Editing X-inactivation (section)

==Mechanism==

=== Cycle of X-chromosome activation in rodents ===
The paragraphs below have to do only with rodents and do not reflect XI in the majority of mammals.
X-inactivation is part of the activation cycle of the X chromosome throughout the female life. The egg and the fertilized zygote initially use maternal transcripts, and the whole embryonic genome is silenced until [[zygotic genome activation]]. Thereafter, all mouse cells undergo an early, [[Imprinting (genetics)|imprinted]] inactivation of the paternally-derived X chromosome in [[mammalian embryogenesis|4–8 cell stage]] [[embryo]]s.<ref>{{cite journal | vauthors = Takagi N, Sasaki M | title = Preferential inactivation of the paternally derived X chromosome in the extra embryonic membranes of the mouse | journal = Nature | volume = 256 | issue = 5519 | pages = 640–2 | date = August 1975 | pmid = 1152998 | doi = 10.1038/256640a0 | bibcode = 1975Natur.256..640T | s2cid = 4190616 }}</ref><ref>{{cite journal | vauthors = Cheng MK, Disteche CM | title = Silence of the fathers: early X inactivation | journal = BioEssays | volume = 26 | issue = 8 | pages = 821–4 | date = August 2004 | pmid = 15273983 | doi = 10.1002/bies.20082 | url = http://www3.interscience.wiley.com/cgi-bin/fulltext/109565168/PDFSTART | doi-access =  | url-access = subscription }}{{dead link|date=February 2019|bot=medic}}{{cbignore|bot=medic}}</ref><ref name="okamoto">{{cite journal | vauthors = Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E | title = Epigenetic dynamics of imprinted X inactivation during early mouse development | journal = Science | volume = 303 | issue = 5658 | pages = 644–9 | date = January 2004 | pmid = 14671313 | doi = 10.1126/science.1092727 | bibcode = 2004Sci...303..644O | s2cid = 26326026 }}</ref><ref name=":2">{{cite journal | vauthors = Deng Q, Ramsköld D, Reinius B, Sandberg R | title = Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells | journal = Science | volume = 343 | issue = 6167 | pages = 193–6 | date = January 2014 | pmid = 24408435 | doi = 10.1126/science.1245316 | bibcode = 2014Sci...343..193D | s2cid = 206552108 }}</ref> The [[extraembryonic tissue]]s (which give rise to the [[placenta]] and other tissues supporting the embryo) retain this early imprinted inactivation, and thus only the maternal X chromosome is active in these tissues.

In the early [[blastocyst]], this initial, imprinted X-inactivation is [[X-chromosome reactivation|reversed]] in the cells of the [[inner cell mass]] (which give rise to the embryo), and in these cells both X chromosomes become active again. Each of these cells then independently and randomly inactivates one copy of the X chromosome.<ref name=okamoto/> This inactivation event is irreversible during the lifetime of the individual, with the exception of the germline. In the female [[germline]] before meiotic entry, X-inactivation is reversed, so that after meiosis all haploid [[oocyte]]s contain a single active X chromosome.

==== Overview ====
The '''Xi''' marks the inactive, '''Xa''' the active X chromosome. '''X<sup>P</sup>''' denotes the paternal, and '''X<sup>M</sup>''' to denotes the maternal X chromosome. When the egg (carrying '''X<sup>M</sup>'''), is fertilized by a sperm (carrying a Y or an '''X<sup>P</sup>''') a diploid zygote forms. From zygote, through adult stage, to the next generation of eggs, the X chromosome undergoes the following changes:

# Xi<sup>P</sup> Xi<sup>M</sup> zygote → undergoing [[zygotic genome activation]], leading to:
# '''Xa<sup>P</sup>''' '''Xa<sup>M</sup>'''  → undergoing '''imprinted''' (paternal) '''X-inactivation''', leading to:
# Xi<sup>P</sup> '''Xa<sup>M</sup>'''  → undergoing '''X-activation''' in the early [[blastocyst]] stage, leading to:
# '''Xa<sup>P</sup> Xa<sup>M</sup>'''  → undergoing '''random X-inactivation''' in the embryonic lineage (inner cell mass) in the blastocyst stage, leading to:
# Xi<sup>P</sup> '''Xa<sup>M</sup>''' OR '''Xa<sup>P</sup>''' Xi<sup>M</sup> → undergoing '''X-reactivation''' in [[primordial germ cells]] before [[meiosis]], leading to:
# '''Xa<sup>M</sup>''' '''Xa<sup>P</sup>''' diploid germ cells in meiotic arrest. As the meiosis I only completes with [[ovulation]], human germ cells exist in this stage from the first weeks of development until puberty. The completion of meiosis leads to:
# '''Xa<sup>M</sup>''' AND '''Xa<sup>P</sup>''' haploid germ cells (eggs).

