Editing Epigenetics (section)

==Molecular basis==
Epigenetic changes modify the activation of certain genes, but not the genetic code sequence of DNA.<ref name="Topart">{{cite journal |vauthors=Topart C, Werner E, Arimondo PB |title=Wandering along the epigenetic timeline |journal=Clin Epigenetics |volume=12 |issue=1 |pages=97 |date=July 2020 |pmid=32616071 |pmc=7330981 |doi=10.1186/s13148-020-00893-7 |doi-access=free |url=}}</ref> The microstructure (not code) of DNA itself or the associated [[chromatin]] proteins may be modified, causing activation or silencing. This mechanism enables differentiated cells in a multicellular organism to express only the genes that are necessary for their own activity. Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism's lifetime; however, these epigenetic changes can be transmitted to the organism's offspring through a process called [[transgenerational epigenetic inheritance]]. Moreover, if gene inactivation occurs in a sperm or egg cell that results in fertilization, this epigenetic modification may also be transferred to the next generation.<ref name="pmid17320501">{{cite journal | vauthors = Chandler VL | title = Paramutation: from maize to mice | journal = Cell | volume = 128 | issue = 4 | pages = 641–5 | date = February 2007 | pmid = 17320501 | doi = 10.1016/j.cell.2007.02.007 | s2cid = 6928707 | doi-access = free }}</ref>

Specific epigenetic processes include [[paramutation]], [[bookmarking]], [[Imprinting (genetics)|imprinting]], [[gene silencing]], [[X-inactivation|X chromosome inactivation]], [[position effect]], [[DNA methylation reprogramming]], [[transvection (genetics)|transvection]], [[maternal effect]]s, the progress of [[carcinogenesis]], many effects of [[teratogen]]s, regulation of [[histone]] modifications and [[heterochromatin]], and technical limitations affecting [[parthenogenesis]] and [[cloning]].<ref>{{cite book | vauthors = Zaidi SK, Lian JB, van Wijnen A, Stein JL, Stein GS | title = RUNX Proteins in Development and Cancer | chapter = Mitotic Gene Bookmarking: An Epigenetic Mechanism for Coordination of Lineage Commitment, Cell Identity and Cell Growth | series = Advances in Experimental Medicine and Biology | volume = 962 | pages = 95–102 | year = 2017 | pmid = 28299653 | pmc = 7233416 | doi = 10.1007/978-981-10-3233-2_7 | isbn = 978-981-10-3231-8 }}</ref><ref>{{cite journal | vauthors = Suter CM, Martin DI | title = Paramutation: the tip of an epigenetic iceberg? | journal = Trends in Genetics | volume = 26 | issue = 1 | pages = 9–14 | date = January 2010 | pmid = 19945764 | pmc = 3137459 | doi = 10.1016/j.tig.2009.11.003 }}</ref><ref>{{cite journal | vauthors = Ferguson-Smith AC | title = Genomic imprinting: the emergence of an epigenetic paradigm | journal = Nature Reviews. Genetics | volume = 12 | issue = 8 | pages = 565–575 | date = July 2011 | pmid = 21765458 | doi = 10.1038/nrg3032 | s2cid = 23630392 }}</ref>

