DNA selection by the master transcription factor PU.1

The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5ʹ-GGAA-3ʹ) flanked by variable sequences, affinity is negotiated by direct readout on the 5ʹ flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3ʹ flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1’s characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins. [Display omitted] •High-resolution PU.1 structures unveil the dynamic basis of DNA specificity•Single evolutionary innovation (Q226) determines PU.1’s distinct target profile•Basis of pathogenic Q226E mutation in Waldenström macroglobulinemia•The structures correct the crystallographic record on PU.1/DNA binding Terrell et al. present 22 high-resolution structures of co-crystal DNA complexes of the master myeloid transcription factor PU.1. They show the role of folded-state dynamics of PU.1 ETS domain in DNA recognition, including strand-specific CpG methylation and recognition of non-canonical targets, over an affinity range of three orders of magnitude.