1
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Jiang J, Wu Q, Rajasekaran S, Wu R. MMP3 at the crossroads: Linking molecular pathways to disease diagnosis and therapy. Pharmacol Res 2025; 216:107750. [PMID: 40311957 DOI: 10.1016/j.phrs.2025.107750] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/17/2025] [Revised: 04/21/2025] [Accepted: 04/27/2025] [Indexed: 05/03/2025]
Abstract
Matrix metalloproteinase 3 (MMP-3) is a multifaceted enzyme that plays a critical role in the regulation of extracellular matrix (ECM) dynamics, influencing both normal physiological and pathological processes. In addition to its established role in ECM degradation, MMP-3 is gaining recognition for modulating cellular behaviors such as inflammation, migration, and proliferation. Recent research has uncovered its capacity to activate latent signaling molecules, release growth factors from the ECM and interact with various cell surface receptors, linking MMP-3 to the progression of various diseases, including inflammatory diseases, infection diseases, cardiovascular diseases, neurodegenerative disorders, and cancer. The review provides an overview of MMP-3's molecular regulation, emphasizing the mechanisms controlling its expression and activity. We discuss MMP3's involvement in both ECM-dependent and independent pathways, and its potential as a diagnostic, prognostic biomarker in various diseases. Additionally, we explore therapeutic strategies targeting MMP-3, summarizing ongoing efforts to develop specific inhibitors and modulate its activity in different pathologic conditions. Through this review, we aim to consolidate the diverse functions of MMP-3 and provide new insights into future research directions, particularly in translating these findings into clinical applications.
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Affiliation(s)
- Jing Jiang
- Section of Cardiology, Department of Medicine, Biological Sciences Division, University of Chicago, Chicago, IL, United States; Binzhou Medical University, Yantai, China
| | - Qiong Wu
- Section of Cardiology, Department of Medicine, Biological Sciences Division, University of Chicago, Chicago, IL, United States
| | - Snekha Rajasekaran
- Section of Cardiology, Department of Medicine, Biological Sciences Division, University of Chicago, Chicago, IL, United States
| | - Rongxue Wu
- Section of Cardiology, Department of Medicine, Biological Sciences Division, University of Chicago, Chicago, IL, United States.
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2
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Luchtel RA. ETS1 Function in Leukemia and Lymphoma. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2024; 1459:359-378. [PMID: 39017852 DOI: 10.1007/978-3-031-62731-6_16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/18/2024]
Abstract
ETS proto-oncogene 1 (ETS1) is a transcription factor (TF) critically involved in lymphoid cell development and function. ETS1 expression is tightly regulated throughout differentiation and activation in T-cells, natural killer (NK) cells, and B-cells. It has also been described as an oncogene in a range of solid and hematologic cancer types. Among hematologic malignancies, its role has been best studied in T-cell acute lymphoblastic leukemia (T-ALL), adult T-cell leukemia/lymphoma (ATLL), and diffuse large B-cell lymphoma (DLBCL). Aberrant expression of ETS1 in these malignancies is driven primarily by chromosomal amplification and enhancer-driven transcriptional regulation, promoting the ETS1 transcriptional program. ETS1 also facilitates aberrantly expressed or activated transcriptional complexes to drive oncogenic pathways. Collectively, ETS1 functions to regulate cell growth, differentiation, signaling, response to stimuli, and viral interactions in these malignancies. A tumor suppressor role has also been indicated for ETS1 in select lymphoma types, emphasizing the importance of cellular context in ETS1 function. Research is ongoing to further characterize the clinical implications of ETS1 dysregulation in hematologic malignancies, to further resolve binding complexes and transcriptional targets, and to identify effective therapeutic targeting approaches.
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Affiliation(s)
- Rebecca A Luchtel
- Division of Hematology and Oncology, Department of Medicine, Northwestern University, Chicago, IL, USA.
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3
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Martin V, Zhuang F, Zhang Y, Pinheiro K, Gordân R. High-throughput data and modeling reveal insights into the mechanisms of cooperative DNA-binding by transcription factor proteins. Nucleic Acids Res 2023; 51:11600-11612. [PMID: 37889068 PMCID: PMC10681739 DOI: 10.1093/nar/gkad872] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 09/21/2023] [Accepted: 10/05/2023] [Indexed: 10/28/2023] Open
Abstract
Cooperative DNA-binding by transcription factor (TF) proteins is critical for eukaryotic gene regulation. In the human genome, many regulatory regions contain TF-binding sites in close proximity to each other, which can facilitate cooperative interactions. However, binding site proximity does not necessarily imply cooperative binding, as TFs can also bind independently to each of their neighboring target sites. Currently, the rules that drive cooperative TF binding are not well understood. In addition, it is oftentimes difficult to infer direct TF-TF cooperativity from existing DNA-binding data. Here, we show that in vitro binding assays using DNA libraries of a few thousand genomic sequences with putative cooperative TF-binding events can be used to develop accurate models of cooperativity and to gain insights into cooperative binding mechanisms. Using factors ETS1 and RUNX1 as our case study, we show that the distance and orientation between ETS1 sites are critical determinants of cooperative ETS1-ETS1 binding, while cooperative ETS1-RUNX1 interactions show more flexibility in distance and orientation and can be accurately predicted based on the affinity and sequence/shape features of the binding sites. The approach described here, combining custom experimental design with machine-learning modeling, can be easily applied to study the cooperative DNA-binding patterns of any TFs.
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Affiliation(s)
- Vincentius Martin
- Department of Computer Science, Durham, NC 27708, USA
- Center for Genomic & Computational Biology, Durham, NC 27708, USA
| | - Farica Zhuang
- Department of Computer Science, Durham, NC 27708, USA
- Center for Genomic & Computational Biology, Durham, NC 27708, USA
| | - Yuning Zhang
- Center for Genomic & Computational Biology, Durham, NC 27708, USA
- Program in Computational Biology & Bioinformatics, Durham, NC 27708, USA
| | - Kyle Pinheiro
- Department of Computer Science, Durham, NC 27708, USA
- Center for Genomic & Computational Biology, Durham, NC 27708, USA
| | - Raluca Gordân
- Department of Computer Science, Durham, NC 27708, USA
- Center for Genomic & Computational Biology, Durham, NC 27708, USA
- Department of Biostatistics & Bioinformatics, Department of Molecular Genetics and Microbiology, Department of Cell Biology, Duke University, Durham, NC 27708, USA
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4
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Ortigosa-Pascual L, Leiding T, Linse S, Pálmadóttir T. Photo-Induced Cross-Linking of Unmodified α-Synuclein Oligomers. ACS Chem Neurosci 2023; 14:3192-3205. [PMID: 37621159 PMCID: PMC10485903 DOI: 10.1021/acschemneuro.3c00326] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Accepted: 08/14/2023] [Indexed: 08/26/2023] Open
Abstract
Photo-induced cross-linking of unmodified proteins (PICUP) has been used in the past to study size distributions of protein assemblies. PICUP may, for example, overcome the significant experimental challenges related to the transient nature, heterogeneity, and low concentration of amyloid protein oligomers relative to monomeric and fibrillar species. In the current study, a reaction chamber was designed, produced, and used for PICUP reaction optimization in terms of reaction conditions and lighting time from ms to s. These efforts make the method more reproducible and accessible and enable the use of shorter reaction times compared to previous studies. We applied the optimized method to an α-synuclein aggregation time course to monitor the relative concentration and size distribution of oligomers over time. The data are compared to the time evolution of the fibril mass concentration, as monitored by thioflavin T fluorescence. At all time points, the smaller the oligomer, the higher its concentration observed after PICUP. Moreover, the total oligomer concentration is highest at short aggregation times, and the decline over time follows the disappearance of monomers. We can therefore conclude that these oligomers form from monomers.
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Affiliation(s)
- Lei Ortigosa-Pascual
- Department of Biochemistry and Structural
Biology, Lund University, 221 00 Lund, Sweden
| | - Thom Leiding
- Department of Biochemistry and Structural
Biology, Lund University, 221 00 Lund, Sweden
| | - Sara Linse
- Department of Biochemistry and Structural
Biology, Lund University, 221 00 Lund, Sweden
| | - Tinna Pálmadóttir
- Department of Biochemistry and Structural
Biology, Lund University, 221 00 Lund, Sweden
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5
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Legrand AJ, Choul-li S, Villeret V, Aumercier M. Poly(ADP-ribose) Polyremase-1 (PARP-1) Inhibition: A Promising Therapeutic Strategy for ETS-Expressing Tumours. Int J Mol Sci 2023; 24:13454. [PMID: 37686260 PMCID: PMC10487777 DOI: 10.3390/ijms241713454] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2023] [Revised: 08/17/2023] [Accepted: 08/28/2023] [Indexed: 09/10/2023] Open
Abstract
ETS transcription factors are a highly conserved family of proteins involved in the progression of many cancers, such as breast and prostate carcinomas, Ewing's sarcoma, and leukaemias. This significant involvement can be explained by their roles at all stages of carcinogenesis progression. Generally, their expression in tumours is associated with a poor prognosis and an aggressive phenotype. Until now, no efficient therapeutic strategy had emerged to specifically target ETS-expressing tumours. Nevertheless, there is evidence that pharmacological inhibition of poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair enzyme, specifically sensitises ETS-expressing cancer cells to DNA damage and limits tumour progression by leading some of the cancer cells to death. These effects result from a strong interplay between ETS transcription factors and the PARP-1 enzyme. This review summarises the existing knowledge of this molecular interaction and discusses the promising therapeutic applications.
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Affiliation(s)
- Arnaud J. Legrand
- CNRS, EMR9002 Integrative Structural Biology, F-59000 Lille, France; (A.J.L.); (V.V.)
- Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167-RID-AGE-Risk Factors and Molecular Deter-minants of Aging-Related Diseases, F-59000 Lille, France
| | - Souhaila Choul-li
- Département de Biologie, Faculté des Sciences, Université Chouaib Doukkali, BP-20, El Jadida 24000, Morocco;
| | - Vincent Villeret
- CNRS, EMR9002 Integrative Structural Biology, F-59000 Lille, France; (A.J.L.); (V.V.)
- Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167-RID-AGE-Risk Factors and Molecular Deter-minants of Aging-Related Diseases, F-59000 Lille, France
| | - Marc Aumercier
- CNRS, EMR9002 Integrative Structural Biology, F-59000 Lille, France; (A.J.L.); (V.V.)
- Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167-RID-AGE-Risk Factors and Molecular Deter-minants of Aging-Related Diseases, F-59000 Lille, France
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6
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Huang K, Xhani S, Albrecht AV, Ha VLT, Esaki S, Poon GMK. Mechanism of cognate sequence discrimination by the ETS-family transcription factor ETS-1. J Biol Chem 2019; 294:9666-9678. [PMID: 31048376 DOI: 10.1074/jbc.ra119.007866] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Revised: 05/01/2019] [Indexed: 12/19/2022] Open
Abstract
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.
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Affiliation(s)
| | | | | | | | | | - Gregory M K Poon
- From the Department of Chemistry and .,Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303
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7
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Transcription Factor ETS-1 and Reactive Oxygen Species: Role in Vascular and Renal Injury. Antioxidants (Basel) 2018; 7:antiox7070084. [PMID: 29970819 PMCID: PMC6071050 DOI: 10.3390/antiox7070084] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2018] [Revised: 06/15/2018] [Accepted: 07/02/2018] [Indexed: 12/24/2022] Open
Abstract
The E26 avian erythroblastosis virus transcription factor-1 (ETS-1) is a member of the ETS family and regulates the expression of a variety of genes including growth factors, chemokines and adhesion molecules. Although ETS-1 was discovered as an oncogene, several lines of research show that it is up-regulated by angiotensin II (Ang II) both in the vasculature and the glomerulus. While reactive oxygen species (ROS) are required for Ang II-induced ETS-1 expression, ETS-1 also regulates the expression of p47phox, which is one of the subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and a major source of ROS in the kidney and vasculature. Thus, there appears to be a positive feedback between ETS-1 and ROS. ETS-1 is also upregulated in the kidneys of rats with salt-sensitive hypertension and plays a major role in the development of end-organ injury in this animal model. Activation of the renin angiotensin system is required for the increased ETS-1 expression in these rats, and blockade of ETS-1 or haplodeficiency reduces the severity of kidney injury in these rats. In summary, ETS-1 plays a major role in the development of vascular and renal injury and is a potential target for the development of novel therapeutic strategies to ameliorate end-organ injury in hypertension.
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8
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Choul-Li S, Legrand AJ, Bidon B, Vicogne D, Villeret V, Aumercier M. Ets-1 interacts through a similar binding interface with Ku70 and Poly (ADP-Ribose) Polymerase-1. Biosci Biotechnol Biochem 2018; 82:1753-1759. [PMID: 29912634 DOI: 10.1080/09168451.2018.1484276] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/14/2022]
Abstract
The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction proteins. We have previously showed that Ets-1 interacted with DNA-dependent protein kinase (DNA-PK) complex including its regulatory subunits, Ku70 and Ku86 and with poly (ADP-ribose) polymerase-1 (PARP-1). In this study, the binding domains for the interaction between Ets-1 and these proteins were reported. We demonstrated that the interaction of Ets-1 with DNA-PK was mediated through the Ku70 subunit and was mapped to the C-terminal region of Ets-1 and the C-terminal part of Ku70 including SAP domain. The interactive domains between Ets-1 and PARP-1 have been mapped to the C-terminal region of Ets-1 and the BRCA1 carboxy-terminal (BRCT) domain of PARP-1. The results presented in this study may advance our understanding of the functional link between Ets-1 and its interaction partners, DNA-PK and PARP-1.
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Affiliation(s)
- Souhaila Choul-Li
- Faculté des Sciences, Département de Biologie , Université Chouaib Doukkali , El Jadida , Maroc
| | | | - Baptiste Bidon
- Groupe d’Etude des Interactions Hôte-Pathogéne (GEIHP - EA 3142), Institut de Biologie en Santé, Université d’Angers, Angers, France
| | - Dorothée Vicogne
- Univ. Lille, CNRS, INRA, UMR8576-UGSF-Unité de Biologie Structurale et Fonctionnelle , Lille , France
| | - Vincent Villeret
- Univ. Lille, CNRS, INRA, UMR8576-UGSF-Unité de Biologie Structurale et Fonctionnelle , Lille , France
| | - Marc Aumercier
- Univ. Lille, CNRS, INRA, UMR8576-UGSF-Unité de Biologie Structurale et Fonctionnelle , Lille , France
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9
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Liu X, Zhang C, Zhang Z, Zhang Z, Ji W, Cao S, Cai X, Lei D, Pan X. E26 Transformation-Specific Transcription Factor ETS2 as an Oncogene Promotes the Progression of Hypopharyngeal Cancer. Cancer Biother Radiopharm 2017; 32:327-334. [PMID: 29111780 DOI: 10.1089/cbr.2017.2296] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The E26 transformation-specific (ETS) family is one of the largest families of transcription factors. Upon activation by MAPK pathway, ETS participates in cell proliferation, differentiation, migration, apoptosis, and metastasis. However, the mechanism by which ETS is deregulated in cancer is unclear. In this study, the authors investigated the role of ETS factor, ETS2, in hypopharyngeal cancer pathogenesis in hypopharyngeal cancer tissues (N = 20) and corresponding non-neoplastic tissues (N = 20). The results showed that expression of ETS2 was increased in cancer tissues as compared with the expression in corresponding non-neoplastic tissues. Analysis of clinicopathological characteristics showed that increased level of ETS2 is associated with III-IV tumor node metastasis stage and lymph node metastasis. In addition, knockdown of ETS2 by siRNA in pharyngeal cancer cell line, FaDu, significantly decreased cell's vitality and colony-forming ability by inducing caspase-3-dependent apoptosis and cell cycle arrest. Furthermore, inhibition of ETS2 could abrogate the migration, invasion, and transforming growth factor-β-induced epithelial mesenchymal transition through the upregulation of E-cadherin, zona occludens protein-1, together with downregulation of vimentin and α-sooth muscle actin. These functions of ETS2 could be associated with the activation of MAPK/p38/ERK/JNK signals. Taken together, the authors opined that ETS2 functions as an oncogene and plays a key role in the progression of hypopharyngeal cancer.
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Affiliation(s)
- Xuejun Liu
- 1 Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Key Laboratory of Otolaryngology, NHFPC (Shandong University) , Jinan, China .,2 Department of Otorhinolaryngology, Second Affiliated Hospital of Wenzhou Medical University , Wenzhou, China
| | - Chuqin Zhang
- 1 Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Key Laboratory of Otolaryngology, NHFPC (Shandong University) , Jinan, China .,2 Department of Otorhinolaryngology, Second Affiliated Hospital of Wenzhou Medical University , Wenzhou, China
| | - Zhonghua Zhang
- 3 Department of Otorhinolaryngology, Affiliated Weihai Second Municipal Hospital of Qingdao University , Weihai, China
| | - Zuping Zhang
- 1 Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Key Laboratory of Otolaryngology, NHFPC (Shandong University) , Jinan, China
| | - Wei Ji
- 1 Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Key Laboratory of Otolaryngology, NHFPC (Shandong University) , Jinan, China
| | - Shengda Cao
- 1 Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Key Laboratory of Otolaryngology, NHFPC (Shandong University) , Jinan, China
| | - Xiaolan Cai
- 1 Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Key Laboratory of Otolaryngology, NHFPC (Shandong University) , Jinan, China
| | - Dapeng Lei
- 1 Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Key Laboratory of Otolaryngology, NHFPC (Shandong University) , Jinan, China
| | - Xinliang Pan
- 1 Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Key Laboratory of Otolaryngology, NHFPC (Shandong University) , Jinan, China
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Peacock J, Jaynes JB. Using competition assays to quantitatively model cooperative binding by transcription factors and other ligands. Biochim Biophys Acta Gen Subj 2017; 1861:2789-2801. [PMID: 28774855 PMCID: PMC5623634 DOI: 10.1016/j.bbagen.2017.07.024] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Revised: 07/27/2017] [Accepted: 07/29/2017] [Indexed: 11/17/2022]
Abstract
BACKGROUND The affinities of DNA binding proteins for target sites can be used to model the regulation of gene expression. These proteins can bind to DNA cooperatively, strongly impacting their affinity and specificity. However, current methods for measuring cooperativity do not provide the means to accurately predict binding behavior over a wide range of concentrations. METHODS We use standard computational and mathematical methods, and develop novel methods as described in Results. RESULTS We explore some complexities of cooperative binding, and develop an improved method for relating in vitro measurements to in vivo function, based on ternary complex formation. We derive expressions for the equilibria among the various complexes, and explore the limitations of binding experiments that model the system using a single parameter. We describe how to use single-ligand binding and ternary complex formation in tandem to determine parameters that have thermodynamic relevance. We develop an improved method for finding both single-ligand dissociation constants and concentrations simultaneously. We show how the cooperativity factor can be found when only one of the single-ligand dissociation constants can be measured. CONCLUSIONS The methods that we develop constitute an optimized approach to accurately model cooperative binding. GENERAL SIGNIFICANCE The expressions and methods we develop for modeling and analyzing DNA binding and cooperativity are applicable to most cases where multiple ligands bind to distinct sites on a common substrate. The parameters determined using these methods can be fed into models of higher-order cooperativity to increase their predictive power.
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Affiliation(s)
- Jacob Peacock
- Dept. of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, United States
| | - James B Jaynes
- Dept. of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, United States.
