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Electrostatic protein/DNA interactions arise from the neutralization of the DNA phosphodiester backbone as well as coupled exchanges by charged protein residues as salt bridges or with mobile ions. Much focus has been and continues to be paid to interfacial ion pairs with DNA. The role of extra-interfacial ionic interactions, particularly as dynamic drivers of DNA sequence selectivity, remain poorly known. The ETS family of transcription factors represents an attractive model for addressing this knowledge gap given their diverse ionic composition in primary structures that fold to a tightly conserved DNA-binding motif. To probe the importance of extra-interfacial salt bridges in DNA recognition, we compared the salt-dependent binding by Elk1 with ETV6, two ETS homologs differing markedly in ionic composition. While both proteins exhibit salt-dependent binding with cognate DNA that corresponds to interfacial phosphate contacts, their nonspecific binding diverges from cognate binding as well as each other. Molecular dynamics simulations in explicit solvent, which generated ionic interactions in agreement with the experimental binding data, revealed distinct salt-bridge dynamics in the nonspecific complexes formed by the two proteins. Impaired DNA contact by ETV6 resulted in fewer backbone contacts in the nonspecific complex, while Elk1 exhibited a redistribution of extra-interfacial salt bridges via residues that are non-conserved between the two ETS relatives. Thus, primary structure variation in ionic residues can encode highly differentiated specificity mechanisms in a highly conserved DNA-binding motif.

DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N-methylpurine-sequencing (NMP-seq), to characterize alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data shows that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs Abf1 and Reb1, but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.

Hairpin polyamides are synthetic small molecules that bind DNA minor groove sequence-selectively and, in many sequences, induce widening of the minor groove and compression of the major groove. The structural distortion of DNA caused by polyamides has enhanced our understanding of the regulation of DNA-binding proteins via polyamides. Polyamides have DNA binding affinities that are comparable to those proteins, therefore, can potentially be used as therapeutic agents to treat diseases caused by aberrant gene expression. In fact, many diseases are characterized by over- or under-expressed genes. PU.1 is a transcription factor that regulates many immune system genes. Aberrant expression of PU.1 has been associated with the development of acute myeloid leukemia (AML). We have, therefore, designed and synthesized ten hairpin polyamides to investigate their capacity in controlling the PU.1-DNA interaction. Our results showed that nine of the polyamides disrupt PU.1-DNA binding and the inhibition capacity strongly correlates with binding affinity. One molecule, FH1024, was observed forming a FH1024-PU.1-DNA ternary complex instead of inhibiting PU.1-DNA binding. This is the first report of a small molecule that is potentially a weak agonist that recruits PU.1 to DNA. This finding sheds light on the design of polyamides that exhibit novel regulatory mechanisms on protein-DNA binding.

Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes. Although the DNA sequence requirements for affinity are well-defined, the dynamic mechanisms that execute cognate recognition are much less resolved. To address this gap, here we examined ETS1, a paradigm developmental transcription factor, as a model for which cognate discrimination remains enigmatic. Using molecular dynamics simulations, we interrogated the DNA-binding domain of murine ETS1 alone and when bound to high-and low-affinity cognate sites or to nonspecific DNA. The results of our analyses revealed collective backbone and side-chain motions that distinguished cognate versus nonspecific as well as high- versus low-affinity cognate DNA binding. Combined with binding experiments with site-directed ETS1 mutants, the molecular dynamics data disclosed a triad of residues that respond specifically to low-affinity cognate DNA. We found that a DNA-contacting residue (Gln-336) specifically recognizes low-affinity DNA and triggers the loss of a distal salt bridge (Glu-343/Arg-378) via a large side-chain motion that compromises the hydrophobic packing of two core helices. As an intact Glu-343/Arg-378 bridge is the default state in unbound ETS1 and maintained in high-affinity and nonspecific complexes, the low-affinity complex represents a unique conformational adaptation to the suboptimization of developmental enhancers.

Eukaryotic transcription factors are versatile mediators of specificity in gene regulation. This versatility is achieved through mutual specification by context-specific DNA binding on the one hand, and identity-specific protein-protein partnerships on the other. This interactivity, known as combinatorial control, enables a repertoire of complex transcriptional outputs that are qualitatively disjoint, or non-continuum, with respect to binding affinity. This feature contrasts starkly with prokaryotic gene regulators, whose activities in general vary quantitatively in step with binding affinity. Biophysical studies on prokaryotic model systems and more recent investigations on transcription factors highlight an important role for folded state dynamics and molecular hydration in protein/DNA recognition. Analysis of molecular models of combinatorial control and recent literature in low-affinity gene regulation suggest that transcription factors harbor unique conformational dynamics that are inaccessible or unused by prokaryotic DNA-binding proteins. Thus, understanding the intrinsic dynamics involved in DNA binding and co-regulator recruitment appears to be a key to understanding how transcription factors mediate non-continuum outcomes in eukaryotic gene expression, and how such capability might have evolved from ancient, structurally conserved counterparts.