The X activation cycle has been best studied in mice, but there are multiple studies in humans. As most of the evidence is coming from mice, the above scheme represents the events in mice. The completion of the meiosis is simplified here for clarity. Steps 1–4 can be studied in in vitro fertilized embryos, and in differentiating stem cells; X-reactivation happens in the developing embryo, and subsequent (6–7) steps inside the female body, therefore much harder to study.

===== Timing =====

The timing of each process depends on the species, and in many cases the precise time is actively debated. [The whole part of the human timing of X-inactivation in this table is highly questionable and should be removed until properly substantiated by empirical data]
{| class="wikitable"
|+Approximate timing of major events in the X chromosome activation cycle
|
|'''Process'''
|'''Mouse'''
|'''Human'''
|-
|1
|Zygotic genome activation
|2–4 cell stage<ref name=":4">{{cite journal | vauthors = Xue Z, Huang K, Cai C, Cai L, Jiang CY, Feng Y, Liu Z, Zeng Q, Cheng L, Sun YE, Liu JY, Horvath S, Fan G | title = Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing | language = En | journal = Nature | volume = 500 | issue = 7464 | pages = 593–7 | date = August 2013 | pmid = 23892778 | pmc = 4950944 | doi = 10.1038/nature12364 | bibcode = 2013Natur.500..593X }}</ref>
|2–8 cell stage<ref name=":4" />
|-
|2
|Imprinted (paternal) X-inactivation
|4–8 cell stage<ref name=":2" /><ref name=":3">{{cite journal|vauthors=Borensztein M, Syx L, Ancelin K, Diabangouaya P, Picard C, Liu T, Liang JB, Vassilev I, Galupa R, Servant N, Barillot E, Surani A, Chen CJ, Heard E|date=March 2017|title=Xist-dependent imprinted X inactivation and the early developmental consequences of its failure|journal=Nature Structural & Molecular Biology|language=En|volume=24|issue=3|pages=226–233|doi=10.1038/nsmb.3365|pmc=5337400|pmid=28134930}}</ref>
|Unclear if it takes place in humans<ref name=":5">{{cite journal | vauthors = Deng X, Berletch JB, Nguyen DK, Disteche CM | title = X chromosome regulation: diverse patterns in development, tissues and disease | language = En | journal = Nature Reviews. Genetics | volume = 15 | issue = 6 | pages = 367–78 | date = June 2014 | pmid = 24733023 | pmc = 4117651 | doi = 10.1038/nrg3687 }}</ref>
|-
|3
|X-activation
|Early blastocyst stage
|Early blastocyst stage
|-
|4
|Random X-inactivation in the embryonic lineage (inner cell mass)
|Late blastocyst stage
|Late blastocyst stage, after implantation<ref name=":5" />
|-
|5
|X-reactivation in primordial germ cells before meiosis
|
|From before developmental week 4 up to week 14<ref>{{cite journal | vauthors = Vértesy Á, Arindrarto W, Roost MS, Reinius B, Torrens-Juaneda V, Bialecka M, Moustakas I, Ariyurek Y, Kuijk E, Mei H, Sandberg R, van Oudenaarden A, Chuva de Sousa Lopes SM | display-authors = 6 | title = Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells | language = En | journal = Nature Communications | volume = 9 | issue = 1 | pages = 1873 | date = May 2018 | pmid = 29760424 | pmc = 5951918 | doi = 10.1038/s41467-018-04215-7 | bibcode = 2018NatCo...9.1873V }}</ref><ref>{{cite journal | vauthors = Guo F, Yan L, Guo H, Li L, Hu B, Zhao Y, Yong J, Hu Y, Wang X, Wei Y, Wang W, Li R, Yan J, Zhi X, Zhang Y, Jin H, Zhang W, Hou Y, Zhu P, Li J, Zhang L, Liu S, Ren Y, Zhu X, Wen L, Gao YQ, Tang F, Qiao J | display-authors = 6 | title = The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells | journal = Cell | volume = 161 | issue = 6 | pages = 1437–52 | date = June 2015 | pmid = 26046443 | doi = 10.1016/j.cell.2015.05.015 | doi-access = free }}</ref>
|}

