Intrinsic Disorder Directs Two Distinct Dimers of the Master Transcription Factor PU.1

Abstract

Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The transcription factor PU.1 presents a well-defined model of Type I intrinsic disorder, the most common configuration of intrinsically disordered regions (IDRs) in transcription factors in which the DNA-binding domain (DBD) is the only structured domain. PU.1 is also a master regulator of hematopoiesis, the physiologic process by which all blood cell lineages develop ultimately from a single population of stem cells in the bone marrow. Dose-response experiments using synthetic reporters and native PU.1 target genes show that the structured DBD of PU.1 regulates gene expression in live cells via a negatively cooperative DNA-bound dimer. Formation of the bound dimer is antagonized, in the absence of DNA, by a conformationally distinct dimeric state under the control of its proximal acidic IDR. The two conformers involve distinct surfaces on the DBD without disorder-to-order contributions from the tethered IDRs. Spectroscopic and calorimetric characterizations show that, unlike DNA-bound complexes, charge-charge contributions from the flanking IDRs make critical contributions in realizing a conformationally destabilized dimer. Dimerization without DNA is further promoted by phosphomimetic (Ser → Asp) substitutions of IDR residues that are physiologically phosphorylated in B-cell activation, and stimulated by acidic crowding agents. The data suggests a novel non-structural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly basic DBDs without DNA. As PU.1 demonstrates, this mechanism renders thermodynamically insulated dimeric states that mutually antagonize to regulate the concentration of functionally active monomer at the protein/DNA level.