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Andrew D. Sharrocks - One of the best experts on this subject based on the ideXlab platform.
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ETS Domain transcription factor elk 1 mediates neuronal survival smn as a potential target
2011Co-Authors: Ozlem Demir, Andrew D. Sharrocks, Nese Aysit, Zeynep Onder, Nezaket Turkel, Gurkan Ozturk, Isil Aksan KurnazAbstract:Elk-1 belongs to the ternary complex factors (TCFs) subfamily of the ETS Domain proteins, and plays a critical role in the expression of immediate-early genes (IEGs) upon mitogen stimulation and activation of the mitogen-activated protein kinase (MAPK) cascade. The association of TCFs with serum response elements (SREs) on IEG promoters has been widely studied and a role for Elk-1 in promoting cell cycle entry has been determined. However, the presence of the ETS Domain transcription factor Elk-1 in axons and dendrites of post-mitotic adult brain neurons has implications for an alternative function for Elk-1 in neurons other than controlling proliferation. In this study, possible alternative roles for Elk-1 in neurons were investigated, and it was demonstrated that blocking TCF-mediated transactivation in neuronal cells leads to apoptosis through a caspase-dependent mechanism. Indeed RNAi-mediated depletion of endogenous Elk-1 results in increased caspase activity. Conversely, overexpression of either Elk-1 or Elk-VP16 fusion proteins was shown to rescue PC12 cells from chemically-induced apoptosis, and that higher levels of endogenous Elk-1 correlated with longer survival of DRGs in culture. It was shown that Elk-1 regulated the Mcl-1 gene expression required for survival, and that RNAi-mediated degradation of endogenous Elk-1 resulted in elimination of the mcl-1 message. We have further identified the survival-of-motor neuron-1 (SMN1) gene as a novel target of Elk-1, and show that the ETS motifs in the SMN1 promoter are involved in this regulation.
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the erk map kinase pea3 etv4 mmp 1 axis is operative in oesophageal adenocarcinoma
2010Co-Authors: Richard Keld, Baoqiang Guo, Paul Downey, Christian Gulmann, Yeng Ang, Andrew D. SharrocksAbstract:Background: Many members of the ETS-Domain transcription factor family are important drivers of tumourigenesis. In this context, their activation by Ras-ERK pathway signaling is particularly relevant to the tumourigenic properties of many ETS-Domain transcription factors. The PEA3 subfamily of ETS-Domain transcription factors have been implicated in tumour metastasis in several different cancers. Results: Here, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in oesophageal adenocarcinomas and determined their role in oesophageal adenocarcinoma cell function. PEA3 plays an important role in controlling both the proliferation and invasive properties of OE33 oesophageal adenocarcinoma cells. A key target gene is MMP-1. The ERK MAP kinase pathway activates PEA3 subfamily members and also plays a role in these PEA3 controlled events, establishing the ERK-PEA3-MMP-1 axis as important in OE33 cells. PEA3 subfamily members are upregulated in human adenocarcinomas and expression correlates with MMP-1 expression and late stage metastatic disease. Enhanced ERK signaling is also more prevalent in late stage oesophageal adenocarcinomas. Conclusions: This study shows that the ERK-PEA3-MMP-1 axis is upregulated in oesophageal adenocarcinoma cells and is a potentially important driver of the metastatic progression of oesophageal adenocarcinomas.
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overlapping promoter targeting by elk 1 and other divergent ETS Domain transcription factor family members
2009Co-Authors: Joanna Boros, Amanda Odonnell, Ian J Donaldson, Aneta Kasza, Leo A H Zeef, Andrew D. SharrocksAbstract:ETS-Domain transcription factors play important roles in controlling gene expression in a variety of different contexts; however, these proteins bind to very similar sites and it is unclear how in vivo specificity is achieved. In silico analysis is unlikely to reveal specific targETS for individual family members and direct experimental approaches are therefore required. Here, we take advantage of an inducible dominant-negative expression system to identify a group of novel target genes for the ETS-Domain transcription factor Elk-1. Elk-1 is thought to mainly function through cooperation with a second transcription factor SRF, but the targETS we identify are largely SRF-independent. Furthermore, we demonstrate that there is a high degree of overlapping, cell type-specific, target gene binding by Elk-1 and other ETS-Domain transcription factors. Our results are therefore consistent with the notion that there is a high degree of functional redundancy in target gene regulation by ETS-Domain transcription factors in addition to the specific target gene regulation that can be dictated through heterotypic interactions exemplified by the Elk-1-SRF complex.