===DNA damage===
DNA damage can also cause epigenetic changes.<ref>{{cite journal | vauthors = Kovalchuk O, Baulch JE | title = Epigenetic changes and nontargeted radiation effects--is there a link? | journal = Environmental and Molecular Mutagenesis | volume = 49 | issue = 1 | pages = 16–25 | date = January 2008 | pmid = 18172877 | doi = 10.1002/em.20361 | bibcode = 2008EnvMM..49...16K | s2cid = 38705208 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Ilnytskyy Y, Kovalchuk O | title = Non-targeted radiation effects-an epigenetic connection | journal = Mutation Research | volume = 714 | issue = 1–2 | pages = 113–25 | date = September 2011 | pmid = 21784089 | doi = 10.1016/j.mrfmmm.2011.06.014 | bibcode = 2011MRFMM.714..113I }}</ref><ref>{{cite journal | vauthors = Friedl AA, Mazurek B, Seiler DM | title = Radiation-induced alterations in histone modification patterns and their potential impact on short-term radiation effects | journal = Frontiers in Oncology | volume = 2 | pages = 117 | year = 2012 | pmid = 23050241 | pmc = 3445916 | doi = 10.3389/fonc.2012.00117 | doi-access = free }}</ref> DNA damage is very frequent, occurring on average about 60,000 times a day per cell of the human body (see [[DNA damage (naturally occurring)]]). These damages are largely repaired, however, epigenetic changes can still remain at the site of DNA repair.<ref name="Cuozzo" /> In particular, a double strand break in DNA can initiate unprogrammed epigenetic gene silencing both by causing DNA methylation as well as by promoting silencing types of histone modifications (chromatin remodeling - see next section).<ref>{{cite journal | vauthors = O'Hagan HM, Mohammad HP, Baylin SB | title = Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island | journal = PLOS Genetics | volume = 4 | issue = 8 | pages = e1000155 | date = August 2008 | pmid = 18704159 | pmc = 2491723 | doi = 10.1371/journal.pgen.1000155 | veditors = Lee JT | doi-access = free }}</ref> In addition, the enzyme [[Poly ADP ribose polymerase|Parp1 (poly(ADP)-ribose polymerase)]] and its product poly(ADP)-ribose (PAR) accumulate at sites of DNA damage as part of the repair process.<ref>{{cite journal | vauthors = Malanga M, Althaus FR | title = The role of poly(ADP-ribose) in the DNA damage signaling network | journal = Biochemistry and Cell Biology | volume = 83 | issue = 3 | pages = 354–64 | date = June 2005 | pmid = 15959561 | doi = 10.1139/o05-038 | url = https://www.zora.uzh.ch/id/eprint/5838/1/RPViewDoc.pdf }}</ref> This accumulation, in turn, directs recruitment and activation of the chromatin remodeling protein, ALC1, that can cause [[nucleosome]] remodeling.<ref>{{cite journal | vauthors = Gottschalk AJ, Timinszky G, Kong SE, Jin J, Cai Y, Swanson SK, Washburn MP, Florens L, Ladurner AG, Conaway JW, Conaway RC | title = Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 106 | issue = 33 | pages = 13770–4 | date = August 2009 | pmid = 19666485 | pmc = 2722505 | doi = 10.1073/pnas.0906920106 | bibcode = 2009PNAS..10613770G | doi-access = free }}</ref> Nucleosome remodeling has been found to cause, for instance, epigenetic silencing of DNA repair gene MLH1.<ref name="isbn0-87969-490-4"/><ref>{{cite journal | vauthors = Lin JC, Jeong S, Liang G, Takai D, Fatemi M, Tsai YC, Egger G, Gal-Yam EN, Jones PA | title = Role of nucleosomal occupancy in the epigenetic silencing of the MLH1 CpG island | journal = Cancer Cell | volume = 12 | issue = 5 | pages = 432–44 | date = November 2007 | pmid = 17996647 | pmc = 4657456 | doi = 10.1016/j.ccr.2007.10.014 }}</ref> DNA damaging chemicals, such as [[benzene]], [[hydroquinone]], [[styrene]], [[carbon tetrachloride]] and [[trichloroethylene]], cause considerable hypomethylation of DNA, some through the activation of oxidative stress pathways.<ref>{{cite journal | vauthors = Tabish AM, Poels K, Hoet P, Godderis L | title = Epigenetic factors in cancer risk: effect of chemical carcinogens on global DNA methylation pattern in human TK6 cells | journal = PLOS ONE | volume = 7 | issue = 4 | pages = e34674 | year = 2012 | pmid = 22509344 | pmc = 3324488 | doi = 10.1371/journal.pone.0034674 | veditors = Chiariotti L | bibcode = 2012PLoSO...734674T | doi-access = free }}</ref>

