Editing Regulatory sequence (section)

==Activation and implementation==

A regulatory DNA sequence does not regulate unless it is activated.  Different regulatory sequences are activated and then implement their regulation by different mechanisms.

===Enhancer activation and implementation===
Expression of genes in mammals can be upregulated when signals are transmitted to the promoters associated with the genes.  [[Cis-regulatory element|''Cis''-regulatory DNA sequences]] that are located in DNA regions distant from the promoters of genes can have very large effects on gene expression, with some genes undergoing up to 100-fold increased expression due to such a ''cis''-regulatory sequence.<ref name=Beagan>{{cite journal | vauthors = Beagan JA, Pastuzyn ED, Fernandez LR, Guo MH, Feng K, Titus KR, Chandrashekar H, Shepherd JD, Phillips-Cremins JE | display-authors = 6 | title = Three-dimensional genome restructuring across timescales of activity-induced neuronal gene expression | journal = Nature Neuroscience | volume = 23 | issue = 6 | pages = 707–717 | date = June 2020 | pmid = 32451484 | pmc = 7558717 | doi = 10.1038/s41593-020-0634-6 }}</ref> These ''cis''-regulatory sequences include [[Enhancer (genetics)|enhancers]], [[Silencer (genetics)|silencers]], [[Insulator (genetics)|insulators]] and tethering elements.<ref name="pmid33102493">{{cite journal | vauthors = Verheul TC, van Hijfte L, Perenthaler E, Barakat TS | title = The Why of YY1: Mechanisms of Transcriptional Regulation by Yin Yang 1 | journal = Frontiers in Cell and Developmental Biology | volume = 8 | issue =  | pages = 592164 | date = 2020 | pmid = 33102493 | pmc = 7554316 | doi = 10.3389/fcell.2020.592164 | doi-access = free }}</ref> Among this constellation of sequences, enhancers and their associated [[transcription factors|transcription factor proteins]] have a leading role in the regulation of gene expression.<ref name="pmid22868264">{{cite journal | vauthors = Spitz F, Furlong EE | title = Transcription factors: from enhancer binding to developmental control | journal = Nature Reviews. Genetics | volume = 13 | issue = 9 | pages = 613–26 | date = September 2012 | pmid = 22868264 | doi = 10.1038/nrg3207 | s2cid = 205485256 }}</ref>

[[Enhancer (genetics)|Enhancers]] are sequences of the genome that are major gene-regulatory elements.  Enhancers control cell-type-specific gene expression programs, most often by looping through long distances to come in physical proximity with the promoters of their target genes.<ref name=Schoenfelder>{{cite journal | vauthors = Schoenfelder S, Fraser P | title = Long-range enhancer-promoter contacts in gene expression control | journal = Nature Reviews. Genetics | volume = 20 | issue = 8 | pages = 437–455 | date = August 2019 | pmid = 31086298 | doi = 10.1038/s41576-019-0128-0 | s2cid = 152283312}}</ref>  In a study of brain cortical neurons, 24,937 loops were found, bringing enhancers to promoters.<ref name=Beagan />  Multiple enhancers, each often at tens or hundreds of thousands of nucleotides distant from their target genes, loop to their target gene promoters and coordinate with each other to control expression of their common target gene.<ref name=Schoenfelder />

