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Luan Y, Min Q, Zhang R, Wen Z, Meng X, Hu Z, Feng X, Yu M, Dong L, Wang JY. EAF2 deficiency attenuates autoimmune disease in Fas lpr mice by modulating B cell activation and apoptosis. iScience 2024; 27:111220. [PMID: 39555413 PMCID: PMC11565555 DOI: 10.1016/j.isci.2024.111220] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Revised: 10/06/2024] [Accepted: 10/17/2024] [Indexed: 11/19/2024] Open
Abstract
MRL/lpr mice develop systemic lupus erythematosus-like autoimmunity due to defective FAS-mediated apoptosis. We generated Fas lpr mice deficient in EAF2, a transcription elongation-associated factor known to promote apoptosis in germinal center (GC) B cells and crucial for preventing autoimmunity. Contrary to expectations, EAF2 deficiency significantly reduced lymphadenopathy and splenomegaly, extended lifespan, and alleviated nephritis by decreasing renal immune complex deposition. Additionally, EAF2 deficiency markedly reduced accumulation of activated B cells, GC B cells, plasma cells, and the abnormal B220+CD3+ T cells in Fas lpr mice. Further analysis revealed that Eaf2 -/- Fas lpr B cells showed hyperactivation upon various stimulations, followed by increased death. RNA sequencing of the B220+CD3+ cells revealed a downregulation in survival-promoting genes such as Bcl-2 and Akt and an upregulation of proapoptotic genes. We conclude that the combined deficiency in FAS- and EAF2-mediated apoptotic pathways leads to B cell hyperactivation and subsequent death, thereby ameliorating systemic autoimmunity in this model.
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Affiliation(s)
- Yingying Luan
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Qing Min
- Department of Clinical Immunology, Children’s Hospital of Fudan University, National Children’s Medical Center, Shanghai, China
- Shanghai Sci-Tech Inno Center for Infection & Immunity, Shanghai, China
| | - Runyun Zhang
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Zichao Wen
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Xin Meng
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Ziying Hu
- Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Xiaoqian Feng
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Meiping Yu
- Department of Clinical Immunology, Children’s Hospital of Fudan University, National Children’s Medical Center, Shanghai, China
| | - Lulu Dong
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Ji-Yang Wang
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
- Department of Clinical Immunology, Children’s Hospital of Fudan University, National Children’s Medical Center, Shanghai, China
- Shanghai Sci-Tech Inno Center for Infection & Immunity, Shanghai, China
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Ji WT, Cui CG, Wang Y. EAF2: a tumor suppressor gene with multi-aspect functions. Front Pharmacol 2024; 15:1440511. [PMID: 39588149 PMCID: PMC11586179 DOI: 10.3389/fphar.2024.1440511] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2024] [Accepted: 10/30/2024] [Indexed: 11/27/2024] Open
Abstract
Since ELL-associated factor 2 (EAF2) was identified in 1997 as an androgen response gene, it has been of medical and scientific interest. Early studies demonstrated the tumor-suppressing function of EAF2 in the prostate. Sequencing studies indicated an association between EAF2 and several other malignant diseases and multiple physiological processes, such as transcription, apoptosis, embryogenesis, and DNA repair. Further understanding of EAF2 will provide new opportunities and therapeutic approaches for cancers, especially prostate cancer. This narrative review summarizes the existing knowledge of EAF2 and outlines its potential significance. To our knowledge, this is the first review of the role of this novel tumor suppressor gene and its possible functions.
