutations and 33 to 66  hitchhiker  or passenger mutations. [ 2 ]  However, in colon tumors compared to adjacent normal-appearing colonic mucosa, there are about 600 to 800 heavily methylated CpG islands in the promoters of genes in the tumors while these CpG islands are not methylated in the adjacent mucosa. [ 3 ] [ 4 ] [ 5 ]  Manipulation of epigenetic alterations holds great promise for cancer prevention, detection, and therapy. [ 6 ] [ 7 ]  In different types of cancer, a variety of epigenetic mechanisms can be perturbed, such as the silencing of  tumor suppressor genes  and  activation  of  oncogenes  by altered  CpG island   methylation  patterns,  histone  modifications, and dysregulation of  DNA binding proteins . There are several medications which have epigenetic impact, that are now used in a number of these diseases.


In somatic cells, patterns of DNA methylation are in general transmitted to daughter cells with high fidelity. [ 8 ]  Typically, this methylation only occurs at cytosines that are located 5' to guanosine in the CpG dinucleotides of higher order eukaryotes. [ 9 ]  However, epigenetic  DNA methylation  differs between normal cells and tumor cells in humans. The "normal"  CpG  methylation profile is often inverted in cells that become tumorigenic. [ 10 ]  In normal cells,  CpG islands  preceding  gene promoters  are generally unmethylated, and tend to be transcriptionally active, while other individual CpG  dinucleotides  throughout the genome tend to be methylated. However, in cancer cells,  CpG islands  preceding  tumor suppressor gene  promoters are often hypermethylated, while CpG methylation of oncogene promoter regions and parasitic  repeat sequences  is often decreased. [ 11 ]


Hypermethylation of tumor suppressor gene promoter regions can result in silencing of those genes. This type of epigenetic mutation allows cells to grow and reproduce uncontrollably, leading to tumorigenesis. [ 10 ]  The addition of methyl groups to cytosines causes the DNA to coil tightly around the histone proteins, resulting in DNA that can not undergo transcription (transcriptionally silenced DNA). Genes commonly found to be transcriptionally silenced due to promoter hypermethylation include: Cyclin-dependent kinase inhibitor  p16 , a cell-cycle inhibitor;  MGMT , a  DNA repair  gene;  APC , a cell cycle regulator;  MLH1 , a DNA-repair gene; and  BRCA1 , another DNA-repair gene. [ 10 ] [ 12 ]  Indeed, cancer cells can become addicted to the transcriptional silencing, due to promoter hypermethylation, of some key tumor suppressor genes, a process known as epigenetic addiction. [ 13 ]


Hypomethylation of CpG dinucleotides in other parts of the genome leads to  chromosome instability  due to mechanisms such as loss of imprinting and reactivation of  transposable elements . [ 14 ] [ 15 ] [ 16 ] [ 17 ]  Loss of imprinting of  insulin-like growth factor  gene (IGF2) increases risk of  colorectal cancer  and is associated with  Beckwith-Wiedemann syndrome  which significantly increases the risk of cancer for newborns. [ 18 ]  In healthy cells, CpG dinucleotides of lower densities are found within coding and  non-coding  intergenic regions. Expression of some repetitive sequences and meiotic recombination at  centromeres  are repressed through methylation  [ 19 ]


The entire genome of a cancerous cell contains significantly less  methylcytosine  than the genome of a healthy cell. In fact, cancer cell genomes have 20-50% less methylation at individual CpG dinucleotides across the genome. [ 14 ] [ 15 ] [ 16 ] [ 17 ]  CpG islands found in promoter regions are usually protected from DNA methylation. In cancer cells CpG islands are hypomethylated  [ 20 ]  The regions flanking CpG islands called CpG island shores are where most DNA methylation occurs in the CpG dinucleotide context. Cancer cells are deferentially methylated at CpG island shores. In cancer cells, hypermethylation in the CpG island shores move into CpG islands, or hypomethylation of CpG islands move into CpG island shores eliminating sharp epigenetic boundaries between these genetic elements. [ 21 ]  In cancer cells "global hypomethylation" due to disruption in  DNA methyltransferases  (DNMTs) may promote  mitotic recombination  and  chromosome rearrangement , ultimately resulting in  aneuploidy  when the chromosomes fail to separate properly during  mitosis . [ 14 ] [ 15 ] [ 16 ] [ 17 ]


CpG island methylation is important in regulation of gene expression, yet cytosine methylation can lead directly to destabilizing genetic mutations and a precancerous cellular state. Methylated cytosines make  hydrolysis  of the  amine group  and spontaneous conversion to  thymine  more favorable. They can cause aberrant recruitment of  chromatin  proteins. Cytosine methylations change the amount of UV light absorption of the nucleotide base, creating  pyrimidine dimers . When mutation results in loss of  heterozygosity  at tumor suppressor gene sites, these genes may become inactive. Single base pair mutations during replication can also have detrimental effects. [ 12 ]


Eukaryotic DNA  has a complex structure. It is generally wrapped around special proteins called  histones  to form a structure called a  nucleosome . A nucleosome consists of 2 sets of 4 histones:  H2A ,  H2B ,  H3 , and  H4 . Additionally,  histone H1  contributes to  DNA packaging  outside of the nucleosome. Certain histone modifying enzymes can add or remove functional groups to the histones, and these modifications influence the level of transcription of the genes wrapped around those histones and the level of DNA replication. Histone modification profiles of healthy and cancerous cells tend to differ.


In comparison to healthy cells, cancerous cells exhibit decreased monoacetylated and trimethylated forms of  histone H4  (decreased H4ac and H4me3). [ 22 ]  Additionally, mouse models have shown that a decrease in histone H4R3 asymmetric dimethylation (H4R3me2a) of the  p19ARF  promoter is correlated with more advanced cases of tumorigenesis and metastasis. [ 23 ]  In mouse models, the loss of histone H4 acetylation and trimethylation increases as tumor growth continues. [ 22 ]  Loss of histone H4 Lysine 16 acetylation ( H4K16ac ), which is a mark of aging at the  telomeres , specifically loses its acetylation. Some scientists hope this particular loss of histone acetylation might be battled with a  histone deacetylase  (HDAC) inhibitor specific for  SIRT1 , an HDAC specific for H4K16. [ 10 ] [ 24 ]


Other histone marks associated with  tumorigenesis  include increased deacetylation (decreased acetylation) of histones H3 and H4, decreased trimethylation of histone H3 Lysine 4 ( H3K4me3 ), and increased monomethylation of histone H3 Lysine 9 ( H3K9me1 ) and trimethylation of histone H3 Lysine 27 ( H3K27me3 ). These histone modifications can silence tumor suppressor genes despite the drop in methylation of the gene's CpG island (an event that normally activates genes). [ 25 ] [ 26 ]


Some research has focused on blocking the action of  BRD4  on acetylated histones, which has been shown to increase the expression of the  Myc  protein, implicated in several cancers. The development process of the drug to bind to BRD4 is noteworthy for the collaborative, open approach the team is taking. [ 27 ]