Foods are known to alter the epigenetics of rats on different diets.<ref>{{cite journal | vauthors = Burdge GC, Hoile SP, Uller T, Thomas NA, Gluckman PD, Hanson MA, [[Karen A. Lillycrop|Lillycrop KA]] | title = Progressive, transgenerational changes in offspring phenotype and epigenotype following nutritional transition | journal = PLOS ONE | volume = 6 | issue = 11 | pages = e28282 | year = 2011 | pmid = 22140567 | pmc = 3227644 | doi = 10.1371/journal.pone.0028282 | veditors = Imhof A | bibcode = 2011PLoSO...628282B | doi-access = free }}</ref> Some food components epigenetically increase the levels of DNA repair enzymes such as [[O-6-methylguanine-DNA methyltransferase|MGMT]] and [[MLH1]]<ref>{{cite journal | vauthors = Fang M, Chen D, Yang CS | title = Dietary polyphenols may affect DNA methylation | journal = The Journal of Nutrition | volume = 137 | issue = 1 Suppl | pages = 223S–228S | date = January 2007 | pmid = 17182830 | doi = 10.1093/jn/137.1.223S | doi-access = free }}</ref> and [[p53]].<ref>{{cite journal | vauthors = Olaharski AJ, Rine J, Marshall BL, Babiarz J, Zhang L, Verdin E, Smith MT | title = The flavoring agent dihydrocoumarin reverses epigenetic silencing and inhibits sirtuin deacetylases | journal = PLOS Genetics | volume = 1 | issue = 6 | pages = e77 | date = December 2005 | pmid = 16362078 | pmc = 1315280 | doi = 10.1371/journal.pgen.0010077 | doi-access = free }}</ref> Other food components can reduce DNA damage, such as soy [[isoflavones]]. In one study, markers for oxidative stress, such as modified nucleotides that can result from DNA damage, were decreased by a 3-week diet supplemented with soy.<ref>{{cite journal | vauthors = Djuric Z, Chen G, Doerge DR, Heilbrun LK, Kucuk O | title = Effect of soy isoflavone supplementation on markers of oxidative stress in men and women | journal = Cancer Letters | volume = 172 | issue = 1 | pages = 1–6 | date = October 2001 | pmid = 11595123 | doi = 10.1016/S0304-3835(01)00627-9 }}</ref> A decrease in oxidative DNA damage was also observed 2 h after consumption of [[anthocyanin]]-rich [[bilberry]] (''[[Vaccinium myrtillus|Vaccinium myrtillius]]'' L.) [[pomace]] extract.<ref>{{cite journal | vauthors = Kropat C, Mueller D, Boettler U, Zimmermann K, Heiss EH, Dirsch VM, Rogoll D, Melcher R, Richling E, Marko D | title = Modulation of Nrf2-dependent gene transcription by bilberry anthocyanins in vivo | journal = Molecular Nutrition & Food Research | volume = 57 | issue = 3 | pages = 545–50 | date = March 2013 | pmid = 23349102 | doi = 10.1002/mnfr.201200504 }}</ref>

===DNA repair===
Damage to DNA is very common and is constantly being repaired. Epigenetic alterations can accompany DNA repair of oxidative damage or double-strand breaks. In human cells, oxidative DNA damage occurs about 10,000 times a day and DNA double-strand breaks occur about 10 to 50 times a cell cycle in somatic replicating cells (see [[DNA damage (naturally occurring)]]). The selective advantage of DNA repair is to allow the cell to survive in the face of DNA damage. The selective advantage of epigenetic alterations that occur with DNA repair is not clear.{{citation needed|date=March 2023}}