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11
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Esaki S, Evich MG, Erlitzki N, Germann MW, Poon GMK. Multiple DNA-binding modes for the ETS family transcription factor PU.1. J Biol Chem 2017; 292:16044-16054. [PMID: 28790174 DOI: 10.1074/jbc.m117.798207] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2017] [Revised: 08/07/2017] [Indexed: 01/17/2023] Open
Abstract
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.
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Affiliation(s)
| | | | | | | | - Gregory M K Poon
- From the Departments of Chemistry and .,the Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303
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12
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Kasahara K, Shiina M, Fukuda I, Ogata K, Nakamura H. Molecular mechanisms of cooperative binding of transcription factors Runx1-CBFβ-Ets1 on the TCRα gene enhancer. PLoS One 2017; 12:e0172654. [PMID: 28231333 PMCID: PMC5322934 DOI: 10.1371/journal.pone.0172654] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2016] [Accepted: 02/07/2017] [Indexed: 11/22/2022] Open
Abstract
Ets1 is an essential transcription factor (TF) for several important physiological processes, including cell proliferation and differentiation. Its recognition of the enhancer region of the TCRα gene is enhanced by the cooperative binding of the Runx1–CBFβ heterodimer, with the cancelation of phosphorylation-dependent autoinhibition. The detailed mechanism of this interesting cooperativity between Ets1 and the Runx1–CBFβ heterodimer is still largely unclear. Here, we investigated the molecular mechanisms of this cooperativity, by using molecular dynamics simulations. Consequently, we detected high flexibility of the loop region between the HI2 and H1 helices of Ets1. Upon Runx1–CBFβ heterodimer binding, this loop transiently adopts various sub-stable conformations in its interactions with the DNA. In addition, a network analysis suggested an allosteric pathway in the molecular assembly and identified some key residues that coincide with previous experimental studies. Our simulations suggest that the cooperative binding of Ets1 and the Runx1–CBFβ heterodimer alters the DNA conformation and induces sub-stable conformations of the HI2–H1 loop of Ets1. This phenomenon increases the flexibility of the regulatory module, including the HI2 helix, and destabilizes the inhibitory form of this module. Thus, we hypothesize that this effect facilitates Ets1–DNA binding and prevents the phosphorylation-dependent DNA binding autoinhibition.
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Affiliation(s)
- Kota Kasahara
- College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan
- * E-mail:
| | - Masaaki Shiina
- Graduate School of Medicine, Yokohama City University, Kanazawa-ku, Yokohama, Kanagawa, Japan
| | - Ikuo Fukuda
- Institute for Protein Research, Osaka University, Suita, Osaka, Japan
| | - Kazuhiro Ogata
- Graduate School of Medicine, Yokohama City University, Kanazawa-ku, Yokohama, Kanagawa, Japan
| | - Haruki Nakamura
- Institute for Protein Research, Osaka University, Suita, Osaka, Japan
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13
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Meckbach C, Tacke R, Hua X, Waack S, Wingender E, Gültas M. PC-TraFF: identification of potentially collaborating transcription factors using pointwise mutual information. BMC Bioinformatics 2015; 16:400. [PMID: 26627005 PMCID: PMC4667426 DOI: 10.1186/s12859-015-0827-2] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2015] [Accepted: 11/17/2015] [Indexed: 01/06/2023] Open
Abstract
Background Transcription factors (TFs) are important regulatory proteins that govern transcriptional regulation. Today, it is known that in higher organisms different TFs have to cooperate rather than acting individually in order to control complex genetic programs. The identification of these interactions is an important challenge for understanding the molecular mechanisms of regulating biological processes. In this study, we present a new method based on pointwise mutual information, PC-TraFF, which considers the genome as a document, the sequences as sentences, and TF binding sites (TFBSs) as words to identify interacting TFs in a set of sequences. Results To demonstrate the effectiveness of PC-TraFF, we performed a genome-wide analysis and a breast cancer-associated sequence set analysis for protein coding and miRNA genes. Our results show that in any of these sequence sets, PC-TraFF is able to identify important interacting TF pairs, for most of which we found support by previously published experimental results. Further, we made a pairwise comparison between PC-TraFF and three conventional methods. The outcome of this comparison study strongly suggests that all these methods focus on different important aspects of interaction between TFs and thus the pairwise overlap between any of them is only marginal. Conclusions In this study, adopting the idea from the field of linguistics in the field of bioinformatics, we develop a new information theoretic method, PC-TraFF, for the identification of potentially collaborating transcription factors based on the idiosyncrasy of their binding site distributions on the genome. The results of our study show that PC-TraFF can succesfully identify known interacting TF pairs and thus its currently biologically uncorfirmed predictions could provide new hypotheses for further experimental validation. Additionally, the comparison of the results of PC-TraFF with the results of previous methods demonstrates that different methods with their specific scopes can perfectly supplement each other. Overall, our analyses indicate that PC-TraFF is a time-efficient method where its algorithm has a tractable computational time and memory consumption. The PC-TraFF server is freely accessible at http://pctraff.bioinf.med.uni-goettingen.de/ Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0827-2) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Cornelia Meckbach
- Institute of Bioinformatics, University of Göttingen, Goldschmidtstr. 1, Göttingen, 37077, Germany.
| | - Rebecca Tacke
- Institute of Bioinformatics, University of Göttingen, Goldschmidtstr. 1, Göttingen, 37077, Germany.
| | - Xu Hua
- Institute of Bioinformatics, University of Göttingen, Goldschmidtstr. 1, Göttingen, 37077, Germany.
| | - Stephan Waack
- Institute of Computer Science, University of Göttingen, Goldschmidtstr. 7, Göttingen, 37077, Germany.
| | - Edgar Wingender
- Institute of Bioinformatics, University of Göttingen, Goldschmidtstr. 1, Göttingen, 37077, Germany.
| | - Mehmet Gültas
- Institute of Bioinformatics, University of Göttingen, Goldschmidtstr. 1, Göttingen, 37077, Germany.
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14
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Dittmer J. The role of the transcription factor Ets1 in carcinoma. Semin Cancer Biol 2015; 35:20-38. [PMID: 26392377 DOI: 10.1016/j.semcancer.2015.09.010] [Citation(s) in RCA: 155] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2015] [Revised: 09/16/2015] [Accepted: 09/16/2015] [Indexed: 12/12/2022]
Abstract
Ets1 belongs to the large family of the ETS domain family of transcription factors and is involved in cancer progression. In most carcinomas, Ets1 expression is linked to poor survival. In breast cancer, Ets1 is primarily expressed in the triple-negative subtype, which is associated with unfavorable prognosis. Ets1 contributes to the acquisition of cancer cell invasiveness, to EMT (epithelial-to-mesenchymal transition), to the development of drug resistance and neo-angiogenesis. The aim of this review is to summarize the current knowledge on the functions of Ets1 in carcinoma progression and on the mechanisms that regulate Ets1 activity in cancer.
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Affiliation(s)
- Jürgen Dittmer
- Clinic for Gynecology, Martin Luther University Halle-Wittenberg, Germany.
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15
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Samorodnitsky D, Szyjka C, Koudelka GB. A Role for Autoinhibition in Preventing Dimerization of the Transcription Factor ETS1. J Biol Chem 2015. [PMID: 26195629 DOI: 10.1074/jbc.m115.671339] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
ETS1 is the archetype of the ETS transcription factor (TF) family. ETS TFs share a DNA-binding domain, the ETS domain. All ETS TFs recognize a core GGA(A/T) binding site, and thus ETS TFs are found to redundantly regulate the same genes. However, each ETS TF has unique targets as well. One prevailing hypotheses explaining this duality is that protein-protein interactions, including homodimerization, allow each ETS TF to display distinct behavior. The behavior of ETS1 is further regulated by autoinhibition. Autoinhibition apparently modulates ETS1 DNA binding affinity, but the mechanism of this inhibition is not completely understood. We sought to characterize the relationship between DNA binding and ETS1 homodimer formation. We find that ETS1 interrogates DNA and forms dimers even when the DNA does not contain an ETS recognition sequence. Mutational studies also link nonspecific DNA backbone contacts with dimer formation, in addition to providing a new role for the recognition helix of ETS1 in maintaining the autoinhibited state. Finally, in showing that residues in the DNA recognition helix affect autoinhibition, we define a new function of ETS1 autoinhibition: maintenance of a monomeric state in the absence of DNA. The conservation of relevant amino acid residues across all ETS TFs indicates that the mechanisms of nonspecific DNA interrogation and protein oligomer formation elucidated here may be common to all ETS proteins that autoinhibit.
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Affiliation(s)
- Daniel Samorodnitsky
- From the Department of Biological Sciences, University at Buffalo, Buffalo, New York 14260
| | - Courtney Szyjka
- From the Department of Biological Sciences, University at Buffalo, Buffalo, New York 14260
| | - Gerald B Koudelka
- From the Department of Biological Sciences, University at Buffalo, Buffalo, New York 14260
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16
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Shen XB, Huang L, Zhang SH, Wang DP, Wu YL, Chen WN, Xu SH, Lin X. Transcriptional regulation of the apolipoprotein F (ApoF) gene by ETS and C/EBPα in hepatoma cells. Biochimie 2015; 112:1-9. [PMID: 25726912 DOI: 10.1016/j.biochi.2015.02.013] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2014] [Accepted: 02/17/2015] [Indexed: 10/23/2022]
Abstract
Apolipoprotein F (ApoF) inhibits cholesteryl ester transfer protein (CETP) activity and plays an important role in lipid metabolism. In the present study, the full-length human ApoF promoter was cloned, and the molecular mechanism of the regulation of ApoF was investigated. The ApoF promoter displayed higher activities in hepatoma cell lines, and the -198 nt to +79 nt promoter region contained the maximum promoter activity. In the promoter region of -198 nt to -2 nt there were four putative binding sites for transcription factors ETS-1/ETS-2 (named EBS-1 to EBS-4) and one for C/EBP. Mutation of EBS-2, EBS4 and the C/EBP binding site abolished the promoter activity, and ETS-1/ETS-2 and C/EBPα could interact with corresponding binding sites. In addition, overexpression of ETS-1/2 or C/EBPα enhanced, while dominant-negative mutants of ETS-1/2 and knockdown of C/EBPα decreased, ApoF promoter activities. ETS-1 and C/EBPα associated physically, and acted synergistically to activate ApoF transcription. These results demonstrated combined activation of the ApoF promoter by liver-enriched and ubiquitous transcription factors. Direct interactions between C/EBPα and ETS-1 were important for high liver-specific expression of ApoF.