The tumor suppressor gene  p53  regulates  DNA repair  and can induce apoptosis in dysregulated cells. E Soto-Reyes and F Recillas-Targa elucidated the importance of the  CTCF  protein in regulating p53 expression. [ 28 ]  CTCF, or CCCTC binding factor, is a  zinc finger  protein that insulates the p53  promoter  from accumulating repressive histone marks. In certain types of cancer cells, the CTCF protein does not bind normally, and the p53 promoter accumulates repressive histone marks, causing p53 expression to decrease. [ 28 ]


Mutations in the epigenetic machinery itself may occur as well, potentially responsible for the changing epigenetic profiles of cancerous cells. The  histone  variants of the H2A family are highly conserved in mammals, playing critical roles in regulating many nuclear processes by altering  chromatin  structure. One of the key H2A variants, H2A.X, marks DNA damage, facilitating the recruitment of DNA repair proteins to restore genomic integrity. Another variant, H2A.Z, plays an important role in both gene activation and repression. A high level of H2A.Z expression is detected in many cancers and is significantly associated with cellular proliferation and genomic instability. [ 11 ]  Histone variant macroH2A1 is important in the pathogenesis of many types of cancers, for instance in hepatocellular carcinoma. [ 29 ]  Other mechanisms include a decrease in H4K16ac may be caused by either a decrease in activity of a  histone acetyltransferases  (HATs) or an increase in deacetylation by SIRT1. [ 10 ]  Likewise, an inactivating  frameshift  mutation in  HDAC2 , a  histone deacetylase  that acts on many histone-tail  lysines , has been associated with cancers showing altered histone acetylation patterns. [ 30 ]  These findings indicate a promising mechanism for altering epigenetic profiles through enzymatic inhibition or enhancement. A new emerging field that captures toxicological epigenetic changes as a result of the exposure to different compounds (drugs, food, and environment) is toxicoepigenetics. In this field, there is growing interest in mapping changes in histone modifications and their possible consequences. [ 31 ]


DNA damage , caused by UV light,  ionizing radiation , environmental toxins, and metabolic chemicals, can also lead to genomic instability and cancer. The DNA damage response to double strand  DNA breaks  (DSB) is mediated in part by histone modifications. At a DSB,  MRE11 - RAD50 - NBS1  (MRN) protein complex recruits  ataxia telangiectasia  mutated (ATM) kinase which phosphorylates Serine 129 of Histone 2A. MDC1, mediator of DNA damage checkpoint 1, binds to the phosphopeptide, and phosphorylation of  H2AX  may spread by a positive feedback loop of MRN-ATM recruitment and phosphorylation.  TIP60  acetylates the  γH2AX , which is then  polyubiquitylated .  RAP80 , a subunit of the DNA repair breast cancer type 1 susceptibility protein complex ( BRCA1 -A), binds  ubiquitin  attached to histones. BRCA1-A activity arrests the  cell cycle  at the  G2/M checkpoint , allowing time for DNA repair, or  apoptosis  may be initiated. [ 32 ]


In mammals,  microRNAs  (miRNAs) regulate about 60% of the  transcriptional  activity of protein-encoding genes. [ 33 ]  Some miRNAs also undergo methylation-associated silencing in cancer cells. [ 34 ] [ 35 ]  Let-7 and miR15/16 play important roles in down-regulating  RAS  and  BCL2   oncogenes , and their silencing occurs in cancer cells. [ 18 ]  Decreased expression of miR-125b1, a miRNA that functions as a  tumor suppressor , was observed in prostate,  ovarian , breast and  glial  cell cancers. In vitro experiments have shown that miR-125b1 targets two genes,  HER2/neu  and  ESR1 , that are linked to breast cancer. DNA methylation, specifically hypermethylation, is one of the main ways that the miR-125b1 is epigenetically silenced. In patients with breast cancer, hypermethylation of CpG islands located proximal to the transcription start site was observed. Loss of  CTCF  binding and an increase in repressive histone marks, H3K9me3 and H3K27me3, correlates with  DNA methylation  and miR-125b1 silencing. Mechanistically, CTCF may function as a boundary element to stop the spread of DNA methylation. Results from experiments conducted by Soto-Reyes et al. [ 36 ]  indicate a negative effect of methylation on the function and expression of miR-125b1. Therefore, they concluded that DNA methylation has a part in silencing the gene. Furthermore, some miRNA's are epigenetically silenced early on in breast cancer, and therefore these miRNA's could potentially be useful as tumor markers. [ 36 ]  The epigenetic silencing of miRNA genes by aberrant DNA methylation is a frequent event in cancer cells; almost one third of miRNA promoters active in normal mammary cells were found hypermethylated in breast cancer cells - that is a several fold greater proportion than is usually observed for protein coding genes. [ 37 ]


Dysregulation of metabolism allows tumor cells to generate needed building blocks as well as to modulate epigenetic marks to support cancer initiation and progression. Cancer-induced metabolic changes alter the epigenetic landscape, especially modifications on histones and DNA, thereby promoting malignant transformation, adaptation to inadequate nutrition, and metastasis. In order to satisfy the biosynthetic demands of cancer cells, metabolic pathways are altered by manipulating oncogenes and tumor suppressive genes concurrently. [ 38 ]  The accumulation of certain metabolites in cancer can target epigenetic enzymes to globally alter the epigenetic landscape. Cancer-related metabolic changes lead to locus-specific recoding of epigenetic marks. Cancer epigenetics can be precisely reprogramed by cellular metabolism through 1) dose-responsive modulation of cancer epigenetics by metabolites; 2) sequence-specific recruitment of metabolic enzymes; and 3) targeting of epigenetic enzymes by nutritional signals. [ 38 ]  In addition to modulating metabolic programming on a molecular level, there are microenvironmental factors that can influence and effect metabolic recoding. These influences include nutritional, inflammatory, and the immune response of malignant tissues.


DNA damage appears to be the primary underlying cause of cancer. [ 39 ] [ 40 ]  If DNA repair is deficient, DNA damage tends to accumulate. Such excess DNA damage can increase  mutational  errors during  DNA replication  due to error-prone  translesion synthesis . Excess DNA damage can also increase  epigenetic  alterations due to errors during DNA repair. [ 41 ] [ 42 ]  Such mutations and epigenetic alterations can give rise to  cancer  (see  malignant neoplasms ).


Germ line  mutations in DNA repair genes cause only 2–5% of  colon cancer  cases. [ 43 ]  However, altered expression of microRNAs, causing DNA repair deficiencies, are frequently associated with cancers and may be an important  causal  factor for these cancers.