Distinct dimeric forms of PU.1 mutually antagonize to control the transcriptionally active dose via a disordered PEST domain., Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The hematopoietic master regulator PU.1 presents a well-defined model of the most common configuration of intrinsically disordered regions (IDRs) in transcription factors. We report that the structured DNA binding domain (DBD) of PU.1 regulates gene expression via antagonistic dimeric states that are reciprocally controlled by cognate DNA on the one hand and by its proximal anionic IDR on the other. The two conformers are mediated by distinct regions of the DBD without structured contributions from the tethered IDRs. Unlike DNA-bound complexes, the unbound dimer is markedly destabilized. Dimerization without DNA is promoted by progressive phosphomimetic substitutions of IDR residues that are phosphorylated in immune activation and stimulated by anionic crowding agents. These results suggest a previously unidentified, nonstructural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly cationic DBDs.

To extend the value of biosensor-SPR in the characterization of DNA recognition by nucleoproteins, we report a comparative analysis of DNA-facilitated target search by two ETS-family transcription factors: Elk1 and ETV6. ETS domains represent an attractive system for developing biosensor-based techniques due to a broad range of physicochemical properties encoded within a highly conserved DNA-binding motif. Building on a biosensor approach in which the protein is quantitatively sequestered and presented to immobilized cognate DNA as nonspecific complexes, we assessed the impact of intrinsic cognate and nonspecific affinities on long-range (intersegmental) target search. The equilibrium constants of DNA-facilitated binding were sensitive to the intrinsic binding properties of the proteins such that their relative specificity for cognate DNA were reinforced when binding occurred by transfer vs. without nonspecific DNA. Direct measurement of association and dissociation kinetics revealed ionic features of the activated complex that evidenced DNA-facilitated dissociation, even though Elk1 and ETV6 harbor only a single DNA-binding surface. At salt concentrations that masked the effects of nonspecific pre-binding at equilibrium, the dissociation kinetics of cognate binding were nevertheless distinct from conditions under which nonspecific DNA was absent. These results further strengthen the significance of long-range DNA-facilitated translocation in the physiologic environment.

The eponymous DNA-binding domain of ETS (E26 transformation specific) transcription factors binds a single sequence-specific site as a monomer over a single helical turn. Following our previous observation by titration calorimetry that the ETS member PU.1 dimerizes sequentially at a single sequence-specific DNA binding site to form a 2:1 complex, we have carried out an extensive spectroscopic and biochemical characterization of site-specific PU.1 ETS complexes. While 10 bp of DNA was sufficient to support PU.1 binding as a monomer, additional flanking bases were required to invoke sequential dimerization of the bound protein. NMR spectroscopy revealed a marked loss of signal intensity in the 2:1 complex, and mutational analysis implicated the distal surface away from the bound DNA as the dimerization interface. Hydroxyl radical DNA footprinting indicated that the site-specifically bound PU.1 dimers occupied an extended DNA interface downstream from the 5'-GGAA-3' core consensus relative to its 1:1 counterpart, thus explaining the apparent site size requirement for sequential dimerization. The site-specifically bound PU.1 dimer resisted competition from nonspecific DNA and showed with affinities similar to other functionally significant PU.1 interactions. As sequential dimerization did not occur with the ETS domain of Ets-1, a close structural homolog of PU.1, 2:1 complex formation may represent an alternative auto-inhibitory mechanism in the ETS family at the protein-DNA level.

