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DC9: Defining the proximal proteome of mutant chromatin proteins associated with CPs-RU

DC9 (Radboud University) will apply an ‘off-the-shelf’ proximity biotinylation enzyme called ProteinA-TurboID that was recently developed in the lab of Michiel Vermeulen (Santos-Barriopedro et al., Nature Commun. 2021, Nature Protocols 2022). This recombinant proximity biotinylation enzyme can be targeted to baits of interest in primary cells using bait-specific antibodies, which are available for all the enzymes studied in this network. This approach thus represents an ideal system to probe the proximal proteome for various mutated chromatin proteins in various chromatinopathies, making use of primary patient cells or patient derived iPSC models without the need for genetic manipulation of the target cells. Identified candidate proximal proteins for various mutated chromatin proteins will be functionally verified using ChIP-seq and a recently developed technology called BANC-seq (Neikes et al., Nature Biotechnolgoy 2023).

Furthermore, functional follow up studies will be pursued such as CRISPR-based functional studies, including degron lines and RNA-seq (i.e Grand et al., Nature 2021). These experiments will reveal the proximal proteome for mutated chromatin proteins that are associated with various chromatinopathies. These interactions will provide critical insights regarding their molecular mechanisms of action and identified
interactions may provide new leads towards novel therapies for these diseases.

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