X chromosome inactivation; linking mechanisms for in cis accumulation of Xist RNA and chromosome silencing.

This proposal aims to understand the fundamental molecular mechanisms that underpin X-inactivation, the process that evolved in mammals to equalise gene dosage in XX females relative to XY males. X-inactivation is orchestrated by a functional non-coding RNA, Xist, which accumulates locally over the chromosome from which it is transcribed and silences underlying genes. We have made significant progress towards understanding Xist-mediated chromosome silencing pathways, but key gaps remain, notably in defining a principal late-acting pathway required to complete silencing of a significant subset of X-linked genes. Additionally, we currently have only a very limited understanding of how Xist RNA accumulates in cis over a single chromosome territory. Thus, we set out a series of experiments to resolve these questions, making use of state of the art methodologies in genomics and proteomics to define the late-acting silencing pathway, and advanced cellular imaging coupled to genetic perturbation to define mechanisms for local accumulation of Xist RNA. Bringing these two goals together, we will determine sufficiency of the known factors and test our models for their mode of action by in vivo expression of synthetic transcripts to which we will tether key factors for silencing and RNA accumulation.