[[File:Regulation of transcription in mammals.jpg|thumb|left|397x397px| '''Regulation of transcription in mammals'''.  An active enhancer regulatory sequence of DNA is enabled to interact with the [[Promoter (genetics)|promoter]] DNA regulatory sequence of its target [[gene]] by formation of a chromosome loop]]The schematic illustration in this section shows an enhancer looping around to come into close physical proximity with the promoter of a target gene.  The loop is stabilized by a dimer of a connector protein (e.g. dimer of [[CTCF]] or [[YY1]]), with one member of the dimer anchored to its binding motif on the enhancer and the other member anchored to its binding motif on the promoter (represented by the red zigzags in the illustration).<ref name="pmid29224777" /> Several cell function specific transcription factor proteins (in 2018 Lambert et al. indicated there were about 1,600 transcription factors in a human cell<ref name="pmid29425488">{{cite journal | vauthors = Lambert SA, Jolma A, Campitelli LF, Das PK, Yin Y, Albu M, Chen X, Taipale J, Hughes TR, Weirauch MT | display-authors = 6 | title = The Human Transcription Factors | journal = Cell | volume = 172 | issue = 4 | pages = 650–665 | date = February 2018 | pmid = 29425488 | doi = 10.1016/j.cell.2018.01.029 | doi-access = free}}</ref>) generally bind to specific motifs on an enhancer<ref name="pmid29987030">{{cite journal | vauthors = Grossman SR, Engreitz J, Ray JP, Nguyen TH, Hacohen N, Lander ES | title = Positional specificity of different transcription factor classes within enhancers | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 115 | issue = 30 | pages = E7222–E7230 | date = July 2018 | pmid = 29987030 | pmc = 6065035 | doi = 10.1073/pnas.1804663115 | doi-access = free | bibcode = 2018PNAS..115E7222G }}</ref> and a small combination of these enhancer-bound transcription factors, when brought close to a promoter by a DNA loop, govern the level of transcription of the target gene.  [[Mediator (coactivator)]] (a complex usually consisting of about 26 proteins in an interacting structure) communicates regulatory signals from enhancer DNA-bound transcription factors directly to the RNA polymerase II (RNAP II) enzyme bound to the promoter.<ref name=Allen2015>{{cite journal | vauthors = Allen BL, Taatjes DJ | title = The Mediator complex: a central integrator of transcription | journal = Nature Reviews. Molecular Cell Biology | volume = 16 | issue = 3 | pages = 155–66 | date = March 2015 | pmid = 25693131 | pmc = 4963239 | doi = 10.1038/nrm3951 }}</ref>

Enhancers, when active, are generally transcribed from both strands of DNA with RNA polymerases acting in two different directions, producing two eRNAs as illustrated in the Figure.<ref name="pmid29378788">{{cite journal | vauthors = Mikhaylichenko O, Bondarenko V, Harnett D, Schor IE, Males M, Viales RR, Furlong EE | title = The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription | journal = Genes & Development | volume = 32 | issue = 1 | pages = 42–57 | date = January 2018 | pmid = 29378788 | pmc = 5828394 | doi = 10.1101/gad.308619.117 }}</ref>  An inactive enhancer may be bound by an inactive transcription factor.  Phosphorylation of the [[transcription factor]] may activate it and that activated transcription factor may then activate the enhancer to which it is bound (see small red star representing phosphorylation of a transcription factor bound to an enhancer in the illustration).<ref name="pmid12514134">{{cite journal | vauthors = Li QJ, Yang SH, Maeda Y, Sladek FM, Sharrocks AD, Martins-Green M | title = MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300 | journal = The EMBO Journal | volume = 22 | issue = 2 | pages = 281–91 | date = January 2003 | pmid = 12514134 | pmc = 140103 | doi = 10.1093/emboj/cdg028 }}</ref>  An activated enhancer begins transcription of its RNA before activating a promoter to initiate transcription of messenger [[RNA]] from its target gene.<ref name="pmid32810208">{{cite journal | vauthors = Carullo NV, Phillips Iii RA, Simon RC, Soto SA, Hinds JE, Salisbury AJ, Revanna JS, Bunner KD, Ianov L, Sultan FA, Savell KE, Gersbach CA, Day JJ | display-authors = 6 | title = Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems | journal = Nucleic Acids Research | volume = 48 | issue = 17 | pages = 9550–9570 | date = September 2020 | pmid = 32810208 | pmc = 7515708 | doi = 10.1093/nar/gkaa671 }}</ref>