====Repair of oxidative DNA damage can alter epigenetic markers====
In the steady state (with endogenous damages occurring and being repaired), there are about 2,400 oxidatively damaged guanines that form [[8-oxo-2'-deoxyguanosine]] (8-OHdG) in the average mammalian cell DNA.<ref name="pmid21163908">{{cite journal |vauthors=Swenberg JA, Lu K, Moeller BC, Gao L, Upton PB, Nakamura J, Starr TB |title=Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment |journal=Toxicol Sci |volume=120 |issue= Suppl 1|pages=S130–45 |date=March 2011 |pmid=21163908 |pmc=3043087 |doi=10.1093/toxsci/kfq371 |url=}}</ref> 8-OHdG constitutes about 5% of the oxidative damages commonly present in DNA.<ref name=Hamilton>{{cite journal |vauthors=Hamilton ML, Guo Z, Fuller CD, Van Remmen H, Ward WF, Austad SN, Troyer DA, Thompson I, Richardson A |title=A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA |journal=Nucleic Acids Res |volume=29 |issue=10 |pages=2117–26 |date=May 2001 |pmid=11353081 |pmc=55450 |doi=10.1093/nar/29.10.2117 |url=}}</ref> The oxidized guanines do not occur randomly among all guanines in DNA. There is a sequence preference for the guanine at a [[DNA methylation|methylated]] [[CpG site]] (a cytosine followed by guanine along its [[Directionality (molecular biology)|5' → 3' direction]] and where the cytosine is methylated (5-mCpG)).<ref name="pmid24571128">{{cite journal |vauthors=Ming X, Matter B, Song M, Veliath E, Shanley R, Jones R, Tretyakova N |title=Mapping structurally defined guanine oxidation products along DNA duplexes: influence of local sequence context and endogenous cytosine methylation |journal=J Am Chem Soc |volume=136 |issue=11 |pages=4223–35 |date=March 2014 |pmid=24571128 |pmc=3985951 |doi=10.1021/ja411636j |bibcode=2014JAChS.136.4223M |url=}}</ref> A 5-mCpG site has the lowest ionization potential for guanine oxidation.{{citation needed|date=March 2023}}

[[File:Initiation of DNA demethylation at a CpG site.svg|thumb|Initiation of [[DNA demethylation]] at a [[CpG site]]. In adult somatic cells DNA methylation typically occurs in the context of CpG dinucleotides ([[CpG sites]]), forming [[5-methylcytosine]]-pG, or 5mCpG. Reactive oxygen species (ROS) may attack guanine at the dinucleotide site, forming [[8-oxo-2'-deoxyguanosine|8-hydroxy-2'-deoxyguanosine]] (8-OHdG), and resulting in a 5mCp-8-OHdG dinucleotide site. The [[base excision repair]] enzyme [[oxoguanine glycosylase|OGG1]] targets 8-OHdG and binds to the lesion without immediate excision. OGG1, present at a 5mCp-8-OHdG site recruits [[Tet methylcytosine dioxygenase 1|TET1]] and TET1 oxidizes the 5mC adjacent to the 8-OHdG. This initiates demethylation of 5mC.<ref name=Zhou>{{cite journal |vauthors=Zhou X, Zhuang Z, Wang W, He L, Wu H, Cao Y, Pan F, Zhao J, Hu Z, Sekhar C, Guo Z |title=OGG1 is essential in oxidative stress-induced DNA demethylation |journal=Cell Signal |volume=28 |issue=9 |pages=1163–1171 |date=September 2016 |pmid=27251462 |doi=10.1016/j.cellsig.2016.05.021 |url=}}</ref>]]

Oxidized guanine has mispairing potential and is mutagenic.<ref name="pmid31993111">{{cite journal |vauthors=Poetsch AR |title=The genomics of oxidative DNA damage, repair, and resulting mutagenesis |journal=Comput Struct Biotechnol J |volume=18 |issue= |pages=207–219 |date=2020 |pmid=31993111 |pmc=6974700 |doi=10.1016/j.csbj.2019.12.013 |url=}}</ref> [[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.<ref name="pmid33171795">{{cite journal |vauthors=D'Augustin O, Huet S, Campalans A, Radicella JP |title=Lost in the Crowd: How Does Human 8-Oxoguanine DNA Glycosylase 1 (OGG1) Find 8-Oxoguanine in the Genome? |journal=Int J Mol Sci |volume=21 |issue=21 |date=November 2020 |page=8360 |pmid=33171795 |pmc=7664663 |doi=10.3390/ijms21218360 |url=|doi-access=free }}</ref> However, OGG1 does not immediately excise 8-OHdG. In HeLa cells half maximum removal of 8-OHdG occurs in 30 minutes,<ref name="pmid15365186">{{cite journal |vauthors=Lan L, Nakajima S, Oohata Y, Takao M, Okano S, Masutani M, Wilson SH, Yasui A |title=In situ analysis of repair processes for oxidative DNA damage in mammalian cells |journal=Proc Natl Acad Sci U S A |volume=101 |issue=38 |pages=13738–43 |date=September 2004 |pmid=15365186 |pmc=518826 |doi=10.1073/pnas.0406048101 |bibcode=2004PNAS..10113738L |url=|doi-access=free }}</ref> and in irradiated mice, the 8-OHdGs induced in the mouse liver are removed with a half-life of 11 minutes.<ref name=Hamilton />