===== Inheritance of inactivation status across cell generations =====

The descendants of each cell which inactivated a particular X chromosome will also inactivate that same chromosome. This phenomenon, which can be observed in the coloration of [[tortoiseshell cat]]s when females are [[heterozygous]] for the [[sex linkage|X-linked]] pigment gene, should not be confused with [[mosaic (genetics)|mosaicism]], which is a term that specifically refers to differences in the [[genotype]] of various cell populations in the same individual; X-inactivation, which is an [[epigenetics|epigenetic]] change that results in a different phenotype, is ''not'' a change at the [[genotype|genotypic]] level. For an individual cell or lineage the inactivation is therefore [[Skewed X-inactivation|skewed]] or '[[Skewed X-inactivation|non-random]]', and this can give rise to mild symptoms in female 'carriers' of [[X-linked]] genetic disorders.<ref>{{cite journal | vauthors = Puck JM, Willard HF | title = X inactivation in females with X-linked disease | journal = The New England Journal of Medicine | volume = 338 | issue = 5 | pages = 325–8 | date = January 1998 | pmid = 9445416 | doi = 10.1056/NEJM199801293380611 }}</ref>

===Selection of one active X chromosome===
Typical females possess two X chromosomes, and in any given cell one chromosome will be active (designated as Xa) and one will be inactive (Xi). However, studies of individuals with [[X chromosome#Role in disease|extra copies of the X chromosome]] show that in cells with more than two X chromosomes there is still only one Xa, and all the remaining X chromosomes are inactivated. This indicates that the default state of the X chromosome in females is inactivation, but one X chromosome is always selected to remain active.<ref name="Tallaksen2023">{{cite journal | vauthors = Tallaksen HB, Johannsen EB, Just J, Viuff MH, Gravholt CH, Skakkebæk A | title = The multi-omic landscape of sex chromosome abnormalities: current status and future directions | journal = Endocrine Connections | volume = 12 | issue = 9 | date = August 2023 | pmid = 37399516 | pmc = 10448593 | doi = 10.1530/EC-23-0011 }}</ref>

It is understood that X-chromosome inactivation is a random process, occurring at about the time of [[gastrulation]] in the [[epiblast]] (cells that will give rise to the embryo). The maternal and paternal X chromosomes have an equal probability of inactivation. This would suggest that women would be expected to suffer from X-linked disorders approximately 50% as often as men (because women have two X chromosomes, while men have only one); however, in actuality, the occurrence of these disorders in females is much lower than that. One explanation for this disparity is that 12–20% <ref>{{cite journal | vauthors = Balaton BP, Cotton AM, Brown CJ | title = Derivation of consensus inactivation status for X-linked genes from genome-wide studies | journal = Biology of Sex Differences | volume = 6 | issue = 35 | pages = 35 | date = 30 December 2015 | pmid = 26719789 | pmc = 4696107 | doi = 10.1186/s13293-015-0053-7 | doi-access = free }}</ref> of genes on the inactivated X chromosome remain expressed, thus providing women with added protection against defective genes coded by the X-chromosome. Some{{who|date=October 2014}} suggest that this disparity must be evidence of preferential (non-random) inactivation. Preferential inactivation of the paternal X-chromosome occurs in both marsupials and in cell lineages that form the membranes surrounding the embryo,<ref>{{cite journal | vauthors = Graves JA | title = Mammals that break the rules: genetics of marsupials and monotremes | journal = Annual Review of Genetics | volume = 30 | pages = 233–60 | year = 1996 | pmid = 8982455 | doi = 10.1146/annurev.genet.30.1.233 }}</ref> whereas in placental mammals either the maternally or the paternally derived X-chromosome may be inactivated in different cell lines.<ref>{{cite journal | vauthors = Lyon MF | title = X-chromosome inactivation and developmental patterns in mammals | journal = Biological Reviews of the Cambridge Philosophical Society | volume = 47 | issue = 1 | pages = 1–35 | date = January 1972 | pmid = 4554151 | doi = 10.1111/j.1469-185X.1972.tb00969.x | s2cid = 39402646 }}</ref>

The time period for X-chromosome inactivation explains this disparity. Inactivation occurs in the epiblast during gastrulation, which gives rise to the embryo.<ref>{{cite journal| vauthors = Migeon, B |title=X chromosome inactivation in human cells|url=http://hstalks.com/main/view_talk.php?t=1676&r=494&c=252|journal=The Biomedical & Life Sciences Collection|publisher=Henry Stewart Talks, Ltd|access-date=15 December 2013 |pages=1–54|year=2010}}</ref> Inactivation occurs on a cellular level, resulting in a mosaic expression, in which patches of cells have an inactive maternal X-chromosome, while other patches have an inactive paternal X-chromosome. For example, a female heterozygous for haemophilia (an X-linked disease) would have about half of her liver cells functioning properly, which is typically enough to ensure normal blood clotting.<ref name="Gartler_2001">{{cite journal | last1 = Gartler | first1 = Stanley M | last2 = Goldman | first2 = Michael A | name-list-style = vanc | title = X-Chromosome Inactivation | url = http://web.udl.es/usuaris/e4650869/docencia/segoncicle/genclin98/recursos_classe_(pdf)/revisionsPDF/XChromoInac.pdf | journal = Encyclopedia of Life Sciences | publisher = Nature Publishing Group | pages = 1–2 | year=2001 }}</ref><ref name="Connallon_2013">{{cite journal | vauthors = Connallon T, Clark AG | title = Sex-differential selection and the evolution of X inactivation strategies | journal = PLOS Genetics | volume = 9 | issue = 4 | pages = e1003440 | date = April 2013 | pmid = 23637618 | pmc = 3630082 | doi = 10.1371/journal.pgen.1003440 | doi-access = free }}</ref> Chance could result in significantly more dysfunctional cells; however, such statistical extremes are unlikely. Genetic differences on the chromosome may also render one X-chromosome more likely to undergo inactivation. Also, if one X-chromosome has a mutation hindering its growth or rendering it non viable, cells which randomly inactivated that X will have a selective advantage over cells which randomly inactivated the normal allele. Thus, although inactivation is initially random, cells that inactivate a normal allele (leaving the mutated allele active) will eventually be overgrown and replaced by functionally normal cells in which nearly all have the same X-chromosome activated.<ref name="Gartler_2001"/>