Transcription factor binding sites within enhancers (see figure above) are usually about 10 base pairs long, though they can vary from just a few to about 20 base pairs.<ref name="pmid33767912">{{cite journal |vauthors=Wang S, Zhang Q, Shen Z, He Y, Chen ZH, Li J, Huang DS |title=Predicting transcription factor binding sites using DNA shape features based on shared hybrid deep learning architecture |journal=Mol Ther Nucleic Acids |volume=24 |issue= |pages=154–163 |date=June 2021 |pmid=33767912 |pmc=7972936 |doi=10.1016/j.omtn.2021.02.014 |url=}}</ref>  Enhancers usually have about 10 transcription factor binding sites within an average enhancer site of about 204 base pairs.<ref name="pmid32728217">{{cite journal |vauthors=Meuleman W, Muratov A, Rynes E, Halow J, Lee K, Bates D, Diegel M, Dunn D, Neri F, Teodosiadis A, Reynolds A, Haugen E, Nelson J, Johnson A, Frerker M, Buckley M, Sandstrom R, Vierstra J, Kaul R, Stamatoyannopoulos J |title=Index and biological spectrum of human DNase I hypersensitive sites |journal=Nature |volume=584 |issue=7820 |pages=244–251 |date=August 2020 |pmid=32728217 |pmc=7422677 |doi=10.1038/s41586-020-2559-3 |bibcode=2020Natur.584..244M |url=}}</ref>  Examining enhancer-gene regulatory interactions occurring in 352 cell types and tissues, more than 13 million active enhancers were found.<ref name="pmid38014075">{{cite journal |vauthors=Gschwind AR, Mualim KS, Karbalayghareh A, Sheth MU, Dey KK, Jagoda E, Nurtdinov RN, Xi W, Tan AS, Jones H, Ma XR, Yao D, Nasser J, Avsec Ž, James BT, Shamim MS, Durand NC, Rao SS, Mahajan R, Doughty BR, Andreeva K, Ulirsch JC, Fan K, Perez EM, Nguyen TC, Kelley DR, Finucane HK, Moore JE, Weng Z, Kellis M, Bassik MC, Price AL, Beer MA, Guigó R, Stamatoyannopoulos JA, Lieberman Aiden E, Greenleaf WJ, Leslie CS, Steinmetz LM, Kundaje A, Engreitz JM |title=An encyclopedia of enhancer-gene regulatory interactions in the human genome |journal=bioRxiv |volume= |issue= |pages= |date=November 2023 |pmid=38014075 |pmc=10680627 |doi=10.1101/2023.11.09.563812 |url=}}</ref>

====Super-enhancer====
{{main|Super-enhancer}}
[[File:Super-enhancer.png|thumb|435x435px|A super-enhancer is a cluster of typical enhancers that drives a high level of transcription of a target gene]]

While enhancers are needed for transcription of genes in a cell above low levels, a cluster of enhancers, known as a super-enhancer, can cause transcription of a target gene at even higher levels. Super-enhancers usually drive genes needed for cell identity to express at high levels.<ref name=Hnisz2013>{{cite journal |vauthors=Hnisz D, Abraham BJ, Lee TI, Lau A, Saint-André V, Sigova AA, Hoke HA, Young RA |title=Super-enhancers in the control of cell identity and disease |journal=Cell |volume=155 |issue=4 |pages=934–47 |date=November 2013 |pmid=24119843 |pmc=3841062 |doi=10.1016/j.cell.2013.09.053 |url=}}</ref><ref name=Hnisz2015>{{cite journal |vauthors=Hnisz D, Schuijers J, Lin CY, Weintraub AS, Abraham BJ, Lee TI, Bradner JE, Young RA |title=Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers |journal=Mol Cell |volume=58 |issue=2 |pages=362–70 |date=April 2015 |pmid=25801169 |pmc=4402134 |doi=10.1016/j.molcel.2015.02.014 |url=}}</ref> In cancers, a super-enhancer may also drive a particular oncogene to express at a high level.<ref name=Hnisz2013 /><ref name=Hnisz2015 />

A super-enhancer is defined as a cluster of typical enhancers in close genomic proximity (within about 9,000<ref name=Hnisz2013 /> to 22,0000<ref name="pmid30169995">{{cite journal |vauthors=Khan A, Mathelier A, Zhang X |title=Super-enhancers are transcriptionally more active and cell type-specific than stretch enhancers |journal=Epigenetics |volume=13 |issue=9 |pages=910–922 |date=2018 |pmid=30169995 |pmc=6284781 |doi=10.1080/15592294.2018.1514231 |url=}}</ref> base pairs in length) that, all together, regulate the expression of a target gene.<ref name=Sengupta>{{cite journal |vauthors=Sengupta S, George RE |title=Super-Enhancer-Driven Transcriptional Dependencies in Cancer |journal=Trends Cancer |volume=3 |issue=4 |pages=269–281 |date=April 2017 |pmid=28718439 |pmc=5546010 |doi=10.1016/j.trecan.2017.03.006 |url=}}</ref>  Super-enhancer-driven genes are expressed at significantly higher levels than the expression of genes under the control of typical enhancers.<ref name=Sengupta />

