Characterisation of a novel NANOG / KDM4B complex to regulate heterochromatin function and chromosome stability in pluripotent stem cells

Pluripotent stem cells (PSC) are unspecialised cells that can form any cell type of the body. There is currently much hope that PSC could be used for cell-based therapies for the treatment of diseases, replacement for worn out tissues as we age, and for better understanding of human development, but there are still several hurdles that must be overcome before these goals are achieved. One of the hurdles is the appearance of genetic instability, and in particular the accumulation of too many chromosomes, that can affect PSC. How these unwanted changes arise remains poorly understood, but scientists are trying to prevent the changes from occurring in order to produce safer and better quality PSC.

We have chosen to study an exciting and mysterious part of our genome called constitutive heterochromatin. In many different cell types, heterochromatin is important for key cellular processes, including the maintenance of genetic stability and control of chromosome number, although it has been relatively poorly studied in PSC so far. In our recent work, we have identified a new pathway through which heterochromatin is controlled in PSC. Unexpectedly, this new pathway uses several well-known stem cell factors but we are now able to assign new functions to them. Importantly, we when use genetic tricks to prevent these factors from functioning in PSC, it leads to defects in heterochromatin organisation, to the associated loss of genetic stability, and to the accumulation of additional chromosomes in the cells. This unanticipated connection between stem cell factors, heterochromatin organisation and the control of chromosome stability is important because it could provide an explanation for how genetic changes appear in PSC and would potentially allow researchers to prevent this from occurring.

Our research has led us to form the hypothesis that heterochromatin is involved directly in the genetic instability of PSC. The overall aim in this research proposal, therefore, is to determine how heterochromatin is controlled in PSC, and what happens when this level of control goes wrong. We have carefully planned three main objectives to test our hypothesis.

The first objective is to define how the stem cell factors control heterochromatin in PSC. Our research strongly suggests the involvement of an additional factor, KDM4B, and so we would like to examine this factor in more detail. We will achieve this by asking whether KDM4B localises to heterochromatin in PSC, and then measure what happens to heterochromatin when we remove Kdm4b from PSC. We predict that heterochromatin will show defects and possibly the appearance of chromosome instability in the PSC.

The second objective is to investigate how defects in heterochromatin lead to chromosome instability in PSC. Previous research from many laboratories has shown that the particular signals that mark and define heterochromatin are required to prevent genetic instability, and we want to take this forward by investigating how these signals are controlled in PSC, especially in light of the new mode of heterochromatin regulation that we have now identified.

The third objective is to use the knowledge that we generate to improve the quality and genetic stability of PSC so that we can remove one of the current hurdles to future applications. We anticipate that if we can understand how heterochromatin organisation is connected to chromosome instability in PSC, then we can, in future, devise ways to prevent this from happening. Understanding the detailed mechanism of how this occurs may lead to improved use of stem cells for regenerative medicine. This knowledge is also important in research outside of PSC, especially in ageing and cancer for example, where the normal process of heterochromatin regulation is disrupted. By better understanding how heterochromatin is controlled in general, we may be able to develop strategies to detect early changes and also to prevent them from happening.

Despite the promise of pluripotent stem cells (PSC), one key concern with the technology is chromosome instability. We propose that an important and unexplored driver of chromosome instability in PSC is aberrant regulation of constitutive heterochromatin domains, including pericentromeric heterochromatin (PCH), which can impact centromere function. In our recent work we have shown that the pluripotency factors Nanog and Sall1 are necessary and sufficient for PCH organisation in mouse PSC. These effects are mediated directly through the epigenetic and transcriptional regulation of major satellite DNA repeats within PCH. Deletion of Nanog or Sall1 leads to mis-regulation of PCH function and to associated chromosome segregation defects and the acquisition of karyotype abnormalities in PSC. These results establish the first direct molecular connection between the pluripotency network and chromatin organisation in PSC, and lead to the conclusion that a distinct PCH identity could have an important role in maintaining chromosome stability in PSC. We now aim to understand more about the involvement of other key co-regulators, and ascertain how they influence chromosome stability in PSC and upon cell reprogramming. Based on our protein-interaction data and the observed changes in H3K9me3 levels, we propose that the H3K9me3-demethylase KDM4B functions together with NANOG / SALL1 to balance H3K9me3 levels at major satellite repeats in PSC, thereby reinforcing the chromatin identity at PCH domains. Through these mechanisms, we suggest that maintaining PCH and centromeric organisation is important to prevent the loss of centromere function and to protect against chromosome mis-segregation that can occur in PSC and upon cellular reprogramming. We will test these hypotheses, anticipating that we will define a new molecular pathway controlling heterochromatin state and chromosome stability in PSC and provide insights to improve reprogramming to a more genetically stable cell type.

The primary impact of our research will come from the advancement of knowledge in mechanisms of genetic stability of pluripotent stem cells, related to regulation of chromatin organisation and chromosome segregation (see Academic Beneficiaries).

Impacts on other stakeholders:

We will contribute to the successful delivery of BBSRC's mission. Our research falls centrally within Strategic Research Priority 3: Bioscience for Health. Specifically:
(i) "Generate new knowledge of the biological mechanisms of development and the maintenance of health across the lifecourse" which we address by examining the role of chromatin organisation in regulating the genetic stability of stem cells and early embryo cell types, and in doing so reveal potential mechanisms relevant to other cell types where heterochromatin is mis-regulated such as during normal ageing.
(ii) "The identification of critical periods during the lifespan which may be particularly susceptible to biological influences/exposures and could potentially inform on the timings of interventions", which we address by examining the hypothesis that early embryo cell types, modeled by pluripotent cells in the first instance, are particularly vulnerable to chromatin perturbation with lasting consequences on chromosome stability.
(iii) "Generating new knowledge to advance regenerative biology, including stem cell research", which we address by investigating the underlying causes of chromosome instability in pluripotent stem cells and taking initial steps towards improving the efficiency and stability of cell reprogramming.
Other relevant areas map to:
(i) 'The 3Rs in research using animals', which we address by using pluripotent stem cells as a means to understand early mammalian development.
(ii) Grand Challenge 3, specifically: "basic molecular and cellular mechanisms responsible for longevity or premature ageing (e.g. triggers of cellular senescence, damage) and how these are modulated by ... developmental factors", which we address by examining the molecular pathways that could lead to genetic instability, with links to developmental transcription factors and epigenetic regulators.

Industry: Knowledge gained through this research could generate important intellectual property, which could be commercialised and exploited leading in the longer term to wealth creation in the UK. In particular, identification of genetic and epigenetic factors that increase the efficiency and stability of cell reprogramming could lead to the improved development of reprogramming reagents, which would be desirable for companies that sell reprogramming products.

In the longer term, the health-care sector, charities and patients. In particular, the research has the potential to generate safer and more stable cell types for regenerative medicine. More broadly, our research will identify pathways that potentially regulate processes involved in disease and ageing, where heterochromatin mis-regulation is an important but poorly understood process. For example, KDM4B is a current anti-cancer target, but little is known about how it functions.

Training: This project will provide professional development for the PI and PDRA in new scientific skills in growth areas (e.g. reprogramming technologies). It will build on Novo's excellent organisational and experimental skills, providing training for her future contribution to UK science.

Science and society: We will contribute to STEM understanding through our public engagement activities. Novo and Rugg-Gunn have been STEM Ambassadors for several years, communicating their knowledge and enthusiasm to the next generation of scientists and informing interested adults through activities such as science exhibitions and science visits to schools and local community groups. These activities are important for informing the public debate about specific areas of research related to this project, e.g. the benefits and challenges of stem cell research.