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. Unlike other ETS homologues, such as Ets-1, DNA recognition by PU.1 is highly sensitive to its osmotic environment due to excess interfacial hydration in the complex. To investigate interfacial hydration in the two homologues, we mutated a conserved tyrosine residue, which is exclusively engaged in coordinating a well-defined water contact between the protein and DNA among ETS proteins, to phenylalanine. The loss of this water-mediated contact blunted the osmotic sensitivity of PU.1/DNA binding, but did not alter binding under normo-osmotic conditions, suggesting that PU.1 has evolved to maximize osmotic sensitivity. The homologous mutation in Ets-1, which was minimally sensitive to osmotic stress due to a sparsely hydrated interface, reduced DNA-binding affinity at normal osmolality but the complex became stabilized by osmotic stress. Molecular dynamics simulations of wildtype and mutant PU.1 and Ets-1 in their free and DNA-bound states, which recapitulated experimental features of the proteins, showed that abrogation of this tyrosine-mediated water contact perturbed the Ets-1/DNA complex not through disruption of interfacial hydration, but by inhibiting local dynamics induced specifically in the bound state. Thus, a configurationally identical water-mediated contact plays mechanistically distinct roles in mediating DNA recognition by structurally homologous ETS transcription factors.

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. DNA target specificity derives from combinatorial interactions with other proteins as well as intrinsic heterogeneity among ETS domains. Emerging evidence suggests molecular hydration as a fundamental feature that defines the intrinsic heterogeneity in DNA target selection and susceptibility to epigenetic DNA modification. This perspective invokes novel hypotheses in the regulation of ETS proteins in physiologic osmotic stress, their pioneering potential in heterochromatin, and the effects of passive and pharmacologic DNA demethylation on ETS regulation.

Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11.

Heterocyclic dications are receiving increasing attention as targeted inhibitors of transcription factors. While many dications act as purely competitive inhibitors, some fail to displace protein efficiently at drug concentrations expected to saturate their DNA target. To achieve a mechanistic understanding of these non-competitive effects, we used a combination of dications, which are intrinsically fluorescent and spectrally-separated fluorescently labeled DNA to dissect complex interactions in multi-component drug/DNA/protein systems. Specifically, we interrogated site-specific binding by the transcription factor PU.1 and its perturbation by DB270, a furan-bisbenzimidazole-diamidine that strongly targets PU.1 binding sites yet poorly inhibits PU.1/DNA complexes. By titrating DB270 and/or cyanine-labeled DNA with protein or unlabeled DNA, and following the changes in their fluorescence polarization, we found direct evidence that DB270 bound protein independently of their mutual affinities for sequence-specific DNA. Each of the three species competed for the other two, and this interplay of mutually dependent equilibria abrogated DB270's inhibitory activity, which was substantively restored under conditions that attenuated DB270/PU.1 binding. PU.1 binding was consistent with DB270's poor inhibitory efficacy of PU.1 in vivo, while its isosteric selenophene analog (DB1976), which did not bind PU.1 and strongly inhibited the PU.1/DNA complex in vitro, fully antagonized PU.1-dependent transactivation in vivo.

Hydration of interfaces is a major determinant of target specificity in protein/DNA interactions. Interfacial hydration is a highly variable feature in DNA recognition by ETS transcription factors and functionally relates to cellular responses to osmotic stress. To understand how hydration is mediated in the conserved ETS/DNA binding interface, secondary structures comprising the DNA contact surface of the strongly hydrated ETS member PU.1 were substituted, one at a time, with corresponding elements from its sparsely hydrated relative Ets-1. The resultant PU.1/Ets-1 chimeras exhibited variably reduced sensitivity to osmotic pressure, indicative of a distributed pattern of interfacial hydration in wildt-ype PU.1. With the exception of the recognition helix H3, the chimeras retained substantially high affinities. Ets-1 residues could therefore offset the loss of favorable hydration contributions in PU.1 via low-water interactions, but at the cost of decreased selectivity at base positions flanking the 5′-GGA-3′ core consensus. Substitutions within H3 alone, which contacts the core consensus, impaired binding affinity and PU.1 transactivation in accordance with the evolutionary separation of the chimeric residues involved. The combined biophysical, bioinformatics and functional data therefore supports hydration as an evolved specificity determinant that endows PU.1 with more stringent sequence selection over its ancestral relative Ets-1.

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., Many factors contribute to mutation hotspots in cancer cells. Here the authors map UV damage at single-nucleotide resolution across the human genome and find that binding sites of ETS transcription factors are especially prone to forming UV lesions, leading to mutation hotspots in melanoma.

The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs' binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs.