A diagram of a super-enhancer is shown in the Figure in this section. In this Figure, the super-enhancer is 12,000 nucleotides long and has four typical enhancers within its length.  Each of the typical enhancers simultaneously contacts the [[Promoter (genetics)|promoter]] region of the same target gene.  Each typical enhancer within the super-enhancer has multiple [[DNA binding site|DNA motifs]] to which transcription factors bind. Each typical enhancer is also bound to a 26-component [[Mediator (coactivator)|mediator complex]] which transmits the signals from the transcription factors bound to the enhancer to the promoter of their joint target gene.  The protein [[BRD4]] forms a complex with each typical enhancer in the super-enhancer and helps to stabilizes the super-enhancer structure.<ref name="pmid29930091">{{cite journal |vauthors=Sabari BR, Dall'Agnese A, Boija A, Klein IA, Coffey EL, Shrinivas K, Abraham BJ, Hannett NM, Zamudio AV, Manteiga JC, Li CH, Guo YE, Day DS, Schuijers J, Vasile E, Malik S, Hnisz D, Lee TI, Cisse II, Roeder RG, Sharp PA, Chakraborty AK, Young RA |title=Coactivator condensation at super-enhancers links phase separation and gene control |journal=Science |volume=361 |issue=6400 |pages= |date=July 2018 |pmid=29930091 |pmc=6092193 |doi=10.1126/science.aar3958 |url=}}</ref>  In addition, the architectural protein YY1 (indicated by paired red zigzags) helps keep the loops together that bring the typical enhancers to their target gene in the super-enhancer.<ref name="pmid29224777">{{cite journal |vauthors=Weintraub AS, Li CH, Zamudio AV, Sigova AA, Hannett NM, Day DS, Abraham BJ, Cohen MA, Nabet B, Buckley DL, Guo YE, Hnisz D, Jaenisch R, Bradner JE, Gray NS, Young RA |title=YY1 Is a Structural Regulator of Enhancer-Promoter Loops |journal=Cell |volume=171 |issue=7 |pages=1573–1588.e28 |date=December 2017 |pmid=29224777 |pmc=5785279 |doi=10.1016/j.cell.2017.11.008 |url=}}</ref> Therefore, there are many proteins in close association at a super-enhancer. These proteins generally have a structured domain as well as a tail with an [[Intrinsically disordered proteins|intrinsically disordered region]] (IDR).<ref name=Boija>{{cite journal |vauthors=Boija A, Klein IA, Sabari BR, Dall'Agnese A, Coffey EL, Zamudio AV, Li CH, Shrinivas K, Manteiga JC, Hannett NM, Abraham BJ, Afeyan LK, Guo YE, Rimel JK, Fant CB, Schuijers J, Lee TI, Taatjes DJ, Young RA |title=Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains |journal=Cell |volume=175 |issue=7 |pages=1842–1855.e16 |date=December 2018 |pmid=30449618 |pmc=6295254 |doi=10.1016/j.cell.2018.10.042 |url=}}</ref> Many of the IDRs of these proteins interact with each other, thereby forming a water-excluding gel or [[biomolecular condensate|phase-separated condensate]] around the super-enhancer.<ref name=Boija />

Some super-enhancers induce very high levels of transcription such as the mouse α-globin super-enhancer<ref name=Blayney>{{cite journal |vauthors=Blayney JW, Francis H, Rampasekova A, Camellato B, Mitchell L, Stolper R, Cornell L, Babbs C, Boeke JD, Higgs DR, Kassouf M |title=Super-enhancers include classical enhancers and facilitators to fully activate gene expression |journal=Cell |volume=186 |issue=26 |pages=5826–5839.e18 |date=December 2023 |pmid=38101409 |pmc=10858684 |doi=10.1016/j.cell.2023.11.030 |url=}}</ref> and the Wap super-enhancer.<ref name=Shin>{{cite journal |vauthors=Shin HY, Willi M, HyunYoo K, Zeng X, Wang C, Metser G, Hennighausen L |title=Hierarchy within the mammary STAT5-driven Wap super-enhancer |journal=Nat Genet |volume=48 |issue=8 |pages=904–911 |date=August 2016 |pmid=27376239 |pmc=4963296 |doi=10.1038/ng.3606 |url=}}</ref>  The mouse α-globin super-enhancer has five typical enhancers within the super-enhancer. Only when acting together, they increase transcription of the ''α-globin gene'' by 450-fold.<ref name=Blayney />  In another example, the mouse Wap super-enhancer includes three typical enhancers.  Only when the three typical enhancers act together do they increase transcription of the ''Wap'' gene by 1000-fold.<ref name=Shin /> 

