Crystallization of the Ets1-Runx1-CBFβ-DNA complex formed on the TCRα gene enhancer (original) (raw)
Related papers
Co-crystallization of an ETS Domain (PU.1) in Complex with DNA
Journal of Biological Chemistry, 1995
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 Å, and  ؍ 111.2°and diffract to at least 2.3 Å. 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.
Cooperative binding of Ets-1 and core binding factor to DNA
Molecular and cellular biology, 1994
Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constrai...
Ets-2, like its closely related homologue Ets-1, is a member of the Ets family of DNA binding transcription factors. Both pro- teins are subject to multiple levels of regulation of their DNA binding and transactivation properties. One such regulatory mechanism is the presence of an autoinhibitory module, which in Ets-1 allosterically inhibits the DNA binding activity. This inhibition can be relieved by interaction with protein partners or cooperative binding to closely separated Ets binding sites in a palindromic arrangement. In this study we describe the 2.5 Å resolution crystal structure of a DNA complex of the Ets-2 Ets domain. The Ets domain crystallized with two distinct species in the asymmetric unit, which closely resemble the autoinhibited and DNA bound forms of Ets-1. This discovery prompted us to re-evaluate the current model for the autoinhibitory mechanism and the structural basis for cooperative DNA binding. In con- trast to Ets-1, in which the autoinhibition is caused by a combi- nation of allosteric and steric mechanisms, we were unable to find clear evidence for the allosteric mechanism in Ets-2. We also demonstrated two possibly distinct types of cooperative binding to substrates with Ets binding motifs separated by four and six base pairs and suggest possible molecular mechanisms for this behavior.
Regulation of the transcription factor Ets-1 by DNA-mediated homo-dimerization
The EMBO Journal, 2008
The function of the Ets-1 transcription factor is regulated by two regions that flank its DNA-binding domain. A previously established mechanism for auto-inhibition of monomeric Ets-1 on DNA response elements with a single ETS-binding site, however, has not been observed for the stromelysin-1 promoter containing two palindromic ETS-binding sites. We present the structure of Ets-1 on this promoter element, revealing a ternary complex in which protein homo-dimerization is mediated by the specific arrangement of the two ETS-binding sites. In this complex, the N-terminal-flanking region is required for ternary protein-DNA assembly. Ets-1 variants, in which residues from this region are mutated, loose the ability for DNA-mediated dimerization and stromelysin-1 promoter transactivation. Thus, our data unravel the molecular basis for relief of auto-inhibition and the ability of Ets-1 to function as a facultative dimeric transcription factor on this site. Our findings may also explain previous data of Ets-1 function in the context of heterologous transcription factors, thus providing a molecular model that could also be valid for Ets-1 regulation by hetero-oligomeric assembly.
Solution structure of the ETS domain from murine Ets-1: A winged helix-turn-helix DNA binding motif
The EMBO Journal
Ets-1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets-1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C-terminus. The ETS domain folds into a helix-turn-helix motif on a fourstranded anti-parallel 13-sheet scaffold. This structure places Ets-1 in the winged helix-turn-helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets-1 with DNA. The C-terminal sequence of Ets-1, which is mutated in the v-Ets oncoprotein, forms an a-helix that packs anti-parallel to the N-terminal helix of the ETS domain. In this position, the C-terminal helix is poised to interact directly with an N-terminal inhibitory region in Ets-1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets-1 DNA binding.
Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev
Ets transcription factors, which share the conserved Ets DNA- binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregula- tion following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein- DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated net- work of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for dis- crimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homo- dimers of Etv1, Etv4, and Etv5 could be reduced to monomers, lead- ing to a 40 –200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.
The Journal of Immunology, 2009
During thymocyte maturation, enhancers of genes encoding for TCRδ (Tcrd) and TCRα (Tcra), Eδ8, and Eα, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an Eα fragment, Tα1-Tα2, which constitutes a well-characterized compact nucleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire Eα or Tα1-Tα4. These experiments indicated that Tα3-Tα4 collaborates with factors bound to Tα1-Tα2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on Tα1-Tα2 explained the molecular basis for requirement of intact Tα2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of Eα, EαMC, in which Eδ myb and runx sites have been substituted for Tα2 runx and ets sites, that argues against the notion of a requirement for strict Eα enhanceosome structure for function. EαMC resulted in a very potent ...
Journal of Molecular Biology, 2010
The Ets family of transcription factors is composed of more than 30 members. One of its members, Elf3, is expressed in virtually all epithelial cells as well as in many tumors, including breast tumors. Several studies observed that the promoter of the type II TGF-β receptor gene (TβR-II) is strongly stimulated by Elf3 via two adjacent Elf3 binding sites, A-site and B-site. Here we report the 2.2 Å resolution crystal structure of a mouse Elf3 C-terminal fragment, containing the DNA-binding Ets domain, in complex with the B-site of mouse type II TGF-β receptor promoter DNA (mTβR-II DNA). Elf3 contacts the core GGAA motif of the B-site from major groove similar to that of known Ets proteins. However, unlike other Ets proteins, Elf3 also contacts sequences of the A-site from the minor groove of the DNA. DNA binding experiments and cell-based transcription studies indicate that minor groove interaction by Arg349 located in the Ets domain is important for Elf3 function. Equally interesting, previous studies have shown that the C-terminal region of Elf3, which flanks the Ets domain, is required for Elf3 binding to DNA. In this study, we determined that Elf3 amino acid residues within this flanking region, including Trp361, are important for the structural integrity of the protein as well as for the Efl3 DNA binding and transactivation activity.