4DCELLFATE

"Developing a global understanding of the PRC and NuRD complexes in stem cell differentiation, in health and disease"

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 Obiettivo del progetto (Objective)

'Embryonic stem (ES) cells have the potential to differentiate into any type of cell as well as to renew indefinitely in culture. They hold great potential for the development of personalized medicines. However, the molecular mechanisms underpinning cell fate decisions by individual cells are poorly understood. There is compelling evidence that two large multi-protein machines, the Nucleosome Remodelling and Deacetylation (NuRD) complex and the Polycomb Repressive Complexes (PRCs), modulate chromatin structure to control stem cell renewal, lineage commitment and differentiation. Moreover, they are clearly implicated in cancer.

The goal of 4DCellFate is to understand how the PRC/NuRD complexes and their plethora of interactions (protein/protein, protein/nucleosome, protein/nucleic acids) regulate cell fate. To obtain this global, quantitative and dynamic – 4D – understanding of the structure/functions of these two multi-protein machines during ES cell differentiation and in different disease states, we propose a large scale multi-disciplinary “data-gathering” approach combining European excellence in interactomics (affinity purification, quantitative mass spectrometry-based proteomics, ChIP-seq analysis, light microscopy), structural biology (X-ray crystallography, NMR, native-state mass spectrometry, Electron Microscopy, biochemistry/biophysics), cellular, tumour, and computational biology. We expect to significantly advance the technology, mainly in the fields of proteomics, structural biology, and small molecule screening, and to make our knowledge, reagents and data publicly available to the scientific community.

The ultimate outcome of 4DCellFate will be to lay the foundations for understanding the role of the PRC/NuRD complexes in ES cell differentiation and cancer, specifically in leukaemogenesis. Three SMEs and one large multi-national pharma will be actively involved ensuring that our findings can be translated into new ways to control complex diseases.'

Descrizione progetto (Article)

A major roadblock to fully exploiting the potential of full-genome sequencing is to truly understand how the information in each genome is actually used. Intriguingly, this is often controlled by so-called epigenetic regulation, which changes the output but not the sequence of the genome. Epigenetic changes can be pre-programmed but can also come from the environment.

The EU-funded http://www.4dcellfate.eu (4DCellFate project) would like to set the basis for understanding the 'epigenetic code'. Such a code guides the epigenetic regulation of cells during differentiation, for instance by telling an embryonic stem cell (ESC) what type of cell it should differentiate into (a muscle cell, a nerve, etc.). Often, such epigenetic guides are misregulated during diseases, such as cancers, and may even have causative effects.

A multidisciplinary consortium comprised of academic and industrial partners has been brought together in 4DCellFate to analyse two major epigenetic regulatory complexes: PCR and NuRD. 4DCellFate scientists will characterise changes in structure and function of the PRC and NuRD complexes in ESC differentiation and cancer. For this, they will use state-of-the-art techniques, including ChIP-seq analysis, X-ray crystallography, mass spectrometry (MS) and electron microscopy, to study the interactions between proteins, nucleosomes and nucleic acids over time and space dimensions.

Several significant discoveries have been made so far. Accurate structure and compositions of NuRD and PRC1/2 complexes were obtained along with potentially useful novel interacting molecules using ChIP-seq in mouse ESCs (mESCs). A flexible, sensitive and high-throughput genetic screening technique was developed for testing cell lines using a small interfering RNA (siRNA) library. This will be used to identify Polycomb regulators in mESCs.

Technical breakthroughs will also help move the project forward. For instance, a novel high-throughput protein crystallisation and X-ray structure determination tool called MultiTRAQ was developed for rapidly characterising protein complexes using MS that considerably shortened processing times from over a year to only a few days. Protocols were also developed to automate the identification of structures in NuRD and PRC1/2 complexes. Finally, multi-protein expression was achieved with a MultiBac-based expression technique along with purification protocols for NuRD and PRC complexes.

Data integration is ongoing for a comprehensive analysis of temporal variations of NuRD and PRC complexes during ESC differentiation. Small molecule screening will be set up to identify potential inhibitors or compounds that can modulate activity in these complexes to alter cell fate decisions.

Research results are disseminated through publications in several peer-reviewed journals as well as on the project website. Elucidating epigenetic complexes could also optimise the selective differentiation of induced pluripotent stem (iPS) cells, providing more options for stem cell cancer therapy and regenerative medicine.