The enhancers within the super-enhancers described above act synergistically.  However, in a second type of super-enhancer, the component enhancers act additively. In a third group, super-enhancers appear to act “logistically” where promoter activity reaches a limit.  One study examined 773 target genes that were paired with near-by groups of possible super-enhancers (with 2–20 enhancers in close proximity likely acting as super-enhancers).  In this study it appeared that 277, 92, and 250 of the likely super-enhancers acted by the additive, synergistic, and logistic models.<ref name="pmid33770473">{{cite journal |vauthors=Choi J, Lysakovskaia K, Stik G, Demel C, Söding J, Tian TV, Graf T, Cramer P |title=Evidence for additive and synergistic action of mammalian enhancers during cell fate determination |journal=eLife |volume=10 |issue= |pages= |date=March 2021 |pmid=33770473 |pmc=8004103 |doi=10.7554/eLife.65381 |doi-access=free |url=}}</ref> 

Super-enhancers may occupy regions of the genome about 10,000 to 60,000 nucleotides long.<ref name="pmid31724731">{{cite journal |vauthors=Wang X, Cairns MJ, Yan J |title=Super-enhancers in transcriptional regulation and genome organization |journal=Nucleic Acids Res |volume=47 |issue=22 |pages=11481–11496 |date=December 2019 |pmid=31724731 |pmc=7145697 |doi=10.1093/nar/gkz1038 |url=}}</ref> while typical enhancers are each about 204 base pairs long.<ref name="pmid32728217">{{cite journal |vauthors=Meuleman W, Muratov A, Rynes E, Halow J, Lee K, Bates D, Diegel M, Dunn D, Neri F, Teodosiadis A, Reynolds A, Haugen E, Nelson J, Johnson A, Frerker M, Buckley M, Sandstrom R, Vierstra J, Kaul R, Stamatoyannopoulos J |title=Index and biological spectrum of human DNase I hypersensitive sites |journal=Nature |volume=584 |issue=7820 |pages=244–251 |date=August 2020 |pmid=32728217 |pmc=7422677 |doi=10.1038/s41586-020-2559-3 |bibcode=2020Natur.584..244M |url=}}</ref> When 8 types of cells were evaluated, super-enhancers constituted between 2.5% to 10.9% of the enhancers driving transcription while typical enhancers were the majority of enhancers driving transcription.  There were between 257 and 1,099 super-enhancers in these eight cell types and between 5,512 and 23,869 typical enhancers.<ref name="pmid26569311">{{cite journal |vauthors=Niederriter AR, Varshney A, Parker SC, Martin DM |title=Super Enhancers in Cancers, Complex Disease, and Developmental Disorders |journal=Genes (Basel) |volume=6 |issue=4 |pages=1183–200 |date=November 2015 |pmid=26569311 |pmc=4690034 |doi=10.3390/genes6041183 |doi-access=free |url=}}</ref>

While super-enhancers are only active at about 2.5% – 10.9 % of actively transcribed sites in a cell, they recruit transcription machinery more actively than at typical single enhancers. The super-enhancers in a cell utilize about 12% to 36% of the RNA polymerases, mediator proteins, BRD4 proteins, and other transcription machinery of the cell.<ref name=Hnisz2013 />

===CpG island methylation and demethylation===

[[File:Cytosine and 5-methylcytosine.svg|thumb|262x262px|A methyl group is added on the carbon at the number 5 position of the ring to form 5-methylcytosine]]