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Affiliation(s)
- Xue-Bin Shen
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China; Department of Cardiology, Affiliated Nanping First Hospital, Fujian Medical University, Nanping, China
| | - Ling Huang
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China; Department of Cardiology, Affiliated Nanping First Hospital, Fujian Medical University, Nanping, China
| | - Shao-Hong Zhang
- Department of Medical Laboratory, Affiliated Nanping First Hospital, Fujian Medical University, Nanping, China
| | - De-Ping Wang
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China; Department of Endocrinology and Metabolism, Hongqi Hospital of MuDanJiang Medical College, Mudanjiang, China
| | - Yun-Li Wu
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
| | - Wan-Nan Chen
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
| | - Shang-Hua Xu
- Department of Cardiology, Affiliated Nanping First Hospital, Fujian Medical University, Nanping, China.
| | - Xu Lin
- Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.
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17
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Newman JA, Cooper CDO, Aitkenhead H, Gileadi O. Structural insights into the autoregulation and cooperativity of the human transcription factor Ets-2. J Biol Chem 2015; 290:8539-49. [PMID: 25670864 PMCID: PMC4375503 DOI: 10.1074/jbc.m114.619270] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Ets-2, like its closely related homologue Ets-1, is a member of the Ets family of DNA binding transcription factors. Both proteins 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 contrast to Ets-1, in which the autoinhibition is caused by a combination 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.
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Affiliation(s)
- Joseph A Newman
- From the Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, United Kingdom
| | - Christopher D O Cooper
- From the Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, United Kingdom
| | - Hazel Aitkenhead
- From the Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, United Kingdom
| | - Opher Gileadi
- From the Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, United Kingdom
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18
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Vaughan CA, Deb SP, Deb S, Windle B. Preferred binding of gain-of-function mutant p53 to bidirectional promoters with coordinated binding of ETS1 and GABPA to multiple binding sites. Oncotarget 2015; 5:417-27. [PMID: 24481480 PMCID: PMC3964217 DOI: 10.18632/oncotarget.1708] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Gain-of-function mutant p53 is thought to induce gene expression in part by binding transcription factors bound to promoters for genes that mediate oncogenesis. We investigated the mechanism of mutant p53 binding by mapping the human genomic binding sites for p53 R273H using ChIP-Seq and showed them to localize to ETS DNA sequence motifs and locations with ETS1 and GABPA binding, both within promoters and distal to promoters. Strikingly, p53 R273H showed statistically significant and substantial binding to bidirectional promoters, which are enriched for inverted repeated ETS DNA sequence motifs. p53 R273H exhibited an exponential increase in probability of binding promoters with a higher number of ETS motifs. Both ETS1 and GABPA also showed an increase in the probability of binding to promoters with a higher number of ETS motifs. However, despite this increase in probability of binding by p53 R273H and ETS1, there was no increase in the binding signal, suggesting that the number of ETS1 and p53 R273H proteins bound per promoter is being limited. In contrast, GABPA did exhibit an increase in binding signal with higher numbers of ETS motifs per promoter. Analysis of the distance between inverted pairs of ETS motifs within promoters and binding by p53 R273H, ETS1 and GABPA, showed a novel coordination of binding for the three proteins. Both ETS1 and p53 R273H exhibited preference for binding promoters with distantly spaced ETS motifs in face-to-face and back-to-back orientations, and low binding preference to promoters with closely spaced ETS motifs. GABPA exhibited the inverse pattern of binding by preferring to bind promoters with closely spaced ETS motifs. Analysis of the helical phase between ETS motifs showed that ETS1 and p53 R273H exhibited a low preference for binding promoters with ETS motifs on the same face of the DNA helix. We propose a model for the binding of ETS1 and p53 R273H in which two inverted ETS motifs on a looped DNA helix are juxtaposed for ETS1 binding as a homodimer, with p53 R273H bound to ETS1. We propose that the formation of this DNA loop and protein-bound complex prevents additional binding of ETS1 and p53 R273H proteins to other proximal binding sites.
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19
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Kumari S, Saradhi M, Rana M, Chatterjee S, Aumercier M, Mukhopadhyay G, Tyagi RK. Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-jun. Exp Cell Res 2015; 330:398-411. [DOI: 10.1016/j.yexcr.2014.09.020] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2014] [Revised: 09/13/2014] [Accepted: 09/15/2014] [Indexed: 11/16/2022]
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20
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Kasahara K, Fukuda I, Nakamura H. A novel approach of dynamic cross correlation analysis on molecular dynamics simulations and its application to Ets1 dimer-DNA complex. PLoS One 2014; 9:e112419. [PMID: 25380315 PMCID: PMC4224484 DOI: 10.1371/journal.pone.0112419] [Citation(s) in RCA: 92] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2014] [Accepted: 10/14/2014] [Indexed: 11/18/2022] Open
Abstract
The dynamic cross correlation (DCC) analysis is a popular method for analyzing the trajectories of molecular dynamics (MD) simulations. However, it is difficult to detect correlative motions that appear transiently in only a part of the trajectory, such as atomic contacts between the side-chains of amino acids, which may rapidly flip. In order to capture these multi-modal behaviors of atoms, which often play essential roles, particularly at the interfaces of macromolecules, we have developed the "multi-modal DCC (mDCC)" analysis. The mDCC is an extension of the DCC and it takes advantage of a Bayesian-based pattern recognition technique. We performed MD simulations for molecular systems modeled from the (Ets1)2-DNA complex and analyzed their results with the mDCC method. Ets1 is an essential transcription factor for a variety of physiological processes, such as immunity and cancer development. Although many structural and biochemical studies have so far been performed, its DNA binding properties are still not well characterized. In particular, it is not straightforward to understand the molecular mechanisms how the cooperative binding of two Ets1 molecules facilitates their recognition of Stromelysin-1 gene regulatory elements. A correlation network was constructed among the essential atomic contacts, and the two major pathways by which the two Ets1 molecules communicate were identified. One is a pathway via direct protein-protein interactions and the other is that via the bound DNA intervening two recognition helices. These two pathways intersected at the particular cytosine bases (C110/C11), interacting with the H1, H2, and H3 helices. Furthermore, the mDCC analysis showed that both pathways included the transient interactions at their intermolecular interfaces of Tyr396-C11 and Ala327-Asn380 in multi-modal motions of the amino acid side chains and the nucleotide backbone. Thus, the current mDCC approach is a powerful tool to reveal these complicated behaviors and scrutinize intermolecular communications in a molecular system.
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Affiliation(s)
- Kota Kasahara
- Institute for Protein Research, Osaka University, Suita, Osaka, Japan
- * E-mail:
| | - Ikuo Fukuda
- Institute for Protein Research, Osaka University, Suita, Osaka, Japan
| | - Haruki Nakamura
- Institute for Protein Research, Osaka University, Suita, Osaka, Japan
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21
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Babayeva ND, Wilder PJ, Shiina M, Mino K, Desler M, Ogata K, Rizzino A, Tahirov TH. Structural basis of Ets1 cooperative binding to palindromic sequences on stromelysin-1 promoter DNA. Cell Cycle 2014. [DOI: 10.4161/cc.9.14.12257] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
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22
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Recent advances in the structural molecular biology of Ets transcription factors: interactions, interfaces and inhibition. Biochem Soc Trans 2014; 42:130-8. [PMID: 24450640 PMCID: PMC3901394 DOI: 10.1042/bst20130227] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
The Ets family of eukaryotic transcription factors is based around the conserved Ets DNA-binding domain. Although their DNA-binding selectivity is biochemically and structurally well characterized, structures of homodimeric and ternary complexes point to Ets domains functioning as versatile protein-interaction modules. In the present paper, we review the progress made over the last decade to elucidate the structural mechanisms involved in modulation of DNA binding and protein partner selection during dimerization. We see that Ets domains, although conserved around a core architecture, have evolved to utilize a variety of interaction surfaces and binding mechanisms, reflecting Ets domains as dynamic interfaces for both DNA and protein interaction. Furthermore, we discuss recent advances in drug development for inhibition of Ets factors, and the roles structural biology can play in their future.
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23
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A novel allosteric mechanism on protein-DNA interactions underlying the phosphorylation-dependent regulation of Ets1 target gene expressions. J Mol Biol 2014; 427:1655-69. [PMID: 25083921 DOI: 10.1016/j.jmb.2014.07.020] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2014] [Revised: 06/27/2014] [Accepted: 07/17/2014] [Indexed: 11/23/2022]
Abstract
Cooperative assemblies of transcription factors (TFs) on target gene enhancers coordinate cell proliferation, fate specification, and differentiation through precise and complicated transcriptional mechanisms. Chemical modifications, such as phosphorylation, of TFs induced by cell signaling further modulate the dynamic cooperativity of TFs. In this study, we found that various Ets1-containing TF-DNA complexes respond differently to calcium-induced phosphorylation of Ets1, which is known to inhibit Ets1-DNA binding. Crystallographic analysis of a complex comprising Ets1, Runx1, and CBFβ at the TCRα enhancer revealed that Ets1 acquires robust binding stability in the Runx1 and DNA-complexed state, via allosteric mechanisms. This allows phosphorylated Ets1 to be retained at the TCRα enhancer with Runx1, in contrast to other Ets1 target gene enhancers including mb-1 and stromelysin-1. This study provides a structure-based model for cell-signaling-dependent regulation of target genes, mediated via chemical modification of TFs.