To find their DNA target sites in complex solution environments containing excess heterogeneous DNA, sequence-specific DNA-binding proteins execute various translocation mechanisms known collectively as facilitated diffusion. For proteins harboring a single DNA contact surface, long-range translocation occurs by jumping between widely spaced DNA segments. We have configured biosensor-based surface plasmon resonance to directly measure the affinity and kinetics of this intersegmental jumping by the ETS-family transcription factor ETS variant 6 (ETV6). To isolate intersegmental target binding in a functionally defined manner, we pre-equilibrated ETV6 with excess salmon sperm DNA, a heterogeneous polymer, before exposing the nonspecifically bound protein to immobilized oligomeric DNA harboring a high-affinity ETV6 site. In this way, the mechanism of ETV6-target association could be toggled electrostatically through varying NaCl concentration in the bulk solution. Direct measurements of association and dissociation kinetics of the site-specific complex indicated that 1) freely diffusive binding by ETV6 proceeds through a nonspecific-like intermediate, 2) intersegmental jumping is rate-limited by dissociation from the nonspecific polymer, and 3) dissociation of the specific complex is independent of the history of complex formation. These results show that target searches by proteins with an ETS domain, such as ETV6, whose single DNA-binding domain cannot contact both source and destination sites simultaneously, are nonetheless strongly modulated by intersegmental jumping in heterogeneous site environments. Our findings establish biosensors as a general technique for directly and specifically measuring target site search by DNA-binding proteins via intersegmental translocation.

ETS family transcription factors regulate diverse genes through binding at cognate DNA sites that overlap substantially in sequence. The DNA-binding domains of ETS proteins (ETS domains) are highly conserved structurally yet share limited amino acid homology. To define the mechanistic implications of sequence diversity within the ETS family, we characterized the thermodynamics and kinetics of DNA site recognition by the ETS domains of Ets-1 and PU.1, which represent the extremes in amino acid divergence among ETS proteins. Even though the two ETS domains bind their optimal sites with similar affinities under physiologic conditions, their nature of site recognition differs strikingly in terms of the role of hydration and counter ion release. The data suggest two distinct mechanisms wherein Ets-1 follows a "dry" mechanism that rapidly parses sites through electrostatic interactions and direct protein-DNA contacts, whereas PU.1 utilizes hydration to interrogate sequence-specific sites and form a long-lived complex relative to the Ets-1 counterpart. The kinetic persistence of the high affinity PU.1 · DNA complex may be relevant to an emerging role of PU.1, but not Ets-1, as a pioneer transcription factor in vivo. In addition, PU.1 activity is critical to the development and function of macrophages and lymphocytes, which present osmotically variable environments, and hydration-dependent specificity may represent an important regulatory mechanism in vivo, a hypothesis that finds support in gene expression profiles of primary murine macrophages.

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.

Transcription factors with structurally homologous DNA-binding domains are conventionally thought to operate similar mechanisms of DNA site recognition, and instead derive their biological specificity from extrinsic interactions such as recruitment of partner proteins and post-translational modifications. The ETS superfamily of transcriptional factors, all of which share structurally conserved, eponymous DNA-binding domains but nonetheless regulate non-redundant genetic networks, exemplify this paradigmic view of eukaryotic gene regulation. Recent genomic studies have defined a hierarchy within the ETS family based on “pioneering” properties: the ability of some ETS-family homologs such as PU.1, but not others such as Ets-1, to autonomously bind nucleosomal and methylated DNA in vivo. To better understand this heterogeneity at a mechanistic level, we used fluorescence anisotropy to explicitly interrogate the recognition by the DNA-binding domains of these two ETS homologs for a single cognate DNA site harboring a defined CpG dinucleotide in which one, the other, or both cytosines have been methylated. We observed that while both ETS domains bind the unmethylated sequence with equally high affinity, they are differentially inhibited at mono-methylated sites in a position-dependent manner. Moreover, whereas PU.1 binds the di-methylated DNA site, Ets-1 is strongly inhibited. Our data indicate that sensitivity to methylated DNA is an intrinsic property of the ETS domain and, more generally, can be encoded into a structurally conserved DNA-binding domain without external control.