When OGG1 is present at an oxidized guanine within a methylated [[CpG site]] it recruits [[TET enzymes|TET1]] to the 8-OHdG lesion (see Figure). This allows TET1 to demethylate an adjacent methylated cytosine. Demethylation of cytosine is an epigenetic alteration.{{citation needed|date=March 2023}}

As an example, when human mammary epithelial cells were treated with H<sub>2</sub>O<sub>2</sub> 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.<ref name=Zhou /> Demethylation of CpGs in a gene promoter by [[TET enzymes|TET enzyme]] activity increases transcription of the gene into messenger RNA.<ref name="pmid24108092">{{cite journal |vauthors=Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM, Tsai SQ, Ho QH, Sander JD, Reyon D, Bernstein BE, Costello JF, Wilkinson MF, Joung JK |title=Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins |journal=Nat. Biotechnol. |volume=31 |issue=12 |pages=1137–42 |date=December 2013 |pmid=24108092 |pmc=3858462 |doi=10.1038/nbt.2726 }}</ref> In cells treated with H<sub>2</sub>O<sub>2</sub>, one particular gene was examined, [[Beta-secretase 1|''BACE1'']].<ref name=Zhou /> The methylation level of the ''BACE1'' [[CpG site#CpG islands|CpG island]] was reduced (an epigenetic alteration) and this allowed about 6.5 fold increase of expression of ''BACE1'' messenger RNA.{{citation needed|date=March 2023}}

While six-hour incubation with H<sub>2</sub>O<sub>2</sub> causes considerable demethylation of 5-mCpG sites, shorter times of H<sub>2</sub>O<sub>2</sub> incubation appear to promote other epigenetic alterations. Treatment of cells with H<sub>2</sub>O<sub>2</sub> 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.<ref name="pmid26186941">{{cite journal |vauthors=Ding N, Bonham EM, Hannon BE, Amick TR, Baylin SB, O'Hagan HM |title=Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage |journal=J Mol Cell Biol |volume=8 |issue=3 |pages=244–54 |date=June 2016 |pmid=26186941 |pmc=4937888 |doi=10.1093/jmcb/mjv050 |url=}}</ref> This could cause increased methylation of cytosines (epigenetic alterations) at these locations.

Jiang et al.<ref name=Jiang>{{cite journal |vauthors=Jiang Z, Lai Y, Beaver JM, Tsegay PS, Zhao ML, Horton JK, Zamora M, Rein HL, Miralles F, Shaver M, Hutcheson JD, Agoulnik I, Wilson SH, Liu Y |title=Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase β and a de novo DNA Methyltransferase |journal=Cells |volume=9 |issue=1 |date=January 2020 |page=225 |pmid=31963223 |pmc=7016758 |doi=10.3390/cells9010225 |url=|doi-access=free }}</ref> 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 [[DNA polymerase|polymerase beta]] localizing to oxidized guanines. Polymerase beta is the main human polymerase in short-patch BER of oxidative DNA damage. Jiang et al.<ref name=Jiang /> 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 (genetics)|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 (genetics)|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|DNA methyltransferase 1 (DNMT1)]] to sites of DNA double-strand breaks.<ref name="pmid15956212">{{cite journal |vauthors=Mortusewicz O, Schermelleh L, Walter J, Cardoso MC, Leonhardt H |title=Recruitment of DNA methyltransferase I to DNA repair sites |journal=Proc Natl Acad Sci U S A |volume=102 |issue=25 |pages=8905–9 |date=June 2005 |pmid=15956212 |pmc=1157029 |doi=10.1073/pnas.0501034102 |bibcode=2005PNAS..102.8905M |url=|doi-access=free }}</ref><ref name=Cuozzo>{{cite journal |vauthors=Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee B, Di Pardo A, Messina S, Iuliano R, Fusco A, Santillo MR, Muller MT, Chiariotti L, Gottesman ME, Avvedimento EV |title=DNA damage, homology-directed repair, and DNA methylation |journal=PLOS Genet |volume=3 |issue=7 |pages=e110 |date=July 2007 |pmid=17616978 |pmc=1913100 |doi=10.1371/journal.pgen.0030110 |url= |doi-access=free }}</ref><ref name="pmid18704159">{{cite journal |vauthors=O'Hagan HM, Mohammad HP, Baylin SB |title=Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island |journal=PLOS Genet |volume=4 |issue=8 |pages=e1000155 |date=August 2008 |pmid=18704159 |pmc=2491723 |doi=10.1371/journal.pgen.1000155 |url= |doi-access=free }}</ref><ref name="pmid20940144">{{cite journal |vauthors=Ha K, Lee GE, Palii SS, Brown KD, Takeda Y, Liu K, Bhalla KN, Robertson KD |title=Rapid and transient recruitment of DNMT1 to DNA double-strand breaks is mediated by its interaction with multiple components of the DNA damage response machinery |journal=Hum Mol Genet |volume=20 |issue=1 |pages=126–40 |date=January 2011 |pmid=20940144 |pmc=3000680 |doi=10.1093/hmg/ddq451 |url=}}</ref> During [[homologous recombination|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 site]]s 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.<ref name="pmid27629060">{{cite journal |vauthors=Russo G, Landi R, Pezone A, Morano A, Zuchegna C, Romano A, Muller MT, Gottesman ME, Porcellini A, Avvedimento EV |title=DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism |journal=Sci Rep |volume=6 |issue= |pages=33222 |date=September 2016 |pmid=27629060 |pmc=5024116 |doi=10.1038/srep33222 |bibcode=2016NatSR...633222R |url=}}</ref>

