Editing Regulatory sequence (section)

===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 />