X-inactivation

The coloration of tortoiseshell cats is a visible manifestation of X-inactivation. The "black" and "orange" alleles of a fur coloration gene reside on the X chromosome. For any given patch of fur, the inactivation of an X chromosome that carries one gene results in the fur color of the other, active gene.

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 packaging in repressive heterochromatin. X-inactivation occurs so that the female, with two X chromosomes, does not have twice as many X chromosome gene products as the male, 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 mice and humans, but once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell. 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 like the autosomes; the other was condensed and heterochromatic (Ohno S, Kaplan WD, Kinosita R: Formation of the sex chromatin by a single X-chromosome in liver cells of rattus norvegicus. Exp Cell Res 18: 415-418, 1959). This finding suggested that one of the X-chromosomes underwent inactivation independently to two groups of investigators. Mary Lyon proposed the random inactivation of one female X chromosome in 1961 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 fertile females. Ernest Beutler, studying heterozygous women for G6PD deficiency independently proposed that there were two red cell populations of erythrocytes in such heterozygotes, deficient cells and normal cells (Beutler E, Yeh M, Fairbanks VF: The normal human female as a mosaic of X-chromosome activity: Studies using the gene for G-6-PD deficiency as a marker. Proc Natl Acad Sci USA 48: 9-16, 1962).

Mechanism

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 leads to mosaicism if a female is heterozygous for a X-linked gene, which can be observed in the coloration of calico cats.

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

Selection of active X chromosomes

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

Xist and Tsix RNAs

The Xist gene encodes a large RNA which is not believed to encode a protein. The Xist RNA is the major effector of X-inactivation. The inactive X chromosome is coated by Xist RNA, whereas the Xa is not. 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 coats 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 is exclusively found on nucleosomes along the Xi.

Barr bodies

Main article: 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. 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. Other genes are expressed equally from the Xa and Xi; mice contain few genes which escape silencing whereas up to a quarter of human X chromosome genes are expressed from the Xi. Many of these genes occur in clusters.

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.

See also

• Sex-determination system
• Dosage compensation
• Barr body
• Heterochromatin
• Epigenetics
• Tortoiseshell cat

Additional Resources

• Carrel L, Willard H (2005). "X-inactivation profile reveals extensive variability in X-linked gene expression in females". Nature. 434 (7031): 400–4. PMID 15772666.
• Chow J, Yen Z, Ziesche S, Brown C (2005). "Silencing of the mammalian X chromosome". Annu Rev Genomics Hum Genet. 6: 69–92. PMID 16124854.CS1 maint: Multiple names: authors list (link)
• Lyon M (2003). "The Lyon and the LINE hypothesis". Semin Cell Dev Biol. 14 (6): 313–8. PMID 15015738.
• Okamoto I, Otte A, Allis C, Reinberg D, Heard E (2004). "Epigenetic dynamics of imprinted X inactivation during early mouse development". Science. 303 (5658): 644–9. PMID 14671313.CS1 maint: Multiple names: authors list (link)
• Plath K, Mlynarczyk-Evans S, Nusinow D, Panning B. "Xist RNA and the mechanism of X chromosome inactivation". Annu Rev Genet. 36: 233–78. PMID 12429693.CS1 maint: Multiple names: authors list (link)
• Huynh KD, Lee JT (2005). "X-chromosome inactivation: a hypothesis linking ontogeny and phylogeny". Nature Rev Genet. 9 (5): 41--8. PMID 15818384.* X-inactivation as a possible cause for autoimmunity