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.<ref name="pmid33267773">{{cite journal |vauthors=Farris MH, Texter PA, Mora AA, Wiles MV, Mac Garrigle EF, Klaus SA, Rosfjord K |title=Detection of CRISPR-mediated genome modifications through altered methylation patterns of CpG islands |journal=BMC Genomics |volume=21 |issue=1 |pages=856 |date=December 2020 |pmid=33267773 |pmc=7709351 |doi=10.1186/s12864-020-07233-2 |url= |doi-access=free }}</ref>

====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.<ref name=Cuozzo /> Further work by Allen et al.<ref name="pmid28423717">{{cite journal |vauthors=Allen B, Pezone A, Porcellini A, Muller MT, Masternak MM |title=Non-homologous end joining induced alterations in DNA methylation: A source of permanent epigenetic change |journal=Oncotarget |volume=8 |issue=25 |pages=40359–40372 |date=June 2017 |pmid=28423717 |pmc=5522286 |doi=10.18632/oncotarget.16122 |url=}}</ref> 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%.<ref name="pmid18704159"/>

===Techniques used to study epigenetics===
Epigenetic research uses a wide range of [[molecular biology|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]].<ref name="verma">{{cite journal | vauthors = Verma M, Rogers S, Divi RL, Schully SD, Nelson S, Joseph Su L, Ross SA, Pilch S, Winn DM, Khoury MJ | title = Epigenetic research in cancer epidemiology: trends, opportunities, and challenges | journal = Cancer Epidemiology, Biomarkers & Prevention | volume = 23 | issue = 2 | pages = 223–33 | date = February 2014 | pmid = 24326628 | pmc = 3925982 | doi = 10.1158/1055-9965.EPI-13-0573 }}</ref> Furthermore, the use of [[bioinformatics]] methods has a role in [[computational epigenetics]].<ref name=verma/>

====Chromatin Immunoprecipitation====
Chromatin Immunoprecipitation (ChIP) has helped bridge the gap between DNA and epigenetic interactions.<ref name="Abcam">{{Cite web|title=Studying epigenetics using ChIP|url=https://www.abcam.com/epigenetics/studying-epigenetics-using-chip | work = Abcam }}</ref> With the use of ChIP, researchers are able to make findings in regards to gene regulation, transcription mechanisms, and chromatin structure.<ref name="Abcam" />