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elucidation of the elk1 target gene network reveals a role in the coordinate regulation of core components of the gene regulation machinery
2009Co-Authors: Joanna Boros, Amanda Odonnell, Ian J Donaldson, Leo A H Zeef, Zaneta Odrowaz, Mathieu Lupien, Clifford A Meyer, Myles Brown, Andrew D. SharrocksAbstract:Transcription factors play an important role in orchestrating the activation of specific networks of genes through targeting their proximal promoter and distal enhancer regions. However, it is unclear how the specificity of downstream responses is maintained by individual members of transcription-factor families and, in most cases, what their target repertoire is. We have used ChIP-chip analysis to identify the target genes of the ETS-Domain transcription factor ELK1. Two distinct modes of ELK1 target gene selection are identified; the first involves redundant promoter binding with other ETS-Domain family members; the second occurs through combinatorial binding with a second transcription factor SRF, which specifies a unique group of target genes. One of the most prominent groups of genes forming the ELK1 target network includes classes involved in core gene expression control, namely, components of the basal transcriptional machinery, the spliceosome and the ribosome. Amongst the set of genes encoding the basal transcription machinery components, are a functionally linked subset of GTFs and TAFs. Our study, therefore, reveals an unsuspected level of coordinate regulation of components of the core gene expression control machinery and also identifies two different modes of promoter targeting through binding with a second transcription factor or redundant binding with other ETS-Domain family members.
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the lim Domain protein lpp is a coactivator for the ETS Domain transcription factor pea3
2006Co-Authors: Baoqiang Guo, Rosemary E Sallis, Amanda Greenall, Marleen M R Petit, Erik Jansen, Leonie S Young, Wim J M Van De Ven, Andrew D. SharrocksAbstract:PEA3 is a member of a subfamily of ETS Domain transcription factors which is regulated by a number of signaling cascades, including the mitogen-activated protein (MAP) kinase pathways. PEA3 activates gene expression and is thought to play an important role in promoting tumor metastasis and also in neuronal development. Here, we have identified the LIM Domain protein LPP as a novel coregulatory binding partner for PEA3. LPP has intrinsic transactivation capacity, forms a complex with PEA3, and is found associated with PEA3-regulated promoters. By manipulating LPP levels, we show that it acts to upregulate the transactivation capacity of PEA3. LPP can also functionally interact in a similar manner with the related family member ER81. Thus, we have uncovered a novel nuclear function for the LIM Domain protein LPP as a transcriptional coactivator. As LPP continually shuttles between the cell periphery and the nucleus, it represents a potential novel link between cell surface events and changes in gene expression.
Barbara J. Graves - One of the best experts on this subject based on the ideXlab platform.
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the biophysical basis for phosphorylation enhanced dna binding autoinhibition of the ETS1 transcription factor
2019Co-Authors: Cecilia Perezborrajero, Barbara J. Graves, Mark Okon, Chang Shenghuei Lin, Karlton Scheu, Michael E P Murphy, Lawrence P. McintoshAbstract:Abstract The eukaryotic transcription factor ETS1 is regulated by an intrinsically disordered serine-rich region (SRR) that transiently associates with the adjacent ETS Domain to inhibit DNA binding. In this study, we further elucidated the physicochemical basis for ETS1 autoinhibition by characterizing the interaction of its ETS Domain with a series of synthetic peptides corresponding to the SRR. Binding is driven by the hydrophobic effect and enhanced electrostatically by phosphorylation of serines adjacent to aromatic residues in the amphipathic SRR. Structural characterization of the dynamic peptide/protein complex by NMR spectroscopy and X-ray crystallography revealed multiple modes of binding that lead to autoinhibition by synergistically blocking the DNA-binding interface of the ETS Domain and stabilizing an appended helical inhibitory module against allosterically induced unfolding. Consistent with these conclusions, the SRR peptide does not interact with DNA-bound ETS1. In addition, we found that the ETS1 SRR phosphopeptide binds to distantly related PU.1 in vitro, indicating that autoinhibition exploits features of the ETS Domain that are conserved across this family of transcription factors.
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structured and disordered regions cooperatively mediate dna binding autoinhibition of ETS factors etv1 etv4 and etv5
2017Co-Authors: Simon L Currie, Jedediah J Doane, Frank G Whitby, Barbara J. Graves, Mark Okon, Lawrence P. McintoshAbstract:: Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS Domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory Domain that packs against the ETS Domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory Domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS Domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein-protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.