In systems biology, molecular titration is a mechanism for introducing sensitivity of genetic networks to their regulatory proteins. Since most eukaryotic transcription factors lack chemical control by low-molecular-weight hormones or metabolites, transcription factors have evolved various strategies, such as cooperative DNA binding and sequestering partner proteins, to generate sensitivity in the expression of the genes that they regulate. The ETS superfamily of transcription factors, which are broadly distributed in the animal kingdom, regulate target genes in an active monomeric state. On the one hand, many ETS members, such as the master transcription factor PU.1, are involved in critical cell-fate decisions and signaling pathways that require highly sensitive output responses, yet no coherent mechanism exists for generating the required sensitivity. On the other hand, dimerization of ETS proteins in both DNA-bound and free states via their DNA-binding domain has been reported, but its purpose and biophysical nature remain obscure. We propose that dimerization serves as a sensitivity-generating mechanism by molecular self-titration: self-association sequesters free ETS proteins in inactive dimers, while dimerization in the DNA-bound state inhibits transactivation by the active protein/DNA complex. With the positive auto-regulation of the PU.1 (Spi1) gene generating self-amplification of PU.1, homodimerization would provide the minimal negative feedback needed to prevent explosion and maintain stable dynamics of PU.1 target genes in the absence of additional interactions. We are defining the thermodynamic and structural parameters of the PU.1 homodimer in the free and DNA-bound states. The data show that dimerization involves previously unexplored molecular surfaces that are distinct from the “orthosteric” protein/DNA contact interface and offer insight into how molecular self-titration would participate in the dynamics of PU.1-dependent gene expression.

Functionally critical decreases in levels or activity of the ETS family transcription factor PU.1 are present in approximately 2/3 of patients with acute myeloid leukemia (AML), across different AML subtypes (Sive, Leukemia 2016) including at the stem cell level (Steidl, Nat Genet 2006; Will, Nat Med 2015). Thus, targeting PU.1 could be an appealing option for treatment. As complete loss of PU.1 leads to stem cell failure (Iwasaki, Blood 2005), we hypothesized that PU.1 inhibition could eradicate leukemic cells harboring already low levels of PU.1, with modest effects on normal cells. We initially tested this hypothesis using 3 different shRNAs, and found that PU.1 inhibition led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested. We demonstrated that these effects were indeed due to decreased PU.1 levels by retroviral add-back experiments. The direct pharmacologic targeting of transcription factors has proven challenging in the past. Besides the core ETS binding motif (GGAA) in the DNA major groove, PU.1 binding to chromatin depends on additional minor groove contacts enriched for AT nucleotides upstream of the ETS motif, which determine selectivity for PU.1. Using an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays, we developed novel small molecules of the heterocyclic diamidine family acting as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibits PU.1 binding via an allosteric mechanism. Consistent with this, the inhibitory effects were selective for PU.1 versus other ETS transcription factors. Treatment with 3 different compounds led to cell growth inhibitory effect with respect to PU.1 level and preferentially affects PU.1low AML cells. Similarly to what we observed with shRNAs, treatment with our novel inhibitors led to decreased proliferation and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cells and a majority of primary AML cell samples. Targeted ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity in AML cells. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over genes regulated by other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonize PU.1-regulated pathways at a genome-wide level. ChIP-seq performed in PU.1low AML cells confirmed a genome-wide decrease of PU.1 peaks after treatment and provides novel insight into the molecular mechanisms mediating the anti-leukemic effects of pharmacological PU.1 inhibition. To test the effects of PU.1 inhibition on normal hematopoiesis, we treated normal hematopoietic stem/progenitors cells (HSPC) in colony forming assays and saw decreased production of mature granulo-monocytic cells, consistent with PU.1's known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on more immature HSPC. Congenic transplantation assays of treated normal bone marrow cells led to no change in myeloid and T-cells and only a modest decrease in B-cell numbers. Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival. To conclude, our study provides proof-of-principle for PU.1 inhibition as a novel therapeutic strategy in AML. Furthermore, we present the development of first-in-class PU.1 inhibitors acting via an allosteric minor groove-mediated mechanism. Our work shows that the specific pharmacological targeting of the DNA interaction of transcription factors such as PU.1 is feasible in principle, and may open the way for targeting of other transcription factors through minor groove-directed approaches. Disclosures Will: Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl: Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Bayer Healthcare: Consultancy.

ETS transcription factors mediate a wide array of cellular functions and are attractive targets for pharmacological control of gene regulation. We report the inhibition of the ETS-family member PU.1 with a panel of novel heterocyclic diamidines. These diamidines are derivatives of furamidine (DB75) in which the central furan has been replaced with selenophene and/or one or both of the bridging phenyl has been replaced with benzimidazole. Like all ETS proteins, PU.1 binds sequence specifically to 10-bp sites by inserting a recognition helix into the major groove of a 5'-GGAA-3' consensus, accompanied by contacts with the flanking minor groove. We showed that diamidines target the minor groove of AT-rich sequences on one or both sides of the consensus and disrupt PU.1 binding. Although all of the diamidines bind to one or both of the expected sequences within the binding site, considerable heterogeneity exists in terms of stoichiometry, site-site interactions and induced DNA conformation. We also showed that these compounds accumulate in live cell nuclei and inhibit PU.1-dependent gene transactivation. This study demonstrates that heterocyclic diamidines are capable of inhibiting PU.1 by targeting the flanking sequences and supports future efforts to develop agents for inhibiting specific members of the ETS family.

