Understanding how RIF1 and KAP1 enable the choice of the future active and inactive X chromosomes: the establishment of functional asymmetry.

In humans, every chromosome is present in two copies, except in men, where there is one copy of each of the sex chromosomes, the X and the Y. Women have two copies of the X chromosome. One of the two X chromosomes in women is inactivated (silenced), so that male and female cells have the same amounts of the gene products coming from the X chromosomes. This process of silencing is called X-inactivation. Either of the two X chromosomes can be inactivated randomly. This means that, in every tissue, half of the cells would have one X chromosome inactive (A) and half would have the other one inactive (B). How is the random choice of one of the two X chromosome initially made? What are the mechanisms that inform one chromosome that other has been chosen? How is a different choice stably maintained in the progeny of each cell, throughout the life of an individual? These are all still unanswered or only partially-answered questions. In addition, in reality, the two copies of the X chromosome are never completely identical and the small differences in their DNA sequences can result in one of the two copies being silenced more easily. As a consequence, for example, if it is easier to silence X chromosome A, then most of the cells in a human organ will have an active B X chromosome, rather than a mixture of cells with an active A and cells with an active B. This is very important when one of the two X chromosomes contains a completely or partially defective gene which can cause a disease. If the X chromosome that is preferentially silenced carries the disease version of the gene, the organ will be functional and the woman will be healthy. But if, instead, the X chromosome, which is preferentially silenced, is the one carrying the normal version of the gene, the organ will not be able to function properly and the woman will suffer from the disease. Identifying the features of the DNA sequences that drive this skew and understanding how X-inactivation works are therefore very important, as they will allow the early identification of the patients at risk of developing severe symptoms, among the women carriers of X-linked diseases. An example of such a disease, where skewed X-inactivation plays a role in the severity of the symptoms, is Duchenne muscular dystrophy, where some female patients are completely asymptomatic and others are severely ill.
In our project, we use mouse embryonic stem cells to investigate three molecules, an RNA and two proteins, that we have shown to be essential for the initial choice of which X chromosome will be inactivated. By studying how these three molecules interact with each other and with the X chromosome DNA, we will identify the molecular mechanism that establishes the choice of the identity of the future active and inactive X chromosomes. In addition, we will explore the role of DNA sequences that can determine skewed X chromosome silencing and investigate how these sequences relate to the unbalanced X chromosome inactivation observed in patients.

The process of random X chromosome inactivation (XCI) equalises the dosage of X-linked genes between males (one X chromosome) and females (two X chromosomes) in placental mammals, by silencing one the two X chromosomes in the females. As a result, female tissues are mosaics of cells carrying either one or the other active X chromosome (Xa), in theory, in equal proportion. However, unknown X-linked genetic elements can influence the probability of X-inactivation, therefore skewing the composition of tissues. As a consequence, a recessive allele of an X-linked disease can display a penetrance much higher than the theoretical 50% and lead to a severe disease in women heterozygous carriers.
The upregulation of the long non-coding (lncRNA) RNA Xist triggers the silencing in cis of the future inactive X chromosome (Xi). Therefore, a skew in the choice of the future Xi is the result of a skewed monoallelic Xist upregulation. By identifying the molecular mechanisms that direct the choice of which Xist allele to upregulate, we will therefore discover the basis of the unequal probability of XCI. For human health, this will be important to design diagnostic methods aimed at the early identification of women heterozygous carriers of X-linked diseases at risk of severe illness.
We have recently shown that a circuit involving the lncRNA Tsix (Xist antisense), and two proteins, RIF1 and KAP1, can establish the asymmetric destiny of the future Xa/Xi, with KAP1 marking the Xist promoter on the future Xa and RIF1 on the future Xi. Using mouse embryonic stem cells as a model system, we will employ both in vitro (DNA-protein bandshifts and RNA-protein pull-downs) and in vivo approaches (RNA crosslinking-immunoprecipitation and sequencing-CLIP, ChIP, FRAP and Cas9/CRIPSR-mediated genome editing), to study the determinants of the mutually exclusive association of RIF1 and KAP1 with both the Xist promoter and Tsix RNA and to identify their roles in the choice of Xi/Xa.