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Signatures of DNA target selectivity by ETS transcription factors.

ABSTRACT: 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.

SUBMITTER: Poon GMK

PROVIDER: S-EPMC5501379 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Signatures of DNA target selectivity by ETS transcription factors.

Poon Gregory M K GMK Kim Hye Mi HM

Transcription 20170316 3

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 perspe ...[more]

PMID: 28301293

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Distinct Roles for Interfacial Hydration in Site-Specific DNA Recognition by ETS-Family Transcription Factors.

Project description: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.

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DNA Occupancy of Polymerizing Transcription Factors: A Chemical Model of the ETS Family Factor Yan.

Project description:Transcription factors use both protein-DNA and protein-protein interactions to assemble appropriate complexes to regulate gene expression. Although most transcription factors operate as monomers or dimers, a few, including the E26 transformation-specific family repressors Drosophila melanogaster Yan and its human homolog TEL/ETV6, can polymerize. Although polymerization is required for both the normal and oncogenic function of Yan and TEL/ETV6, the mechanisms by which it influences the recruitment, organization, and stability of transcriptional complexes remain poorly understood. Further, a quantitative description of the DNA occupancy of a polymerizing transcription factor is lacking, and such a description would have broader applications to the conceptually related area of polymerizing chromatin regulators. To expand the theoretical basis for understanding how the oligomeric state of a transcriptional regulator influences its chromatin occupancy and function, we leveraged the extensive biochemical characterization of E26 transformation-specific factors to develop a mathematical model of Yan occupancy at chemical equilibrium. We find that spreading condensation from a specific binding site can take place in a path-independent manner given reasonable values of the free energies of specific and non-specific DNA binding and protein-protein cooperativity. Our calculations show that polymerization confers upon a transcription factor the unique ability to extend occupancy across DNA regions far from specific binding sites. In contrast, dimerization promotes recruitment to clustered binding sites and maximizes discrimination between specific and non-specific sites. We speculate that the association with non-specific DNA afforded by polymerization may enable regulatory behaviors that are well-suited to transcriptional repressors but perhaps incompatible with precise activation.

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Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors.

Project description: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.

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Differential sensitivity to methylated DNA by ETS-family transcription factors is intrinsically encoded in their DNA-binding domains.

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Development of Mithramycin Analogues with Increased Selectivity toward ETS Transcription Factor Expressing Cancers.

Project description:Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.

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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.

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Potential downstream target genes of aberrant ETS transcription factors are differentially affected in Ewing's sarcoma and prostate carcinoma.

Project description:FLI1 and ERG, the major ETS transcription factors involved in rearrangements in the Ewing's sarcoma family of tumors (ESFT) and in prostate carcinomas (PCa), respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT) and choose four genes among the top-most differentially expressed between PCa with (PCa ERG+) and without (PCa ETS-) ETS fusion genes (HIST1H4L, KCNN2, ECRG4 and LDOC1), as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (NR0B1, CAV1, IGFBP3 and TGFBR2). Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS), we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa ERG+ and PCa ETS-, CAV1, IGFBP3 and TGFBR2 were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for ECRG4 and LDOC1, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both HIST1H4L and KCNN2 are significantly overexpressed in PCa ERG+ and that ERG binds to the promoter of these genes. Conversely, KCNN2 was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.

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Combinatorial function of ETS transcription factors in the developing vasculature.

Project description:Members of the ETS family of transcription factors are among the first genes expressed in the developing vasculature, but loss-of-function experiments for individual ETS factors in mice have not uncovered important early functional roles for these genes. However, multiple ETS factors are expressed in spatially and temporally overlapping patterns in the developing vasculature, suggesting possible functional overlap. We have taken a comprehensive approach to exploring the function of these factors during vascular development by employing the genetic and experimental tools available in the zebrafish to analyze four ETS family members expressed together in the zebrafish vasculature; fli1, fli1b, ets1, and etsrp. We isolated and characterized an ENU-induced mutant with defects in trunk angiogenesis and positionally cloned the defective gene from this mutant, etsrp. Using the etsrp morpholinos targeting each of the four genes, we show that the four ETS factors function combinatorially during vascular and hematopoietic development. Reduction of etsrp or any of the other genes alone results in either partial or no defects in endothelial differentiation, while combined reduction in the function of all four genes causes dramatic loss of endothelial cells. Our results demonstrate that combinatorial ETS factor function is essential for early endothelial specification and differentiation.

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Structural insights into target DNA recognition by R2R3-MYB transcription factors.

Project description:As the largest group of MYB family transcription factors, R2R3-MYB proteins play essential roles during plant growth and development. However, the structural basis underlying how R2R3-MYBs recognize the target DNA remains elusive. Here, we report the crystal structure of Arabidopsis WEREWOLF (WER), an R2R3-MYB protein, in complex with its target DNA. Structural analysis showed that the third α-helices in both the R2 and R3 repeats of WER fit in the major groove of the DNA, specifically recognizing the DNA motif 5'-AACNGC-3'. In combination with mutagenesis, in vitro binding and in vivo luciferase assays, we showed that K55, N106, K109 and N110 are critical for the function of WER. Although L59 of WER is not involved in DNA binding in the structure, ITC analysis suggested that L59 plays an important role in sensing DNA methylation at the fifth position of cytosine (5mC). Like 5mC, methylation at the sixth position of adenine (6mA) in the AAC element also inhibits the interaction between WER and its target DNA. Our study not only unravels the molecular basis of how WER recognizes its target DNA, but also suggests that 5mC and 6mA modifications may block the interaction between R2R3-MYB transcription factors and their target genes.

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