The ETS family of transcription factors is widely distributed among the metazoan phyla and regulates the expression of a wide range of genes. Despite their functional diversity, all ETS proteins share a structurally conserved DNA-binding (or ETS) domain. Given the highly overlapping sequence preferences among ETS members, it is as yet unclear how ETS proteins achieve functional specificity, a problem known as the “specificity conundrum.” Compounding this problem is a current lack of understanding of the biophysical mechanism of sequence selectivity among ETS binding sites. We hypothesize that the structural conservation among ETS domains disguises physicochemical heterogeneity in their mechanisms of sequence recognition. We have previously demonstrated that the ETS-family member PU.1 (Spi-1) recruits a cooperative network of water-mediated contacts along the protein-DNA interface for high-affinity binding. We have now compared the thermodynamics and kinetics of sequence recognition between the ETS domains of PU.1 and ETS1 which represent extremes of sequence divergence (∼30% homology) in the ETS family. We found that the thermodynamics and kinetics between the two structurally conserved ETS domains are highly differentiated under physiological conditions. More precisely, whereas high-affinity PU.1 ETS-DNA binding is enthalpically driven against an entropic penalty, ETS1 ETS-DNA binding is entropically driven. Kinetically, whereas ETS1 ETS associates rapidly with a high-affinity cognate site (ka > 107 M−1 s−1), PU.1 ETS is strikingly slow (ka ∼104 M−1 s−1). This profound difference in association rate constants means that the high-affinity PU.1 ETS-DNA complex, despite being somewhat thermodynamically less stable than the corresponding complex with ETS1, is significantly longer-lived. If these differences that underlie the intrinsic heterogeneity in site recognition by ETS proteins extend to protein-protein and domain-domain interactions, they offer one potential biophysical resolution to the specificity conundrum.

The current paradigm of ETS transcription factors holds that their DNA-binding (ETS) domain binds to a single sequence-specific site with strict 1:1 stoichiometry. PU.1 (Spi-1) is a lineage-restricted member of the ETS family that is essential in normal hematopoietic development. Characterization of the binding properties of the ETS domain of PU.1 by isothermal titration calorimetry revealed that it binds a single sequence-specific binding site with 1:1 and 2:1 stoichiometry in a discrete, sequential, and negatively cooperative manner. While both high-affinity- and low-affinity-specific sites exhibit this behavior, the thermodynamics for each complex are highly differentiated. In the unbound state, the PU.1 ETS domain exists as a weak noncovalent homodimer that dissociates and unfolds cooperatively. Thus, the PU.1 ETS domain exists as a monomeric and dimeric species in both DNA-bound and free states. Structural characterization of the protein-DNA interface by quantitative DNA footprinting revealed new minor groove contacts and changes in the core consensus suggestive of increased DNA distortion in the 2:1 complex. Together, the structural and thermodynamic data support a model in which DNA binding dissociates a PU.1 ETS dimer to a 1:1 protein-DNA complex followed by, at higher concentrations, an asymmetric 2:1 complex. The implications of distinct monomeric and dimeric states on the known structural biology of ETS domains as well as potential ETS-protein interactions are discussed.

PU.1 is an essential transcription factor in normal hematopoietic lineage development. It recognizes a large number of promoter sites differing only in bases flanking a core consensus of 5′-GGAA-3′. DNA binding is mediated by its ETS domain, whose sequence selectivity directly corresponds to the transactivational activity and frequency of binding sites for full-length PU.1 in vivo. To better understand the basis of sequence discrimination, we characterized its binding properties to a high affinity and low affinity site. Despite sharing a homologous structural framework as confirmed by DNase I and hydroxyl radical footprinting, the two complexes exhibit striking heterogeneity in terms of hydration properties. High affinity binding is destabilized by osmotic stress, whereas low affinity binding is insensitive. Dimethyl sulfate footprinting showed that the major groove at the core consensus is protected in the high affinity complex but accessible in the low affinity one. Finally, destabilization of low affinity binding by salt is in quantitative agreement with the number of phosphate contacts but is substantially attenuated in high affinity binding. These observations support a mechanism of sequence discrimination wherein specifically bound water molecules couple flanking backbone contacts with base-specific interactions in a sequestered cavity at the core consensus. The implications of this model with respect to other ETS paralogs are discussed.