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24
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Furumatsu T, Matsumoto-Ogawa E, Tanaka T, Lu Z, Ozaki T. ROCK inhibition enhances aggrecan deposition and suppresses matrix metalloproteinase-3 production in human articular chondrocytes. Connect Tissue Res 2014; 55:89-95. [PMID: 24111521 DOI: 10.3109/03008207.2013.852544] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Homeostasis of articular cartilage is maintained by a balance between catabolism and anabolism. Matrix metalloproteinase-3 (MMP-3) catabolism of cartilaginous extracellular matrix (ECM), including aggrecan (AGN), is an important factor in osteoarthritis progression. We previously reported that inhibition of Rho-associated coiled-coil forming kinase (ROCK), an effector of Rho family GTPases, activates the chondrogenic transcription factor SRY-type high-mobility-group box (SOX) 9 and prevents dedifferentiation of monolayer-cultured chondrocytes. We hypothesized that ROCK inhibition prevents chondrocyte dedifferentiation by altering the transcriptional balance between MMP-3 and AGN. Normal human articular chondrocytes were cultured in the presence or absence of ROCK inhibitor (ROCKi, Y-27632). Expression of MMP-3 and AGN during monolayer cultivation was assessed by quantitative real-time PCR and western blot analysis. Chondrogenic redifferentiation potential of ROCKi-treated chondrocytes was evaluated by immunohistological analysis of pellet cultures. ROCKi treatment suppressed MMP-3 expression in monolayer- and pellet-cultured chondrocytes but increased AGN expression. Chromatin immunoprecipitation revealed that the association between transcription factors E26 transformation specific (ETS)-1 and SOX9 and their target genes MMP-3 and AGN, respectively, was affected by ROCKi treatment. ROCKi decreased the association between ETS-1 and its binding sites on the MMP-3 promoter, whereas ROCKi promoted the interaction between SOX9 and the AGN promoter. Our results suggest that ROCK inhibition may have an important role in modulating the balance between degradation and synthesis of cartilaginous ECM, a finding that may facilitate development of techniques to prepare differentiated chondrocytes for cartilage regeneration therapy.
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Affiliation(s)
- Takayuki Furumatsu
- Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences , Kitaku, Okayama , Japan
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Shrivastava T, Mino K, Babayeva ND, Baranovskaya OI, Rizzino A, Tahirov TH. Structural basis of Ets1 activation by Runx1. Leukemia 2014; 28:2040-8. [PMID: 24646888 PMCID: PMC4169772 DOI: 10.1038/leu.2014.111] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2013] [Revised: 03/11/2014] [Accepted: 03/13/2014] [Indexed: 11/23/2022]
Abstract
Runx1 is required for definitive hematopoiesis and is well-known for its frequent chromosomal translocations and point mutations in leukemia. Runx1 regulates a variety of genes via Ets1 activation on an Ets1•Runx1 composite DNA sequence. The structural basis of such regulation remains unresolved. To address this problem, we determined the crystal structure of the ternary complex containing Runx11-242 and Ets1296-441 bound to T cell receptor alpha (TCRα) enhancer DNA. In the crystal, an Ets1-interacting domain of Runx1 is bound to the Ets1 DNA-binding domain and displaced an entire autoinhibitory module of Ets1, revealing a novel mechanism of Ets1 activation. The DNA binding and transcriptional studies with a variety of structure-guided Runx1 mutants confirmed a critical role of direct Ets1•Runx1 interaction in Ets1 activation. More importantly, the discovered mechanism provides a plausible explanation for how the Ets1•Runx1 interaction effectively activates not only a wild-type Ets1, but also a highly inhibited phosphorylated form of Ets1.
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Affiliation(s)
- T Shrivastava
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
| | - K Mino
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
| | - N D Babayeva
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
| | - O I Baranovskaya
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
| | - A Rizzino
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
| | - T H Tahirov
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
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Findlay VJ, LaRue AC, Turner DP, Watson PM, Watson DK. Understanding the role of ETS-mediated gene regulation in complex biological processes. Adv Cancer Res 2014; 119:1-61. [PMID: 23870508 DOI: 10.1016/b978-0-12-407190-2.00001-0] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Ets factors are members of one of the largest families of evolutionarily conserved transcription factors, regulating critical functions in normal cell homeostasis, which when perturbed contribute to tumor progression. The well-documented alterations in ETS factor expression and function during cancer progression result in pleiotropic effects manifested by the downstream effect on their target genes. Multiple ETS factors bind to the same regulatory sites present on target genes, suggesting redundant or competitive functions. The anti- and prometastatic signatures obtained by examining specific ETS regulatory networks will significantly improve our ability to accurately predict tumor progression and advance our understanding of gene regulation in cancer. Coordination of multiple ETS gene functions also mediates interactions between tumor and stromal cells and thus contributes to the cancer phenotype. As such, these new insights may provide a novel view of the ETS gene family as well as a focal point for studying the complex biological control involved in tumor progression. One of the goals of molecular biology is to elucidate the mechanisms that contribute to the development and progression of cancer. Such an understanding of the molecular basis of cancer will provide new possibilities for: (1) earlier detection, as well as better diagnosis and staging of disease; (2) detection of minimal residual disease recurrences and evaluation of response to therapy; (3) prevention; and (4) novel treatment strategies. Increased understanding of ETS-regulated biological pathways will directly impact these areas.
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Affiliation(s)
- Victoria J Findlay
- Department of Pathology and Laboratory Medicine, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina, USA
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27
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Legrand AJ, Choul-Li S, Spriet C, Idziorek T, Vicogne D, Drobecq H, Dantzer F, Villeret V, Aumercier M. The level of Ets-1 protein is regulated by poly(ADP-ribose) polymerase-1 (PARP-1) in cancer cells to prevent DNA damage. PLoS One 2013; 8:e55883. [PMID: 23405229 PMCID: PMC3566071 DOI: 10.1371/journal.pone.0055883] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2012] [Accepted: 01/03/2013] [Indexed: 12/24/2022] Open
Abstract
Ets-1 is a transcription factor that regulates many genes involved in cancer progression and in tumour invasion. It is a poor prognostic marker for breast, lung, colorectal and ovary carcinomas. Here, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel interaction partner of Ets-1. We show that Ets-1 activates, by direct interaction, the catalytic activity of PARP-1 and is then poly(ADP-ribosyl)ated in a DNA-independent manner. The catalytic inhibition of PARP-1 enhanced Ets-1 transcriptional activity and caused its massive accumulation in cell nuclei. Ets-1 expression was correlated with an increase in DNA damage when PARP-1 was inhibited, leading to cancer cell death. Moreover, PARP-1 inhibitors caused only Ets-1-expressing cells to accumulate DNA damage. These results provide new insight into Ets-1 regulation in cancer cells and its link with DNA repair proteins. Furthermore, our findings suggest that PARP-1 inhibitors would be useful in a new therapeutic strategy that specifically targets Ets-1-expressing tumours.
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Affiliation(s)
- Arnaud J Legrand
- CNRS USR 3078, Institut de Recherche Interdisciplinaire, Campus CNRS de la Haute Borne, Université Lille Nord de France, IFR 147, BP 70478, Villeneuve d'Ascq, France
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Review of Ets1 structure, function, and roles in immunity. Cell Mol Life Sci 2013; 70:3375-90. [PMID: 23288305 DOI: 10.1007/s00018-012-1243-7] [Citation(s) in RCA: 156] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2012] [Revised: 11/20/2012] [Accepted: 12/11/2012] [Indexed: 10/27/2022]
Abstract
The Ets1 transcription factor is a member of the Ets gene family and is highly conserved throughout evolution. Ets1 is known to regulate a number of important biological processes in normal cells and in tumors. In particular, Ets1 has been associated with regulation of immune cell function and with an aggressive behavior in tumors that express it at high levels. Here we review and summarize the general features of Ets1 and describe its roles in immunity and autoimmunity, with a focus on its roles in B lymphocytes. We also review evidence that suggests that Ets1 may play a role in malignant transformation of hematopoietic malignancies including B cell malignancies.
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Pallai R, Bhaskar A, Sodi V, Rice LM. Ets1 and Elk1 transcription factors regulate cancerous inhibitor of protein phosphatase 2A expression in cervical and endometrial carcinoma cells. Transcription 2012; 3:323-35. [PMID: 23117818 DOI: 10.4161/trns.22518] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Cancerous inhibitor of protein phosphatase 2A (CIP2A) has been identified as a proto-oncogene that is overexpressed in various types of human cancers. CIP2A acts by inhibiting protein phosphatase 2A-dependent destabilization of c-Myc, resulting in increased cell proliferation. Here, we have characterized the proximal promoter region of the human CIP2A gene in cervical, endometrial and liver carcinoma cells. The 5' flanking minimal proximal promoter of the CIP2A gene consists of putative binding sites for Ets1 and Elk1 in forward and reverse orientations. Here, we show that Ets1 and Elk1 binding is essential for CIP2A basal expression in several urogenital cancer cell lines. Interestingly, both Ets1 and Elk1 are required together for CIP2A expression, as siRNA knockdown of Ets1 and Elk1 together decreased CIP2A gene transcription, whereas knockdown of Ets1 or Elk1 alone had no effect. Moreover, ectopic expression of Ets1 and Elk1 together increased CIP2A expression. To gain physiological significance of the Ets1 and Elk1 regulation we observed, a panel of matched human cervical carcinoma samples was analyzed for the expression of CIP2A and Ets1 and/or Elk1. We found a direct correlation between the levels of CIP2A and the levels of Ets1 and Elk1. Our results suggest that the binding of Ets1 and Elk1 together to the proximal CIP2A promoter is absolutely required for CIP2A expression in cervical, endometrial and liver carcinoma cell lines. Thus, different factors regulate CIP2A expression in a cell-type specific manner. As previous work has shown a requirement for only Ets1 in prostate and gastric carcinomas, our results now indicate that CIP2A regulation is more complex than previously determined.
