Biophysical Principles of Lineage Factor PU.1 Binding Revealed by NextPBMs

Determining the biophysical principles that shape transcription factor (TF) binding in a cell-specific manner is key to quantitative models of gene expression. High-throughput (HT) in vitro methods measuring protein-DNA binding are invaluable for relating TF binding affinity to genome-wide binding; however, the impact of cell-specific post-translational modifications (PTMs) and cofactors are not routinely assessed. To address these limitations, we describe a new HT approach, called nextPBMs (nuclear extract protein-binding microarrays), to characterize TF binding that accounts for PTMs and endogenous cofactors. We use nextPBMs to examine the DNA binding of the lineage factor PU.1/Spi1 and IRF8 in human monocytes. We identify two binding modes for PU.1 in monocytes - autonomous binding unaffected by PTMs and cooperative binding with IRF8, and identify a single cooperative mode for IRF8. We characterize the DNA binding of PU.1:IRF8 complexes, and show how nextPBMs can be used to discover cell-specific cofactors and characterize TF cooperativity at single-nucleotide resolution. We show that chromatin state and cofactors both influence the affinity requirements for PU.1 binding sites. Furthermore, we find that the influences of cooperative (IRF8) and collaborative (C/EBPα) cofactors on PU.1-binding site affinity are independent and additive.