X-inactivation



X-inactivation (also called lyonization) is a process by which one of the two copies of the X chromosome present in female mammals is inactivated. The inactive X chromosome is silenced by its being packaged in such a way that it has a transcriptionally inactive structure called heterochromatin. As female mammals have two X chromosomes, X-inactivation prevents them from having twice as many X chromosome gene products as males, which only possess a single copy of the X chromosome (see dosage compensation). The choice of which X chromosome will be inactivated is random in placental mammals such as humans, but once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and its descendants in the organism. Unlike the random X-inactivation in placental mammals, inactivation in marsupials applies exclusively to the paternally derived X chromosome.

History
In 1959 Susumu Ohno showed that the two X-chromosomes of mammals were different: one appeared similar to the autosomes; the other was condensed and heterochromatic. This finding suggested, independently to two groups of investigators, that one of the X-chromosomes underwent inactivation. In 1961, Mary Lyon proposed the random inactivation of one female X chromosome to explain the mottled phenotype of female mice heterozygous for coat color genes. The Lyon hypothesis also accounted for the findings that one copy of the X chromosome in female cells was highly condensed, and that mice with only one copy of the X chromosome developed as infertile females. This suggested to Ernest Beutler, studying heterozygous females for Glucose-6-phosphate dehydrogenase (G6PD) deficiency, that there were two red cell populations of erythrocytes in such heterozygotes: deficient cells and normal cells, depending on whether the inactivated X chromosome contains the normal or defective G6PD allele.

The Lyon hypothesis became the Lyon Law on July 22, 2011 at the EMBO 50-years of X-inactivation conference in Oxford.

Timing
All mouse cells undergo an early, imprinted inactivation of the paternally-derived X chromosome in two-cell or four-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. This inactivation event is irreversible during the lifetime of the cell, so all the descendants of a cell which inactivated a particular X chromosome will also inactivate that same chromosome. This phenomenon, which can be observed in the coloration of tortoiseshell cats when females are heterozygous for the X-linked gene, should not be confused with 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 epigenetic change that results in a different phenotype, is not a change at the genotypic level. For an individual cell or lineage the inactivation is therefore skewed or 'non-random', and this can give rise to mild symptoms in female 'carriers' of X-linked genetic disorders.

X-inactivation is reversed in the female germline, so that all oocytes contain an active X chromosome.

Selection of one active X chromosome
Normal 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 having 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 reality the occurrence is much lower than that in females. One explanation for this disparity is that over 25% 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 proof 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, but does not occur in the vast majority of our cells.

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. Statistics could make the ratio of properly to unproperly functioning cells unfavorable, 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.

It is hypothesized that there is an autosomally-encoded 'blocking factor' which binds to the X chromosome and prevents its inactivation. 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.

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 XIC contains four non-translated RNA genes, Xist, Tsix, Jpx and Ftx, which are involved in X-inactivation. The XIC also contains binding sites for both known and unknown regulatory proteins.

Xist and Tsix RNAs
The X-inactive specific transcript (Xist) gene encodes a large non-coding RNA that is responsible for mediating the specific silencing of the X chromosome from which it is transcribed. The inactive X chromosome is coated by Xist RNA, whereas the Xa is not (See Figure to the right). The Xist gene is the only gene which is expressed from the Xi but not from the Xa. X chromosomes which 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; the Xist RNA does not localize to the Xa. The 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 transcribed on the opposite strand of DNA from the Xist gene. 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.

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, all of which are associated with gene silencing. 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. 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.

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 abnormal numbers of the X chromosome, such as Turner syndrome (X0) or Klinefelter syndrome (XXY). Theoretically, X-inactivation should eliminate the differences in gene dosage between affected individuals and individuals with a normal chromosome complement, but in affected individuals the dosage of these non-silenced genes will differ as they escape X-inactivation.

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
Stanley Michael Gartler used X chromosome inactivation to demonstrate the clonal origin of cancers. Examining normal tissues and tumors from females heterozygous for isoenzymes of the sex-linked G6PD gene demonstrated that tumor cells from such individuals express only one form of G6PD, whereas normal tissues are composed of a nearly equal mixture of cells expressing the two different phenotypes. This pattern suggests that a single cell, and not a population, grows into a cancer.

Researchers have also investigated using X-chromosome inactivation to silence the activity of autosomal chromosomes. For example, Jiang et al. inserted a copy of the Xist gene into one copy of chromosome 21 in stem cells derived from an individual with trisomy 21 (Down's syndrome). The inserted Xist gene induces Barr body formation, triggers stable heterochromatin modifications, and silences most of the genes on the extra copy of chromosome 21. In these modified stem cells, the Xist-mediated gene silencing seems to reverse some of the defects associated with Down's syndrome.