It is hypothesized that there is an autosomally-encoded 'blocking factor' which binds to the X chromosome and prevents its inactivation.<ref>{{Cite journal |last1=Avner |first1=Philip |last2=Heard |first2=Edith |date=January 2001 |title=X-chromosome inactivation: counting, choice and initiation |url=https://www.nature.com/articles/35047580 |journal=Nature Reviews Genetics |language=en |volume=2 |issue=1 |pages=59–67 |doi=10.1038/35047580 |pmid=11253071 |s2cid=5234164 |issn=1471-0064|url-access=subscription }}</ref> The model postulates that there is a limiting blocking factor, so once the available blocking factor molecule binds to one X chromosome the remaining X chromosome(s) are not protected from inactivation. This model is supported by the existence of a single Xa in cells with many X chromosomes and by the existence of two active X chromosomes in cell lines with twice the normal number of autosomes.<ref>{{cite book | first1 = Tahsin Stefan | last1 = Barakat | first2 = Joost | last2 = Gribnau | name-list-style = vanc | chapter = X Chromosome Inactivation and Embryonic Stem Cells | chapter-url = https://www.ncbi.nlm.nih.gov/books/NBK45037/ | title = The Cell Biology of Stem Cells | editor-first1 = Eran | editor-last1 = Meshorer | editor-first2 = Kathrin | editor-last2 = Plath | year = 2010 | publisher = Landes Bioscience and Springer Science+Business Media }}</ref>

Sequences at the '''X inactivation center''' ('''XIC'''), present on the X chromosome, control the silencing of the X chromosome. The hypothetical blocking factor is predicted to bind to sequences within the XIC.

=== Expression of X-linked disorders in heterozygous females ===
The effect of female X heterozygosity is apparent in some localized traits, such as the unique coat pattern of a [[calico cat]]. It can be more difficult, however, to fully understand the expression of un-localized traits in these females, such as the expression of disease.

Since males only have one copy of the X chromosome, all expressed X-chromosomal [[gene]]s (or [[allele]]s, in the case of multiple variant forms for a given gene in the population) are located on that copy of the chromosome. Females, however, will primarily express the genes or alleles located on the X-chromosomal copy that remains active. Considering the situation for one gene or multiple genes causing individual differences in a particular [[Phenotypic trait|phenotype]] (i.e., causing variation observed in the population for that phenotype), in homozygous females it does not particularly matter which copy of the chromosome is inactivated, as the alleles on both copies are the same. However, in females that are heterozygous at the causal genes, the inactivation of one copy of the chromosome over the other can have a direct impact on their phenotypic value. Because of this phenomenon, there is an observed increase in phenotypic variation in females that are heterozygous at the involved gene or genes than in females that are homozygous at that gene or those genes.<ref>{{cite journal | vauthors = Ma L, Hoffman G, Keinan A | title = X-inactivation informs variance-based testing for X-linked association of a quantitative trait | journal = BMC Genomics | volume = 16 | pages = 241 | date = March 2015 | issue = 1 | pmid = 25880738 | pmc = 4381508 | doi = 10.1186/s12864-015-1463-y | doi-access = free }}</ref> There are many different ways in which the phenotypic variation can play out. In many cases, heterozygous females may be asymptomatic or only present minor symptoms of a given disorder, such as with [[Adrenoleukodystrophy|X-linked adrenoleukodystrophy.]]<ref>{{cite journal | vauthors = Habekost CT, Pereira FS, Vargas CR, Coelho DM, Torrez V, Oses JP, Portela LV, Schestatsky P, Felix VT, Matte U, Torman VL, Jardim LB | title = Progression rate of myelopathy in X-linked adrenoleukodystrophy heterozygotes | journal = Metabolic Brain Disease | volume = 30 | issue = 5 | pages = 1279–84 | date = October 2015 | pmid = 25920484 | doi = 10.1007/s11011-015-9672-2 | s2cid = 11375978 }}</ref>