Over-expression of certain miRNAs may directly reduce expression of specific DNA repair proteins. Wan et al. [ 44 ]  referred to 6 DNA repair genes that are directly targeted by the miRNAs indicated in parentheses:  ATM  (miR-421),  RAD52  (miR-210, miR-373),  RAD23B  (miR-373),  MSH2  (miR-21),  BRCA1  (miR-182) and  P53  (miR-504, miR-125b). More recently, Tessitore et al. [ 45 ]  listed further DNA repair genes that are directly targeted by additional miRNAs, including  ATM  (miR-18a, miR-101),  DNA-PK  (miR-101),  ATR  (miR-185),  Wip1  (miR-16),  MLH1, MSH2  and  MSH6  (miR-155),  ERCC3  and  ERCC4  (miR-192) and  UNG2  (mir-16, miR-34c and miR-199a). Of these miRNAs, miR-16, miR-18a, miR-21, miR-34c, miR-125b, miR-101, miR-155, miR-182, miR-185 and miR-192 are among those identified by Schnekenburger and Diederich [ 46 ]  as over-expressed in colon cancer through epigenetic hypomethylation. Over expression of any one of these miRNAs can cause reduced expression of its target DNA repair gene.


Up to 15% of the  MLH1 -deficiencies in sporadic colon cancers appeared to be due to over-expression of the microRNA  miR-155 , which represses MLH1 expression. [ 47 ]  However, the majority of 68 sporadic colon cancers with reduced expression of the  DNA mismatch repair  protein  MLH1  were found to be deficient due to  epigenetic methylation  of the CpG island of the  MLH1  gene. [ 48 ]


In 28% of glioblastomas, the MGMT DNA repair protein is deficient but the  MGMT  promoter is not methylated. [ 49 ]  In the glioblastomas without methylated  MGMT  promoters, the level of microRNA miR-181d is  inversely correlated  with protein expression of MGMT and the direct target of miR-181d is the MGMT  mRNA  3'UTR (the  three prime untranslated region  of MGMT mRNA). [ 49 ]  Thus, in 28% of glioblastomas, increased expression of miR-181d and reduced expression of DNA repair enzyme MGMT may be a causal factor. In 29–66% [ 49 ] [ 50 ]  of  glioblastomas , DNA repair is deficient due to epigenetic methylation of the  MGMT  gene, which reduces protein expression of MGMT.


High mobility group A ( HMGA ) proteins, characterized by an  AT-hook , are small, nonhistone, chromatin-associated proteins that can modulate transcription. MicroRNAs control the expression of  HMGA  proteins, and these proteins ( HMGA1  and  HMGA2 ) are architectural chromatin transcription-controlling elements. Palmieri et al. [ 51 ]  showed that, in normal tissues,  HGMA1  and  HMGA2  genes are targeted (and thus strongly reduced in expression) by  miR-15 ,  miR-16 ,  miR-26a ,  miR-196a2  and  Let-7a .


HMGA expression is almost undetectable in differentiated adult tissues but is elevated in many cancers. HGMA proteins are polypeptides of ~100 amino acid residues characterized by a modular sequence organization. These proteins have three highly positively charged regions, termed  AT hooks , that bind the minor groove of AT-rich DNA stretches in specific regions of DNA. Human neoplasias, including thyroid, prostatic, cervical, colorectal, pancreatic and ovarian carcinoma, show a strong increase of HMGA1a and HMGA1b proteins. [ 52 ]  Transgenic mice with HMGA1 targeted to lymphoid cells develop aggressive lymphoma, showing that high HMGA1 expression is not only associated with cancers, but that the  HMGA1  gene can act as an oncogene to cause cancer. [ 53 ]  Baldassarre et al., [ 54 ]  showed that HMGA1 protein binds to the promoter region of DNA repair gene  BRCA1  and inhibits  BRCA1  promoter activity. They also showed that while only 11% of breast tumors had hypermethylation of the  BRCA1  gene, 82% of aggressive breast cancers have low BRCA1 protein expression, and most of these reductions were due to chromatin remodeling by high levels of HMGA1 protein.


HMGA2 protein specifically targets the promoter of  ERCC1 , thus reducing expression of this DNA repair gene. [ 55 ]  ERCC1 protein expression was deficient in 100% of 47 evaluated colon cancers (though the extent to which HGMA2 was involved is unknown). [ 56 ]


Palmieri et al. [ 51 ]  showed that each of the miRNAs that target  HMGA  genes are drastically reduced in almost all human pituitary adenomas studied, when compared with the normal pituitary gland. Consistent with the down-regulation of these HMGA-targeting miRNAs, an increase in the HMGA1 and HMGA2-specific mRNAs was observed. Three of these microRNAs (miR-16, miR-196a and Let-7a) [ 46 ] [ 57 ]  have methylated promoters and therefore low expression in colon cancer. For two of these, miR-15 and miR-16, the coding regions are epigenetically silenced in cancer due to  histone deacetylase  activity. [ 58 ]  When these microRNAs are expressed at a low level, then HMGA1 and HMGA2 proteins are expressed at a high level. HMGA1 and HMGA2 target (reduce expression of)  BRCA1  and  ERCC1  DNA repair genes. Thus DNA repair can be reduced, likely contributing to cancer progression. [ 40 ]


The chart in this section shows some frequent DNA damaging agents, examples of DNA lesions they cause, and the pathways that deal with these DNA damages. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes. [ 59 ]  Of these, 83 are directly employed in repairing the 5 types of DNA damages illustrated in the chart.


Some of the more well studied genes central to these repair processes are shown in the chart. The gene designations shown in red, gray or cyan indicate genes frequently epigenetically altered in various types of cancers. Wikipedia articles on each of the genes highlighted by red, gray or cyan describe the epigenetic alteration(s) and the cancer(s) in which these epimutations are found. Two broad experimental survey articles [ 60 ] [ 61 ]  also document most of these epigenetic DNA repair deficiencies in cancers.


Red-highlighted genes are frequently reduced or silenced by epigenetic mechanisms in various cancers. When these genes have low or absent expression, DNA damages can accumulate. Replication errors past these damages (see  translesion synthesis ) can lead to increased mutations and, ultimately, cancer. Epigenetic repression of DNA repair genes in  accurate  DNA repair pathways appear to be central to  carcinogenesis .


The two gray-highlighted genes  RAD51  and  BRCA2 , are required for  homologous recombinational  repair. They are sometimes epigenetically over-expressed and sometimes under-expressed in certain cancers. As indicated in the Wikipedia articles on  RAD51  and  BRCA2 , such cancers ordinarily have epigenetic deficiencies in other DNA repair genes. These repair deficiencies would likely cause increased unrepaired DNA damages. The over-expression of  RAD51  and  BRCA2  seen in these cancers may reflect selective pressures for compensatory  RAD51  or  BRCA2  over-expression and increased homologous recombinational repair to at least partially deal with such excess DNA damages. In those cases where  RAD51  or  BRCA2  are under-expressed, this would itself lead to increased unrepaired DNA damages. Replication errors past these damages (see  translesion synthesis ) could cause increased mutations and cancer, so that under-expression of  RAD51  or  BRCA2  would be carcinogenic in itself.


