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

4DCellFate Project Overview

The 4DCellFate project aims to understand how the PRC and NuRD complexes function to control cell fate by systematically analyzing all aspects of the Polycomb repressive complex (PRC) and the NuRD complex. For this, we have established an integrated approach and will explore, among other things, the structure of the complexes, their functional roles in various cellular contexts, and their interactions with other regulatory factors. This information can then be used to ultimately develop small molecule inhibitors and activators of the complexes.


SUBPROJECT 1: Polycomb repressive complex & NuRD INTERACTOME

leader: Di Croce

  • Proteomics over time

  • Integration of proteomics with genome-wide analysis and single molecule fluorescence microscopy

  • High-throughput genetic screening for components involved in PRC and NuRD-dependent transcription

We will generate a comprehensive, quantitative, and dynamic description of the PRC/NuRD complexes and their interactions during different stages of ES cell differentiation. This will be achieved by carrying out gene expression profiling, in vivo protein tagging, mass spectrometry, next-generation sequencing, and high-throughput genetic screens. We will also study the interactions of the complexes with nucleic acids and proteins to obtain a detailed molecular insight into their function.





SUBPROJECT 2: Polycomb repressive complex & NuRD STRUCTURAL BIOLOGY

leader: Laue

  • Structural biology of PRC and NuRD complexes

  • Measuring binding of PRC and NURD complexes to nucleosomes

  • Complex sample preparation and characterisation

  • Small molecule screening

We aim to elucidate the structure of the PRC1 and NuRD complexes and their components. This is a daunting challenge due to the flexibility, size, and heterogeneity of the complexes involved. An unprecedented integrative approach that combines native-state mass spectrometry (MS), negative-staining and cryo- electron microscopy (EM), X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and binding affinity measurements will allow us to build a comprehensive and precise 3D understanding of how PRC/NuRD complexes assemble, how they are recruited to particular genomic locations, and how they are regulated. The structures we generate will permit us to then design small molecules that can precisely interfere with the functions of these complexes.



SUBPROJECT 3: Polycomb repressive complex & NuRD DATA INTEGRATION

leader: Lehner

  • Data integration and open access

We will integrate the different types of data generated by the project with other available public resources, to provide a 4D view of how the expression, structure, interactions, and activity of the PRC and NuRD complexes vary over time during embryonic stem cell differentiation.








leader: Hendrich

  • PRC & NuRD biology in disease

Finally, we will analyze how the NuRD and PRC complexes function in stem cell differentiation, and how altering these complex functions can contribute to disease. We anticipate that understanding how PRC and NuRD complexes control stem cell differentiation will allow us to control the seemingly random process of ex vivo lineage commitment and differentiation. Achieving this could potentially revolutionize medicine.

We are also investigating to which degree specific interactions, functions, and post-translational modifications of NuRD/PRC components are different in leukemic cells from their non-cancer counterparts. If we detect differences, we will analyze whether we can restore or alter aberrant PRC or NuRD functions with therapeutic effects. The importance of “cancer stem cells” (CSCs) in tumor initiation has been firmly established in leukemia. Likewise, misregulation of PRC components and PRC target genes is known to contribute to leukemogenesis. We are thus taking advantage of the knowledge we gain from investigating these complexes in ES cells to identify novel components and novel modifications that might specifically regulate PRC and/or NuRD complexes in cancer cells.