What Controls the Controller: Structure and Function Characterizations of Transcription Factor PU.1 Uncover Its Regulatory Mechanism

The ETS family transcription factor PU.1/Spi-1 is a master regulator of the self-renewal of hematopoietic stem cells and their differentiation along both major lymphoid and myeloid branches. PU.1 activity is determined in a dosage-dependent manner as a function of both its expression and real-time regulation at the DNA level. While control of PU.1 expression is well established, the molecular mechanisms of its real-time regulation remain elusive. Our work is focused on discovering a complete regulatory mechanism that governs the molecular interactions of PU.1. Structurally, PU.1 exhibits a classic transcription factor architecture in which intrinsically disordered regions (IDR), consisting of 66% of its primary structure, are tethered to a well-structured DNA binding domain. The transcriptionally active form of PU.1 is a monomer that binds target DNA sites as a 1:1 complex. Our investigations show that IDRs of PU.1 reciprocally control two separate inactive dimeric forms, with and without DNA. At high concentrations, PU.1 forms a non-canonical 2:1 complex at a single DNA specific site. In the absence of DNA, PU.1 also forms a dimer, but it is incompatible with DNA binding. The DNA-free PU.1 dimer is further promoted by phosphomimetic mutants of IDR residues that are phosphorylated in B-lymphocytic activation. These results lead us to postulate a model of real-time PU.1 regulation, unknown in the ETS family, where independent dimeric forms antagonize each other to control the dosage of active PU.1 monomer at its target DNA sites. To demonstrate the biological relevance of our model, cellular assays probing PU.1-specific reporters and native target genes show that PU.1 transactivation exhibits a distinct dose response consistent with negative feedback. In summary, we have established the first model for the general real-time regulation of PU.1 at the DNA/protein level, without the need for recruiting specific binding partners. These novel interactions present potential therapeutic targets for correcting de-regulated PU.1 dosage in hematologic disorders, including leukemia, lymphoma, and myeloma