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steric mechanism of auto inhibitory regulation of specific and non specific dna binding by the ETS transcriptional repressor etv6
2014Co-Authors: Anson C K Chan, Barbara J. Graves, Mark Okon, Jerome H Coyne, Niraja Bhachech, Michael E P Murphy, Ulrike Hermsdorf, Lawrence P. McintoshAbstract:DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS Domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus (5')GGAA(3') recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS Domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS Domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing (5')GGAA(3')versus(5')GGAT(3'). These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.
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autoinhibition of etv6 tel dna binding appended helices sterically block the ETS Domain
2012Co-Authors: Jerome H Coyne, Barbara J. Graves, Mark Okon, Sean M Green, Niraja Bhachech, Lawrence P. McintoshAbstract:ETV6 (or TEL), a transcriptional repressor belonging to the ETS family, is frequently involved in chromosomal translocations linked with human cancers. It displays a DNA-binding mode distinct from other ETS proteins due to the presence of a self-associating PNT Domain. In this study, we used NMR spectroscopy to dissect the structural and dynamic bases for the autoinhibition of ETV6 DNA binding by sequences C-terminal to its ETS Domain. The C-terminal inhibitory Domain (CID) contains two helices, H4 and H5, which sterically block the DNA-binding interface of the ETS Domain. Importantly, these appended helices are only marginally stable as revealed by amide hydrogen exchange and (15)N relaxation measurements. The CID is thus poised to undergo a facile conformational change as required for DNA binding. The CID also dampens millisecond timescale motions of the ETS Domain hypothesized to be critical for the recognition of specific ETS target sequences. This work illustrates the use of appended sequences on conserved structural Domains to generate biological diversity and complements previous studies of the allosteric mechanism of ETS1 autoinhibition to reveal both common and divergent features underlying the regulation of DNA binding by ETS transcription factors.
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dna binding by the ETS protein tel etv6 is regulated by autoinhibition and self association
2010Co-Authors: Sean M Green, Jerome H Coyne, Lawrence P. Mcintosh, Barbara J. GravesAbstract:The ETS protein TEL, a transcriptional repressor, contains a PNT Domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (K(d) = 2.8 x 10(-8) M). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory Domain (CID) that attenuates DNA binding by approximately 10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two alpha-helices, one of which appears to block the DNA binding surface of the TEL ETS Domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT Domain and DNA-binding ETS Domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.
Lawrence P. Mcintosh - One of the best experts on this subject based on the ideXlab platform.
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the biophysical basis for phosphorylation enhanced dna binding autoinhibition of the ETS1 transcription factor
2019Co-Authors: Cecilia Perezborrajero, Barbara J. Graves, Mark Okon, Chang Shenghuei Lin, Karlton Scheu, Michael E P Murphy, Lawrence P. McintoshAbstract:Abstract The eukaryotic transcription factor ETS1 is regulated by an intrinsically disordered serine-rich region (SRR) that transiently associates with the adjacent ETS Domain to inhibit DNA binding. In this study, we further elucidated the physicochemical basis for ETS1 autoinhibition by characterizing the interaction of its ETS Domain with a series of synthetic peptides corresponding to the SRR. Binding is driven by the hydrophobic effect and enhanced electrostatically by phosphorylation of serines adjacent to aromatic residues in the amphipathic SRR. Structural characterization of the dynamic peptide/protein complex by NMR spectroscopy and X-ray crystallography revealed multiple modes of binding that lead to autoinhibition by synergistically blocking the DNA-binding interface of the ETS Domain and stabilizing an appended helical inhibitory module against allosterically induced unfolding. Consistent with these conclusions, the SRR peptide does not interact with DNA-bound ETS1. In addition, we found that the ETS1 SRR phosphopeptide binds to distantly related PU.1 in vitro, indicating that autoinhibition exploits features of the ETS Domain that are conserved across this family of transcription factors.
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structured and disordered regions cooperatively mediate dna binding autoinhibition of ETS factors etv1 etv4 and etv5
2017Co-Authors: Simon L Currie, Jedediah J Doane, Frank G Whitby, Barbara J. Graves, Mark Okon, Lawrence P. McintoshAbstract:: Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS Domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory Domain that packs against the ETS Domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory Domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS Domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein-protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.
