The nucleosome remodelling and deacetylase (NuRD) complex is a repressive chromatin-associating protein complex that regulates gene expression and plays a major role in cell fate decisions. Although efforts have been made to characterise the stoichiometry and structure of the multi-protein and multi-paralogue NuRD complex, our understanding of the full composition and complexity has remained elusive due to the dynamic nature of the complex. More recently, Cyclin-Dependent Kinase 2-Associated Protein 1 (CDK2AP1), a cell cycle regulator that is associated with various oral and colorectal cancers, has been speculated to be a member of the NuRD complex. CDK2AP1 and the NuRD complex are co-localised at methylated sections of DNA and different studies have shown the association of CDK2AP1 with NuRD subunits. However, the mechanism by which CDK2AP1 interacts with the NuRD complex and subsequently manipulates epigenetic regulation is yet to be uncovered.
Here, we have established a protein pulldown assay using HEK293 cells, followed by liquid chromatography‐tandem mass spectrometry, to map the interaction of the different NuRD components with CDK2AP1. Mass spectrometry of pulled down proteins (>40 kDa) indicated the association of MTA1,2, RbBP4,7, HDAC1,2, and P66α, β proteins and CHD4 with CDK2AP1. To further pinpoint the specific interaction partners of CDK2AP1, pull down assay was carried out using NuRD proteins expressed individually using a in vitro transcription‐translation system. The results intriguingly uncover an unprecedented trimeric interaction where the CHD4 and P66β components of the NuRD complex combinatorically capture CDK2AP1. Our findings provide insight into molecular mechanism behind the NuRD complex mediated recruitment of CDK2AP1 to target genes.