X Chromosome: X Inactivation

Figure 1: Calico cat.

The patchy distribution of color on calico cats results from the random inactivation of one X chromosome in females.

Ksmith4f via Wikimedia Commons.

Unlike the gene-poor Y chromosome, the X chromosome contains over 1,000 genes that are essential for proper development and cell viability. However, females carry two copies of the X chromosome, resulting in a potentially toxic double dose of X-linked genes. To correct this imbalance, mammalian females have evolved a unique mechanism of dosage compensation distinct from that used by organisms such as flies and worms. In particular, by way of the process called X-chromosome inactivation (XCI), female mammals transcriptionally silence one of their two Xs in a complex and highly coordinated manner (Lyon, 1961). The inactivated X chromosome then condenses into a compact structure called a Barr body, and it is stably maintained in a silent state (Boumil & Lee, 2001).

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.

X inactivation exists in two different forms: random and imprinted. Although both forms utilize the same RNAs and silencing enzymes, they differ in terms of their developmental timing and mechanism of action.

As previously mentioned, RNA plays an important role in X inactivation. Specifically, two noncoding, complementary RNAs—XIST and TSIX—initiate and control the inactivation process.

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.

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.

© 2005 Nature Publishing Group Huynh, K. D. et al. X-chromosome inactivation: a hypothesis linking ontogeny and phylogeny. Nature Reviews Genetics 6, 10 (2005). All rights reserved.

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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).

X inactivation has been studied for over half a century, yet several unresolved questions remain. For example, how exactly does the cell correctly count its number of Xs? How is silencing achieved and maintained at the molecular level? Why do noncoding RNAs play such a large role in XCI? How do some crucial genes manage to escape from the pervasive influence of XIST on the inactive X?

The study of X inactivation may also provide insight into cancer biology, as two active Xs have been found in many human breast and ovarian tumors (Liao et al., 2003). Additionally, the X-inactivation process can extend beyond the scope of X-linked genes and be applied to many human disorders involving imprinted genes—genes expressed from only one of two parental chromosomes that are apparently also regulated by noncoding RNAs (Sleutels & Barlow, 2002). Without a doubt, X inactivation represents a great model system with which to study a broad range of developmental and epigenetic processes—those involving stable gene expression without changes to the underlying DNA sequence.