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,
imprinted inactivation of the paternally-derived X chromosome in
4–8 cell stage embryos. The
extraembryonic tissues (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
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.
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
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. 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% of genes on the inactivated X chromosome remain expressed, thus providing women with added protection against defective genes coded by the X-chromosome. Some 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, whereas in placental mammals either the maternally or the paternally derived X-chromosome may be inactivated in different cell lines. The time period for X-chromosome inactivation explains this disparity. Inactivation occurs in the epiblast during gastrulation, which gives rise to the embryo. 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. 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. 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. 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
tortoiseshell 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
genes (or
alleles, 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
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. 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
X-linked adrenoleukodystrophy. 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. On average, each X chromosome is inactivated in half of the cells, although 5-20% of women display X-inactivation skewing. 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.
Chromosomal component The X-inactivation center (or simply XIC) on the X chromosome is
necessary and sufficient to cause X-inactivation.
Chromosomal translocations which place the XIC on an autosome lead to inactivation of the autosome, and X chromosomes lacking the XIC are not inactivated. The inactive X chromosome is coated by Xist RNA, whereas the Xa is not (See Figure to the right). X chromosomes that lack the Xist gene cannot be inactivated. Artificially placing and expressing the Xist gene on another chromosome leads to silencing of that chromosome. 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; 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.
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
methylation, and high levels of histone H3 lysine-9 methylation and H3 lysine-27 methylation mark which is placed by the
PRC2 complex recruited by Xist, all of which are associated with gene silencing.
PRC2 regulates
chromatin compaction and
chromatin remodeling in several processes including the
DNA damage response. Additionally, a histone variant called macroH2A (
H2AFY) is exclusively found on
nucleosomes along the Xi.
Barr bodies 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. The Barr body is generally located on the periphery of the
nucleus, is late
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. 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. Up to one quarter of genes on the human Xi are capable of escape.) 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. 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. ==Uses in experimental biology==