Final Report Summary - EPIGENETIX (Epigenetic regulation and monoallelic gene expression: the X-inactivation paradigm and beyond)
Over the last century, progress in our understanding of the molecular basis of heredity and of how our genetic information is stored, read and replicated through DNA has been remarkable. Our comprehension of the way that the genome is packaged into the nucleus in eukaryotes and of how some genes are expressed or “read” in some cells but not in others in order to produce cellular diversity, has been more challenging. This ERC project set out to study how differential gene expression can occur not only between cell types, but even between two copies of a gene within the same cell. To do this we have studied X-chromosome inactivation, where one of the two X chromosomes becomes silenced during early development in females. The process of X inactivation is remarkable in at least two ways. First it entails the differential treatment of two identical chromosomes in the same nucleus. Second, the memory of which of the two X chromosomes has been inactivated is stably propagated across hundreds of cell divisions, despite the numerous opportunities for this state to be “forgotten” or reversed, for example during DNA replication. Thanks to a combination of super-resolution microscopy and state of the art molecular techniques that the funding by the ERC provided, we discovered that chromosomes are folded into domains spanning several hundreds of thousands of base pairs of DNA, known as topologically associated domains (TADs), and that the dynamic fluctuations in DNA interactions within these TADs, can probably lead to fluctuations in gene expression. These fluctuations may in turn lead to unequal expression states between two copies of the same gene. Such asymmetric expression states can then sometimes be memorised, leading to populations of cells during development where either one or other of the two copies is expressed. In the case of the non-coding Xist gene, which is the trigger of X-chromosome wide silencing, its asymmetric expression may be initiated by such fluctuations in chromatin folding. We believe that this could explain, at least partly, how the two X chromosomes become differently expressed early on in development. This project led us not only to discover the TAD organisation that hosts the Xist gene but also to identify some of the DNA sequences that likely control the folding within TADs and ensure monoallelic expression. In the course of the ERC programme, we also demonstrated that this type of monoallelic gene expression is not just a characteristic of the X chromosome but can also be found at several hundred loci on autosomes. Importantly, we also showed that such monoallelic expression can be seen in vivo and that some of the genes affected have been associated with human autosomal dominant disorders. This suggests that if monoallelic expression becomes clonally heritable it might sometimes have severe consequences, because if one copy of a gene is mutated, while the other normal copy is silent, then some cells will not express the gene at all and this could be deleterious in a cell or organ. If this is the case, then reversal of the silent state of the normal gene could help to treat such diseases, which is an avenue we are actively researching. Our research, also led to the discovery that embryonic stem cells with two X chromosomes are delayed in their early development when compared to cells with only one X chromosome, at least until X inactivation has been triggered. This reveals that a double dose of some X chromosome genes must provide a checkpoint, to ensure that dosage compensation is achieved, in order for development to proceed normally. Finally we also identified some of the specific molecules that associate with the X chromosome, and that can act either to trigger its silencing or to ensure the cellular memory of the inactive state. These discoveries have provided important insights into the mechanisms by which chromosomes can be differentially expressed in development and disease.