Myeloid differentiation is a tightly regulated process and defects can lead to hematologic disorders including cancer. The pioneering transcription factor C/EBPα plays a lineage-instructing role during myelopoiesis and also regulates proliferation and differentiation in many other cell types. The CEBPA gene is expressed as two isoforms, the full-length isoform P42 and the truncated isoform P30 that lacks N-terminal transactivating regions and is therefore considered an inhibitor of P42 function. C/EBPα comprises a modular structure, is intrinsically disordered and extensively post-translationally modified, all attributing functional plasticity to the protein and posing a challenge to protein interaction studies. In order to map the post-translational modification (PTM) dependent interactome and to unravel C/EBPα functions that are connected to intrinsic disorder we employed two alternative mass spectrometry based methods for protein interaction studies, biotin proximity labeling (BioID) and a protein interaction screen on a peptide matrix (PRISMA). In BioID experiments, the myeloid cell line NB4 was transduced with C/EBPα fused to a promiscuous biotin ligase and upon addition of biotin proteins in close proximity of C/EBPα were biotinylated and subsequently enriched and quantified. BioID experiments identified known and novel C/EBPα interactors as well as isoform specific interaction partners connected to the biological functions of the transcription factor. While BioID is a powerful technique for protein interaction studies it does not provide information about the site of interaction and the influence of PTMs on the protein interaction. To this end we probed over 140 C/EBPα derived peptides, including post translationally modified peptides for protein interactions with NB4 nuclear extract using PRISMA. Combined with BioID, PRISMA produced a detailed interaction map across the C/EBPα primary sequence in NB4 cells and revealed footprints of interacting proteins and complexes in a PTM dependent fashion. Hotspots of protein interactions correlate with conserved molecular recognition features within disordered regions and comparison with previously published data revealed related binding profiles of homologous C/EBP regions. The data presented in this thesis provides evidence that a conserved region within the N-terminus of P30 contains transactivating potential and that the function of P30 is more complex than a mere inhibitor of full length C/EBPα.
|Publication status||Published - 2020|