Cyan-highlighted genes are in the  microhomology-mediated end joining  (MMEJ) pathway and are up-regulated in cancer. MMEJ is an additional error-prone  inaccurate  repair pathway for double-strand breaks. In MMEJ repair of a double-strand break, an homology of 5-25 complementary base pairs between both paired strands is sufficient to align the strands, but mismatched ends (flaps) are usually present. MMEJ removes the extra nucleotides (flaps) where strands are joined, and then ligates the strands to create an intact DNA double helix. MMEJ almost always involves at least a small deletion, so that it is a mutagenic pathway. [ 62 ]   FEN1 , the flap endonuclease in MMEJ, is epigenetically increased by promoter hypomethylation and is over-expressed in the majority of cancers of the breast, [ 63 ]  prostate, [ 64 ]  stomach, [ 65 ] [ 66 ]  neuroblastomas, [ 67 ]  pancreas, [ 68 ]  and lung. [ 69 ]  PARP1 is also over-expressed when its promoter region  ETS  site is epigenetically hypomethylated, and this contributes to progression to endometrial cancer, [ 70 ]  BRCA-mutated ovarian cancer, [ 71 ]  and BRCA-mutated serous ovarian cancer. [ 72 ]  Other genes in the  MMEJ  pathway are also over-expressed in a number of cancers (see  MMEJ  for summary), and are also shown in blue.


Deficiencies in DNA repair proteins that function in accurate DNA repair pathways increase the risk of mutation. Mutation rates are strongly increased in cells with mutations in  DNA mismatch repair [ 73 ] [ 74 ]  or in  homologous recombinational  repair (HRR). [ 75 ]  Individuals with inherited mutations in any of 34 DNA repair genes are at increased risk of cancer (see  DNA repair defects and increased cancer risk ).


In sporadic cancers, a deficiency in DNA repair is occasionally found to be due to a mutation in a DNA repair gene, but much more frequently reduced or absent expression of DNA repair genes is due to epigenetic alterations that reduce or silence gene expression. For example, for 113 colorectal cancers examined in sequence, 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 (an epigenetic alteration). [ 76 ]  Similarly, out of 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene  PMS2  expression, PMS2 protein was deficient in 6 due to mutations in the  PMS2  gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1). [ 48 ]  In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1. [ 47 ]


Epigenetic defects in DNA repair genes are frequent in cancers. In the table, multiple cancers were evaluated for reduced or absent expression of the DNA repair gene of interest, and the frequency shown is the frequency with which the cancers had an epigenetic deficiency of gene expression. Such epigenetic deficiencies likely arise early in  carcinogenesis , since they are also frequently found (though at somewhat lower frequency) in the  field defect  surrounding the cancer from which the cancer likely arose (see Table).


It appears that cancers may frequently be initiated by an epigenetic reduction in expression of one or more DNA repair enzymes. Reduced DNA repair likely allows accumulation of DNA damages. Error prone  translesion synthesis  past some of these DNA damages may give rise to a mutation with a selective advantage. A clonal patch with a selective advantage may grow and out-compete neighboring cells, forming a  field defect . While there is no obvious  selective advantage  for a cell to have reduced DNA repair, the  epimutation  of the DNA repair gene may be carried along as a passenger when the cells with the selectively advantageous mutation are replicated. In the cells carrying both the epimutation of the DNA repair gene and the mutation with the selective advantage, further DNA damages will accumulate, and these could, in turn, give rise to further mutations with still greater selective advantages. Epigenetic defects in DNA repair may thus contribute to the characteristic high frequency of mutations in the genomes of cancers, and cause their carcinogenic progression.


Cancers have high levels of  genome instability , associated with a high frequency of  mutations . A high frequency of genomic mutations increases the likelihood of particular mutations occurring that activate oncogenes and inactivate tumor suppressor genes, leading to  carcinogenesis . On the basis of  whole genome sequencing , cancers are found to have thousands to hundreds of thousands of mutations in their whole genomes. [ 87 ]  (Also see  Mutation frequencies in cancers .) By comparison, the mutation frequency in the whole genome between generations for humans (parent to child) is about 70 new mutations per generation. [ 88 ] [ 89 ]  In the protein coding regions of the genome, there are only about 0.35 mutations between parent/child generations (less than one mutated protein per generation). [ 90 ]  Whole genome sequencing in blood cells for a pair of identical twin 100-year-old centenarians only found 8 somatic differences, though somatic variation occurring in less than 20% of blood cells would be undetected. [ 91 ]


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. [ 41 ] [ 42 ] [ 92 ] [ 93 ]  The DNA damages that accumulate due to epigenetic DNA repair defects 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. [ 94 ]  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 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 epigenetic promoter methylation alterations in tumors. Some of these epigenetic alterations may contribute to cancer progression.


A variety of compounds are considered as epigenetic  carcinogens —they result in an increased incidence of tumors, but they do not show  mutagen  activity (toxic compounds or pathogens that cause tumors incident to increased regeneration should also be excluded). Examples include  diethylstilbestrol ,  arsenite ,  hexachlorobenzene , and  nickel  compounds.


Many  teratogens  exert specific effects on the fetus by epigenetic mechanisms. [ 95 ] [ 96 ]  While epigenetic effects may preserve the effect of a teratogen such as  diethylstilbestrol  throughout the life of an affected child, the possibility of birth defects resulting from exposure of fathers or in second and succeeding generations of offspring has generally been rejected on theoretical grounds and for lack of evidence. [ 97 ]  However, a range of male-mediated abnormalities have been demonstrated, and more are likely to exist. [ 98 ]  FDA label information for Vidaza, a formulation of  5-azacitidine  (an unmethylatable analog of cytidine that causes hypomethylation when incorporated into DNA) states that "men should be advised not to father a child" while using the drug, citing evidence in treated male mice of reduced fertility, increased  embryo loss , and abnormal embryo development. [ 99 ]  In rats, endocrine differences were observed in offspring of males exposed to morphine. [ 100 ]  In mice, second generation effects of diethylstilbesterol have been described occurring by epigenetic mechanisms. [ 101 ]


Melanoma  is a deadly skin cancer that originates from melanocytes. Several  epigenetic alterations  are known to play a role in the transition of melanocytes to melanoma cells. This includes DNA methylation that can be inherited without making changes to the DNA sequence, as well as silencing the tumor suppressor genes in the epidermis that have been exposed to UV radiation for periods of time. [ 102 ]  The silencing of tumor suppressor genes leads to  photocarcinogenesis  which is associated to epigenetic alterations in DNA methylation, DNA methyltransferases, and histone acetylation. [ 102 ]  These alterations are the consequence of deregulation of their corresponding enzymes. Several histone methyltransferases and demethylases are among these enzymes. [ 103 ]


Prostate cancer  kills around 35,000 men yearly, and about 220,000 men are diagnosed with prostate cancer per year, in North America alone. [ 104 ]  Prostate cancer is the second leading cause of cancer-caused fatalities in men, and within a man's lifetime, one in six men will have the disease. [ 104 ]  Alterations in histone acetylation and DNA methylation occur in various genes influencing prostate cancer, and have been seen in genes involved in hormonal response. [ 105 ]  More than 90% of prostate cancers show  gene silencing  by  CpG island hypermethylation  of the  GSTP1  gene  promoter , which protects  prostate  cells from genomic damage that is caused by different oxidants or  carcinogens . [ 106 ]  Real-time methylation-specific  polymerase chain reaction  (PCR) suggests that many other genes are also hypermethylated. [ 106 ]  Gene expression in the prostate may be modulated by nutrition and lifestyle changes. [ 107 ]


