Editing Cellular differentiation (section)

===Mechanisms of epigenetic regulation===

====Pioneer factors (Oct4, Sox2, Nanog)====
Three transcription factors, OCT4, SOX2, and [[Homeobox protein NANOG|NANOG]] – the first two of which are used in induced pluripotent stem cell (iPSC) reprogramming, along with [[Klf4]] and [[c-Myc]] – are highly expressed in undifferentiated embryonic stem cells and are necessary for the maintenance of their [[pluripotency]].<ref name = "Christophersen">{{cite journal | vauthors = Christophersen NS, Helin K | title = Epigenetic control of embryonic stem cell fate | journal = The Journal of Experimental Medicine | volume = 207 | issue = 11 | pages = 2287–2295 | date = October 2010 | pmid = 20975044 | pmc = 2964577 | doi = 10.1084/jem.20101438 }}</ref> It is thought that they achieve this through alterations in [[chromatin]] structure, such as [[histone modification]] and DNA methylation, to restrict or permit the transcription of target genes. While highly expressed, their levels require a precise balance to maintain pluripotency, perturbation of which will promote differentiation towards different lineages based on how the gene expression levels change. Differential regulation of [[Oct-4]] and [[SOX2]] levels have been shown to precede germ layer fate selection.<ref name="ReferenceA">{{cite journal | vauthors = Thomson M, Liu SJ, Zou LN, Smith Z, Meissner A, Ramanathan S | title = Pluripotency factors in embryonic stem cells regulate differentiation into germ layers | journal = Cell | volume = 145 | issue = 6 | pages = 875–889 | date = June 2011 | pmid = 21663792 | pmc = 5603300 | doi = 10.1016/j.cell.2011.05.017 }}</ref> Increased levels of Oct4 and decreased levels of Sox2 promote a [[Gastrulation|mesendodermal]] fate, with Oct4 actively suppressing genes associated with a neural [[neurulation|ectodermal]] fate. Similarly, increased levels of Sox2 and decreased levels of Oct4 promote differentiation towards a neural ectodermal fate, with Sox2 inhibiting differentiation towards a mesendodermal fate. Regardless of the lineage cells differentiate down, suppression of NANOG has been identified as a necessary prerequisite for differentiation.<ref name="ReferenceA"/>

====Polycomb repressive complex (PRC2)====
In the realm of [[gene silencing]], [[PRC2|Polycomb repressive complex 2]], one of two classes of the [[Polycomb-group proteins|Polycomb group]] (PcG) family of proteins, catalyzes the di- and tri-methylation of histone H3 lysine 27 (H3K27me2/me3).<ref name= "Christophersen"/><ref name="Jiang">{{cite journal | vauthors = Zhu J, Adli M, Zou JY, Verstappen G, Coyne M, Zhang X, Durham T, Miri M, Deshpande V, De Jager PL, Bennett DA, Houmard JA, Muoio DM, Onder TT, Camahort R, Cowan CA, Meissner A, Epstein CB, Shoresh N, Bernstein BE | title = Genome-wide chromatin state transitions associated with developmental and environmental cues | journal = Cell | volume = 152 | issue = 3 | pages = 642–654 | date = January 2013 | pmid = 23333102 | pmc = 3563935 | doi = 10.1016/j.cell.2012.12.033 }}</ref><ref name = "Guenther">{{cite journal | vauthors = Guenther MG, Young RA | title = Transcription. Repressive transcription | journal = Science | volume = 329 | issue = 5988 | pages = 150–151 | date = July 2010 | pmid = 20616255 | pmc = 3006433 | doi = 10.1126/science.1193995 | bibcode = 2010Sci...329..150G }}</ref> By binding to the H3K27me2/3-tagged nucleosome, PRC1 (also a complex of PcG family proteins) catalyzes the mono-ubiquitinylation of histone H2A at lysine 119 (H2AK119Ub1), blocking [[RNA polymerase II]] activity and resulting in transcriptional suppression.<ref name= "Christophersen"/> PcG knockout ES cells do not differentiate efficiently into the three germ layers, and deletion of the PRC1 and PRC2 genes leads to increased expression of lineage-affiliated genes and unscheduled differentiation.<ref name= "Christophersen"/> Presumably, PcG complexes are responsible for transcriptionally repressing differentiation and development-promoting genes.

====Trithorax group proteins (TrxG)====
Alternately, upon receiving differentiation signals, PcG proteins are recruited to promoters of pluripotency transcription factors. PcG-deficient ES cells can begin differentiation but cannot maintain the differentiated phenotype.<ref name= "Christophersen"/> Simultaneously, differentiation and development-promoting genes are activated by Trithorax group (TrxG) chromatin regulators and lose their repression.<ref name= "Christophersen"/><ref name = "Guenther"/> TrxG proteins are recruited at regions of high transcriptional activity, where they catalyze the trimethylation of histone H3 lysine 4 ([[H3K4me3]]) and promote gene activation through histone acetylation.<ref name = "Guenther"/> PcG and TrxG complexes engage in direct competition and are thought to be functionally antagonistic, creating at differentiation and development-promoting loci what is termed a "bivalent domain" and rendering these genes sensitive to rapid induction or repression.<ref name = "Meissner">{{cite journal | vauthors = Meissner A | title = Epigenetic modifications in pluripotent and differentiated cells | journal = Nature Biotechnology | volume = 28 | issue = 10 | pages = 1079–1088 | date = October 2010 | pmid = 20944600 | doi = 10.1038/nbt.1684 | s2cid = 205274850 }}</ref>