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Affiliation(s)
- Rajash Pallai
- Department of Cancer Signaling and Cell Cycle, Venenum Biodesign, L.L.C, Hamilton, NJ, USA
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Identification of signaling pathways mediating cell cycle arrest and apoptosis induced by Porphyromonas gingivalis in human trophoblasts. Infect Immun 2012; 80:2847-57. [PMID: 22689813 DOI: 10.1128/iai.00258-12] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Epidemiological and interventional studies of humans have revealed a close association between periodontal diseases and preterm delivery of low-birth-weight infants. Porphyromonas gingivalis, a periodontal pathogen, can translocate to gestational tissues following oral-hematogenous spread. We previously reported that P. gingivalis invades extravillous trophoblast cells (HTR-8) derived from the human placenta and inhibits proliferation through induction of arrest in the G(1) phase of the cell cycle. The purpose of the present study was to identify signaling pathways mediating cellular impairment caused by P. gingivalis. Following P. gingivalis infection, the expression of Fas was induced and p53 accumulated, responses consistent with response to DNA damage. Ataxia telangiectasia- and Rad3-related kinase (ATR), an essential regulator of DNA damage checkpoints, was shown to be activated together with its downstream signaling molecule Chk2, while the p53 degradation-related protein MDM2 was not induced. The inhibition of ATR prevented both G(1) arrest and apoptosis caused by P. gingivalis in HTR-8 cells. In addition, small interfering RNA (siRNA) knockdown of p53 abrogated both G(1) arrest and apoptosis. The regulation of apoptosis was associated with Ets1 activation. HTR-8 cells infected with P. gingivalis exhibited activation of Ets1, and knockdown of Ets1 with siRNA diminished both G(1) arrest and apoptosis. These results suggest that P. gingivalis activates cellular DNA damage signaling pathways that lead to G(1) arrest and apoptosis in trophoblasts.
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Florkowska M, Tymoszuk P, Balwierz A, Skucha A, Kochan J, Wawro M, Stalinska K, Kasza A. EGF activates TTP expression by activation of ELK-1 and EGR-1 transcription factors. BMC Mol Biol 2012; 13:8. [PMID: 22433566 PMCID: PMC3342124 DOI: 10.1186/1471-2199-13-8] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2011] [Accepted: 03/20/2012] [Indexed: 01/04/2023] Open
Abstract
Background Tristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described. Results Our results demonstrate the induction of the gene coding TTP (ZFP36) by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates ZFP36 expression by two mechanisms: by binding the ZFP36 promoter directly through ETS-binding site (+ 883 to +905 bp) and by inducing expression of EGR-1, which in turn increases ZFP36 expression through sequences located between -111 and -103 bp. Conclusions EGF activates TTP expression via ELK-1 and EGR-1 transcription factors.
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Structural basis of Ets1 cooperative binding to widely separated sites on promoter DNA. PLoS One 2012; 7:e33698. [PMID: 22432043 PMCID: PMC3303851 DOI: 10.1371/journal.pone.0033698] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2012] [Accepted: 02/17/2012] [Indexed: 11/19/2022] Open
Abstract
Ets1 is a member of the Ets family of transcription factors. Ets1 is expressed in autoinhibited form and its DNA binding depends on partner proteins bound to adjacent sequences or the relative positioning of a second Ets-binding site (EBS). The autoinhibition of Ets1 is mediated by structural coupling of regions flanking the DNA-binding domain. The NMR structure of Ets1 revealed that the inhibitory regions comprised of helices HI1 and HI2 and H4 are packed together on the Ets domain to form an inhibitory module. The crystal structure of Ets1 unexpectedly revealed a homodimer in which homodimerisation occurs via swapping of HI1 helices. Modeling of DNA binding indicates that the Ets1 dimer can bind to two antiparallel pieces of DNA. To verify this, we crystallized and solved the structure of the complex comprised of Ets1 dimer and two pieces of DNA. DNA binding by Ets1 dimer resulted in formation of additional intermolecular protein•DNA interactions, implying that the complex formation is cooperative.
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Kato T, Fujita Y, Nakane K, Kojima T, Nozawa Y, Deguchi T, Ito M. ETS1 promotes chemoresistance and invasion of paclitaxel-resistant, hormone-refractory PC3 prostate cancer cells by up-regulating MDR1 and MMP9 expression. Biochem Biophys Res Commun 2011; 417:966-71. [PMID: 22206665 DOI: 10.1016/j.bbrc.2011.12.047] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2011] [Accepted: 12/13/2011] [Indexed: 12/14/2022]
Abstract
ETS1, which belongs to the ETS transcription factor family, plays important roles in diverse aspects of cancer such as drug resistance and metastasis. In the present study, we examined the functional roles of ETS1 in paclitaxel resistance and invasion using human prostate cancer PC3 cells and paclitaxel-resistant PC3PR cells established from PC3 cells. Our results showed that ETS1mRNA and protein expression was markedly up-regulated in paclitaxel-resistant PC3PR cells compared with paclitaxel-sensitive PC3 cells. The mRNA levels of MDR1 as well as MMP1, MMP3, MMP9 and uPA were positively correlated with that of ETS1. In PC3PR cells, silencing of ETS1 expression by siRNAs inhibited the activity of the MDR1 promoter containing ETS binding sites, reduced the mRNA and protein levels of MDR1 and suppressed paclitaxel resistance. Furthermore, ETS1 knockdown decreased secretion of MMP9 as well as its intracellular mRNA level, and dramatically inhibited invasion of PC3PR cells. Our results suggest that ETS1 promotes paclitaxel resistance and invasion in part by up-regulating MDR1 and MMP9 expression. Taken together, a novel therapeutic strategy targeting the ETS1 gene could be designed to overcome chemoresistance and metastasis of taxane-resistant, hormone-refractory prostate cancer.
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Affiliation(s)
- Taku Kato
- Department of Urology, Gifu University Graduate School of Medicine, Japan
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Hollenhorst PC, McIntosh LP, Graves BJ. Genomic and biochemical insights into the specificity of ETS transcription factors. Annu Rev Biochem 2011; 80:437-71. [PMID: 21548782 DOI: 10.1146/annurev.biochem.79.081507.103945] [Citation(s) in RCA: 384] [Impact Index Per Article: 27.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
ETS proteins are a group of evolutionarily related, DNA-binding transcriptional factors. These proteins direct gene expression in diverse normal and disease states by binding to specific promoters and enhancers and facilitating assembly of other components of the transcriptional machinery. The highly conserved DNA-binding ETS domain defines the family and is responsible for specific recognition of a common sequence motif, 5'-GGA(A/T)-3'. Attaining specificity for biological regulation in such a family is thus a conundrum. We present the current knowledge of routes to functional diversity and DNA binding specificity, including divergent properties of the conserved ETS and PNT domains, the involvement of flanking structured and unstructured regions appended to these dynamic domains, posttranslational modifications, and protein partnerships with other DNA-binding proteins and coregulators. The review emphasizes recent advances from biochemical and biophysical approaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells.
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Affiliation(s)
- Peter C Hollenhorst
- Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana 47405, USA.
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Berger I, Blanco AG, Boelens R, Cavarelli J, Coll M, Folkers GE, Nie Y, Pogenberg V, Schultz P, Wilmanns M, Moras D, Poterszman A. Structural insights into transcription complexes. J Struct Biol 2011; 175:135-46. [DOI: 10.1016/j.jsb.2011.04.015] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2010] [Revised: 04/09/2011] [Accepted: 04/27/2011] [Indexed: 01/24/2023]
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Couillard J, Estève PO, Pradhan S, St-Pierre Y. 5-Aza-2′-deoxycytidine and interleukin-1 cooperate to regulate matrix metalloproteinase-3 gene expression. Int J Cancer 2011; 129:2083-92. [DOI: 10.1002/ijc.25865] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2010] [Accepted: 11/30/2010] [Indexed: 12/31/2022]
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Babayeva ND, Wilder PJ, Shiina M, Mino K, Desler M, Ogata K, Rizzino A, Tahirov TH. Structural basis of Ets1 cooperative binding to palindromic sequences on stromelysin-1 promoter DNA. Cell Cycle 2010; 9:3054-62. [PMID: 20686355 PMCID: PMC2928650 DOI: 10.4161/cc.9.15.12257] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2010] [Accepted: 05/05/2010] [Indexed: 11/19/2022] Open
Abstract
Ets1 is a member of the Ets family of transcription factors. Ets1 is autoinhibited and its activation requires heterodimerization with a partner protein or DNA-mediated homodimerization for cooperative DNA binding. In the latter case, Ets1 molecules bind to palindromic sequences in which two Ets-binding sites (EBS) are separated by four base pairs, for example in the promoters of stromelysin-1 and p53. Interestingly, counteraction of autoinhibition requires the autoinhibitory region encoded by exon VII of the gene. The structural basis for the requirement of autoinhibitory sequences for Ets1 binding to palindromic EBS still remains unresolved. Here we report the crystal structure of two Ets1 molecules bound to an EBS palindrome of the stromelysin-1 promoter DNA, providing a plausible explanation for the requirement of exon VII-encoded sequences for Ets1 cooperative DNA binding. The proposed mechanism was verified both in vitro by surface plasmon resonance and in vivo by transcription-based assays.
