A prime example of X inactivation is in the coat-color patterning of tortoiseshell or calico cats (Figure 1). In cats, the fur pigmentation gene is X-linked, and depending on which copy of the X chromosome each cell chooses to leave active, either an orange or black coat color results. X inactivation only occurs in cells with multiple X chromosomes, which explains why almost all calico cats are female.
Imprinted X Inactivation: Preferential Silencing of the Father
As mentioned earlier in the article, there are two types of X inactivation: imprinted and random. During imprinted X inactivation, the paternal (father's) X chromosome is preferentially silenced in the placenta (an extraembryonic tissue) of eutherian mammals, as well as in all cells of earlier marsupial mammals, such as opossums and kangaroos (Graves, 2006; Huynh & Lee, 2005). Interestingly, XIST and TSIX are both important for imprinted XCI in the mouse (a eutherian), but marsupials do not have an XIST gene homologue (Duret et al., 2006; Lee, 2000; Marahrens et al., 1997; Sado et al., 2001). This raises the question of how imprinted inactivation is achieved in marsupials without using XIST. One possible mechanism involves pre-silencing of the paternal X in the male germ line during a process whereby both sex chromosomes are inactivated during meiosis; here, the inactive state of the X could be transmitted to the next generation (Hornecker et al., 2007; Huynh & Lee, 2005; Namekawa et al., 2007). From an evolutionary point of view, such a form of inherited XCI would have easily and economically achieved dosage compensation in female embryos, because only females inherit an X chromosome from their fathers.
Interestingly, remnants of this type of mechanism may still exist in eutherian mammals, such as the mouse (Huynh & Lee, 2005). While debate remains concerning the details of how and when dosage compensation is achieved, it is clear that imprinted XCI first occurs in all cells of early eutherian mammalian embryos prior to implantation into the uterus (Huynh & Lee, 2005; Okamoto & Heard, 2006). Together with the fact that earlier mammals, such as marsupials, also only silence their paternal Xs, imprinted XCI may represent an evolutionarily ancestral form of X inactivation. Occurring before the appearance of XIST and TSIX, imprinted XCI led to the evolution of random XCI in eutherian mammals.
Random X Inactivation
Random X inactivation occurs in the early female embryo, where both the maternal and the paternal X chromosome have an equal chance of becoming inactivated (Figure 4). Each female cell has the difficult task of trying to distinguish between two X chromosomes within the same nucleus, then designating one as an active X chromosome and the other as an inactive X. This complex process of silencing is accomplished independently in each cell, largely by XIST and TSIX.
Embryonic stem (ES) cells can undergo random X inactivation when differentiated in vitro (Martin et al., 1978; Rastan & Robertson, 1985), and they thus serve as a good model system with which to study this phenomenon. In fact, the use of ES cells along with early mouse embryos has enabled geneticists to dissect the different phases of the random XCI pathway. It seems that each cell first counts its number of X chromosomes, then randomly chooses one X to remain active, and, finally, silences the future inactive X (Bourmil & Lee, 2001). Whole-chromosome silencing involves the recruitment of many specialized factors, such as histone variants and chromatin modifiers (Lucchesi et al., 2005).
In addition to silencing one of the two Xs, the cell must also make sure that the other X remains active. Thus, there must be a way for the two Xs to communicate with each other to designate mutually exclusive fates. Interestingly, recent evidence suggests that this communication is mediated by protein- and transcription-dependent pairing between the Xs during early development (Bacher et al., 2006; Xu et al., 2006, 2007). The random XCI story becomes even more complex with the discovery of various enhancers and modifiers that can alter or skew inactivation of one X chromosome over the other.
The ontogeny of X-chromosome inactivation in the mouse: two current views.
Figure 4: The ontogeny of X-chromosome inactivation in the mouse: two current views.
a) The de novo inactivation model requires many rounds of inactivation and reactivation: the paternal germline initiates meiotic sex-chromosome inactivation, but the X chromosome is completely reactivated after meiosis. The zygote inherits two fully active X chromosomes and begins re-inactivation of the paternal X chromosome (XP) at the 4- to 8-cell stage. In the trophectoderm (extra-embryonic cells, shown in blue), XP silencing is maintained, therefore accounting for the imprinted form of X-chromosome inactivation. By contrast, in the epiblast (green cells), yet another round of reactivation takes place in preparation for a final round of inactivation in the form of random X-chromosome inactivation. b) In the pre-inactivation model, the female zygote inherits a partially silent XP and maintains the silent state throughout pre-implantation development. Silencing becomes globalized and complete in extra-embryonic tissues. This accounts for the imprinted form of X-chromosome inactivation. By contrast, the epiblast cells of the inner cell mass (ICM) undergo a single round of reactivation followed by a random form of X-chromosome inactivation.
Why Evolve Random X Inactivation?
This comparison of imprinted versus random X inactivation raises an important question: If all cells of the early preimplantation embryo already inactivate the paternal X, why is there a need to reactivate it, only to randomly inactivate an X chromosome again? One hypothesis is that perhaps random X inactivation evolved in placental mammals in order to have an additional chance to cope with X-linked mutations. Specifically, if the paternal X is always inactivated, this places the burden of all X-chromosome gene expression on the mother. Placental mammals may have therefore evolved random X inactivation to alleviate the burden of maternal X-chromosome mutations. In Rett's syndrome, for instance, females that carry a mutated copy of the MECP2 gene on the maternal X are able to survive (although with variable symptoms), because the paternal X has a normal copy that remains active in some cells (Ham et al., 2005). Random XCI is also mechanistically more complete than imprinted XCI, which tends to be very leaky, or still exhibit some normal phenotype, in marsupials (Graves, 2006).
Although random XCI is remarkably complete, a significant minority of eutherian genes can escape inactivation and remain active, particularly in humans, despite the fact that XIST RNA spreads along the entire X chromosome (Carrel & Willard, 2005; Johnston et al., 2008). The fact that certain genes can escape silencing directly impacts certain human conditions in which there is an abnormal number of X chromosomes. For example, XO females (Turner's syndrome), XXX females, and XXY males (Klinefelter's syndrome) all display developmental defects (including infertility and congenital heart problems), even though supernumerary Xs are silenced in XXX and XXY individuals (Figure 2), while the single X of the Turner's syndrome female remains active (Ham et al., 2005). These syndromes may be attributed to the fact that X-linked genes that normally escape XCI are not expressed at the correct dosage.
