Understanding how the NuRD complex regulates ES cell differentiation using single molecule fluorescence imaging

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 cell in the body.

The project brings together a unique combination of skill sets, in biochemistry/structural biology, single molecule imaging, and stem cell biology, to develop single molecule approaches for studying nuclear processes in mammalian cells. Up to now most work in this field has focussed on studies of processes occurring on either the cell surface or in bacterial cells. We have carried out a great deal of preliminary work to demonstrate feasibility (and thus reduce the risk), such that we can now envisage a highly inter-disciplinary research program to study the way the NuRD complex interacts with chromatin, and how this controls gene expression during ES cell differentiation.

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. In a related project we are attempting to develop small molecule inhibitors and activators of NuRD complexes to control chromatin structure. Our research may thus in the long-term facilitate our ability to directly influence gene expression profiles, stem cell differentiation and disease.

In preliminary studies we have obtained high quality 3D Photo-Activated Light Microscopy (PALM) images of both nuclear proteins and DNA probes targeting particular nascent mRNAs (genes) in ES cells at a single molecule level using mEos3- and Halo-tagged proteins. We now envisage the development of two types of experiment: 1) 3D Stochastic Optical Reconstruction Microscopy (STORM) to study co-localisation of NuRD with different chromatin associated proteins/transcription factors in fixed cells; and 2) 3D PALM for single molecule tracking of particular proteins/complexes in live cells. Both types of experiment will exploit a novel 3D super-resolution instrument that we have designed in Cambridge using strategic funding from the MRC, which uses reflected light sheet activation and a double-helix point spread function. For multi-colour 3D STORM of NuRD complexes, we will visualise NuRD components labelled with pairs of organic dyes, either attached to antibodies or to ligands targeting Halo- and SNAP-tagged proteins. Single molecule tracking of CHD4, MBD3 etc. will exploit mEos3-/Halo-tagged proteins. We will also explore the use of novel fluorescence resonance energy transfer (FRET) cassettes that we have developed, which consist of a photo-switchable fluorophore such as mEos3 linked to either the Halo-/SNAP-tags. In preliminary experiments we have tested different organic fluorophores for use in live cells and have shown that the use of FRET cassettes reduces photo bleaching. This will allow us to study the dynamics and track NuRD proteins or transcription factors for longer periods of time in live cell experiments, e.g. to compare the dynamics of CHD4 on its own (i.e. in Mbd3-/- cells) and in NuRD complexes. We will also explore whether it is possible to use FRET, between e.g. mEos3 attached to one component and Halo-tag attached to the other, to track complexes in live cell experiments. Our ongoing structural studies will be crucial for designing these experiments.

Who will benefit from this research, both directly and indirectly?

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 Germany, nurturing stronger relations with the European research community.

How will 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 study of the mechanistic behaviour of the NuRD complex will provide key functional 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.