Re-defining the paradigm of X-chromosome inactivation

In humans, and most mammals, the sex of an individual is determined by the presence of a pair of sex chromosomes: XX in females, XY in males. The X chromosome carries about 800 genes which contain genetic information that is essential for the body to function. The normal, healthy 'dosage' of genes is delivered through the presence of one chromosome. In males, who carry one X and one Y chromosome (XY), this genetic dosage is naturally delivered. In females, who carry two X chromosomes (XX), the correct genetic dosage is obtained through silencing of one of their X chromosomes, in a process known as 'X-Chromosome Inactivation'.

Since this discovery of X chromosome inactivation in 1961, the exact mechanisms underpinning this process have been the focus of extensive study. My group recently made the surprising discovery that males also undergo X-chromosome inactivation in early embryonic development, albeit transiently. This finding has the potential to change the scientific understanding of how X-chromosome inactivation works.

In the present study, we propose to use naïve pluripotent stem cells from mice and humans to study X-chromosome inactivation. Naïve pluripotent stem cells are cells that represent the very earliest stage embryonic development, and have the ability to self-replicate exactly or to change into any cell type in the body. Our research will reveal how changes in these naïve cells at key embryonic developmental stages initiates X chromosome inactivation in both males and females. Establishing these processes is central to our understanding of early developmental stem cell biology, and will shift the current accepted paradigm that X chromosome inactivation is a female-only phenomenon.

In the medical context, random reactivation of X-linked genes has been reported during ageing and in cancerous cells. Understanding the process of X chromosome inactivation and reactivation in mechanistic detail may uncover pathways and processes that will enhance our understanding of pathological processes associated with ageing and cancer.

Naïve pluripotent stem cells are also widely used as a platform for early developmental research, with much investment being focussed on both understanding how cells maintain their naïve state and how they commit to becoming specific cell types. The proposed research will provide insights into how we might be able to stabilise stem cells in this naïve state, providing more stable stem cell platforms for use as a research tool, in drug discovery programmes and in regenerative medicine applications.

Our research group has a strong track record in stem cell biology and has the necessary expertise to successfully compete this important project. Further, working within the Cambridge Stem Cell Institute puts us in a strategically strong position, with close collaborators including Professor Austin Smith, who can provide access to new stem cell lines to facilitate pioneering research.

A hallmark of naïve cell identity is the presence of two active X-chromosomes in females. However, it was unknown if the naïve gene network prevented the initiation of X-chromosome-inactivation (XCI). In work currently under review at Cell Stem Cell we demonstrated that robust naïve pluripotent stem cell (nPSC) self-renewal abolishes expression of Xist, the master regulator of XCI. Intriguingly, at the onset of differentiation when nPSCs become Nanog-negative, they activate Xist biallelically in females and monoallelically in males. Surprisingly, we found that XCI occurs in males, as characterised by Xist RNA spreading, appearance of a repressive chromatin signature and partial X-linked gene silencing, albeit transiently and rapidly. We proposed that initiation of XCI is gender independent and triggered by the destabilization of the naïve identity, which suggests that gender-specific mechanisms follow rather than precede XCI initiation. Because of its impact for the paradigm of XCI the work under review opens new avenues to investigate how this fundamental process in biology is regulated. The pertinent questions I now want to address include: 1- Using Xist reporter cell lines I want to define what are the expression dynamics of Xist in self-renewing or differentiating mouse and human naïve pluripotent stem cells? 2- What are the molecular events and mechanisms associated with Xist upregulation? 3- I also want to define what are the molecular events associated to the reactivation of the male inactive X. In conclusion, the overarching goal of the proposed research project is to re-define the paradigm of X-chromosome inactivation (XCI).

Stem cell biology and regenerative medicine has enormous potential in advancing our fundamental understanding of cell biology and in advancing treatment options for a range of diseases through the ability to repair, replace and regenerate damaged cells, tissues and ultimately organs. As such, stem cell biology is a priority area for investment in the UK. The research proposed in this application has the potential to significantly advance our understanding of stem cell biology, with direct impact on the academic community, biotechnology and pharmaceutical industries, clinicians and patient groups and the wider public more generally.

Academic Community: This project will advance our understanding of X-chromosome inactivation (XCI), a fundamental process in cell biology. Establishing the mechanisms of this cellular process in male as well as female cells will drive a paradigm shift in the field and will impact the field of developmental biology, where there is ever increasing interest in human embryonic development. For regenerative medicine research, naïve pluripotent stem cells are a fundamental research tool for understanding cell fate decisions, therefore the more detailed understanding we have of these cells, the further we can develop this platform for regenerative therapies. The specific development of a Xist reporter system may generate new Intellectual property and will be made available for use in the wider research community.

Biotechnology and Pharmaceutical Industries: Increasingly, biotechnology and pharmaceutical industries are using stem cell platforms as a research tool in drug discovery and regenerative medicine. The delivery of more in-depth understanding of the process underpinning XCI will lead to more control within stem cell platforms and the generation of better pluripotent stem cells feeding in to more reproducible drug discovery systems.

General Public: As outlined in the communications plan, we will share our research findings with the public who fund our research and with the wider world. Through direct interaction at public events, and via online and print communications, we aim to increase awareness of stem cell research and relay the positive impacts that this research can have on public health and people's lives.