The DNA-binding domain of the ETS family of transcription factors interacts with DNA in a sequence-selective manner, yet it tolerates remarkable variation in the recognition sequence. The biophysical basis for this sequence selectivity is currently unknown. Previous investigations have found significant differences in the thermodynamics of strong and weak ETS-DNA complexes. However, the ETS domain is structurally similar in both DNA-bound and unbound states. In the absence of significant coupled folding by either protein or DNA, selectivity may be linked to differences in conformational dynamics in the protein-DNA complex. Specifically, sequence selectivity may arise from differential reductions in conformational dynamics upon complex formation. We hypothesize that time-averaged differences in conformation dynamics can be resolved through their equilibrium hydration properties. The rationale for our hypothesis is that folded but conformationally mobile segments should be more solvent-accessible than segments that are relatively immobilized. To test this hypothesis, we are probing the hydration of various ETS-DNA complexes by high-resolution DNA and protein footprinting. We expect that this approach will provide a quantitative measure of a complex's overall conformational dynamics as well as identify structural elements that become conformationally stabilized upon complex formation.

The ETS domain of the transcription factor PU.1 tolerates a large number of DNA cognate variants that differ exclusively in the sequences flanking a critical central consensus, 5'-GGAA-3'. We investigated the thermodynamics of site selection by the DNA-binding domain by following the PU.1 ETS/DNA equilibrium with a large set of cognate variants under various temperature and salt conditions by filter binding. Our results indicate that the stability of the ETS/DNA complex is quantitatively tied to variations in the change in heat capacity. Thermodynamic effects induced by changing Na(+) concentrations from 150 mM to 250 mM are complex and not readily interpreted by polyelectrolyte theory. We also extended our understanding of data from our previous investigation on energetic base-neighbour coupling, by dissecting the thermodynamic contributions underlying the observed free-energy coupling. In conjunction with available structural and biochemical data, we propose that site selectivity by the PU.1 ETS domain arises from differential protein/DNA contacts in the flanking sequences that modulate the orientation of the ETS recognition helix and trigger a coupled reduction in the flexibility observed in the unbound ETS domain.

Eukaryotic transcription factors are characterized by large families that share structurally homologous DNA-binding domains and similar DNA site preferences. The molecular mechanism by which these functionally non-interchangeable homologs achieve specificity remains poorly known. The ETS-family of transcription factors represent an excellent model system that embodies this “specificity conundrum.” The 28 human ETS paralogs share a structurally conserved DNA-binding domain (known as the ETS domain) and recognize sites harboring a central 5'-GGAA/T-3' consensus. Although ETS domains are highly conserved structurally, they share low amino acid homology. We hypothesize that this apparent sequence space encodes divergent physicochemical properties that confers specificity to site discrimination at the protein-DNA level. We compared the solution properties of site discrimination by the ETS domains of PU.1 and Ets-1, two highly structurally conserved by sequence-divergent proteins. The proteins exhibit profound differences in interfacial hydration: whereas Ets-1 is weakly sensitive to perturbation in water activity, PU.1 is strongly destabilized by osmotic stress. The contrasting thermodynamics and kinetics of site recognition by PU.1 and Ets-1 indicate significant differences in their mechanisms of selectivity. Specifically, PU.1 extensively integrates structural water into high-affinity binding, a feature that renders site specificity by PU.1 highly sensitive to the solution osmotic status. PU.1 is expressed only in immune and closely-related cell lineages, all of which develop and function in osmotically variable environments. PU.1 and Ets-1 direct distinct responses in cells in which they are co-expressed. The strong sensitivity to osmotic stress in sequence discrimination by which PU.1 may represent a mechanism by which it functions distinctly from other ETS members such as Ets-1 in cells that must adapt to physiological osmotic stress.

