This is the situation in ES cells, where both X chromosomes are active. Once the ES cells begin to differentiate, one of the pair stops expressing Tsix. This allows expression of Xist from that X chromosome, which drives X inactivation.
Tsix alone is probably not enough to keep Xist repressed. In ES cells, the proteins Oct4, Sox2 and Nanog bind to the first intron of Xist and suppress its expression[112]. Oct4 and Sox2 were two of the four factors used by Shinya Yamanaka when he reprogrammed somatic cells to the pluripotent iPS cell type. Subsequent experiments showed that Nanog (named after the mythical Celtic land of everlasting youth) can also work as a reprogramming factor. Oct4, Sox2 and Nanog are highly expressed in undifferentiated cells like ES cells, but their levels fall as cells start to differentiate. When this happens in differentiating female ES cells, Oct4, Sox2 and Nanog stop binding to the Xist intron. This removes some of the barriers to Xist expression. Conversely, when female somatic cells are reprogrammed using the Yamanaka approach, the inactive X chromosome is reactivated[113]. The only other time the inactive X is reactivated is during the formation of primordial germ cells in development, which is why the zygote starts out with two active X chromosomes.
We are still a bit vague as to why X inactivation is so mutually exclusive between the pair of chromosomes. One theory is that it’s all down to what happens when the X chromosomes kiss. This happens at a developmental point where Tsix levels are starting to fall, and the levels of the Yamanaka factors are also declining. The theory is that the pair of chromosomes reaches some sort of compromise. Rather than each ending up with a sub-optimal amount of non-coding RNAs and other factors, the binding molecules all get shunted together onto one of the pair. There’s not a great deal of clarity on how this happens. It could be that one of the pair of chromosomes just by chance carries slightly more of a key factor than the other. This makes it slightly more attractive to certain proteins. Complexes may build up in a self-sustaining way, so that the more of a complex one chromosome starts with, the more it can drag off its partner. The rich get richer, the poor get poorer …
It’s quite remarkable how many gaps remain in our understanding of X inactivation, 50 years after Mary Lyon’s formative work. We don’t even really understand how the Xist RNA ends up coating the chromosome from which it is expressed, or how it recruits all those negative repressive epigenetic enzymes and modifications. So perhaps it’s timely to move off the shifting sands and step back onto more solid ground.
Let’s return to this statement from earlier in the chapter: ‘Once a cell has switched off one of a pair of X chromosomes, that particular copy of the X stays switched off in all the daughter cells for the rest of that woman’s life, even if she lives to over a hundred years of age.’ How do we know that? How can we be so certain that X inactivation is stable in somatic cells? It is now possible to perform genetic manipulation to show this in species like mice. But long before that became feasible scientists were already pretty certain this was the case. For this piece of information we thank not mice, but cats.
Not just any old cats, but specifically tortoiseshell ones. You probably know how to recognise a classic tortoiseshell cat. It’s the one that’s a mixture of black and ginger splodges, sometimes on a white background. The colour of each hair in a cat’s coat is caused by cells called melanocytes that produce pigment. Melanocytes are found in the skin, and develop from special stem cells. When melanocyte stem cells divide, the daughter cells stay close to each other, forming a little patch of clonal cells from the same parent stem cell.
Now, here’s an amazing thing: if a cat’s colour is tortoiseshell, it’s a female.
There is a gene for coat colour that encodes either black pigment or orange pigment. This gene is carried on the X chromosome. A cat may receive the black version of the gene on the X chromosome inherited from her mother and the orange version on the X chromosome inherited from her father (or vice versa). Figure 9.5 shows what happens next.
Figure 9.5 In female tortoiseshell cats, the genes for orange and black fur are carried on the X chromosome. Depending on the pattern of X chromosome inactivation in the skin, clonal patches of cells will give rise to discrete patterns of orange and black fur.
So the tortoiseshell cat ends up with patches of orange and patches of black, depending on the X chromosome that was randomly inactivated in the melanocyte stem cell. The pattern won’t change as the cat gets older, it stays the same throughout its life. That tells us that the X inactivation stays the same in the cells that create this coat pattern.
We know that tortoiseshell cats are always female because the gene for the coat colour is only on the X chromosome, not the Y. A male cat only has one X chromosome, so it could have black fur or ginger fur, but never both.
Something rather similar happens in a rare human disorder called X-linked hypohidrotic ectodermal dysplasia. This condition is caused by mutations in a gene called ECTODYSPLASIN-A, carried on the X chromosome[114]. A male with a mutation in his sole copy of ECTODYSPLASIN-A on his single X chromosome has a variety of symptoms, including a total lack of sweat glands. This might sound socially advantageous, but is actually incredibly dangerous. Sweating is one of the major routes by which we lose excess heat, and men with this condition are at serious risk of tissue damage or even death as a result of heat stroke[115].
Females have two copies of the ECTODYSPLASIN-A gene, one on each of their X chromosomes. In female carriers of X-linked hypohidrotic ectodermal dysplasia, one X carries a normal copy of the gene, and one a mutated version. There will be random inactivation of one X chromosome in different cells. This means some cells will express a normal copy of ECTODYSPLASIN-A. Other cells will randomly shut down the X carrying the normal copy of the gene, and won’t be able to express the ECTODYSPLASIN-A protein. Because of the clonal way in which areas of skin develop, just like in the tortoiseshell cat, these women have some patches of skin that express ECTODYSPLASIN-A and some that don’t. Where there’s no ECTODYSPLASIN-A, the skin can’t form sweat glands. As a consequence, these women have patches of skin that can sweat and cool down, and others that can’t.
Random X inactivation can significantly influence how females are affected by mutations in genes on the X chromosome. This depends not just on the type of gene that is mutated but also on the tissues that express and require the protein encoded by that gene. The disease called mucopolysaccharidosis II (MPSII) is caused by mutations in the LYSOSOMAL IDURONATE-2-SULFATASE gene, on the X chromosome. Boys with this mutation on their single X chromosome are unable to break down certain large molecules and these build up to toxic levels in cells. The main symptoms include airway infections, short stature and enlargement of the spleen and liver. Severely affected boys also suffer mental retardation, and may die in their teenage years.