====Fluorescent ''in situ'' hybridization====
Fluorescent ''in situ'' hybridization (FISH) is very important to understand epigenetic mechanisms.<ref name="Chaumeil_2008">{{cite book | vauthors = Chaumeil J, Augui S, Chow JC, Heard E | chapter = Combined Immunofluorescence, RNA Fluorescent in Situ Hybridization, and DNA Fluorescent in Situ Hybridization to Study Chromatin Changes, Transcriptional Activity, Nuclear Organization, and X-Chromosome Inactivation | title = The Nucleus | series = Methods in Molecular Biology | location = Clifton, N.J. | publisher = Springer | volume = 463 | pages = 297–308 | date = 2008 | pmid = 18951174 | doi = 10.1007/978-1-59745-406-3_18 | isbn = 978-1-58829-977-2 | chapter-url = }}</ref> FISH can be used to find the location of genes on chromosomes, as well as finding noncoding RNAs.<ref name="Chaumeil_2008" /><ref name="O'Connor_2008">{{Cite journal | vauthors = O'Connor C | title = Fluorescence in situ hybridization (FISH). | journal = Nature Education | date = 2008 | volume = 1 | issue = 1 | page = 171 |url= https://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-327/ }}</ref> FISH is predominantly used for detecting chromosomal abnormalities in humans.<ref name="O'Connor_2008" />

====Methylation-sensitive restriction enzymes====
Methylation sensitive restriction enzymes paired with PCR is a way to evaluate methylation in DNA - specifically the CpG sites.<ref name="Hashimoto_2007">{{cite journal | vauthors = Hashimoto K, Kokubun S, Itoi E, Roach HI | title = Improved quantification of DNA methylation using methylation-sensitive restriction enzymes and real-time PCR | journal = Epigenetics | volume = 2 | issue = 2 | pages = 86–91 | year = 2007 | pmid = 17965602 | doi = 10.4161/epi.2.2.4203 | s2cid = 26728480 | doi-access = free }}</ref> If DNA is methylated, the restriction enzymes will not cleave the strand.<ref name="Hashimoto_2007" /> Contrarily, if the DNA is not methylated, the enzymes will cleave the strand and it will be amplified by PCR.<ref name="Hashimoto_2007" />

====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.<ref name="Hashimoto_2007" /><ref name="Li-Byarlay et al 2020">{{cite journal | vauthors = Li-Byarlay H, Boncristiani H, Howell G, Herman J, Clark L, Strand MK, Tarpy D, Rueppell O | title = Transcriptomic and Epigenomic Dynamics of Honey Bees in Response to Lethal Viral Infection | journal = Frontiers in Genetics | volume = 11 | pages = 566320 | date = 24 September 2020 | pmid = 33101388 | pmc = 7546774 | doi = 10.3389/fgene.2020.566320 | doi-access = free }}</ref><ref name="ReferenceC">{{cite journal | vauthors = Li-Byarlay H, Li Y, Stroud H, Feng S, Newman TC, Kaneda M, Hou KK, Worley KC, Elsik CG, Wickline SA, Jacobsen SE, Ma J, Robinson GE | title = RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 110 | issue = 31 | pages = 12750–12755 | date = July 2013 | pmid = 23852726 | pmc = 3732956 | doi = 10.1073/pnas.1310735110 | doi-access = free | bibcode = 2013PNAS..11012750L }}</ref>

====Nanopore sequencing====
Certain sequencing methods, such as [[nanopore sequencing]], allow sequencing of native DNA. Native (=unamplified) DNA retains the epigenetic modifications which would otherwise be lost during the amplification step. Nanopore basecaller models can distinguish between the signals obtained for epigenetically modified bases and unaltered based and provide an epigenetic profile in addition to the sequencing result.<ref>{{cite journal | vauthors = Simpson JT, Workman RE, Zuzarte PC, David M, Dursi LJ, Timp W | title = Detecting DNA cytosine methylation using nanopore sequencing | journal = Nature Methods | volume = 14 | issue = 4 | pages = 407–410 | date = April 2017 | pmid = 28218898 | doi = 10.1038/nmeth.4184 | s2cid = 16152628 }}</ref>