The second most common malignant tumor in women is invasive  cervical cancer  (ICC) and more than 50% of all  invasive cervical cancer  (ICC) is caused by  oncongenic   human papillomavirus  16 ( HPV16 ). [ 108 ]  Furthermore,  cervix intraepithelial neoplasia  (CIN) is primarily caused by oncogenic HPV16. [ 108 ]  As in many cases, the causative factor for cancer does not always take a direct route from infection to the development of cancer. Genomic methylation patterns have been associated with invasive cervical cancer. Within the  HPV16L1 region , 14 tested CpG sites have significantly higher methylation in  CIN3+  than in HPV16 genomes of women without  CIN3 . [ 108 ]  Only 2/16 CpG sites tested in HPV16 upstream regulatory region were found to have association with increased methylation in CIN3+. [ 108 ]  This suggests that the direct route from infection to cancer is sometimes detoured to a precancerous state in cervix intraepithelial neoplasia. Additionally, increased CpG site methylation was found in low levels in most of the five host nuclear genes studied, including 5/5  TERT , 1/4  DAPK1 , 2/5  RARB ,  MAL , and  CADM1 . [ 108 ]  Furthermore, 1/3 of CpG sites in  mitochondrial DNA  were associated with increased methylation in CIN3+. [ 108 ]  Thus, a correlation exists between CIN3+ and increased methylation of CpG sites in the HPV16 L1 open reading frame. [ 108 ]  This could be a potential  biomarker  for future screens of cancerous and precancerous cervical disease. [ 108 ]


Recent studies have shown that the  mixed-lineage leukemia  (MLL) gene causes  leukemia  by rearranging and fusing with other genes in different chromosomes, which is a process under epigenetic control. [ 109 ]  Mutations in MLL block the correct regulatory regions in leukemia associated translocations or insertions causing malignant transformation controlled by HOX genes. [ 110 ]  This is what leads to the increase in white blood cells. Leukemia related genes are managed by the same pathways that control epigenetics, signaling transduction, transcriptional regulation, and energy metabolism. It was indicated that infections, electromagnetic fields and increased birth weight can contribute to being the causes of leukemia. [ 111 ]


There are about 15,000 new cases of sarcoma in the US each year, and about 6,200 people were projected to die of sarcoma in the US in 2014. [ 112 ]  Sarcomas comprise a large number of rare, histogenetically heterogeneous mesenchymal tumors that, for example, include chondrosarcoma, Ewing's sarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, synovial sarcoma, and (alveolar and embryonal) rhabdomyosarcoma. Several oncogenes and tumor suppressor genes are epigenetically altered in sarcomas. These include APC, CDKN1A, CDKN2A, CDKN2B, Ezrin, FGFR1, GADD45A, MGMT, STK3, STK4, PTEN, RASSF1A, WIF1, as well as several miRNAs. [ 113 ]  Expression of epigenetic modifiers such as that of the BMI1 component of the PRC1 complex is deregulated in chondrosarcoma, Ewing's sarcoma, and osteosarcoma, and expression of the EZH2 component of the PRC2 complex is altered in Ewing's sarcoma and rhabdomyosarcoma. Similarly, expression of another epigenetic modifier, the LSD1 histone demethylase, is increased in chondrosarcoma, Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma. Drug targeting and inhibition of EZH2 in Ewing's sarcoma, [ 114 ]  or of LSD1 in several sarcomas, [ 115 ]  inhibits tumor cell growth in these sarcomas.


Lung cancer is the second most common type of cancer and leading cause of death in men and women in the United States, it is estimated that there is about 216,000 new cases and 160,000 deaths due to lung cancer. [ 116 ]


Initiation and progression of lung carcinoma is the result of the interaction between genetic, epigenetic and environmental factors. Most cases of lung cancer are because of genetic mutations in  EGFR ,  KRAS ,  STK11  (also known as  LKB1 ),  TP53  (also known as  p53 ), and  CDKN2A  (also known as  p16  or  INK4a ) [ 117 ] [ 118 ] [ 119 ]  with the most common type of lung cancer being an inactivation at p16. p16 is a tumor suppressor protein that occurs in mostly in humans the functional significance of the mutations was tested on many other species including mice, cats, dogs, monkeys and cows the identification of these multiple nonoverlapping clones was not entirely surprising since the reduced stringency hybridization of a zoo blot with the same probe also revealed 10-15 positive EcoRI fragments in all species tested. [ 120 ]


Previously, epigenetic profiles were limited to individual genes under scrutiny by a particular research team. Recently, however, scientists have been moving toward a more genomic approach to determine an entire genomic profile for cancerous versus healthy cells. [ 10 ]


Popular approaches for measuring CpG methylation in cells include:


Since bisulfite sequencing is considered the gold standard for measuring CpG methylation, when one of the other methods is used, results are usually confirmed using bisulfite sequencing[1].
Popular approaches for determining  histone modification  profiles in cancerous versus healthy cells include: [ 10 ]


Researchers are hoping to identify specific epigenetic profiles of various types and subtypes of cancer with the goal of using these profiles as tools to diagnose individuals more specifically and accurately. [ 10 ]  Since epigenetic profiles change, scientists would like to use the different epigenomic profiles to determine the stage of development or level of aggressiveness of a particular cancer in patients. For example, hypermethylation of the genes coding for  Death-Associated Protein Kinase  (DAPK), p16, and  Epithelial Membrane Protein 3  (EMP3) have been linked to more aggressive forms of  lung ,  colorectal , and  brain cancers . [ 17 ]  This type of knowledge can affect the way that doctors will diagnose and choose to treat their patients.


Another factor that will influence the treatment of patients is knowing how well they will respond to certain treatments. Personalized epigenomic profiles of cancerous cells can provide insight into this field. For example,  MGMT  is an enzyme that reverses the addition of  alkyl groups  to the nucleotide  guanine . [ 121 ]  Alkylating guanine, however, is the mechanism by which several  chemotherapeutic drugs  act in order to disrupt DNA and cause  cell death . [ 122 ] [ 123 ] [ 124 ] [ 125 ]  Therefore, if the gene encoding MGMT in cancer cells is hypermethylated and in effect silenced or repressed, the chemotherapeutic drugs that act by methylating guanine will be more effective than in cancer cells that have a functional MGMT enzyme.


