The Epigenetics Revolution

One interesting consequence of X inactivation is that (epigenetically) females are more complicated than males. Males only have one X chromosome in their cells, so they don’t carry out X inactivation. But females randomly inactivate an X chromosome in all their cells. Consequently, at a very fundamental level, all cells in a female body can be split into two camps depending on which X chromosome they inactivated. The expression for this is that females are epigenetic mosaics.

This sophisticated epigenetic control in females is a complicated and highly regulated process, and that’s where Mary Lyon’s predictions have provided such a useful conceptual framework. They can be paraphrased as the following four steps:

Counting: cells from the normal female would contain only one active X chromosome;

Choice: X inactivation would occur early in development;

Initiation: the inactive X could be either maternally or paternally derived, and the inactivation would be random in any one cell;

Maintenance: X inactivation would be irreversible in a somatic cell and all its descendants.

Unravelling the mechanisms behind these four processes has kept researchers busy for nearly 50 years, and this effort is continuing today. The processes are incredibly complicated and sometimes involve mechanisms that had barely been imagined by any scientists. That’s not really surprising, because Lyonisation is quite extraordinary – X inactivation is a procedure where a cell treats two identical chromosomes in diametrically opposite and mutually exclusive ways.

Experimentally, X inactivation is challenging to investigate. It is a finely balanced system in cells, and slight variations in technique may have a major impact on the outcome of experiments. There’s also considerable debate about the most appropriate species to study. Mouse cells have traditionally been used as the experimental system of choice, but we are now realising that mouse and human cells aren’t identical with respect to X inactivation[99]. However, even allowing for these ambiguities, a fascinating picture is beginning to emerge.

Counting chromosomes

Mammalian cells must have a mechanism to count how many X chromosomes they contain. This prevents the X chromosome from being switched off in male cells. The importance of this was shown in the 1980s by Davor Solter. He created embryos by transferring male pronuclei into fertilised eggs. Males have an XY karyotype, and when they produce gametes each individual sperm will contain either an X or a Y. By taking pronuclei from different sperm and injecting them into ‘empty’ eggs, it was possible to create XX, XY or YY zygotes. None of these resulted in live births, because a zygote requires both maternal and paternal inputs, as we have already seen. But the results still told us something very interesting, and are summarised in Figure 9.3.

^{Figure 9.3 Donor egg reconstitution experiments were performed in which the donor egg received a male and female pronucleus or two pronuclei from males. Just as in Figure 7.2, the embryos derived from two male pronuclei failed to develop to term. When the nuclei each contained a Y chromosome, and no X chromosome, the embryos failed at a very early stage. Embryos derived from two male pronuclei where at least one contained an X chromosome developed further before they also died.}

The earliest loss of embryos occurred in those that had been reconstituted from two male pronuclei which each contained a Y chromosome as the sole sex chromosome[100]. In these embryos there was no X chromosome at all, and this was associated with exceptionally early developmental failure. This shows that the X chromosome is clearly essential for viability. This is why male (XY) cells need to be able to count, so that they can recognise that they only contain one X, and thus avoid inactivating it. Turning off the solitary X would be disastrous for the cell.

Having counted the number of X chromosomes, there must be a mechanism in female cells by which one X is randomly selected for inactivation. Having selected a chromosome, the cell starts the inactivation procedure.