===Structural inheritance===
{{further|Structural inheritance}}
In [[ciliate]]s such as ''[[Tetrahymena]]'' and ''[[Paramecium]]'', genetically identical cells show heritable differences in the patterns of ciliary rows on their cell surface. Experimentally altered patterns can be transmitted to daughter cells. It seems existing structures act as templates for new structures. The mechanisms of such inheritance are unclear, but reasons exist to assume that multicellular organisms also use existing cell structures to assemble new ones.<ref name="pmid1804215">{{cite book |doi=10.1007/978-1-4615-6823-0_11 |pmid=1804215 |chapter=Concepts of Organization the Leverage of Ciliate Protozoa |title=A Conceptual History of Modern Embryology |series=Developmental Biology |volume=7 |pages=229–258 |year=1991 | vauthors = Sapp J |isbn=978-1-4615-6825-4 }}</ref><ref name="isbn0-19-515619-6">{{cite book | vauthors=Sapp J | title=Genesis: the evolution of biology | publisher=Oxford University Press | location=Oxford | year=2003 | isbn=978-0-19-515619-5 | url-access=registration | url=https://archive.org/details/genesisevolution00sapp }}</ref><ref name="isbn0-262-65063-0">{{cite book |vauthors=Gray RD, Oyama S, Griffiths PE | title=Cycles of Contingency: Developmental Systems and Evolution (Life and Mind: Philosophical Issues in Biology and Psychology) | publisher=The MIT Press | location=Cambridge, Massachusetts | year=2003 | isbn=978-0-262-65063-2 }}</ref>

===Nucleosome positioning===
Eukaryotic genomes have numerous [[nucleosomes]]. Nucleosome position is not random, and determine the accessibility of DNA to regulatory proteins. Promoters active in different tissues have been shown to have different nucleosome positioning features.<ref>{{Cite journal| vauthors = Serizay J, Dong Y, Jänes J, Chesney M, Cerrato C, Ahringer J |date=2020-02-20|title=Tissue-specific profiling reveals distinctive regulatory architectures for ubiquitous, germline and somatic genes |journal=bioRxiv |pages=2020.02.20.958579|doi=10.1101/2020.02.20.958579|s2cid=212943176|doi-access=free}}</ref> This determines differences in gene expression and cell differentiation. It has been shown that at least some nucleosomes are retained in sperm cells (where most but not all histones are replaced by [[protamines]]). Thus nucleosome positioning is to some degree inheritable. Recent studies have uncovered connections between nucleosome positioning and other epigenetic factors, such as DNA methylation and hydroxymethylation.<ref name=Teif_2014>{{cite journal | vauthors = Teif VB, Beshnova DA, Vainshtein Y, Marth C, Mallm JP, Höfer T, Rippe K | title = Nucleosome repositioning links DNA (de)methylation and differential CTCF binding during stem cell development | journal = Genome Research | volume = 24 | issue = 8 | pages = 1285–95 | date = August 2014 | pmid = 24812327 | pmc = 4120082 | doi = 10.1101/gr.164418.113 }}</ref>

===Histone variants===
Different [[histone variants]] are incorporated into specific regions of the genome non-randomly. Their differential biochemical characteristics can affect genome functions via their roles in gene regulation,<ref>{{cite journal | vauthors = Buschbeck M, Hake SB | title = Variants of core histones and their roles in cell fate decisions, development and cancer | journal = Nature Reviews. Molecular Cell Biology | volume = 18 | issue = 5 | pages = 299–314 | date = May 2017 | pmid = 28144029 | doi = 10.1038/nrm.2016.166 | url = https://www.nature.com/articles/nrm.2016.166 | s2cid = 3307731 }}</ref> and maintenance of chromosome structures.<ref>{{cite journal | vauthors = Jang CW, Shibata Y, Starmer J, Yee D, Magnuson T | title = Histone H3.3 maintains genome integrity during mammalian development | journal = Genes & Development | volume = 29 | issue = 13 | pages = 1377–92 | date = July 2015 | pmid = 26159997 | pmc = 4511213 | doi = 10.1101/gad.264150.115 }}</ref>

===Genomic architecture===
The three-dimensional configuration of the genome (the 3D genome) is complex, dynamic and crucial for regulating genomic function and nuclear processes such as DNA replication, transcription and DNA-damage repair.<ref>{{Cite web|title=The 3D genome|url=https://www.nature.com/collections/rsxlmsyslk/|access-date=2021-09-26|website=www.nature.com|date=2 September 2019 |language=en}}</ref>