[[5-methylcytosine|5-Methylcytosine]] (5-mC) is a [[methylation|methylated]] form of the [[DNA]] base [[cytosine]] (see figure).  5-mC is an [[Epigenetics|epigenetic]] marker found predominantly on cytosines within CpG dinucleotides, which consist of a cytosine is followed by a guanine reading in the 5' to 3' direction along the DNA strand ([[CpG sites]]).  About 28 million CpG dinucleotides occur in the human genome.<ref name="pmid26932361">{{cite journal | vauthors = Lövkvist C, Dodd IB, Sneppen K, Haerter JO | title = DNA methylation in human epigenomes depends on local topology of CpG sites | journal = Nucleic Acids Research | volume = 44 | issue = 11 | pages = 5123–32 | date = June 2016 | pmid = 26932361 | pmc = 4914085 | doi = 10.1093/nar/gkw124 }}</ref> In most tissues of mammals, on average, 70% to 80% of CpG cytosines are methylated (forming 5-methyl-CpG, or 5-mCpG).<ref name="pmid15177689">{{cite journal | vauthors = Jabbari K, Bernardi G | title = Cytosine methylation and CpG, TpG (CpA) and TpA frequencies | journal = Gene | volume = 333 | issue =  | pages = 143–9 | date = May 2004 | pmid = 15177689 | doi = 10.1016/j.gene.2004.02.043 }}</ref>  Methylated cytosines within CpG sequences often occur in groups, called [[CpG site#CpG islands|CpG islands]]. About 59% of promoter sequences have a CpG island while only about 6% of enhancer sequences have a CpG island.<ref name="pmid32338759">{{cite journal | vauthors = Steinhaus R, Gonzalez T, Seelow D, Robinson PN | title = Pervasive and CpG-dependent promoter-like characteristics of transcribed enhancers | journal = Nucleic Acids Research | volume = 48 | issue = 10 | pages = 5306–5317 | date = June 2020 | pmid = 32338759 | pmc = 7261191 | doi = 10.1093/nar/gkaa223 }}</ref> CpG islands constitute regulatory sequences, since if CpG islands are methylated in the promoter of a gene this can reduce or silence gene expression.<ref name="pmid11782440">{{cite journal | vauthors = Bird A | title = DNA methylation patterns and epigenetic memory | journal = Genes & Development | volume = 16 | issue = 1 | pages = 6–21 | date = January 2002 | pmid = 11782440 | doi = 10.1101/gad.947102 | doi-access = free }}</ref>

DNA methylation regulates gene expression through interaction with methyl binding domain (MBD) proteins, such as MeCP2, MBD1 and MBD2.  These MBD proteins bind most strongly to highly methylated [[CpG site#CpG islands|CpG islands]].<ref name=Du>{{cite journal | vauthors = Du Q, Luu PL, Stirzaker C, Clark SJ | title = Methyl-CpG-binding domain proteins: readers of the epigenome | journal = Epigenomics | volume = 7 | issue = 6 | pages = 1051–73 | date = 2015 | pmid = 25927341 | doi = 10.2217/epi.15.39 | doi-access = free }}</ref>  These MBD proteins have both a methyl-CpG-binding domain and a transcriptional repression domain.<ref name=Du />  They bind to methylated DNA and guide or direct protein complexes with chromatin remodeling and/or histone modifying activity to methylated CpG islands. MBD proteins generally repress local chromatin by means such as catalyzing the introduction of repressive histone marks or creating an overall repressive chromatin environment through [[nucleosome]] remodeling and chromatin reorganization.<ref name=Du />

[[Transcription factors]] are proteins that bind to specific DNA sequences in order to regulate the expression of a given gene.  The binding sequence for a transcription factor in DNA is usually about 10 or 11 nucleotides long.  There are approximately 1,400 different transcription factors encoded in the human genome, and they constitute about 6% of all human protein coding genes.<ref name="pmid19274049">{{cite journal | vauthors = Vaquerizas JM, Kummerfeld SK, Teichmann SA, Luscombe NM | title = A census of human transcription factors: function, expression and evolution | journal = Nature Reviews. Genetics | volume = 10 | issue = 4 | pages = 252–63 | date = April 2009 | pmid = 19274049 | doi = 10.1038/nrg2538 | s2cid = 3207586 }}</ref>  About 94% of transcription factor binding sites that are associated with signal-responsive genes occur in enhancers while only about 6% of such sites occur in promoters.<ref name="pmid29987030"/>