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Affiliation(s)
- Nigar D Babayeva
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, NE, USA
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Son O, Hur YS, Kim YK, Lee HJ, Kim S, Kim MR, Nam KH, Lee MS, Kim BY, Park J, Park J, Lee SC, Hanada A, Yamaguchi S, Lee IJ, Kim SK, Yun DJ, Söderman E, Cheon CI. ATHB12, an ABA-Inducible Homeodomain-Leucine Zipper (HD-Zip) Protein of Arabidopsis, Negatively Regulates the Growth of the Inflorescence Stem by Decreasing the Expression of a Gibberellin 20-Oxidase Gene. ACTA ACUST UNITED AC 2010; 51:1537-47. [DOI: 10.1093/pcp/pcq108] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
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Shukla AA, Jain M, Chauhan SS. Ets-1/Elk-1 is a critical mediator of dipeptidyl-peptidase III transcription in human glioblastoma cells. FEBS J 2010; 277:1861-75. [PMID: 20236318 DOI: 10.1111/j.1742-4658.2010.07603.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
Dipetidyl-peptidase III is a metallopeptidase involved in a number of physiological processes and its expression has been reported to increase with the histological aggressiveness of human ovarian primary carcinomas. Because no information regarding the regulation of its expression was available, experiments were designed to clone, define and characterize the promoter region of the human dipeptidyl-peptidase III (DPP-III) gene. In this study, we cloned a 1038 bp 5'-flanking DNA fragment of the human DPP-III gene for the first time and demonstrated strong promoter activity in this region. Deletion analysis revealed that as few as 45 nucleotides proximal to the transcription start site retained approximately 40% of the activity of the full-length promoter. This promoter lacked the TATA box but contained multiple GC boxes and a single CAAT box. Similarly, two Ets-1/Elk-1-binding motifs are present in the first 25 nucleotides from the transcription start site. Binding of Ets-1/Elk-1 proteins to these motifs was visualized by electrophoretic mobility shift and chromatin immunoprecipitation assays. Mutations of these binding sites abolished not only binding of the Ets protein, but also the intrinsic promoter activity. Increased DNA-binding activity of Ets-1/Elk-1 by v-Ha-ras also augmented the mRNA level and promoter activity of this gene. Similarly, co-transfection of DPP-III promoter-reporter constructs with Ets-1 expression vector led to a significant increase in promoter activity. From these results, we conclude that Ets-1/Elk-1 plays a critical role in transcription of the human DPP-III gene.
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Affiliation(s)
- Abhay A Shukla
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
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40
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Caspase cleavage of Ets-1 p51 generates fragments with transcriptional dominant-negative function. Biochem J 2010; 426:229-41. [PMID: 20001963 DOI: 10.1042/bj20090877] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Ets-1 is a transcription factor that plays an important role in various physiological and pathological processes, such as development, angiogenesis, apoptosis and tumour invasion. In the present study, we have demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed in a caspase-dependent manner in Jurkat T-leukaemia cells undergoing apoptosis, resulting in three C-terminal fragments Cp20, Cp17 and Cp14 and a N-terminal fragment, Np36. In vitro cleavage of Ets-1 p51 by caspase 3 produces fragments consistent with those observed in cells undergoing apoptosis. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p51. This region is absent from the Ets-1 p42 isoform, which therefore cannot be cleaved by caspases. In Ets-1 p51, cleavage generates C-terminal fragments containing the DNA-binding domain, but lacking the transactivation domain. The Cp17 fragment, the major cleavage product generated during apoptosis, is devoid of transcriptional activity and inhibits Ets-1 p51-mediated transactivation of target genes by competing with Ets-1 p51 for binding to Ets-binding sites present in the target promoters. In the present study, we have demonstrated that caspase cleavage of Ets-1 within the exon VII-encoded region leads to specific down-regulation of the Ets-1 p51 isoform during apoptosis. Furthermore, our results establish that caspase cleavage generates a stable C-terminal fragment that acts as a natural dominant-negative form of the full-length Ets-1 p51 protein.
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Agarkar VB, Babayeva ND, Wilder PJ, Rizzino A, Tahirov TH. Crystal structure of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-beta receptor promoter DNA. J Mol Biol 2010; 397:278-89. [PMID: 20079749 DOI: 10.1016/j.jmb.2010.01.017] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2009] [Revised: 01/05/2010] [Accepted: 01/06/2010] [Indexed: 12/21/2022]
Abstract
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-beta receptor gene (TbetaR-II) is strongly stimulated by Elf3 via two adjacent Elf3 binding sites, the A-site and the B-site. Here, we report the 2.2 A 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-beta receptor promoter DNA (mTbetaR-II(DNA)). Elf3 contacts the core GGAA motif of the B-site from a 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.
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Affiliation(s)
- Vinod B Agarkar
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 987696 Nebraska Medical Center, Omaha, NE 68198-7696, USA
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Choul-li S, Drobecq H, Aumercier M. DNA-dependent protein kinase is a novel interaction partner for Ets-1 isoforms. Biochem Biophys Res Commun 2009; 390:839-44. [PMID: 19836356 DOI: 10.1016/j.bbrc.2009.10.059] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2009] [Accepted: 10/13/2009] [Indexed: 10/20/2022]
Abstract
The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction partner proteins. We used our previously developed, recombinant biotinylated Ets-1 that conserves native Ets-1 properties to identify new interaction partners. Here, based on results from streptavidin pull-down assays, mass spectrometry and co-immunoprecipitation, we report a novel interaction partner for Ets-1 isoforms: a heterotrimeric complex of DNA-dependent protein kinase (DNA-PK), made up of Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs). Kinase assays performed in vitro showed that DNA-PK phosphorylates the Ets-1 protein. Furthermore, we demonstrated that Ku86, but not Ku70 or DNA-PKcs, down-regulated the transcriptional activity of Ets-1 when analysed using a reporter gene assay. These results illustrate how detecting novel molecular interactions may provide new clues for understanding the diverse functions of Ets-1.
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Affiliation(s)
- Souhaila Choul-li
- CNRS UMR 8161, Institut de Biologie de Lille, Institut Pasteur de Lille, Univ Lille Nord de France, IFR 142, BP 447, 1 rue du Pr Calmette, 59021 Lille Cedex, France
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Leprivier G, Baillat D, Begue A, Hartmann B, Aumercier M. Ets-1 p51 and p42 isoforms differentially modulate Stromelysin-1 promoter according to induced DNA bend orientation. Nucleic Acids Res 2009; 37:4341-52. [PMID: 19465391 PMCID: PMC2715226 DOI: 10.1093/nar/gkp307] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
The Stromelysin-1 gene promoter contains a palindrome of two Ets-binding sites (EBS) that bind the p51 and p42 isoforms of the human Ets-1-transcription factor. A previous study established that full gene transactivation is associated with a ternary complex consisting of two p51 bound to the two EBS on the promoter. p42, only able to bind one of the two EBS, induces only very weak activity. Here, we investigate the mechanism by which the Stromelysin-1 promoter discriminates between p51 and p42. The differential stoichiometry of the two Ets-1 isoforms arises from the Stromelysin-1 EBS palindrome. The ternary complex requires the presence of two inhibitory domains flanking the DNA-binding domain and the ability to form an intramolecular autoinhibition module. Most importantly, the p51-ternary and the p42-binary complexes induce DNA curvatures with opposite orientations. These results establish that differential DNA bending, via p51 and p42 differential binding, is correlated with the Stromelysin-1 promoter activation process.
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Affiliation(s)
- Gabriel Leprivier
- CNRS UMR 8161, Institut de Biologie de Lille, Université de Lille 1 and Lille 2, IFR 142, BP 447, 59021 Lille Cedex, France
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Laitem C, Leprivier G, Choul-Li S, Begue A, Monte D, Larsimont D, Dumont P, Duterque-Coquillaud M, Aumercier M. Ets-1 p27: a novel Ets-1 isoform with dominant-negative effects on the transcriptional properties and the subcellular localization of Ets-1 p51. Oncogene 2009; 28:2087-99. [PMID: 19377509 DOI: 10.1038/onc.2009.72] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The transcription factor Ets-1 is implicated in various physiological processes and invasive pathologies. We identified a novel variant of ets-1, ets-1Delta(III-VI), resulting from the alternative splicing of exons III to VI. This variant encodes a 27 kDa isoform, named Ets-1 p27. Ets-1 p27 lacks the threonine-38 residue, the Pointed domain and the transactivation domain, all of which are required for the transactivation of Ets-1 target genes. Both inhibitory domains surrounding the DNA-binding domain are conserved, suggesting that Ets-1 p27, like the full-length Ets-1 p51 isoform, is autoinhibited for DNA binding. We showed that Ets-1 p27 binds DNA in the same way as Ets-1 p51 does and that it acts both at a transcriptional and a subcellular localization level, thereby constituting a dual-acting dominant negative of Ets-1 p51. Ets-1 p27 blocks Ets-1 p51-mediated transactivation of target genes and induces the translocation of Ets-1 p51 from the nucleus to the cytoplasm. Furthermore, Ets-1 p27 overexpression represses the tumor properties of MDA-MB-231 mammary carcinoma cells in correlation with the known implication of Ets-1 in various cellular mechanisms. Thus the dual-acting dominant-negative function of Ets-1 p27 gives to the Ets-1 p27/Ets-1 p51 ratio a determining effect on cell fate.
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Affiliation(s)
- C Laitem
- CNRS Unité Mixte de Recherche 8161, Institut de Biologie de Lille, Institut Pasteur de Lille, Universités de Lille 1 and Lille 2, IFR 142, Lille, France
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Nagarajan P, Parikh N, Garrett-Sinha LA, Sinha S. Ets1 induces dysplastic changes when expressed in terminally-differentiating squamous epidermal cells. PLoS One 2009; 4:e4179. [PMID: 19142229 PMCID: PMC2615206 DOI: 10.1371/journal.pone.0004179] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2008] [Accepted: 11/28/2008] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Ets1 is an oncogene that functions as a transcription factor and regulates the activity of many genes potentially important for tumor initiation and progression. Interestingly, the Ets1 oncogene is over-expressed in many human squamous cell cancers and over-expression is highly correlated with invasion and metastasis. Thus, Ets1 is believed to mainly play a role in later stages of the oncogenic process, but not early events. METHODOLOGY/PRINCIPAL FINDINGS To better define the role of Ets1 in squamous cell carcinogenesis, we generated a transgenic mouse model in which expression of the Ets1 oncogene could be temporally and spatially regulated. Upon Ets1 induction in differentiating cells of stratified squamous epithelium, these mice exhibited dramatic changes in epithelial organization including increased proliferation and blocked terminal differentiation. The phenotype was completely reversed when Ets1 expression was suppressed. In mice where Ets1 expression was re-induced at a later age, the phenotype was more localized and the lesions that developed were more invasive. Many potential Ets1 targets were upregulated in the skin of these mice with the most dramatic being the metalloprotease MMP13, which we demonstrate to be a direct transcriptional target of Ets1. CONCLUSIONS/SIGNIFICANCE Collectively, our data reveal that upregulation of Ets1 can be an early event that promotes pre-neoplastic changes in epidermal tissues via its regulation of key genes driving growth and invasion. Thus, the Ets1 oncogene may be important for oncogenic processes in both early and late stages of tumor development.
