2020 – DIRECT PHARMACOLOGICAL REDISTRIBUTION OF THE MASTER TRANSCRIPTION FACTOR PU.1

Pharmacological targeting of aberrant transcriptional networks, such as PU.1 in Acute Myeloid Leukemia, is a highly desirable yet currently unrealized prospect. First-in-class PU.1 binding site inhibitors have been reported with promising therapeutic potential, however the exact molecular and transcriptomic consequences of PU.1 binding site inhibition have not been characterized. We use one of these novel inhibitors, DB2115, as a molecular probe to study PU.1-chromatin binding dynamics, and identify the consequences of PU.1 binding site blockade upon PU.1-driven gene transcription and chromatin accessibility.We found, unexpectedly, that DB2115 not only led to inhibition of expression of some canonical PU.1 targets but also mediated concurrent increases of other PU.1 targets. This two-sided response correlated strongly with a robust redistribution of PU.1 chromatin binding detected by CUT&Tag. Kinetic analyses of PU.1 redistribution reveal that PU.1 losses occur rapidly after DB2115 exposure, whereas PU.1 gains occur more slowly suggestive of a loci searching phase prior to novel site binding. Development of a novel approach combining CLICK-chemistry with PU.1 CUT&Tag identified selective drug binding at displaced PU.1 binding sites compared to unchanged sites, with both lost and gained sites being locally enriched for distinct surrounding nucleotide sequences including A/T enrichment. Furthermore, we have functionally validated the importance of specific gained and lost PU.1 binding sites for driving expression of target genes via CRISPRd and eGFP-reporter assays.Pharmacological transcription factor redistribution via genomic binding site inhibition represents an unprecedented therapeutic and investigative approach, and allows for the study of complex and fast transcription factor dynamics without disrupting the structure or levels of the factor itself.