The differentiation of phenotype in heterozygous females is furthered by the presence of X-inactivation skewing. Typically, each X-chromosome is silenced in half of the cells, but this process is skewed when preferential inactivation of a chromosome occurs. It is thought that skewing happens either by chance or by a physical characteristic of a chromosome that may cause it to be silenced more or less often, such as an unfavorable mutation.<ref name=":0">{{cite journal | vauthors = Belmont JW | title = Genetic control of X inactivation and processes leading to X-inactivation skewing | journal = American Journal of Human Genetics | volume = 58 | issue = 6 | pages = 1101–8 | date = June 1996 | pmid = 8651285 | pmc = 1915050 }}</ref><ref name=":1">{{cite journal | vauthors = Holle JR, Marsh RA, Holdcroft AM, Davies SM, Wang L, Zhang K, Jordan MB | title = Hemophagocytic lymphohistiocytosis in a female patient due to a heterozygous XIAP mutation and skewed X chromosome inactivation | journal = Pediatric Blood & Cancer | volume = 62 | issue = 7 | pages = 1288–90 | date = July 2015 | pmid = 25801017 | doi = 10.1002/pbc.25483 | s2cid = 5516967 }}</ref>

On average, each X chromosome is inactivated in half of the cells, although 5-20% of women display X-inactivation skewing.<ref name=":0" /> In cases where skewing is present, a broad range of symptom expression can occur, resulting in expression varying from minor to severe depending on the skewing proportion. An extreme case of this was seen where monozygotic female twins had extreme variance in expression of [[Menkes disease]] (an X-linked disorder) resulting in the death of one twin while the other remained asymptomatic.<ref>{{cite journal | vauthors = Burgemeister AL, Zirn B, Oeffner F, Kaler SG, Lemm G, Rossier E, Büttel HM | title = Menkes disease with discordant phenotype in female monozygotic twins | journal = American Journal of Medical Genetics. Part A | volume = 167A | issue = 11 | pages = 2826–9 | date = November 2015 | pmid = 26239182 | pmc = 6475897 | doi = 10.1002/ajmg.a.37276 }}</ref>

It is thought that X-inactivation skewing could be caused by issues in the mechanism that causes inactivation, or by issues in the chromosome itself.<ref name=":0" /><ref name=":1" /> However, the link between phenotype and skewing is still being questioned, and should be examined on a case-by-case basis. A study looking at both symptomatic and asymptomatic females who were heterozygous for [[Duchenne muscular dystrophy|Duchenne]] and Becker muscular dystrophies (DMD) found no apparent link between transcript expression and skewed X-Inactivation. The study suggests that both mechanisms are independently regulated, and there are other unknown factors at play.<ref name="pmid22894145">{{cite journal | vauthors = Brioschi S, Gualandi F, Scotton C, Armaroli A, Bovolenta M, Falzarano MS, Sabatelli P, Selvatici R, D'Amico A, Pane M, Ricci G, Siciliano G, Tedeschi S, Pini A, Vercelli L, De Grandis D, Mercuri E, Bertini E, Merlini L, Mongini T, Ferlini A | title = Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype | journal = BMC Medical Genetics | volume = 13 | pages = 73 | date = August 2012 | pmid = 22894145 | pmc = 3459813 | doi = 10.1186/1471-2350-13-73 | doi-access = free }}</ref>

===Chromosomal component===
The X-inactivation center (or simply XIC) on the X chromosome is [[necessary and sufficient]] to cause X-inactivation. [[Chromosomal translocation]]s which place the XIC on an autosome lead to inactivation of the autosome, and X chromosomes lacking the XIC are not inactivated.<ref name=":6" /><ref name=":7" />

The XIC contains four non-[[translation (genetics)|translated]] [[RNA]] genes, [[Xist]], [[Xist#Tsix antisense transcript|Tsix]], [[Jpx (gene)|Jpx]] and [[Ftx (gene)|Ftx]], which are involved in X-inactivation. The XIC also contains binding sites for both known and unknown [[regulatory protein]]s.<ref name=":8" />

