Although geneticists knew that mammalian females had two X chromosomes, whereas males had only one X and one Y chromosome, nobody had proposed a mechanism for equalizing the gene dosage until a seminal paper by Mary Lyon in 1961. She realized that if mice with only one X and no Y chromosome (XO mice) could develop as females and live, then one of the two X chromosomes in mammals might not be needed and could be inactivated during development, explaining the darkly staining bundle of sex chromatin seen only in female cells. Combining this with observations of mouse coat colour, she suggested that the inactivation of paternal versus maternal X was random in groups of cells, producing a mottled coat pattern when the colour gene was on the X chromosome. A similar pattern is seen in the tortoiseshell cat, as Lyon points out in her paper.
© Ruhina Ahmed
This revolutionary idea was supported by direct evidence from humans 2 years later. Ronald Davidson and colleagues confirmed, using gel electrophoresis, that some females had two forms of the enzyme glucose-6-phosphate dehydrogenase (G6PD), encoded by alleles on the two X chromosomes. However, when individual cells were cloned and tested, each clone now only expressed one form or the other, not both. So, females clearly expressed only one G6PD gene in each cell, and the other appeared to be silenced. The authors cited data from contemporary papers showing that the entire second X might not be inactivated, which has now been confirmed by numerous direct surveys of gene activity.
Work on X-chromosome inactivation continued, and it was soon shown that the X inactivation signal seemed to emanate in cis from a centre in approximately the middle of the human chromosome, named the XIC. However, it was not until 1991 that Carolyn Brown et al. from the group of Huntington Willard characterized the first gene expressed exclusively from the inactive X, specifically from the XIC. They could not find any evidence of protein made from this gene and therefore named it X (inactive)-specific transcript (XIST). When examined in cells with multiple inactive X chromosomes, the gene was expressed from all of the inactive X chromosomes, and certainly met the criteria for a cis-acting signal for inactivation. Their follow-up paper, a year later, confirmed that XIST was active as a large RNA molecule, not as a protein, and it could be seen to coat the inactive X chromosome in female cells.
These amazing papers helped open the way for the field of epigenetics and highlighted the need to think about more than DNA to truly understand the conversion of genotype to phenotype. Intensive studies now focus on how the XIST RNA comes to coat the soon-to-be inactive X and interacts with recently described components of the chromatin machinery to effectively silence almost an entire chromosome.
