Mechanisms of Cistromic Repositioning of the Transcription Factor PU.1

Aberrant transcriptional networks are a hallmark of cancer, yet our knowledge of the intricacies of transcription factor behavior is poor. PU.1 is an ETS family transcription factor that is essential for hematopoiesis, however more than 50% of AML patients display a disruption of the PU.1 transcriptional network. In this study we implement novel molecular probes to competitively displace PU.1 from canonical DNA binding sites, allowing us to understand PU.1-chromatin binding dynamics, as well as identify the consequences of PU.1 binding site blockade upon PU.1-driven gene transcription and chromatin accessibility.

We treated human PU.1^low AML cells with a tool PU.1-DNA binding inhibitor, DB2115, and performed PU.1 CUT&Tag, ATAC sequencing and transcriptional profiling. We found, unexpectedly, that DB2115 not only led to inhibition 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 rather than a global inhibition - and included losses at regulatory regions of MYC, POMP and gains at CSF1R and TREM2. In fact, most of these redistributed sites (78% of PU.1 gained sites) display subsequent increases in chromatin accessibility and elevated target gene expression, highlighting the pioneering and transcriptional control exerted by repositioned PU.1. Kinetic analyses of PU.1 redistribution reveal that PU.1 losses occur rapidly after DB2115 exposure (1-4hrs), whereas PU.1 gains occur more slowly (4-12hrs) indicative of a loci searching phase prior to novel site binding. Development of an experimental approach combining CLICK-chemistry compound mapping with PU.1 CUT&Tag identified selective drug binding at displaced PU.1 binding sites compared to unchanged/gained sites, with both lost and gained regions being locally enriched for specific and distinct surrounding nucleotide sequences including A/T enrichment. Furthermore, CRISPR-dCas9 blockade and binding site-driven reporter investigations into specific PU.1 cistromic elements revealed important PU.1-mediated direct and rapid control of the POMP, CSF1R and STRAP genes.

Overall, PU.1-DNA binding inhibition causes a robust perturbation of PU.1 transcriptional circuits via a novel phenomenon we describe as "Pharmacological transcription factor repositioning". Further investigations with binding site inhibitors such as these represent an unprecedented investigative approach to study complex and fast transcription factor dynamics without disrupting the structure or levels of the factor itself. Furthermore, exploitation of the pharmacological TF repositioning phenomenon may provide novel avenues for therapeutic intervention in PU.1-driven hematologic disorders and other transcriptionally-aberrant diseases.

No relevant conflicts of interest to declare.