Understanding how the NuRD complex assembles and functions in mouse embryonic stem cells (mESC's)

The Nucleosome Remodeling and Deacetylation (NuRD) protein complex plays a key role in controlling the way our genomes are packaged inside the cell into a structure called chromatin. This packaging in turn controls whether particular genes (sequences of DNA) are/are not expressed. In particular, the NuRD complex controls gene expression as embryonic stem (ES) cells first start to differentiate into all the different types of specialised cells in the body. Without some of the NuRD complex components, ES cells cannot differentiate at all, clearly demonstrating its importance. So how does the NuRD complex form? And how does it affect gene expression in ES cells? These are the questions we hope to answer.
Up until now, we (and the field as a whole) have focussed on trying to understand which components make up the NuRD complex, which regions of the genome it interacts with, and which genes are affected by it. However, we have recently shown that we can make the individual components and assemble the NuRD complex outside of a cell. This will allow us to study its structure and how it interacts with the small regions of the genome to which it binds. We have shown using cutting-edge imaging that we can track single NuRD complex components inside a cell and watch in real-time how they assemble on chromatin. We can also study how the NuRD complex affects the binding of other proteins, and ultimately gene expression. We now envisage a highly inter-disciplinary research program that combines these approaches to determine the structure of the NuRD complex, understand how NuRD complexes assemble and interact with different parts of the genome, and how they control gene expression.

Our long-term goal is to use this understanding to control the differentiation of stem cells. This understanding when applied to either ES cells, or adult cells that have been induced to become stem cells (iPS cells), could have enormous potential - e.g. for providing a source of human tissue to study disease progression, or to develop drugs for personalised molecular therapies. We will also attempt to develop small molecule inhibitors and activators of NuRD complexes to control chromatin structure. Our research may in the long-term facilitate our ability to directly influence gene expression profiles, stem cell differentiation and disease.

We have developed a novel chromosome conformation capture (Hi-C) method to determine genome structure in single mouse ES cells. We find that highly NuRD-regulated genes form clusters (which we confirmed using super-resolution microscopy) that associate with active enhancers/promoters, which bind key transcription factors such as KLF4. These results suggest that NuRD may regulate the binding of key transcription factors. We now want to understand how NuRD assembles and how it affects the binding of e.g. KLF4 to regulate gene expression.

To study NuRD assembly we have purified a core NuRD complex, with deacetylase but not chromatin remodelling activity, and have carried out preliminary cryo-EM and cross-linking mass spectrometry (CL-MS). We have also purified larger NuRD complexes with the chromatin remodeller CHD4, as well as complexes of CHD4 and mono-/di-nucleosomes. To understand how the various components contribute to NuRD activity, we will use a deacetylation assay and a single-molecule Förster Resonance Energy Transfer (FRET) assay for nucleosome remodelling. We will also use these assays to study small molecules that have been found by our collaborators to bind to NuRD, or to regulate Human stem cell self-renewal, and utilise them in the single-molecule imaging approaches described below. Finally, we will determine structures of NuRD/nucleosome complexes by cryo-EM/CL-MS, and later by X-ray crystallography after generating smaller constructs from limited proteolysis and CL-MS analysis.

In parallel, we have developed approaches for single-molecule tracking of proteins in live ES cells and we have developed a novel single-molecule FRET approach for the tracking of protein complexes. These studies have suggested cell-cycle dependent assembly of NuRD. We would now like to use these approaches to study how NuRD assembles at specific genomic loci and how it affects the binding of proteins like KLF4.

Who might benefit from this research?

Beneficiaries or 'users' within the commercial private sector who will gain from our research include pharmaceutical and small biotech companies who are either interested in the NuRD complex and targeting this for therapeutic benefit, or who might apply the technology we develop to understanding how their own proteins of interest assemble and function as complexes inside living cells. Our research specifically involves collaborations with groups in Belgium, Germany, France, The Netherlands and Spain, nurturing stronger relations with the European research community.

How might they benefit from this research?

It is increasingly clear that epigenetic regulation of chromatin structure and gene expression is a dynamic and reversible process, which underlies normal development. When it malfunctions, however, it can contribute to many diseases. The NuRD complex brings together key proteins that mediate epigenetic regulation through the deacetylation and demethylation of histones as well as proteins that remodel nucleosomes. Several of the proteins/enzymes and protein interactions in this complex are emerging as potential drug targets, and it will be important to understand how it interacts with and remodels chromatin structure, if we are to design appropriate therapeutic approaches.
Perhaps more importantly, however, the NuRD complex has a central role in the reprogramming of adult cells into induced pluripotent stem (iPS) cells and the differentiation of stem cells towards a committed lineage. Our long-term goal is to understand how the NuRD complex controls these early steps of differentiation of ES cells - there is already evidence that small molecule inhibitors of HDACs, lysine demethylases and the G9a methyl-transferase may help in the generation of iPS cells and their directed differentiation along particular lineages.

We envisage that our structures and studies of the mechanistic behaviour of the NuRD complex will provide key information for future structure based drug design and may attract R&D investment. Our protocols for the production and assembly of the recombinant complex will be readily accessible to industrial and academic groups for small molecule screening programmes. Such screens could lead to the development of drugs or therapies, which may subsequently progress to clinical trials. In this way, we hope to advance the economic competitiveness of the U.K.

As mentioned above, our long-term goal is to use the understanding we gain, to develop approaches to control ES cell differentiation. This understanding, when applied to embryonic and induced pluripotent stem cells, will have enormous potential for providing a source of human tissue to study disease progression, and to develop drugs for personalised molecular therapies. In addition, aberrant gene regulation by these complexes is clearly implicated in cancer. Cures and ameliorative therapies for many diseases may thus be possible through the manipulation of transcriptional profiles and the control of stem cell differentiation. The development of small molecule inhibitors and activators of the NuRD complex to control chromatin structure may facilitate our ability to directly influence transcriptional profiles, stem cell renewal/differentiation and cancer. There are important opportunities in the treatment of diabetes and neurodegenerative disorders (for example, Parkinson's and Alzheimer's Disease) as well as cancer.

Transferrable skills gained by staff employed over the duration of the project will include the ability to communicate scientific findings to the wider public, presenting research in the form of posters and talks, writing up scientific conclusions in the form of manuscripts, writing grant proposals and training in the management and supervision of PhD and undergraduate students.