Recurrent mutations are frequently associated with transcription factor (TF) binding sites (TFBS) in melanoma, but the mechanism driving mutagenesis at TFBS is unclear. Here, we use a method called CPD-seq to map the distribution of UV-induced cyclobutane pyrimidine dimers (CPDs) across the human genome at single nucleotide resolution. Our results indicate that CPD lesions are elevated at active TFBS, an effect that is primarily due to E26 transformation-specific (ETS) TFs. We show that ETS TFs induce a unique signature of CPD hotspots that are highly correlated with recurrent mutations in melanomas, despite high repair activity at these sites. ETS1 protein renders its DNA binding targets extremely susceptible to UV damage in vitro, due to binding-induced perturbations in the DNA structure that favor CPD formation. These findings define a mechanism responsible for recurrent mutations in melanoma and reveal that DNA binding by ETS TFs is inherently mutagenic in UV-exposed cells.

The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class small-molecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either PU.1^lo mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro–B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro– to pre–B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1’s critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.

Transactivation by the ETS family of transcription factors, whose members share structurally conserved DNA-binding domains, is variably sensitive to methylation of their target genes. The mechanism by which DNA methylation controls ETS proteins remains poorly understood. Uncertainly also pervades the effects of hemi-methylated DNA, which occurs following DNA replication and in response to hypomethylating agents, on site recognition by ETS proteins. To address these questions, we measured the affinities of two sequence-divergent ETS homologs, PU.1 and Ets-1, to DNA sites harboring a hemi- and fully methylated CpG dinucleotide. While the two proteins bound unmethylated DNA with indistinguishable affinity, their affinities to methylated DNA are markedly heterogeneous and exhibit major energetic coupling between the two CpG methylcytosines. Analysis of simulated DNA and existing co-crystal structures revealed that hemi-methylation induced non-local backbone and groove geometries that were not conserved in the fully methylated state. Indirect readout of these perturbations was differentially achieved by the two ETS homologs, with the distinctive interfacial hydration in PU.1/DNA binding moderating the inhibitory effects of DNA methylation on binding. This data established a biophysical basis for the pioneering properties associated with PU.1, which robustly bound fully methylated DNA, but not Ets-1, which was substantially inhibited.