Epigenetic  biomarkers  can also be utilized as tools for molecular prognosis. In primary tumor and  mediastinal   lymph node   biopsy  samples, hypermethylation of both  CDKN2A  and  CDH13  serves as the marker for increased risk of faster cancer relapse and higher death rate of patients. [ 126 ]


Epigenetic control of the proto-onco regions and the tumor suppressor sequences by conformational changes in histones plays a role in the formation and progression of cancer. [ 127 ]  Pharmaceuticals that reverse epigenetic changes might have a role in a variety of cancers. [ 105 ] [ 127 ] [ 128 ]


Recently, it is evidently known that associations between specific cancer histotypes and epigenetic changes can facilitate the development of novel epi-drugs. [ 129 ]  Drug development has focused mainly on modifying  DNA methyltransferase ,  histone acetyltransferase  (HAT) and  histone deacetylase  (HDAC). [ 130 ]


Drugs that specifically target the inverted methylation pattern of cancerous cells include the  DNA methyltransferase  inhibitors  azacitidine [ 131 ] [ 132 ]  and  decitabine . [ 133 ] [ 134 ]  These hypomethylating agents are used to treat  myelodysplastic syndrome , [ 135 ]  a  blood cancer  produced by abnormal  bone marrow stem cells . [ 12 ]  These agents inhibit all three types of active DNA methyltransferases, and had been thought to be highly toxic, but proved to be effective when used in low dosage, reducing progression of myelodysplastic syndrome to  leukemia . [ 136 ]


Histone deacetylase  (HDAC) inhibitors show efficacy in treatment of  T cell lymphoma . two HDAC inhibitors,  vorinostat  and  romidepsin , have been approved by the  Food and Drug Administration . [ 137 ] [ 138 ]  However, since these HDAC inhibitors alter the  acetylation  state of many proteins in addition to the histone of interest, knowledge of the underlying mechanism at the molecular level of patient response is required to enhance the efficiency of using such inhibitors as treatment. [ 18 ]  Treatment with HDAC inhibitors has been found to promote  gene reactivation  after DNA methyl-transferases inhibitors have repressed transcription. [ 139 ]   Panobinostat  is approved for certain situations in  myeloma . [ 140 ]


Other pharmaceutical targets in research are  histone lysine methyltransferases  (KMT) and protein arginine methyltransferases (PRMT). [ 141 ]  Preclinical study has suggested that  lunasin  may have potentially beneficial epigenetic effects. [ 142 ]


Epigenetic therapy of cancer has shown to be a promising and possible treatment of cancerous cells. Epigenetic inactivation is an ideal target for cancerous cells because it targets genes imperative for controlling cell growth, specifically cancer cell growth. It is crucial for these genes to be reactivated in order to suppress tumor growth and sensitize the cells to cancer curing therapies. [ 143 ]  Typical chemotherapy aims to kill and eliminate cancer cells in the body. Cancer initiated by genetic alterations of cells are typically permanent and nearly impossible to reverse, this differs from epigenetic cancer because the cancer causing epigenetic aberrations have the capability of being reversed, and the cells being returned to normal function. The ability for epigenetic mechanisms to be reversed is attributed to the fact that the coding of the genes being silenced through histone and DNA modification is not being altered. [ 144 ]


There are two primary types of epigenetic alterations in cancer cells, these are known as DNA methylation and Histone modification. It is the goal of epigenetic therapies to inhibit these alterations. DNA Methyltransferases (DNMTs) and Histone Deacetylases (HDAC) are the primary catalyzes of the epigenetic modifications of cancer cells. [ 145 ]  The goal for epigenetic therapies is to repress this methylation and reverse these modifications in order to create a new epigenome where cancer cells no longer thrive and tumor suppression is the new function. Synthetic drugs are used as tools in epigenetic therapies due to their ability to inhibit enzymes causing histone modifications and DNA methylations. Combination therapy is one method of epigenetic therapy which involves the use of more than one synthetic drug, these drugs include a low dose DNMT inhibitor as well as an HDAC inhibitor. Together, these drugs are able to target the linkage between DNA methylation and Histone modification. [ 146 ]


The goal of epigenetic therapies for cancer in relation to DNA methylation is to both decrease the methylation of DNA and in turn decrease the silencing of genes related to tumor suppression. [ 147 ]  The term associated with decreasing the methylation of DNA will be known as hypomethylation. The Food and Drug Administration (FDA) has currently approved one hypomethylating agent which, through the conduction of clinical trials, has shown promising results when utilized to treat patients with Myelodysplastic Syndrome (MDS). [ 148 ]  This hypomethylating agent is known as the doozy analogue of 5-azacytidine and works to promote hypomethylation by targeting all DNA methyltransferases for degradation. [ 147 ]


Chromatin  assembly factor-1  ( CAF-1 ) is a  protein complex  — including  Chaf1a  (p150),   Chaf1b  (p60), and p48 subunits in humans, or Cac1, Cac2, and Cac3, respectively, in yeast— that assembles  histone  tetramers onto  replicating DNA  during the S phase of the cell cycle. [ 1 ] [ 2 ] [ 3 ] [ 4 ]


CAF-1 functions as a histone  chaperone  that mediates the first step in  nucleosome  formation by tetramerizing and depositing newly synthesized histone  H3 / H4  onto DNA rapidly behind  replication forks . [ 5 ] [ 6 ] [ 7 ]   H3 and H4 are synthesized in the cytoplasm. [ 4 ]  Several studies have shown that the interaction between CAF-1 and  PCNA  (proliferating cell nuclear antigen, which stabilizes CAF-1 at replication forks, is important for CAF-1's role in nucleosome assembly [ 8 ]


The three subunits work together to make the complex function. The human subunit (p150) interacts with PCNA, which acts as a sliding clamp, to help the CAF-1 complex interact with the DNA replication fork. Additionally, p150 along with PCNA performs nucleotide excision repair to fix any damaged DNA. P60 interacts with ASF1a/b, which is a histone chaperone for H3/H4. p48 has roles outside of CAF-1, but when involved with the complex, it binds to H4. [ 4 ]


p60 attracts ASF1a/b which is a chaperone for H3/H4 and this is in the complex with p150 which interacts with PCNA to attach to the replication fork. The CAF-1 complex adds the histones to the DNA ahead of the replication fork. [ 4 ]


A mutation in p150 that results in a loss of function would lead to double stranded breaks, interruptions in the replication fork and translocations. In p60, loss of function would mean the histone chaperone for H3/H4 would not interact with the complex. A mutation like this in either subunit would result in loss of function for the CAF-1 complex as a whole. However, loss of function in p48 would alter how well the complex is able to chaperone chromatin, but would not stop it as a whole. [ 4 ]


CAF-1 is required for the spatial organization and  epigenetic  marking of  heterochromatin  domains in pluripotent  embryonic  cells, creating a cellular memory of  somatic cell  identity during  cellular differentiation . [ 9 ]


Cells resembling 2-cell-stage mouse embryos ( totipotent  cells) can be induced in vitro through downregulation of the chromatin-assembly activity of CAF-1 in  embryonic stem cells . [ 10 ]


CAF-1 forms a deadenylase complex with  CCR4-Not , which should not be confused with the unrelated  CCR4 . The CAF-1/CCR4-Not complex cooperates with the  release factor  eRF3 and  PABPC1  to shorten the poly(A) tail of  mRNA  during  translation. [ 11 ]


Chromatin remodeling  is the dynamic modification of  chromatin  architecture to allow access of condensed genomic DNA to the regulatory  transcription machinery proteins , and thereby control gene expression. Such remodeling is principally carried out by 1) covalent  histone modifications  by specific enzymes, e.g., histone acetyltransferases (HATs), deacetylases, methyltransferases, and kinases, and 2) ATP-dependent chromatin remodeling complexes which either move, eject or restructure  nucleosomes . [ 1 ]  Besides actively regulating gene expression, dynamic remodeling of chromatin imparts an epigenetic regulatory role in several key biological processes, egg cells DNA replication and repair; apoptosis; chromosome segregation as well as development and pluripotency. Aberrations in chromatin remodeling proteins are found to be associated with human diseases, including cancer. Targeting chromatin remodeling pathways is currently evolving as a major therapeutic strategy in the treatment of several cancers.