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Affiliation(s)
- Priyadharsini Nagarajan
- Department of Biochemistry, State University of New York at Buffalo, Center for Excellence in Bioinformatics and Life Sciences, Buffalo, New York, United States of America
| | - Neha Parikh
- Department of Biochemistry, State University of New York at Buffalo, Center for Excellence in Bioinformatics and Life Sciences, Buffalo, New York, United States of America
| | - Lee Ann Garrett-Sinha
- Department of Biochemistry, State University of New York at Buffalo, Center for Excellence in Bioinformatics and Life Sciences, Buffalo, New York, United States of America
- * E-mail: (LAG-S); (SS)
| | - Satrajit Sinha
- Department of Biochemistry, State University of New York at Buffalo, Center for Excellence in Bioinformatics and Life Sciences, Buffalo, New York, United States of America
- * E-mail: (LAG-S); (SS)
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Baillat D, Laitem C, Leprivier G, Margerin C, Aumercier M. Ets-1 binds cooperatively to the palindromic Ets-binding sites in the p53 promoter. Biochem Biophys Res Commun 2008; 378:213-7. [PMID: 19022222 DOI: 10.1016/j.bbrc.2008.11.035] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2008] [Accepted: 11/06/2008] [Indexed: 11/25/2022]
Abstract
Due to its autoinhibition for DNA binding, the Ets-1 transcription factor must interact with partners to enhance its affinity for DNA. In a study on the stromelysin-1 promoter, we showed that Ets-1 binds cooperatively to two Ets-binding sites (EBS) organized in palindrome, thereby circumventing the need for a binding partner to counteract autoinhibition. This leads to the formation of an Ets-1-DNA-Ets-1 ternary complex necessary for promoter activation. Here we show that Ets-1 also binds cooperatively to the EBS palindrome of the human p53 promoter, despite the presence of a degenerate EBS to which Ets-1 cannot otherwise bind. Transcriptional transactivation through this palindrome fully correlates to Ets-1 binding. Thus, the cooperative binding model that we initially proposed for the stromelysin-1 promoter may be a general mechanism of Ets-1 binding to palindromic EBS separated by 4bp and a way to counteract binding site degeneracy.
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Affiliation(s)
- David Baillat
- CNRS Unité Mixte de Recherche 8161, Institut de Biologie de Lille, Institut Pasteur de Lille, Universités de Lille 1 and Lille 2, IFR 142, B.P. 447, 1 rue du Pr. Calmette, 59021 Lille Cedex, France
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Grenningloh R, Miaw SC, Moisan J, Graves BJ, Ho IC. Role of Ets-1 phosphorylation in the effector function of Th cells. Eur J Immunol 2008; 38:1700-5. [PMID: 18465773 DOI: 10.1002/eji.200738112] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The transcription factor Ets-1 critically regulates differentiation and function of T helper (Th) cells. In vitro studies have demonstrated that DNA binding and transcriptional activity of Ets-1 are regulated by phosphorylation. Depending on the site of phosphorylation, Ets-1 function can either be increased or inhibited. In addition, a splice variant lacking several inhibitory phosphorylation sites has been identified, raising the possibility that this splice variant may function differently from the full-length Ets-1. However, it is unclear how the activating and inhibitory phosphorylation events of Ets-1 are coordinated during Th cell activation. Furthermore, the biological consequences of Ets-1 phosphorylation and alternative splicing in regulating the function of Th cells are unknown. We report here that both activating and inhibitory phosphorylation events of Ets-1 occur simultaneously and independently of each other during Th cell activation. We further demonstrate that the effect of Ets-1 phosphorylation is very modest and that full-length Ets-1 and its splice variant are functionally interchangeable in the regulation of cytokine production in Th cells.
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Affiliation(s)
- Roland Grenningloh
- Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA
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Armata IA, Ananthanarayanan M, Balasubramaniyan N, Shashidharan P. Regulation of DYT1 gene expression by the Ets family of transcription factors. J Neurochem 2008; 106:1052-65. [PMID: 18466338 PMCID: PMC2668265 DOI: 10.1111/j.1471-4159.2008.05465.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The DYT1 gene encodes for torsinA, a protein with widespread tissue distribution, involved in early onset dystonia (EOD). Numerous studies have focused on torsinA function but no information is available on its transcriptional regulation. We cloned mouse and human 5'-upstream DYT1 DNA fragments, exhibiting high transcriptional activity, as well as tissue specificity. We identified a proximal minimal DYT1 promoter within -141 bp for mouse and -191 bp for human with respect to the ATG codon. Primer extension analysis indicated multiple transcription start sites. In silico analysis of approximately 500 bp 5'-upstream DYT1 fragment demonstrated lack of a classical TATA or CAAT box and the presence of a highly conserved direct repeat of two Ets binding cores within -86 bp to -77 bp and -78 bp to -69 bp of the mouse and human DYT1 gene, respectively. A single or a two base nucleotide alteration within the downstream Ets core resulted in approximately 90% (mouse) or 45-60% (human) drop in activity. Interestingly, a 3-bp distance increase between the two Ets cores dramatically decreased transcriptional activity which was partially restored when the distance was increased up to 10 bp. Ets-like dominant negatives confirmed the Ets factors as DYT1 transcriptional activators.
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Affiliation(s)
- Ioanna A Armata
- Department of Neurology, Laboratory of Developmental and Molecular Hepatology, Mount Sinai School of Medicine, New York 10029, USA
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Lee GM, Pufall MA, Meeker CA, Kang HS, Graves BJ, McIntosh LP. The affinity of Ets-1 for DNA is modulated by phosphorylation through transient interactions of an unstructured region. J Mol Biol 2008; 382:1014-30. [PMID: 18692067 DOI: 10.1016/j.jmb.2008.07.064] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2008] [Revised: 07/24/2008] [Accepted: 07/24/2008] [Indexed: 11/15/2022]
Abstract
Binding of the transcription factor Ets-1 to DNA is allosterically regulated by a serine-rich region (SRR) that modulates the dynamic character of the adjacent structured DNA-binding ETS domain and its flanking autoinhibitory elements. Multi-site phosphorylation of the flexible SRR in response to Ca(2+) signaling mediates variable regulation of Ets-1 DNA-binding affinity. In this study, we further investigated the mechanism of this regulation. First, thermal and urea denaturation experiments demonstrated that phosphorylation of the predominantly unstructured SRR imparts enhanced thermodynamic stability on the well-folded ETS domain and its inhibitory module. We next identified a minimal fragment (residues 279-440) that exhibits both enhanced autoinhibition of Ets-1 DNA-binding and allosteric reinforcement by phosphorylation. To test for intramolecular interactions between the SRR and the rest of the fragment that were not detectable by (1)H-(1)H NOE measurements, paramagnetic relaxation enhancements were performed using Cu(2+) bound to the N-terminal ATCUN motif. Increased relaxation detected for specific amide and methyl groups revealed a preferential interaction surface for the flexible SRR extending from the inhibitory module to the DNA-binding interface. Phosphorylation enhanced the localization of the SRR to this surface. We therefore hypothesize that the positioning of the SRR at the DNA-binding interface and its role in shifting Ets-1 to an inhibited conformation are linked. In particular, transient interactions dampen the conformational flexibility of the ETS domain and inhibitory module required for high-affinity binding, as well as possibly occlude the DNA interaction site. Surprisingly, the phosphorylation-dependent effects were relatively insensitive to changes in ionic strength, suggesting that electrostatic forces are not the dominant mechanism for mediating these interactions. The results of this study highlight the role of flexibility and transient binding in the variable regulation of Ets-1 activity.
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Affiliation(s)
- Gregory M Lee
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
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50
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Lamber EP, Vanhille L, Textor LC, Kachalova GS, Sieweke MH, Wilmanns M. Regulation of the transcription factor Ets-1 by DNA-mediated homo-dimerization. EMBO J 2008; 27:2006-17. [PMID: 18566588 PMCID: PMC2486274 DOI: 10.1038/emboj.2008.117] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2007] [Accepted: 05/23/2008] [Indexed: 01/07/2023] Open
Abstract
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.
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Affiliation(s)
| | - Laurent Vanhille
- Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Marseille, France,Institut National de la Santé et de la Recherche Médicale, Marseille, France,Centre National de la Recherche Scientifique, Parc scientifique de Luminy, Marseille, France
| | | | - Galina S Kachalova
- Max-Planck Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany
| | - Michael H Sieweke
- Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Marseille, France,Institut National de la Santé et de la Recherche Médicale, Marseille, France,Centre National de la Recherche Scientifique, Parc scientifique de Luminy, Marseille, France
| | - Matthias Wilmanns
- EMBL-Hamburg, c/o DESY, Hamburg, Germany,EMBL Hamburg Outstation, EMBL c/o DESY, Notkestrasse 85, Building 25A, Hamburg D-22603, Germany. Tel.: +49 40 899 021 26; Fax: +49 40 899 021 49; E-mail:
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