5-methylcytosine-pg, or 5mcpg. reactive oxygen species (ros) may attack guanine at the dinucleotide site, forming 8-hydroxy-2'-deoxyguanosine (8-ohdg), and resulting in a 5mcp-8-ohdg dinucleotide site. the base excision repair enzyme ogg1 targets 8-ohdg and binds to the lesion without immediate excision. ogg1, present at a 5mcp-8-ohdg site recruits tet1 and tet1 oxidizes the 5mc adjacent to the 8-ohdg. this initiates demethylation of 5mc. oxidized guanine has mispairing potential and is mutagenic. oxoguanine glycosylase (ogg1) is the primary enzyme responsible for the excision of the oxidized guanine during dna repair. ogg1 finds and binds to an 8-ohdg within a few seconds. however, ogg1 does not immediately excise 8-ohdg. in hela cells half maximum removal of 8-ohdg occurs in 30 minutes, and in irradiated mice, the 8-ohdgs induced in the mouse liver are removed with a half-life of 11 minutes. when ogg1 is present at an oxidized guanine within a methylated cpg site it recruits tet1 to the 8-ohdg lesion (see figure). this allows tet1 to demethylate an adjacent methylated cytosine. demethylation of cytosine is an epigenetic alteration. as an example, when human mammary epithelial cells were treated with h2o2 for six hours, 8-ohdg increased about 3.5-fold in dna and this caused about 80% demethylation of the 5-methylcytosines in the genome. demethylation of cpgs in a gene promoter by tet enzyme activity increases transcription of the gene into messenger rna. in cells treated with h2o2, one particular gene was examined, bace1. the methylation level of the bace1 cpg island was reduced (an epigenetic alteration) and this allowed about 6.5 fold increase of expression of bace1 messenger rna. while six-hour incubation with h2o2 causes considerable demethylation of 5-mcpg sites, shorter times of h2o2 incubation appear to promote other epigenetic alterations. treatment of cells with h2o2 for 30 minutes causes the mismatch repair protein heterodimer msh2-msh6 to recruit dna methyltransferase 1 (dnmt1) to sites of some kinds of oxidative dna damage. this could cause increased methylation of cytosines (epigenetic alterations) at these locations. jiang et al. treated hek 293 cells with agents causing oxidative dna damage, (potassium bromate (kbro3) or potassium chromate (k2cro4)). base excision repair (ber) of oxidative damage occurred with the dna repair enzyme polymerase beta localizing to oxidized guanines. polymerase beta is the main human polymerase in short-patch ber of oxidative dna damage. jiang et al. also found that polymerase beta recruited the dna methyltransferase protein dnmt3b to ber repair sites. they then evaluated the methylation pattern at the single nucleotide level in a small region of dna including the promoter region and the early transcription region of the brca1 gene. oxidative dna damage from bromate modulated the dna methylation pattern (caused epigenetic alterations) at cpg sites within the region of dna studied. in untreated cells, cpgs located at −189, −134, −29, −19, +16, and +19 of the brca1 gene had methylated cytosines (where numbering is from the messenger rna transcription start site, and negative numbers indicate nucleotides in the upstream promoter region). bromate treatment-induced oxidation resulted in the loss of cytosine methylation at −189, −134, +16 and +19 while also leading to the formation of new methylation at the cpgs located at −80, −55, −21 and +8 after dna repair was allowed. homologous recombinational repair alters epigenetic markers at least four articles report the recruitment of dna methyltransferase 1 (dnmt1) to sites of dna double-strand breaks. during homologous recombinational repair (hr) of the double-strand break, the involvement of dnmt1 causes the two repaired strands of dna to have different levels of methylated cytosines. one strand becomes frequently methylated at about 21 cpg sites downstream of the repaired double-strand break. the other dna strand loses methylation at about six cpg sites that were previously methylated downstream of the double-strand break, as well as losing methylation at about five cpg sites that were previously methylated upstream of the double-strand break. when the chromosome is replicated, this gives rise to one daughter chromosome that is heavily methylated downstream of the previous break site and one that is unmethylated in the region both upstream and downstream of the previous break site. with respect to the gene that was broken by the double-strand break, half of the progeny cells express that gene at a high level and in the other half of the progeny cells expression of that gene is repressed. when clones of these cells were maintained for three years, the new methylation patterns were maintained over that time period. in mice with a crispr-mediated homology-directed recombination insertion in their genome there were a large number of increased methylations of cpg sites within the double-strand break-associated insertion. non-homologous end joining can cause some epigenetic marker alterations non-homologous end joining (nhej) repair of a double-strand break can cause a small number of demethylations of pre-existing cytosine dna methylations downstream of the repaired double-strand break. further work by allen et al. showed that nhej of a dna double-strand break in a cell could give rise to some progeny cells having repressed expression of the gene harboring the initial double-strand break and some progeny having high expression of that gene due to epigenetic alterations associated with nhej repair. the frequency of epigenetic alterations causing repression of a gene after an nhej repair of a dna double-strand break in that gene may be about 0.9%. techniques used to study epigenetics epigenetic research uses a wide range of molecular biological techniques to further understanding of epigenetic phenomena. these techniques include chromatin immunoprecipitation (together with its large-scale variants chip-on-chip and chip-seq), fluorescent in situ hybridization, methylation-sensitive restriction enzymes, dna adenine methyltransferase identification (damid) and bisulfite sequencing. furthermore, the use of bioinformatics methods has a role in computational epigenetics. chromatin immunoprecipitation chromatin immunoprecipitation (chip) has helped bridge the gap between dna and epigenetic interactions. with the use of chip, researchers are able to make findings in regards to gene regulation, transcription mechanisms, and chromatin structure. fluorescent in situ hybridization fluorescent in situ hybridization (fish) is very important to understand epigenetic mechanisms. fish can be used to find the location of genes on chromosomes, as well as finding noncoding rnas. fish is predominantly used for detecting