The transcriptional regulation of the genome is controlled primarily at the  preinitiation stage  by binding of the core transcriptional machinery proteins (namely, RNA polymerase, transcription factors, and activators and repressors) to the core promoter sequence on the coding region of the DNA. However, DNA is tightly packaged in the nucleus with the help of packaging proteins, chiefly histone proteins to form repeating units of  nucleosomes  which further bundle together to form condensed chromatin structure.  Such condensed structure occludes many DNA regulatory regions, not allowing them to interact with transcriptional machinery proteins and regulate gene expression. To overcome this issue and allow dynamic access to condensed DNA, a process known as chromatin remodeling alters nucleosome architecture to expose or hide regions of DNA for transcriptional regulation.


By definition, chromatin remodeling is the enzyme-assisted process to facilitate access of nucleosomal DNA by remodeling the structure, composition and positioning of nucleosomes.


Access to nucleosomal DNA is governed by two major classes of protein complexes:


Specific protein complexes, known as histone-modifying complexes catalyze addition or removal of various chemical elements on histones. These enzymatic modifications include  acetylation ,  methylation ,  phosphorylation , and  ubiquitination  and primarily occur at N-terminal histone tails. Such modifications affect the binding affinity between histones and DNA, and thus loosening or tightening the condensed DNA wrapped around histones, e.g., Methylation of specific lysine residues in H3 and H4 causes further condensation of DNA around histones, and thereby prevents binding of transcription factors to the DNA that lead to gene repression. On the contrary, histone acetylation relaxes chromatin condensation and exposes DNA for TF binding, leading to increased gene expression. [ 3 ]


Well characterized modifications to histones include: [ 4 ]


Both lysine and arginine residues are known to be methylated. Methylated lysines are the best understood marks of the histone code, as specific methylated lysine match well with gene expression states. Methylation of lysines H3K4 and H3K36 is correlated with transcriptional activation while demethylation of H3K4 is correlated with silencing of the genomic region. Methylation of lysines H3K9 and H3K27 is correlated with transcriptional repression. [ 5 ]  Particularly, H3K9me3 is highly correlated with constitutive heterochromatin. [ 6 ]


Acetylation tends to define the 'openness' of  chromatin  as acetylated histones cannot pack as well together as deacetylated histones.


However, there are many more histone modifications, and sensitive  mass spectrometry  approaches have recently greatly expanded the catalog. [ 7 ]


The  histone code  is a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, primarily on their unstructured ends.  Together with similar modifications such as  DNA methylation  it is part of the  epigenetic code .


Cumulative evidence suggests that such code is written by specific enzymes which can (for example) methylate or acetylate DNA ('writers'), removed by other enzymes having demethylase or deacetylase activity ('erasers'), and finally readily identified by proteins ('readers') that are recruited to such histone modifications and bind via specific domains, e.g., bromodomain, chromodomain. These triple action of 'writing', 'reading' and 'erasing' establish the favorable local environment for transcriptional regulation, DNA-damage repair, etc. [ 8 ]


The critical concept of the  histone code hypothesis  is that the histone modifications serve to recruit other proteins by specific recognition of the modified histone via  protein domains  specialized for such purposes, rather than through simply stabilizing or destabilizing the interaction between histone and the underlying DNA. These recruited proteins then act to alter chromatin structure actively or to promote transcription.


A very basic summary of the histone code for gene expression status is given below (histone nomenclature is described  here ):


ATP-dependent chromatin-remodeling complexes regulate gene expression by either moving, ejecting or restructuring nucleosomes. These protein complexes have a common ATPase domain and energy from the hydrolysis of ATP allows these remodeling complexes to reposition nucleosomes (often referred to as "nucleosome sliding") along the DNA, eject or assemble histones on/off of DNA or facilitate exchange of histone variants, and thus creating nucleosome-free regions of DNA for gene activation. [ 13 ]  Also, several remodelers have DNA-translocation activity to carry out specific remodeling tasks. [ 14 ]


All  ATP-dependent chromatin-remodeling complexes possess a sub unit of ATPase that  belongs to the SNF2 superfamily of proteins. In association to the sub unit's identity, two main groups have been classified for these proteins. These are known as the SWI2/SNF2 group and the imitation SWI (ISWI) group. The third class of ATP-dependent complexes that has been recently described contains a Snf2-like ATPase and also demonstrates deacetylase activity. [ 15 ]


There are at least four families of chromatin remodelers in eukaryotes:  SWI/SNF ,  ISWI ,  NuRD /Mi-2/ CHD , and INO80 with first two remodelers being very well studied so far, especially in the yeast model.  Although all of remodelers share common ATPase domain, their functions are specific based on several biological processes (DNA repair, apoptosis, etc.). This is due to the fact that each remodeler complex has unique protein domains ( Helicase ,  bromodomain , etc.) in their catalytic ATPase region and also has different recruited subunits.


Chromatin remodeling plays a central role in the regulation of gene expression by providing the transcription machinery with dynamic access to an otherwise tightly packaged genome. Further, nucleosome movement by chromatin remodelers is essential to several important biological processes, including chromosome assembly and segregation, DNA replication and repair, embryonic development and pluripotency, and cell-cycle progression. Deregulation of chromatin remodeling causes loss of transcriptional regulation at these critical check-points required for proper cellular functions, and thus causes various disease syndromes, including cancer.


Chromatin relaxation is one of the earliest cellular responses to DNA damage. [ 16 ]  Several experiments have been performed on the recruitment  kinetics  of proteins involved in the response to DNA damage. The relaxation appears to be initiated by  PARP1 , whose accumulation at DNA damage is half complete by 1.6 seconds after DNA damage occurs. [ 17 ]  This is quickly followed by accumulation of chromatin remodeler  Alc1 , which has an  ADP-ribose –binding domain, allowing it to be quickly attracted to the product of PARP1. The maximum recruitment of Alc1 occurs within 10 seconds of DNA damage. [ 16 ]  About half of the maximum chromatin relaxation, presumably due to action of Alc1, occurs by 10 seconds. [ 16 ]  PARP1 action at the site of a double-strand break allows recruitment of the two DNA repair enzymes  MRE11  and  NBS1 . Half maximum recruitment of these two  DNA repair  enzymes takes 13 seconds for MRE11 and 28 seconds for NBS1. [ 17 ]


Another process of chromatin relaxation, after formation of a DNA double-strand break, employs γH2AX, the phosphorylated form of the  H2AX  protein.  The  histone  variant H2AX constitutes about 10% of the H2A histones in human chromatin. [ 18 ]  γH2AX (phosphorylated on serine 139 of H2AX) was detected at 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurred in one minute. [ 18 ]  The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break. [ 18 ]


