AbstractThe pioneering transcription factor C/EBPα coordinates cell fate and cell differentiation. C/EBPα represents an intrinsically disordered protein with multiple short linear motifs and extensive post-translational side chain modifications (PTM), reflecting its modularity and functional plasticity. Here, we combined arrayed peptide matrix screening (PRISMA) with biotin ligase proximity labeling proteomics (BioID) to generate a linear, isoform specific and PTM-dependent protein interaction map of C/EBPα in myeloid cells. The C/EBPα interactome comprises promiscuous and PTM-regulated interactions with protein machineries involved in gene expression, epigenetics, genome organization, DNA replication, RNA processing, and nuclear transport as the basis of functional C/EBPα plasticity. Protein interaction hotspots were identified that coincide with homologous conserved regions of the C/EBP family and revealed interaction motifs that score as molecular recognition features (MoRF). PTMs alter the interaction spectrum of multi-valent C/EBP-motifs to configure a multimodal transcription factor hub that allows interaction with multiple co-regulatory components, including BAF/SWI-SNF or Mediator complexes. Combining PRISMA and BioID acts as a powerful strategy to systematically explore the interactomes of intrinsically disordered proteins and their PTM-regulated, multimodal capacity.Key pointsIntegration of proximity labeling and arrayed peptide screen proteomics refines the interactome of C/EBPα isoformsHotspots of protein interactions in C/EBPα mostly occur in conserved short linear motifsInteractions of the BAF/SWI-SNF complex with C/EBPα are modulated by arginine methylation and isoform statusThe integrated experimental strategy suits systematic interactome studies of intrinsically disordered proteins
Proteome-wide analysis of human disease mutations in short linear motifs: neglected players in cancer?