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steric mechanism of auto inhibitory regulation of specific and non specific dna binding by the ETS transcriptional repressor etv6
2014Co-Authors: Anson C K Chan, Barbara J. Graves, Mark Okon, Jerome H Coyne, Niraja Bhachech, Michael E P Murphy, Ulrike Hermsdorf, Lawrence P. McintoshAbstract:DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS Domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus (5')GGAA(3') recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS Domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS Domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing (5')GGAA(3')versus(5')GGAT(3'). These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.
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autoinhibition of etv6 tel dna binding appended helices sterically block the ETS Domain
2012Co-Authors: Jerome H Coyne, Barbara J. Graves, Mark Okon, Sean M Green, Niraja Bhachech, Lawrence P. McintoshAbstract:ETV6 (or TEL), a transcriptional repressor belonging to the ETS family, is frequently involved in chromosomal translocations linked with human cancers. It displays a DNA-binding mode distinct from other ETS proteins due to the presence of a self-associating PNT Domain. In this study, we used NMR spectroscopy to dissect the structural and dynamic bases for the autoinhibition of ETV6 DNA binding by sequences C-terminal to its ETS Domain. The C-terminal inhibitory Domain (CID) contains two helices, H4 and H5, which sterically block the DNA-binding interface of the ETS Domain. Importantly, these appended helices are only marginally stable as revealed by amide hydrogen exchange and (15)N relaxation measurements. The CID is thus poised to undergo a facile conformational change as required for DNA binding. The CID also dampens millisecond timescale motions of the ETS Domain hypothesized to be critical for the recognition of specific ETS target sequences. This work illustrates the use of appended sequences on conserved structural Domains to generate biological diversity and complements previous studies of the allosteric mechanism of ETS1 autoinhibition to reveal both common and divergent features underlying the regulation of DNA binding by ETS transcription factors.
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dna binding by the ETS protein tel etv6 is regulated by autoinhibition and self association
2010Co-Authors: Sean M Green, Jerome H Coyne, Lawrence P. Mcintosh, Barbara J. GravesAbstract:The ETS protein TEL, a transcriptional repressor, contains a PNT Domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (K(d) = 2.8 x 10(-8) M). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory Domain (CID) that attenuates DNA binding by approximately 10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two alpha-helices, one of which appears to block the DNA binding surface of the TEL ETS Domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT Domain and DNA-binding ETS Domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.
Richard A Maki - One of the best experts on this subject based on the ideXlab platform.
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mutants of ETS Domain pu 1 and ggaa t recognition free energies and kinetics
1999Co-Authors: Frederic Pio, Nuria Assamunt, Richard A Maki, Juan YguerabideAbstract:The ETS family members display specific DNA binding site preferences. As an example, PU.1 and ETS-1 recognize different DNA sequences with a core element centered over 5'-GGAA-3' and 5'-GGAA/T-3', respectively. To understand the molecular basis of this recognition, we carried out site-directed mutagenesis experiments followed by DNA binding studies that use electrophoretic mobility shift assay (EMSA) and surface plasmon resonance methods. EMSA experiments identified amino acid changes A231S and/or N236Y as being important for PU.1 recognition of both 5'-GGAA-3' and 5'-GGAT-3' containing oligonucleotides. To confirm these data and obtain accurate binding parameters, we performed kinetic studies using surface plasmon resonance on these mutants. The N236Y substitution revealed a weak protein-DNA interaction with the 5'-GGAA-3' containing oligonucleotide caused by a faster release of the protein from the DNA (k(off) tenfold higher than the wild-type protein). With the double mutant A231S-N236Y, we obtained an increase in binding affinity and stability toward both 5'-GGAA-3' and 5'-GGAT-3' containing oligonucleotides. We propose that substitution of alanine for serine introduces an oxygen atom that can accept hydrogen and interact with potential water molecules or other atoms to make an energetically favorable hydrogen bond with both 5'-GGAA-3' and 5'-GGAT-3' oligonucleotides. The free energy of dissociation for the double mutant A231S-N236Y with 5'-GGAA-3' (delta deltaG((A231S-N236Y) - (N236Y)) = -1.2 kcal mol confirm the stabilizing effect of this mutant in the protein-DNA complex formation. We conclude that N236Y mutation relaxes the specificity toward 5'-GGAA-3' and 5'-GGAT-3' sequences, while A231S mutation modulates the degree of specificity toward 5'-GGAA-3' and 5'GGAT-3' sequences. This study explains why wild-type PU.1 does not recognize 5'-GGAT-3' sequences and in addition broadens our understanding of 5'-GGAA/T-3' recognition by ETS protein family members.