The ETS family of transcription factors participate in a broad array of developmental and disease processes. ETS proteins are united by a conserved DNA-binding domain that targets 10-bp sequences harboring a central 5’-GGAA/T-3’ consensus. A primary recognition helix confers specificity for the consensus by direct readout of the major groove while adjoining mobile elements contact backbone contacts of flanking 5' and 3' sequences. Although flanking contacts play a secondary role in specifying the ETS binding site, they profoundly influence its affinity and transactivational activity. Elucidating the biophysical basis for the intrinsic selectivity of the ETS domain is therefore essential to understanding other interactions within the transcription regulatory complex. We have characterized the structure and thermodynamics of the ETS complexes with a high-, low-affinity, and nonspecific sequence. Calorimetric measurements reveal that the unbound ETS domain is a noncovalent, entropically-driven dimer. DNA binding induces ETS dissociation with sequence-dependent thermodynamics, suggesting distinct conformational arrangements accompanying the binding of each sequence variant. Footprinting titrations show that hypersensitivity to DNase I at the consensus is an experimental hallmark of sequence-specific ETS binding, regardless of affinity, indicating the importance of DNA distortion triggered by indirect readout. High- and low-affinity ETS cognates can be readily differentiated in terms of sensitivity to methylation in the latter. Based on this data, we present a model for ETS-DNA interactions in which indirect and direct readout are structurally linked to achieve sequence-specific binding. Complex formation in turn induces rheostatic rearrangements of ETS conformation (as manifest in terms of complex stability) that represent specific permissive or inhibitory states for protein-protein interactions (as manifest in ETS dimerization). Thus, the sequence identity of an ETS binding site may serve a functional role in initiating regulatory functions as well as an address for localization in the genome.

Specific inhibition or activation of enzymes and other proteins with small molecules is a widely exploited approach to modulating cellular pathways. Targeting the expression of these proteins at the level of transcription has remained relatively unexplored, however, and may offer significant biotechnological and therapeutic potential. We are interested in pharmacological modulation of genes controlled by members of the ETS family of transcription factors. ETS proteins are widely distributed among metazoan phyla and regulate a functionally diverse array of genes. As a model system, we have characterized the inhibition of sequence-specific binding of the ETS domain of PU.1 (Spi-1) to a natural high-affinity site by distamycin. Using previously established thermodynamic data on this protein-DNA system, we have refined the experimental conditions for surface-plasmon resonance (SPR) overcome mass transfer and other issues that have limited the use of SPR in protein-DNA interactions under physiologically relevant conditions. We found that distamycin potently inhibits PU.1 ETS binding with an IC50 of 2.5 nM under physiologic conditions. Since distamycin binds in the minor groove and ETS domains recognize their consensus motif in the major groove, this is an example of allosteric inhibition of a DNA-binding protein. We are now extending our studies to explore the potential of inhibiting the ptgs2 promoter, a native ETS-dependent promoter sequence, in live cells. We have engineered a reporter system based on a bright but destabilized green fluorescent protein (GFP) with a 2-hour biological half-life. We are characterizing this system in various cell lines that respond to various stimuli by inducing expression of the ptgs2 gene. We expect the combination of SPR and GFP reporter to serve as a useful platform for screening and developing ETS-targeting drug candidates.

Despite increasing awareness of their biological importance, mechanisms of DNA site discrimination by structurally homologous and functionally non-redundant transcription factors remain poorly defined concepts. Members of the ETS family of transcription factors, which regulate the differentiation of hematopoietic stem cells to the gamut of blood cell lineages, exemplify this conundrum. While the ETS-family members PU.1 and Ets-1 share structurally superimposable DNA-binding domains, the two homologs direct distinct cohorts of genes and induce mutually exclusive outcomes in hematopoietic cell-fate decisions. Using an array of biochemical, spectroscopic, and calorimetric techniques, we have compared DNA site recognition by PU.1 and Ets-1. The data indicate a startling level of structural, dynamic, thermodynamic, and kinetic heterogeneity associated with DNA site recognition by these proteins, which may be traced to their differential coupling of molecular hydration and counter-ion release with site-specific binding. Whereas site recognition by Ets-1 appears homogeneous with respect to DNA sequence identity, high- and low-affinity sites unmask major heterogeneity in PU.1 binding by all experimental observations. These differences are intrinsic to the two homologous DNA-binding domains; additional intramolecular interactions in adjacent domains exert no effect on the apparent homogeneity in Ets-1/DNA binding. The emerging evidence suggests a strong coupling between molecular hydration, DNA curvature, conformational dynamics, electrostatics as a mechanism for PU.1 to enforce different sequence preferences and kinetic behavior relative to Ets-1, and offer new insight into the functional differences between the two homologs. [This investigation is supported by NIH R15GM112002-01 to GMKP, NIH R01AI083803 to JKB, and NSF MCB1411502 to GMKP and WDW.]