===Xist and Tsix RNAs===
{{Main|Xist}}
The X-inactive specific transcript ([[Xist]]) gene encodes a large [[NcRNA|non-coding RNA]] that is responsible for mediating the specific silencing of the X chromosome from which it is transcribed.<ref>{{cite journal | vauthors = Hoki Y, Kimura N, Kanbayashi M, Amakawa Y, Ohhata T, Sasaki H, Sado T | title = A proximal conserved repeat in the Xist gene is essential as a genomic element for X-inactivation in mouse | journal = Development | volume = 136 | issue = 1 | pages = 139–46 | date = January 2009 | pmid = 19036803 | doi = 10.1242/dev.026427 | doi-access = free }}</ref> The inactive X chromosome is coated by Xist RNA,<ref>{{cite journal | vauthors = Ng K, Pullirsch D, Leeb M, Wutz A | title = Xist and the order of silencing | journal = EMBO Reports | volume = 8 | issue = 1 | pages = 34–9 | date = January 2007 | pmid = 17203100 | pmc = 1796754 | doi = 10.1038/sj.embor.7400871 | format = Review Article | quote = [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1796754/ Figure 1 Xist RNA encompasses the X from which it is transcribed.] }}</ref> whereas the Xa is not (See Figure to the right). X chromosomes that lack the Xist gene cannot be inactivated.<ref>{{cite journal | vauthors = Penny GD, Kay GF, Sheardown SA, Rastan S, [[Neil Brockdorff|Brockdorff N]] | title = Requirement for Xist in X chromosome inactivation | journal = Nature | volume = 379 | issue = 6561 | pages = 131–7 | year = 1996 | pmid = 8538762 | doi = 10.1038/379131a0 | bibcode = 1996Natur.379..131P | s2cid = 4329368 }}</ref> Artificially placing and expressing the Xist gene on another chromosome leads to silencing of that chromosome.<ref name="Herzing">{{cite journal | vauthors = Herzing LB, Romer JT, Horn JM, Ashworth A | title = Xist has properties of the X-chromosome inactivation centre | journal = Nature | volume = 386 | issue = 6622 | pages = 272–5 | date = March 1997 | pmid = 9069284 | doi = 10.1038/386272a0 | bibcode = 1997Natur.386..272H | s2cid = 4371247 }}</ref><ref name=":6">{{cite journal | vauthors = Lee JT, Jaenisch R | title = Long-range cis effects of ectopic X-inactivation centres on a mouse autosome | journal = Nature | volume = 386 | issue = 6622 | pages = 275–9 | date = March 1997 | pmid = 9069285 | doi = 10.1038/386275a0 | bibcode = 1997Natur.386..275L | s2cid = 10899129 }}</ref>

Prior to inactivation, both X chromosomes weakly express Xist RNA from the Xist gene. During the inactivation process, the future Xa ceases to express Xist, whereas the future Xi dramatically increases Xist RNA production. On the future Xi, the Xist RNA progressively coats the chromosome, spreading out from the XIC;<ref name=Herzing/> the Xist RNA does not localize to the Xa. The [[gene silencing|silencing of genes]] along the Xi occurs soon after coating by Xist RNA.

Like Xist, the [[Tsix]] gene encodes a large RNA which is not believed to encode a protein. The Tsix RNA is transcribed [[antisense]] to Xist, meaning that the Tsix gene overlaps the Xist gene and is [[transcription (genetics)|transcribed]] on the opposite strand of [[DNA]] from the Xist gene.<ref name=":7">{{cite journal | vauthors = Lee JT, Davidow LS, Warshawsky D | title = Tsix, a gene antisense to Xist at the X-inactivation centre | journal = Nature Genetics | volume = 21 | issue = 4 | pages = 400–4 | date = April 1999 | pmid = 10192391 | doi = 10.1038/7734 | s2cid = 30636065 }}</ref> Tsix is a negative regulator of Xist; X chromosomes lacking Tsix expression (and thus having high levels of Xist transcription) are inactivated much more frequently than normal chromosomes.

Like Xist, prior to inactivation, both X chromosomes weakly express Tsix RNA from the Tsix gene. Upon the onset of X-inactivation, the future Xi ceases to express Tsix RNA (and increases Xist expression), whereas Xa continues to express Tsix for several days.

Rep A is a long non coding RNA that works with another long non coding RNA, Xist, for X inactivation. Rep A inhibits the function of Tsix, the antisense of Xist, in conjunction with eliminating expression of Xite. It promotes methylation of the Tsix region by attracting PRC2 and thus inactivating one of the X chromosomes.<ref name=":8">Mercer, T.R., Dinger, M.E., Mattick, J.S., (2009). Long non-coding RNAs: insight into functions. Nature Reviews Genetics. (10) 155–159.</ref>

===Silencing===
The inactive X chromosome does not express the majority of its genes, unlike the active X chromosome. This is due to the silencing of the Xi by repressive [[heterochromatin]], which compacts the Xi DNA and prevents the expression of most genes.