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new insights on dna recognition by ETS proteins from the crystal structure of the pu 1 ETS Domain dna complex
1996Co-Authors: Frederic Pio, Scott R Mckercher, Michael J Klemsz, Richard A Maki, Ramadurgam Kodandapani, William Shepard, Kathryn R ElyAbstract:Transcription factors belonging to the ETS family regulate gene expression and share a conserved ETS DNA-binding Domain that binds to the core sequence 5'-(C/A)GGA(A/T)-3'. The Domain is similar to alpha+beta ("winged") helix-turn-helix DNA-binding proteins. The crystal structure of the PU.1 ETS Domain complexed to a 16-base pair oligonucleotide revealed a pattern for DNA recognition from a novel loop-helix-loop architecture (Kodandapani, R., Pio, F., Ni. C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460). Correlation of this model with mutational analyses and chemical shift data on other ETS proteins confirms this complex as a paradigm for ETS DNA recognition. The second helix in the helix-turn-helix motif lies deep in the major groove with specific contacts with bases in both strands in the core sequence made by conserved residues in alpha3. On either side of this helix, two loops contact the phosphate backbone. The DNA is bent (8 degrees) but uniformly curved without distinct kinks. ETS Domains bind DNA as a monomer yet make extensive DNA contacts over 30 A. DNA bending likely results from phosphate neutralization of the phosphate backbone in the minor groove by both loops in the loop-helix-loop motif. Contacts from these loops stabilize DNA bending and may mediate specific base interactions by inducing a bend toward the protein.
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a new pattern for helix turn helix recognition revealed by the pu 1 ETS Domain dna complex
1996Co-Authors: Ramadurgam Kodandapani, Frederic Pio, Scott R Mckercher, Michael J Klemsz, Richard A Maki, Gennaro Piccialli, Kathryn R ElyAbstract:THE ETS family of transcription factors, of which there are now about 35 members1,2, regulate gene expression during growth and development. They share a conserved Domain of around 85 amino acids3 which binds as a monomer to the DNA sequence 5'-C/ AGGAA/T-3'. We have determined theocrystal structure of an ETS Domain complexed with DNA, at 2.3-A resolution. The Domain is similar to α+β (winged) 'helix–turn–helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8°. The Domain contacts the DNA by a novel loop–helix–loop architecture. Four of the amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix–turn–helix' motif, which binds downstream and on the opposite strand.
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co crystallization of an ETS Domain pu 1 in complex with dna engineering the length of both protein and oligonucleotide
1995Co-Authors: Frederic Pio, Richard S Mitchell, John Knight, Scott R Mckercher, Michael J Klemsz, Angela Lombardo, Richard A Maki, Kathryn R ElyAbstract:The PU.1 transcription factor is a member of the ETS gene family of regulatory proteins. These molecules play a role in normal development and also have been implicated in malignant processes such as the development of erythroid leukemia. The ETS proteins share a conserved DNA-binding Domain (the ETS Domain) that recognizes a purine-rich sequence with the core sequence: 5′-C/AGGAA/T-3′. This Domain binds to DNA as a monomer, unlike many other DNA-binding proteins. The ETS Domain of the PU.1 transcription factor has been crystallized in complex with a 16-base pair oligonucleotide that contains the recognition sequence. The crystals formed in the space group C2 with a = 89.1, b = 101.9, c = 55.6 A, and β = 111.2° and diffract to at least 2.3 A. There are two complexes in the asymmetric unit. Production of large usable crystals was dependent on the length of both protein and DNA components, the use of oligonucleotides with unpaired A and T bases at the termini, and the presence of polyethylene glycol and zinc acetate in the crystallization solutions. This is the first ETS Domain to be crystallized, and the strategy used to crystallize this complex may be useful for other members of the ETS family.