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Affiliation(s)
- Wen-Tong Ji
- Urology 2nd Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
| | - Chun-Guo Cui
- Galactophore Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
| | - Yao Wang
- Urology 2nd Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
- Jilin Key Laboratory of Molecular Diagnosis of Urologic Neoplasms, Urology 2nd Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
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Zhang Z, Xu J, Liu J, Wang J, Lei L. SEC: A core hub during cell fate alteration. FASEB J 2024; 38:e23680. [PMID: 38758186 DOI: 10.1096/fj.202400514r] [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: 03/06/2024] [Revised: 04/18/2024] [Accepted: 05/07/2024] [Indexed: 05/18/2024]
Abstract
Pol II pause release is a rate-limiting step in gene transcription, influencing various cell fate alterations. Numerous proteins orchestrate Pol II pause release, thereby playing pivotal roles in the intricate process of cellular fate modulation. Super elongation complex (SEC), a large assembly comprising diverse protein components, has garnered attention due to its emerging significance in orchestrating physiological and pathological cellular identity changes by regulating the transcription of crucial genes. Consequently, SEC emerges as a noteworthy functional complex capable of modulating cell fate alterations. Therefore, a comprehensive review is warranted to systematically summarize the core roles of SEC in different types of cell fate alterations. This review focuses on elucidating the current understanding of the structural and functional basis of SEC. Additionally, we discuss the intricate regulatory mechanisms governing SEC in various models of cell fate alteration, encompassing both physiological and pathological contexts. Furthermore, leveraging the existing knowledge of SEC, we propose some insightful directions for future research, aiming to enhance our mechanistic and functional comprehension of SEC within the diverse landscape of cell fate alterations.
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Affiliation(s)
- Zhijing Zhang
- College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province, China
- Department of Histology and Embryology, Harbin Medical University, Harbin, Heilongjiang Province, China
| | - Jingyi Xu
- Department of Histology and Embryology, Harbin Medical University, Harbin, Heilongjiang Province, China
| | - Jiqiang Liu
- Department of Histology and Embryology, Harbin Medical University, Harbin, Heilongjiang Province, China
| | - Jiaqiang Wang
- College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province, China
| | - Lei Lei
- Department of Histology and Embryology, Harbin Medical University, Harbin, Heilongjiang Province, China
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Chen Y, Cramer P. RNA polymerase II elongation factors use conserved regulatory mechanisms. Curr Opin Struct Biol 2024; 84:102766. [PMID: 38181687 DOI: 10.1016/j.sbi.2023.102766] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Revised: 12/11/2023] [Accepted: 12/13/2023] [Indexed: 01/07/2024]
Abstract
RNA polymerase II (Pol II) transcription is regulated by many elongation factors. Among these factors, TFIIF, PAF-RTF1, ELL and Elongin stimulate mRNA chain elongation by Pol II. Cryo-EM structures of Pol II complexes with these elongation factors now reveal some general principles on how elongation factors bind Pol II and how they stimulate transcription. All four elongation factors contact Pol II at domains external 2 and protrusion, whereas TFIIF and ELL additionally bind the Pol II lobe. All factors apparently stabilize cleft-flanking elements, whereas RTF1 and Elongin additionally approach the active site with a latch element and may influence catalysis or translocation. Due to the shared binding sites on Pol II, factor binding is mutually exclusive, and thus it remains to be studied what determines which elongation factors bind at a certain gene and under which condition.
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Affiliation(s)
- Ying Chen
- Department of Molecular Biology, Max Planck Institute for Multidisciplinary Sciences, Am Fassberg 11, 37077 Göttingen, Germany.
| | - Patrick Cramer
- Department of Molecular Biology, Max Planck Institute for Multidisciplinary Sciences, Am Fassberg 11, 37077 Göttingen, Germany.
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Ghosh A, Chakraborty P, Biswas D. Fine tuning of the transcription juggernaut: A sweet and sour saga of acetylation and ubiquitination. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2023; 1866:194944. [PMID: 37236503 DOI: 10.1016/j.bbagrm.2023.194944] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/17/2023] [Revised: 04/26/2023] [Accepted: 05/18/2023] [Indexed: 05/28/2023]
Abstract
Among post-translational modifications of proteins, acetylation, phosphorylation, and ubiquitination are most extensively studied over the last several decades. Owing to their different target residues for modifications, cross-talk between phosphorylation with that of acetylation and ubiquitination is relatively less pronounced. However, since canonical acetylation and ubiquitination happen only on the lysine residues, an overlap of the same lysine residue being targeted for both acetylation and ubiquitination happens quite frequently and thus plays key roles in overall functional regulation predominantly through modulation of protein stability. In this review, we discuss the cross-talk of acetylation and ubiquitination in the regulation of protein stability for the functional regulation of cellular processes with an emphasis on transcriptional regulation. Further, we emphasize our understanding of the functional regulation of Super Elongation Complex (SEC)-mediated transcription, through regulation of stabilization by acetylation, deacetylation and ubiquitination and associated enzymes and its implication in human diseases.