Compared to the Xa, the Xi has high levels of [[DNA methylation]], low levels of [[histone acetylation]], low levels of [[histone H3]] lysine-4 [[histone methylation|methylation]], and high levels of histone H3 lysine-9 methylation and H3 lysine-27 methylation mark which is placed by the [[Polycomb recruitment in X chromosome inactivation|PRC2 complex recruited by Xist]], all of which are associated with gene silencing.<ref>{{cite journal | vauthors = Ng K, Pullirsch D, Leeb M, Wutz A | title = Xist and the order of silencing | journal = EMBO Reports | volume = 8 | issue = 1 | pages = 34–9 | date = January 2007 | pmid = 17203100 | pmc = 1796754 | doi = 10.1038/sj.embor.7400871 | format = Review Article | quote = [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1796754/ Table 1 Features of the inactive X territory] }} – Originated from;<br />{{cite journal | vauthors = Chow JC, Yen Z, Ziesche SM, Brown CJ | title = Silencing of the mammalian X chromosome | journal = Annual Review of Genomics and Human Genetics | volume = 6 | pages = 69–92 | year = 2005 | pmid = 16124854 | doi = 10.1146/annurev.genom.6.080604.162350 }}<br />{{cite journal | vauthors = Lucchesi JC, Kelly WG, Panning B | title = Chromatin remodeling in dosage compensation | journal = Annual Review of Genetics | volume = 39 | pages = 615–51 | year = 2005 | pmid = 16285873 | doi = 10.1146/annurev.genet.39.073003.094210 | citeseerx = 10.1.1.328.2992 }}</ref> [[PRC2]] regulates [[chromatin]] compaction and [[chromatin remodeling]] in several processes including the [[DNA damage (naturally occurring)|DNA damage]] response.<ref name="pmid28758948">{{cite journal |vauthors=Veneti Z, Gkouskou KK, Eliopoulos AG |title=Polycomb Repressor Complex 2 in Genomic Instability and Cancer |journal=Int J Mol Sci |volume=18 |issue=8 |pages= 1657|date=July 2017 |pmid=28758948 |pmc=5578047 |doi=10.3390/ijms18081657 |doi-access=free }}</ref> Additionally, a histone variant called macroH2A ([[H2AFY]]) is exclusively found on [[nucleosome]]s along the Xi.<ref name="Costanzi1998">{{cite journal | vauthors = Costanzi C, Pehrson JR | title = Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals | journal = Nature | volume = 393 | issue = 6685 | pages = 599–601 | date = June 1998 | pmid = 9634239 | doi = 10.1038/31275 | bibcode = 1998Natur.393..599C | s2cid = 205001095 }}</ref><ref name="Costanzi2000">{{cite journal | vauthors = Costanzi C, Stein P, Worrad DM, Schultz RM, Pehrson JR | title = Histone macroH2A1 is concentrated in the inactive X chromosome of female preimplantation mouse embryos | journal = Development | volume = 127 | issue = 11 | pages = 2283–9 | date = June 2000 | doi = 10.1242/dev.127.11.2283 | pmid = 10804171 | url = http://dev.biologists.org/cgi/reprint/127/11/2283.pdf }}</ref>

===Barr bodies===
{{main|Barr body}}

DNA packaged in heterochromatin, such as the Xi, is more condensed than DNA packaged in [[euchromatin]], such as the Xa. The inactive X forms a discrete body within the nucleus called a [[Barr body]].<ref name="barr 1949">{{cite journal | vauthors = Barr ML, Bertram EG | title = A morphological distinction between neurones of the male and female, and the behaviour of the nucleolar satellite during accelerated nucleoprotein synthesis | journal = Nature | volume = 163 | issue = 4148 | pages = 676–677 | date = April 1949 | pmid = 18120749 | doi = 10.1038/163676a0 | bibcode = 1949Natur.163..676B | s2cid = 4093883 }}</ref> The Barr body is generally located on the periphery of the [[cell nucleus|nucleus]], is late [[DNA replication|replicating]] within the [[cell cycle]], and, as it contains the Xi, contains heterochromatin modifications and the Xist RNA.

====Expressed genes on the inactive X chromosome====
A fraction of the genes along the X chromosome escape inactivation on the Xi. The Xist gene is expressed at high levels on the Xi and is not expressed on the Xa.<ref name="Plath">{{cite journal | vauthors = Plath K, Mlynarczyk-Evans S, Nusinow DA, Panning B | title = Xist RNA and the mechanism of X chromosome inactivation | journal = Annual Review of Genetics | volume = 36 | pages = 233–78 | year = 2002 | pmid = 12429693 | doi = 10.1146/annurev.genet.36.042902.092433 }}</ref> Many other genes escape inactivation; some are expressed equally from the Xa and Xi, and others, while expressed from both chromosomes, are still predominantly expressed from the Xa.<ref name="Carrel L, Willard H 2005 400–404">{{cite journal | vauthors = Carrel L, Willard HF | title = X-inactivation profile reveals extensive variability in X-linked gene expression in females | journal = Nature | volume = 434 | issue = 7031 | pages = 400–4 | date = March 2005 | pmid = 15772666 | doi = 10.1038/nature03479 | bibcode = 2005Natur.434..400C | s2cid = 4358447 }}</ref><ref name="Calabrese JM, Sun W, Song L, Mugford JW, Williams L, Yee D, Starmer J, Mieczkowski P, Crawford GE, Magnuson T 2012 951–63">{{cite journal | vauthors = Calabrese JM, Sun W, Song L, Mugford JW, Williams L, Yee D, Starmer J, Mieczkowski P, Crawford GE, Magnuson T | title = Site-specific silencing of regulatory elements as a mechanism of X inactivation | journal = Cell | volume = 151 | issue = 5 | pages = 951–63 | date = November 2012 | pmid = 23178118 | pmc = 3511858 | doi = 10.1016/j.cell.2012.10.037 }}</ref><ref name="Yang F, Babak T, Shendure J, Disteche CM 2010 614–22">{{cite journal | vauthors = Yang F, Babak T, Shendure J, Disteche CM | title = Global survey of escape from X inactivation by RNA-sequencing in mouse | journal = Genome Research | volume = 20 | issue = 5 | pages = 614–22 | date = May 2010 | pmid = 20363980 | pmc = 2860163 | doi = 10.1101/gr.103200.109 }}</ref> Up to one quarter of genes on the human Xi are capable of escape.<ref name="Carrel L, Willard H 2005 400–404"/> Studies in the mouse suggest that in any given cell type, 3% to 15% of genes escape inactivation, and that escaping gene identity varies between tissues.<ref name="Calabrese JM, Sun W, Song L, Mugford JW, Williams L, Yee D, Starmer J, Mieczkowski P, Crawford GE, Magnuson T 2012 951–63"/><ref name="Yang F, Babak T, Shendure J, Disteche CM 2010 614–22"/>

Many of the genes which escape inactivation are present along regions of the X chromosome which, unlike the majority of the X chromosome, contain genes also present on the [[Y chromosome]]. These regions are termed [[pseudoautosomal]] regions, as individuals of either sex will receive two copies of every gene in these regions (like an autosome), unlike the majority of genes along the sex chromosomes. Since individuals of either sex will receive two copies of every gene in a [[pseudoautosomal region]], no dosage compensation is needed for females, so it is postulated that these regions of DNA have evolved mechanisms to escape X-inactivation. The genes of pseudoautosomal regions of the Xi do not have the typical modifications of the Xi and have little Xist RNA bound.

The existence of genes along the inactive X which are not silenced explains the defects in humans with atypical numbers of the X chromosome, such as [[Turner syndrome]] (X0, caused by SHOX gene<ref>{{Cite web |title=Turner syndrome: MedlinePlus Genetics |url=https://medlineplus.gov/genetics/condition/turner-syndrome/ |access-date=10 February 2023 |website=medlineplus.gov |language=en}}</ref>) or [[Klinefelter syndrome]] (XXY). Theoretically, X-inactivation should eliminate the differences in gene dosage between affected individuals and individuals with a typical chromosome complement. In affected individuals, however, X-inactivation is incomplete and the dosage of these non-silenced genes will differ as they escape X-inactivation, similar to an autosomal [[aneuploidy]].

The precise mechanisms that control escape from X-inactivation are not known, but silenced and escape regions have been shown to have distinct chromatin marks.<ref name="Calabrese JM, Sun W, Song L, Mugford JW, Williams L, Yee D, Starmer J, Mieczkowski P, Crawford GE, Magnuson T 2012 951–63"/><ref>{{cite journal | vauthors = Berletch JB, Yang F, Disteche CM | title = Escape from X inactivation in mice and humans | journal = Genome Biology | volume = 11 | issue = 6 | pages = 213 | date = June 2010 | pmid = 20573260 | pmc = 2911101 | doi = 10.1186/gb-2010-11-6-213 | doi-access = free }}</ref> It has been suggested that escape from X-inactivation might be mediated by expression of [[long non-coding RNA]] (lncRNA) within the escaping chromosomal domains.<ref name="pmid21047393">{{cite journal | vauthors = Reinius B, Shi C, Hengshuo L, Sandhu KS, Radomska KJ, Rosen GD, Lu L, Kullander K, Williams RW, Jazin E | title = Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse | journal = BMC Genomics | volume = 11 | pages = 614 | date = November 2010 | pmid = 21047393 | pmc = 3091755 | doi = 10.1186/1471-2164-11-614 | doi-access = free }}</ref>