Transcriptional control of cell fate decisions by chromatin remodelling proteins

A cell's identity is defined by its genes. A cell located in the heart, for example, will express heart genes, but not genes important for liver or brain function. Conversely, liver cells express liver genes but not heart or lung genes. During embryonic development animals are formed by different groups of stem cells, which are able to give rise to the myriad of different cell types found in an adult organism. When stem cells undergo the process of differentiating into a new cell type, it is critically important that they turn on genes appropriate for the new cell type, but also turn off the genes that define a 'stem cell' identity and also keep off any genes associated with other cell types. Failure to properly regulate expression of all these different kinds of genes can result in the wrong decisions being made, or the inability to make any decision when required. The consequences of such failure can be developmental abnormalities or cancer. Unsurprisingly, then, our cells contain many proteins whose jobs are to ensure the proper control of gene expression. This proposal focusses on two such proteins, called Chd4 and Brg1. These proteins control the way genes get packaged within the cell. Though at a mechanistic level these two proteins appear to have similar activities, a number of studies have found that they actually tend to work in opposition. We know that loss of either protein results in failed development from very early stages, and both proteins are often found to be deleted or mutated in cancer. We propose that it is precisely this balance of opposing activities between Chd4 and Brg1 that fine tunes gene expression patterns, and that this balance is crucial for cells to successfully make developmental decisions. Yet exactly how the biochemical activities of these proteins controls gene expression, and whether the two really directly control the expression of the same genes during development is not well understood. In the work proposed herein we will use cutting edge technologies to very accurately define the function of these proteins in stem cells and in early embryonic cells as they make their very first decisions. We will show how loss of one either protein impacts the function of other key regulatory proteins, and how this leads to developmental failure. We will then examine how these proteins function during the very first cell fate decision in mammalian embryogenesis. Together this project will reveal how gene expression is controlled as cells make decisions, and how the cells use these proteins to precisely control cell fate choices during normal development. This will inform the creation of new methods for controlling gene expression in mammalian cells. Such methods could be of considerable benefit in efforts to prevent the progression of cancer, and to enable more efficient and effective protocols to use stem cells in regenerative medicine.

A cell's identity is defined by its gene expression patterns, and gene expression potential is encoded in chromatin. Changes to cell state which occur during normal development require very precise changes in chromatin structure and gene expression. Failure to properly regulate gene expression patterns can prevent successful execution of developmental decisions, but can also lead to tumourigenesis. Chd4 and Brg1 are two mammalian chromatin remodelling proteins which use energy derived from ATP hydrolysis to alter the structure of chromatin and thereby influence gene expression patterns. Both proteins play essential roles in early mammalian development, and both proteins have been found to be mutated in a wide variety of different tumour types. A body of evidence indicates that these two proteins often act in opposition. I hypothesise that these two proteins normally act in opposition to precisely correlate gene expression patterns. This proposal will test this hypothesis in pluripotent cells undergoing cell fate decisions both in culture and during early mouse embryogenesis. We will provide precise molecular details of how control of enhancer chromatin structure by Chd4 and Brg1 impacts the binding and subsequent activities of enhancer-binding transcription factors, and how this then influences the activity of RNA polymerase at target promoters. Using super resolution microscopy, we will determine how chromatin remodellers control transcription factor binding kinetics in live cells in culture and in preimplantation stage embryos undergoing the first cell fate decision. We will further define how these chromatin remodelling systems are used by embryonic cells to drive developmental transitions. This project combines cutting edge imaging and single cell biology to reveal fundamental transcriptional mechanisms at unprecedented molecular detail, greatly increasing the resolution of our understanding of how cell fate decisions are enacted in mammalian cells.

The work proposed herein will define at a molecular level how chromatin remodelling proteins impact gene expression, and how this activity is finely balanced to enable lineage decisions. We will also develop reagents and methodologies to study transcription factor behaviour in live cells. What sets our work apart from what has been done in the past is the inclusion of cutting edge imaging methodology (the development of which in the Laue lab was funded largely by the MRC) and validation of our in-depth molecular analyses in an in vivo system, the early mouse embryo. This will be of direct relevance to a broad range of researchers interested in chromatin biology, stem cells, development, and oncogenesis (See 'Academic Beneficiaries' section). More broadly, improved understanding of the complexities of gene expression, cellular differentiation and cancer progression is potentially relevant to specialists in different fields including history, archaeology and anthropology.

We will ensure the impact of our work is felt as widely as possible by promoting it at national and international meetings, and on social media, which can immediately reach thousands of scientists worldwide. Exposure and impact will be maximised by depositing manuscripts on the BioRxiv preprint server prior to publication, and by ensuring all published work is open access from the date of publication.

The work proposed is to investigate the functions of protein complexes known to play important roles in cancer initiation and/or progression, and therefore will contribute towards our understanding of how cells become tumourigeneic. This will provide mechanistic insights which could be exploited in the design of cancer therapeutics, thereby benefitting the commercial sector. Similarly, insights gained into the control of developmental decisions in pluripotent cells will be useful to those working to increase the utility of pluripotent stem cell-based applications. The indirect and long-term impact on therapeutics and patient care could be considerable.

Aside from our potential impact on future healthcare, we also plan for our research to have a more immediate impact on the wider public through a commitment to effective communication and public engagement. Many different non-specialists have a thirst for the latest information about gene expression; be they families with a history of a genetic condition, schools teaching developmental biology, or groups interested in genealogy. On the broadest level, public understanding of genetics is a wide-reaching social, cultural and political issue. It is important that as a society we have access to the latest research that may influence our thinking. The details of our commitments to sharing our research in an accessible and appropriate way are outlined in the attached 'Pathway to Impact'.