[[EGR1]] is a transcription factor important for regulation of methylation of CpG islands.  An EGR1 transcription factor binding site is frequently located in enhancer or promoter sequences.<ref name=SunZ>{{cite journal | vauthors = Sun Z, Xu X, He J, Murray A, Sun MA, Wei X, Wang X, McCoig E, Xie E, Jiang X, Li L, Zhu J, Chen J, Morozov A, Pickrell AM, Theus MH, Xie H | display-authors = 6 | title = EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity | journal = Nature Communications | volume = 10 | issue = 1 | pages = 3892 | date = August 2019 | pmid = 31467272 | pmc = 6715719 | doi = 10.1038/s41467-019-11905-3 | bibcode = 2019NatCo..10.3892S }}</ref>  There are about 12,000 binding sites for EGR1 in the mammalian genome and about half of EGR1 binding sites are located in promoters and half in enhancers.<ref name=SunZ />  The binding of EGR1 to its target DNA binding site is insensitive to cytosine methylation in the DNA.<ref name=SunZ />

While only small amounts of EGR1 protein are detectable in cells that are un-stimulated, EGR1 translation into protein at one hour after stimulation is markedly elevated.<ref name=Kubosaki>{{cite journal | vauthors = Kubosaki A, Tomaru Y, Tagami M, Arner E, Miura H, Suzuki T, Suzuki M, Suzuki H, Hayashizaki Y | display-authors = 6 | title = Genome-wide investigation of in vivo EGR-1 binding sites in monocytic differentiation | journal = Genome Biology | volume = 10 | issue = 4 | pages = R41 | date = 2009 | pmid = 19374776 | pmc = 2688932 | doi = 10.1186/gb-2009-10-4-r41 | doi-access = free }}</ref>  Expression of EGR1 in various types of cells can be stimulated by growth factors, neurotransmitters, hormones, stress and injury.<ref name=Kubosaki />  In the brain, when neurons are activated, EGR1 proteins are upregulated, and they bind to (recruit) pre-existing TET1 enzymes, which are highly expressed in neurons.  [[TET enzymes]] can catalyze demethylation of 5-methylcytosine.  When EGR1 transcription factors bring TET1 enzymes to EGR1 binding sites in promoters, the TET enzymes can [[DNA demethylation|demethylate]] the methylated CpG islands at those promoters.  Upon demethylation, these promoters can then initiate transcription of their target genes.  Hundreds of genes in neurons are differentially expressed after neuron activation through EGR1 recruitment of TET1 to methylated regulatory sequences in their promoters.<ref name=SunZ />

===Activation by double- or single-strand breaks===
About 600 regulatory sequences in promoters and about 800 regulatory sequences in enhancers appear to depend on double-strand breaks initiated by [[TOP2B|topoisomerase 2β]] (TOP2B) for activation.<ref name="pmid31110352">{{cite journal | vauthors = Dellino GI, Palluzzi F, Chiariello AM, Piccioni R, Bianco S, Furia L, De Conti G, Bouwman BA, Melloni G, Guido D, Giacò L, Luzi L, Cittaro D, Faretta M, Nicodemi M, Crosetto N, Pelicci PG | display-authors = 6 | title = Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations | journal = Nature Genetics | volume = 51 | issue = 6 | pages = 1011–1023 | date = June 2019 | pmid = 31110352 | doi = 10.1038/s41588-019-0421-z | s2cid = 159041612 | url = https://www.openaccessrepository.it/record/76042 | archive-url = https://web.archive.org/web/20220525082707/https://www.openaccessrepository.it/record/76042 | url-status = dead | archive-date = May 25, 2022 }}</ref><ref name="pmid32029477">{{cite journal |vauthors=Singh S, Szlachta K, Manukyan A, Raimer HM, Dinda M, Bekiranov S, Wang YH |title=Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks |journal=J Biol Chem |volume=295 |issue=12 |pages=3990–4000 |date=March 2020 |pmid=32029477 |pmc=7086017 |doi=10.1074/jbc.RA119.011665 |url=|doi-access=free }}</ref>  The induction of particular double-strand breaks is specific with respect to the inducing signal.  When neurons are activated ''in vitro'', just 22 TOP2B-induced double-strand breaks occur in their genomes.<ref name=Madabhushi>{{cite journal | vauthors = Madabhushi R, Gao F, Pfenning AR, Pan L, Yamakawa S, Seo J, Rueda R, Phan TX, Yamakawa H, Pao PC, Stott RT, Gjoneska E, Nott A, Cho S, Kellis M, Tsai LH | display-authors = 6 | title = Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes | journal = Cell | volume = 161 | issue = 7 | pages = 1592–605 | date = June 2015 | pmid = 26052046 | pmc = 4886855 | doi = 10.1016/j.cell.2015.05.032 }}</ref>  However, when [[fear conditioning|contextual fear conditioning]] is carried out in a mouse, this conditioning causes hundreds of gene-associated DSBs in the medial prefrontal cortex and hippocampus, which are important for learning and memory.<ref>{{cite journal |vauthors=Stott RT, Kritsky O, Tsai LH |title=Profiling DNA break sites and transcriptional changes in response to contextual fear learning |journal=PLOS ONE |volume=16 |issue=7 |pages=e0249691 |date=2021 |pmid=34197463 |pmc=8248687 |doi=10.1371/journal.pone.0249691 |bibcode=2021PLoSO..1649691S |url=|doi-access=free }}</ref>

