PRISMA and BioID disclose a motifs-based interactome of the intrinsically disordered transcription factor C/EBPalpha.

C/EBPalpha represents a paradigm intrinsically disordered transcription factor containing short linear motifs and post-translational modifications (PTM). Unraveling C/EBPalpha protein interaction networks is a prerequisite for understanding the multi-modal functions of C/EBPalpha in hematopoiesis and leukemia. Here, we combined arrayed peptide matrix screening (PRISMA) with BioID to generate an in vivo validated and isoform specific interaction map of C/EBPalpha. The myeloid C/EBPalpha interactome comprises promiscuous and PTM-regulated interactions with protein machineries involved in gene expression, epigenetics, genome organization, DNA replication, RNA processing, and nuclear transport. C/EBPalpha interaction hotspots coincide with homologous conserved regions of the C/EBP family that also score as molecular recognition features. PTMs alter the interaction spectrum of C/EBP-motifs to configure a multi-valent transcription factor hub that interacts with multiple co-regulatory components, including BAF/SWI-SNF or Mediator complexes. Combining PRISMA and BioID is a powerful strategy to systematically explore the PTM-regulated interactomes of intrinsically disordered transcription factors.