====DNA methylation====
Regulation of gene expression is further achieved through DNA methylation, in which the [[DNA methyltransferase]]-mediated methylation of cytosine residues in CpG dinucleotides maintains heritable repression by controlling DNA accessibility.<ref name = "Meissner"/> The majority of CpG sites in embryonic stem cells are unmethylated and appear to be associated with H3K4me3-carrying nucleosomes.<ref name= "Christophersen"/> Upon differentiation, a small number of genes, including OCT4 and NANOG,<ref name = "Meissner"/> are methylated and their promoters repressed to prevent their further expression. Consistently, DNA methylation-deficient embryonic stem cells rapidly enter [[apoptosis]] upon in vitro differentiation.<ref name= "Christophersen"/>

====Nucleosome positioning====
While the [[nucleic acid sequence|DNA sequence]] of most cells of an organism is the same, the binding patterns of transcription factors and the corresponding gene expression patterns are different. To a large extent, differences in transcription factor binding are determined by the chromatin accessibility of their binding sites through [[histone modification]] and/or [[pioneer factor]]s. In particular, it is important to know whether a [[nucleosome]] is covering a given genomic binding site or not. This can be determined using a [[chromatin immunoprecipitation]] assay.<ref>{{Cite web|url=http://www.bio.brandeis.edu/haberlab/jehsite/chIP.html|title=Chromatin Immuprecipitation|website=www.bio.brandeis.edu|access-date=2016-12-26|archive-date=2017-11-25|archive-url=https://web.archive.org/web/20171125204418/http://www.bio.brandeis.edu/haberlab/jehsite/chIP.html|url-status=dead}}</ref>

=====Histone acetylation and methylation=====
DNA-nucleosome interactions are characterized by two states: either tightly bound by nucleosomes and transcriptionally inactive, called [[heterochromatin]], or loosely bound and usually, but not always, transcriptionally active, called [[euchromatin]]. The epigenetic processes of histone methylation and acetylation, and their inverses demethylation and deacetylation primarily account for these changes. The effects of acetylation and deacetylation are more predictable. An acetyl group is either added to or removed from the positively charged Lysine residues in histones by enzymes called [[histone acetyltransferase]]s or [[histone deactylase]]s, respectively. The acetyl group prevents Lysine's association with the negatively charged DNA backbone. Methylation is not as straightforward, as neither methylation nor demethylation consistently correlate with either gene activation or repression. However, certain methylations have been repeatedly shown to either activate or repress genes. The trimethylation of lysine 4 on histone 3 (H3K4Me3) is associated with gene activation, whereas trimethylation of lysine 27 on histone 3 represses genes.<ref name="pmid12667454">{{cite journal | vauthors = Krogan NJ, Dover J, Wood A, Schneider J, Heidt J, Boateng MA, Dean K, Ryan OW, Golshani A, Johnston M, Greenblatt JF, Shilatifard A | title = The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation | journal = Molecular Cell | volume = 11 | issue = 3 | pages = 721–729 | date = March 2003 | pmid = 12667454 | doi = 10.1016/S1097-2765(03)00091-1 | doi-access = free }}</ref><ref name="pmid12667453">{{cite journal | vauthors = Ng HH, Robert F, Young RA, Struhl K | title = Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity | journal = Molecular Cell | volume = 11 | issue = 3 | pages = 709–719 | date = March 2003 | pmid = 12667453 | doi = 10.1016/S1097-2765(03)00092-3 | doi-access = free }}</ref><ref name="pmid15680324">{{cite journal | vauthors = Bernstein BE, Kamal M, Lindblad-Toh K, Bekiranov S, Bailey DK, Huebert DJ, McMahon S, Karlsson EK, Kulbokas EJ, Gingeras TR, Schreiber SL, Lander ES | title = Genomic maps and comparative analysis of histone modifications in human and mouse | journal = Cell | volume = 120 | issue = 2 | pages = 169–181 | date = January 2005 | pmid = 15680324 | doi = 10.1016/j.cell.2005.01.001 | s2cid = 7193829 | doi-access = free }}</ref>

=====In stem cells=====
{{further|Stem cell}}

During differentiation, stem cells change their gene expression profiles. Recent studies have implicated a role for nucleosome positioning and histone modifications during this process.<ref name = "Teif_et_al">{{cite journal | vauthors = Teif VB, Vainshtein Y, Caudron-Herger M, Mallm JP, Marth C, Höfer T, Rippe K | title = Genome-wide nucleosome positioning during embryonic stem cell development | journal = Nature Structural & Molecular Biology | volume = 19 | issue = 11 | pages = 1185–1192 | date = November 2012 | pmid = 23085715 | doi = 10.1038/nsmb.2419 | s2cid = 34509771 }}</ref> There are two components of this process: turning off the expression of embryonic stem cell (ESC) genes, and the activation of cell fate genes. Lysine specific demethylase 1 ([[LSD1|KDM1A]]) is thought to prevent the use of [[enhancer (genetics)|enhancer]] regions of pluripotency genes, thereby inhibiting their transcription.<ref name="ReferenceB">{{cite journal | vauthors = Whyte WA, Bilodeau S, Orlando DA, Hoke HA, Frampton GM, Foster CT, Cowley SM, Young RA | title = Enhancer decommissioning by LSD1 during embryonic stem cell differentiation | journal = Nature | volume = 482 | issue = 7384 | pages = 221–225 | date = February 2012 | pmid = 22297846 | pmc = 4144424 | doi = 10.1038/nature10805 | bibcode = 2012Natur.482..221W }}</ref> It interacts with [[NuRD|Mi-2/NuRD complex]] (nucleosome remodelling and histone deacetylase) complex,<ref name="ReferenceB"/> giving an instance where methylation and acetylation are not discrete and mutually exclusive, but intertwined processes.