chromosomal abnormalities in humans. methylation-sensitive restriction enzymes methylation sensitive restriction enzymes paired with pcr is a way to evaluate methylation in dna - specifically the cpg sites. if dna is methylated, the restriction enzymes will not cleave the strand. contrarily, if the dna is not methylated, the enzymes will cleave the strand and it will be amplified by pcr. bisulfite sequencing bisulfite sequencing is another way to evaluate dna methylation. cytosine will be changed to uracil from being treated with sodium bisulfite, whereas methylated cytosines will not be affected. mechanisms epigenetic mechanisms are sensitive to environmental effects, and key participants in shaping an adult phenotype. several types of epigenetic inheritance systems may play a role in what has become known as cell memory, note however that not all of these are universally accepted to be examples of epigenetics. covalent modifications covalent modification of either dna (e.g. cytosine methylation and hydroxymethylation) or of histone proteins (e.g. lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation) play central roles in many types of epigenetic inheritance. therefore, the word "epigenetics" is sometimes used as a synonym for these processes. however, this can be misleading. chromatin remodeling is not always inherited, and not all epigenetic inheritance involves chromatin remodeling. in 2019, a further lysine modification appeared in the scientific literature linking epigenetics modification to cell metabolism, i.e. lactylation dna associates with histone proteins to form chromatin. because the phenotype of a cell or individual is affected by which of its genes are transcribed, heritable transcription states can give rise to epigenetic effects. there are several layers of regulation of gene expression. one way that genes are regulated is through the remodeling of chromatin. chromatin is the complex of dna and the histone proteins with which it associates. if the way that dna is wrapped around the histones changes, gene expression can change as well. chromatin remodeling is accomplished through two main mechanisms: the first way is post translational modification of the amino acids that make up histone proteins. histone proteins are made up of long chains of amino acids. if the amino acids that are in the chain are changed, the shape of the histone might be modified. dna is not completely unwound during replication. it is possible, then, that the modified histones may be carried into each new copy of the dna. once there, these histones may act as templates, initiating the surrounding new histones to be shaped in the new manner. by altering the shape of the histones around them, these modified histones would ensure that a lineage-specific transcription program is maintained after cell division. the second way is the addition of methyl groups to the dna, mostly at cpg sites, to convert cytosine to 5-methylcytosine. 5-methylcytosine performs much like a regular cytosine, pairing with a guanine in double-stranded dna. however, when methylated cytosines are present in cpg sites in the promoter and enhancer regions of genes, the genes are often repressed. when methylated cytosines are present in cpg sites in the gene body (in the coding region excluding the transcription start site) expression of the gene is often enhanced. transcription of a gene usually depends on a transcription factor binding to a (10 base or less) recognition sequence at the enhancer that interacts with the promoter region of that gene (gene expression# enhancers, transcription factors, mediator complex and dna loops in mammalian transcription). about 22% of transcription factors are inhibited from binding when the recognition sequence has a methylated cytosine. in addition, presence of methylated cytosines at a promoter region can attract methyl-cpg-binding domain (mbd) proteins. all mbds interact with nucleosome remodeling and histone deacetylase complexes, which leads to gene silencing. in addition, another covalent modification involving methylated cytosine is its demethylation by tet enzymes. hundreds of such demethylations occur, for instance, during learning and memory forming events in neurons. there is frequently a reciprocal relationship between dna methylation and histone lysine methylation. for instance, the methyl binding domain protein mbd1, attracted to and associating with methylated cytosine in a dna cpg site, can also associate with h3k9 methyltransferase activity to methylate histone 3 at lysine 9. on the other hand, dna maintenance methylation by dnmt1 appears to partly rely on recognition of histone methylation on the nucleosome present at the dna site to carry out cytosine methylation on newly synthesized dna. there is further crosstalk between dna methylation carried out by dnmt3a and dnmt3b and histone methylation so that there is a correlation between the genome-wide distribution of dna methylation and histone methylation. mechanisms of heritability of histone state are not well understood; however, much is known about the mechanism of heritability of dna methylation state during cell division and differentiation. heritability of methylation state depends on certain enzymes (such as dnmt1) that have a higher affinity for 5-methylcytosine than for cytosine. if this enzyme reaches a "hemimethylated" portion of dna (where 5-methylcytosine is in only one of the two dna strands) the enzyme will methylate the other half. however, it is now known that dnmt1 physically interacts with the protein uhrf1. uhrf1 has been recently recognized as essential for dnmt1-mediated maintenance of dna methylation. uhrf1 is the protein that specifically recognizes hemi-methylated dna, therefore bringing dnmt1 to its substrate to maintain dna methylation. some acetylations and some methylations of lysines (symbol k) are activation signals for transcription when present on a nucleosome, as shown in the top figure. some methylations on lysines or arginine (r) are repression signals for transcription when present on a nucleosome, as shown in the bottom figure. nucleosomes consist of four pairs of histone proteins in a tightly assembled core region plus up to 30% of each histone remaining in a loosely organized tail (only one tail of each pair is shown). dna is wrapped around the histone core proteins in chromatin. the lysines (k) are designated with a number showing their position as, for instance (k4), indicating lysine as the 4th amino acid from the amino (n) end of the tail in the histone protein. methylations , and acetylations are common post-translational modifications on the lysines of the histone tails. although histone modifications occur throughout the entire sequence, the unstructured n-termini of histones