γH2AX does not, by itself, cause chromatin decondensation, but within seconds of irradiation the protein "Mediator of the DNA damage checkpoint 1" ( MDC1 ) specifically attaches to γH2AX. [ 19 ] [ 20 ]  This is accompanied by simultaneous accumulation of  RNF8  protein and the  DNA repair  protein  NBS1  which bind to  MDC1  as MDC1 attaches to γH2AX. [ 21 ]  RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with  CHD4  protein, [ 22 ]  a component of the nucleosome remodeling and deacetylase complex  NuRD . CHD4 accumulation at the site of the double-strand break is rapid, with half-maximum accumulation occurring by 40 seconds after irradiation. [ 23 ]


The fast initial chromatin relaxation upon DNA damage (with rapid initiation of DNA repair) is followed by a slow recondensation, with chromatin recovering a compaction state close to its pre-damage level in ~ 20 min. [ 16 ]


Chromatin remodeling provides fine-tuning at crucial cell growth and division steps, like cell-cycle progression, DNA repair and chromosome segregation, and therefore exerts tumor-suppressor function. Mutations in such chromatin remodelers and deregulated covalent histone modifications potentially favor self-sufficiency in cell growth and escape from growth-regulatory cell signals - two important hallmarks of  cancer . [ 24 ]


Rapid advance in  cancer genomics  and high-throughput  ChIP-chip ,  ChIP-Seq  and  Bisulfite sequencing  methods are providing more insight into role of chromatin remodeling in transcriptional regulation and role in cancer.


Epigenetic instability caused by deregulation in chromatin remodeling is studied in several cancers, including breast cancer, colorectal cancer, pancreatic cancer. Such instability largely cause widespread silencing of genes with primary impact on tumor-suppressor genes. Hence, strategies are now being tried to overcome epigenetic silencing with synergistic combination of  HDAC inhibitors or HDI  and  DNA-demethylating agents .
HDIs are primarily used as adjunct therapy in several cancer types. [ 36 ] [ 37 ]  HDAC inhibitors can induce  p21  (WAF1) expression, a regulator of  p53 's  tumor suppressoractivity . HDACs are involved in the pathway by which the  retinoblastoma protein  (pRb) suppresses  cell proliferation . [ 38 ]  Estrogen is well-established as a  mitogenic factor  implicated in the tumorigenesis and progression of  breast cancer  via its binding to the  estrogen receptor alpha  (ERα).  Recent data indicate that chromatin inactivation mediated by HDAC and DNA methylation is a critical component of ERα silencing in human breast cancer cells. [ 39 ]


Current front-runner candidates for new drug targets are  Histone Lysine Methyltransferases  (KMT) and Protein Arginine Methyltransferases (PRMT). [ 44 ]


Chromatin architectural remodeling is implicated in the process of  cellular senescence , which is related to, and yet distinct from,  organismal aging . Replicative cellular senescence refers to a permanent  cell cycle  arrest where post- mitotic  cells continue to exist as metabolically active cells but fail to  proliferate. [ 47 ] [ 48 ]  Senescence can arise due to  age associated degradation ,  telomere attrition ,  progerias ,  pre-malignancies , and other forms of  damage  or disease. Senescent cells undergo distinct repressive phenotypic changes, potentially to prevent the proliferation of damaged or cancerous cells, with modified  chromatin organization , fluctuations in remodeler abundance, and changes in  epigenetic modifications. [ 49 ] [ 50 ] [ 47 ]  Senescent cells undergo  chromatin landscape  modifications as constitutive  heterochromatin  migrates to the center of the nucleus and displaces  euchromatin  and facultative heterochromatin to regions at the edge of the nucleus. This disrupts chromatin- lamin  interactions and inverts of the pattern typically seen in a mitotically active cell. [ 51 ] [ 49 ]  Individual Lamin-Associated Domains (LADs) and  Topologically Associating Domains  (TADs) are disrupted by this migration which can affect  cis interactions  across the genome. [ 52 ]  Additionally, there is a general pattern of canonical  histone  loss, particularly in terms of the  nucleosome  histones  H3  and  H4  and the linker histone  H1. [ 51 ]  Histone variants with two exons are upregulated in senescent cells to produce modified nucleosome assembly which contributes to chromatin permissiveness to senescent changes. [ 52 ]  Although transcription of variant histone proteins may be elevated, canonical histone proteins are not expressed as they are only made during the  S phase  of the cell cycle and senescent cells are post-mitotic. [ 51 ]  During senescence, portions of  chromosomes  can be exported from the nucleus for  lysosomal degradation  which results in greater organizational disarray and disruption of chromatin interactions. [ 50 ]


Chromatin remodeler abundance may be implicated in cellular senescence as  knockdown  or  knockout  of ATP-dependent remodelers such as NuRD, ACF1, and SWI/SNP can result in DNA damage and senescent phenotypes in yeast,  C. elegans,  mice, and human cell cultures. [ 53 ] [ 50 ] [ 54 ]   ACF1 and NuRD are downregulated in senescent cells which suggests that chromatin remodeling is essential for maintaining a mitotic phenotype. [ 53 ] [ 54 ]  Genes involved in signaling for senescence can be silenced by chromatin confirmation and polycomb repressive complexes as seen in PRC1/PCR2 silencing of  p16 . [ 55 ] [ 56 ]  Specific remodeler depletion results in activation of proliferative genes through a failure to maintain silencing. [ 50 ]   Some remodelers act on enhancer regions of genes rather than the specific loci to prevent re-entry into the cell cycle by forming regions of dense heterochromatin around regulatory regions. [ 56 ]


Senescent cells undergo widespread fluctuations in epigenetic modifications in specific chromatin regions compared to mitotic cells. Human and murine cells undergoing replicative senescence experience a general global decrease in methylation; however, specific loci can differ from the general trend. [ 57 ] [ 52 ] [ 50 ] [ 55 ]  Specific chromatin regions, especially those around the promoters or enhancers of proliferative loci, may exhibit elevated methylation states with an overall imbalance of repressive and activating histone modifications. [ 49 ]   Proliferative genes may show increases in the repressive mark  H3K27me3   while genes involved in silencing or aberrant histone products may be enriched with the activating modification  H3K4me3 . [ 52 ]  Additionally, upregulating histone deacetylases, such as members of the  sirtuin  family, can delay senescence by removing acetyl groups that contribute to greater chromatin accessibility. [ 58 ]  General loss of methylation, combined with the addition of acetyl groups results in a more accessible chromatin conformation with a propensity towards disorganization when compared to mitotically active cells. [ 50 ]  General loss of histones precludes addition of histone modifications and contributes changes in enrichment in some chromatin regions during senescence. [ 51 ]


Chromodomain helicase DNA-binding (CHD) proteins  is a subfamily of  ATP-dependent chromatin remodeling  complexes (remodelers). All remodelers fall under the umbrella of RNA/DNA helicase superfamily 2. In yeast, CHD complexes are primarily responsible for  nucleosome  assembly and organization. These complexes play an additional role in multicellular eukaryotes, assisting in chromatin access and nucleosome editing. [ 1 ]