[[File:TOP2B NHEJ RNAP II double-strand break 2.jpg|thumb|500px|Regulatory sequence in a promoter at a transcription start site with a paused RNA polymerase and a TOP2B-induced double-strand break]]

Such TOP2B-induced double-strand breaks are accompanied by at least four enzymes of the [[non-homologous end joining|non-homologous end joining (NHEJ) DNA repair pathway]] (DNA-PKcs, KU70, KU80 and DNA LIGASE IV) (see figure). These enzymes repair the double-strand breaks within about 15 minutes to 2 hours.<ref name=Madabhushi /><ref name=Ju>{{cite journal | vauthors = Ju BG, Lunyak VV, Perissi V, Garcia-Bassets I, Rose DW, Glass CK, Rosenfeld MG | title = A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription | journal = Science | volume = 312 | issue = 5781 | pages = 1798–802 | date = June 2006 | pmid = 16794079 | doi = 10.1126/science.1127196 | bibcode = 2006Sci...312.1798J | s2cid = 206508330 }}</ref>  The double-strand breaks in the promoter are thus associated with TOP2B and at least these four repair enzymes.  These proteins are present simultaneously on a single promoter nucleosome (there are about 147 nucleotides in the DNA sequence wrapped around a single nucleosome) located near the transcription start site of their target gene.<ref name=Ju />

The double-strand break introduced by TOP2B apparently frees the part of the promoter at an RNA polymerase–bound transcription start site to physically move to its associated enhancer.  This allows the enhancer, with its bound transcription factors and mediator proteins, to directly interact with the RNA polymerase that had been paused at the transcription start site to start transcription.<ref name=Madabhushi /><ref name=Allen2015/>

Similarly, topoisomerase I (TOP1) enzymes appear to be located at many enhancers, and those enhancers become activated when TOP1 introduces a single-strand break.<ref name=Puc>{{cite journal | vauthors = Puc J, Kozbial P, Li W, Tan Y, Liu Z, Suter T, Ohgi KA, Zhang J, Aggarwal AK, Rosenfeld MG | display-authors = 6 | title = Ligand-dependent enhancer activation regulated by topoisomerase-I activity | journal = Cell | volume = 160 | issue = 3 | pages = 367–80 | date = January 2015 | pmid = 25619691 | pmc = 4422651 | doi = 10.1016/j.cell.2014.12.023 }}</ref>  TOP1 causes single-strand breaks in particular enhancer DNA regulatory sequences when signaled by a specific enhancer-binding transcription factor.<ref name=Puc />  Topoisomerase I breaks are associated with different DNA repair factors than those surrounding TOP2B breaks.  In the case of TOP1, the breaks are associated most immediately with DNA repair enzymes [[MRE11A|MRE11]], [[RAD50 (gene)|RAD50]] and [[Ataxia telangiectasia and Rad3 related|ATR]].<ref name=Puc />