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Affiliation(s)
- Avik Ghosh
- Laboratory of Transcription Biology Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 32, India
| | - Poushali Chakraborty
- Laboratory of Transcription Biology Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 32, India
| | - Debabrata Biswas
- Laboratory of Transcription Biology Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 32, India.
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Liu W, Lin S, Li L, Tai Z, Liu JX. Zebrafish ELL-associated factors Eaf1/2 modulate erythropoiesis via regulating gata1a expression and WNT signaling to facilitate hypoxia tolerance. CELL REGENERATION (LONDON, ENGLAND) 2023; 12:10. [PMID: 37002435 PMCID: PMC10066051 DOI: 10.1186/s13619-022-00154-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Accepted: 11/28/2022] [Indexed: 04/04/2023]
Abstract
EAF1 and EAF2, the eleven-nineteen lysine-rich leukemia (ELL)-associated factors which can assemble to the super elongation complex (AFF1/4, AF9/ENL, ELL, and P-TEFb), are reported to participate in RNA polymerase II to actively regulate a variety of biological processes, including leukemia and embryogenesis, but whether and how EAF1/2 function in hematopoietic system related hypoxia tolerance during embryogenesis remains unclear. Here, we unveiled that deletion of EAF1/2 (eaf1-/- and eaf2-/-) caused reduction in hypoxia tolerance in zebrafish, leading to reduced erythropoiesis during hematopoietic processes. Meanwhile, eaf1-/- and eaf2-/- mutants showed significant reduction in the expression of key transcriptional regulators scl, lmo2, and gata1a in erythropoiesis at both 24 h post fertilization (hpf) and 72 hpf, with gata1a downregulated while scl and lmo2 upregulated at 14 hpf. Mechanistically, eaf1-/- and eaf2-/- mutants exhibited significant changes in the expression of epigenetic modified histones, with a significant increase in the binding enrichment of modified histone H3K27me3 in gata1a promoter rather than scl and lmo2 promoters. Additionally, eaf1-/- and eaf2-/- mutants exhibited a dynamic expression of canonical WNT/β-catenin signaling during erythropoiesis, with significant reduction in p-β-Catenin level and in the binding enrichment of both scl and lmo2 promoters with the WNT transcriptional factor TCF4 at 24 hpf. These findings demonstrate an important role of Eaf1/2 in erythropoiesis in zebrafish and may have shed some light on regeneration medicine for anemia and related diseases and on molecular basis for fish economic or productive traits, such as growth, disease resistance, hypoxia tolerance, and so on.
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Affiliation(s)
- WenYe Liu
- grid.35155.370000 0004 1790 4137College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, 430070 Wuhan, China
| | - ShuHui Lin
- grid.35155.370000 0004 1790 4137College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, 430070 Wuhan, China
| | - LingYa Li
- grid.35155.370000 0004 1790 4137College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, 430070 Wuhan, China
| | - ZhiPeng Tai
- grid.35155.370000 0004 1790 4137College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, 430070 Wuhan, China
| | - Jing-Xia Liu
- grid.35155.370000 0004 1790 4137College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, 430070 Wuhan, China
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Wu X, Xie Y, Zhao K, Lu J. Targeting the super elongation complex for oncogenic transcription driven tumor malignancies: Progress in structure, mechanisms and small molecular inhibitor discovery. Adv Cancer Res 2023; 158:387-421. [PMID: 36990537 DOI: 10.1016/bs.acr.2022.12.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Oncogenic transcription activation is associated with tumor development and resistance derived from chemotherapy or target therapy. The super elongation complex (SEC) is an important complex regulating gene transcription and expression in metazoans closely related to physiological activities. In normal transcriptional regulation, SEC can trigger promoter escape, limit proteolytic degradation of transcription elongation factors and increase the synthesis of RNA polymerase II (POL II), and regulate many normal human genes to stimulate RNA elongation. Dysregulation of SEC accompanied by multiple transcription factors in cancer promotes rapid transcription of oncogenes and induce cancer development. In this review, we summarized recent progress in understanding the mechanisms of SEC in regulating normal transcription, and importantly its roles in cancer development. We also highlighted the discovery of SEC complex target related inhibitors and their potential applications in cancer treatment.
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Affiliation(s)
- Xinyu Wu
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China; Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
| | - Yanqiu Xie
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China; Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
| | - Kehao Zhao
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China.
| | - