Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structure-dependent inhibition of the ETS-family transcription factor PU.1 by novel heterocyclic diamidines.

ABSTRACT: 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.

SUBMITTER: Munde M

PROVIDER: S-EPMC3902942 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Multiple DNA-binding modes for the ETS family transcription factor PU.1.

Project description: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. Whereas 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 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 autoinhibitory mechanism in the ETS family at the protein-DNA level.

| S-EPMC5625037 | biostudies-literature

Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines.

Project description:Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site.

| S-EPMC3592449 | biostudies-literature

Mechanism of cognate sequence discrimination by the ETS-family transcription factor ETS-1.

Project description: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.

| S-EPMC6597803 | biostudies-literature

Sequence discrimination by DNA-binding domain of ETS family transcription factor PU.1 is linked to specific hydration of protein-DNA interface.

Project description: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.

| S-EPMC3365747 | biostudies-literature

Pharmacological inhibition of the transcription factor PU.1 in leukemia.

Project description: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.1lo 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.

| S-EPMC5707147 | biostudies-literature

Mechanistic heterogeneity in site recognition by the structurally homologous DNA-binding domains of the ETS family transcription factors Ets-1 and PU.1.

Project description: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.

| S-EPMC4118120 | biostudies-literature

Pharmacologic efficacy of PU.1 inhibition by heterocyclic dications: a mechanistic analysis.

Project description: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.

| S-EPMC4872103 | biostudies-literature

The Sfpi-1 proviral integration site of Friend erythroleukemia encodes the ets-related transcription factor Pu.1.

Project description:Previous studies identified a common site (Sfpi-1) for proviral integration in immortalized Friend erythroleukemias. cDNAs corresponding to a 1.5-kb Sfpi-1 mRNA were isolated and sequenced. These were larger than an independently isolated Sfpi-1 cDNA described by researchers from another laboratory, and they contained common differences from that sequence, including in the coding region four extra nucleotides that altered the reading frame. The properly translated protein is identical to Pu.1, a transcription activation factor that is related to the ets oncogene family. Genetic methods were used to map Sfpi-1 with respect to other loci on mouse chromosome 2. Our results suggest that Pu.1 blocks erythroblast differentiation and thereby causes immortalization.

| S-EPMC240539 | biostudies-other

Mapping interfacial hydration in ETS-family transcription factor complexes with DNA: a chimeric approach.

Project description: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.

| S-EPMC6237740 | biostudies-literature

Genome-wide identification and evolutionary analysis of the animal specific ETS transcription factor family.

Project description:The ETS proteins are a family of transcription factors (TFs) that regulate a variety of biological processes. We made genome-wide analyses to explore the classification of the ETS gene family. We identified 207 ETS genes which encode 321 ETS TFs from ten animal species. Of the 321 ETS TFs, 155 contain only an ETS domain, about 50% contain a ETS_PEA3_N or a SAM_PNT domain in addition to an ETS domain, the rest (only four) contain a second ETS domain or a second ETS_PEA3_N domain or an another domain (AT_hook or DNA_pol_B). A Neighbor-Joining phylogenetic tree was constructed using the amino acid sequences of the ETS domain of the ETS TFs. The results revealed that the ETS genes of the ten species can be divided into two distinct groups. Group I contains one nematode ETS gene and 18 vertebrate animal ETS genes. Group II contains the majority of the ETS TFs and can be further divided into eleven subgroups. The sequence motifs outside the DNA-binding domain and the conservation of the exon-intron structural patterns of the ETS TFs in human, cattle, and chicken further support the phylogenetic classification among these ETS TFs. Extensive duplication of the ETS genes was found in the genome of each species. The duplicated ETS genes account for ~69% of the total of ETS genes. Furthermore, we also found there are ETS gene clusters in all of the ten animal species. Statistical analysis of the Gene Ontology annotations of the ETS genes showed that the ETS proteins tend to be related to RNA biosynthetic process, biopolymer metabolic process and macromolecule metabolic process expected from the common GO categories of transcriptional factors. We also discussed the functional conservation and diversification of ETS TFs.

| S-EPMC2789578 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data