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regulation of lymphoid specific immunoglobulin mu heavy chain gene enhancer by ETS Domain proteins
1993Co-Authors: Barbara Nelsen, Richard A Maki, Gang Tian, Batu Erman, Jacqueline Gregoire, Barbara J. GravesAbstract:The enhancer for the immunoglobulin mu heavy chain gene (IgH) activates a heterologous gene at the pre-B cell stage of B lymphocyte differentiation. A lymphoid-specific element, microB, is necessary for enhancer function in pre-B cells. A microB binding protein is encoded by the PU.1/Spi-1 proto-oncogene. Another sequence element, microA, was identified in the mu enhancer that binds the product of the ETS-1 proto-oncogene. The microA motif was required for microB-dependent enhancer activity, which suggests that a minimal B cell-specific enhancer is composed of both the PU.1 and ETS-1 binding sites. Co-expression of both PU.1 and ETS-1 in nonlymphoid cells trans-activated reporter plasmids that contained the minimal mu enhancer. These results implicate two members of the ETS family in the activation of IgH gene expression.
Kathryn R Ely - One of the best experts on this subject based on the ideXlab platform.
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new insights on dna recognition by ETS proteins from the crystal structure of the pu 1 ETS Domain dna complex
1996Co-Authors: Frederic Pio, Scott R Mckercher, Michael J Klemsz, Richard A Maki, Ramadurgam Kodandapani, William Shepard, Kathryn R ElyAbstract:Transcription factors belonging to the ETS family regulate gene expression and share a conserved ETS DNA-binding Domain that binds to the core sequence 5'-(C/A)GGA(A/T)-3'. The Domain is similar to alpha+beta ("winged") helix-turn-helix DNA-binding proteins. The crystal structure of the PU.1 ETS Domain complexed to a 16-base pair oligonucleotide revealed a pattern for DNA recognition from a novel loop-helix-loop architecture (Kodandapani, R., Pio, F., Ni. C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460). Correlation of this model with mutational analyses and chemical shift data on other ETS proteins confirms this complex as a paradigm for ETS DNA recognition. The second helix in the helix-turn-helix motif lies deep in the major groove with specific contacts with bases in both strands in the core sequence made by conserved residues in alpha3. On either side of this helix, two loops contact the phosphate backbone. The DNA is bent (8 degrees) but uniformly curved without distinct kinks. ETS Domains bind DNA as a monomer yet make extensive DNA contacts over 30 A. DNA bending likely results from phosphate neutralization of the phosphate backbone in the minor groove by both loops in the loop-helix-loop motif. Contacts from these loops stabilize DNA bending and may mediate specific base interactions by inducing a bend toward the protein.
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a new pattern for helix turn helix recognition revealed by the pu 1 ETS Domain dna complex
1996Co-Authors: Ramadurgam Kodandapani, Frederic Pio, Scott R Mckercher, Michael J Klemsz, Richard A Maki, Gennaro Piccialli, Kathryn R ElyAbstract:THE ETS family of transcription factors, of which there are now about 35 members1,2, regulate gene expression during growth and development. They share a conserved Domain of around 85 amino acids3 which binds as a monomer to the DNA sequence 5'-C/ AGGAA/T-3'. We have determined theocrystal structure of an ETS Domain complexed with DNA, at 2.3-A resolution. The Domain is similar to α+β (winged) 'helix–turn–helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8°. The Domain contacts the DNA by a novel loop–helix–loop architecture. Four of the amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix–turn–helix' motif, which binds downstream and on the opposite strand.
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co crystallization of an ETS Domain pu 1 in complex with dna engineering the length of both protein and oligonucleotide
1995Co-Authors: Frederic Pio, Richard S Mitchell, John Knight, Scott R Mckercher, Michael J Klemsz, Angela Lombardo, Richard A Maki, Kathryn R ElyAbstract:The PU.1 transcription factor is a member of the ETS gene family of regulatory proteins. These molecules play a role in normal development and also have been implicated in malignant processes such as the development of erythroid leukemia. The ETS proteins share a conserved DNA-binding Domain (the ETS Domain) that recognizes a purine-rich sequence with the core sequence: 5′-C/AGGAA/T-3′. This Domain binds to DNA as a monomer, unlike many other DNA-binding proteins. The ETS Domain of the PU.1 transcription factor has been crystallized in complex with a 16-base pair oligonucleotide that contains the recognition sequence. The crystals formed in the space group C2 with a = 89.1, b = 101.9, c = 55.6 A, and β = 111.2° and diffract to at least 2.3 A. There are two complexes in the asymmetric unit. Production of large usable crystals was dependent on the length of both protein and DNA components, the use of oligonucleotides with unpaired A and T bases at the termini, and the presence of polyethylene glycol and zinc acetate in the crystallization solutions. This is the first ETS Domain to be crystallized, and the strategy used to crystallize this complex may be useful for other members of the ETS family.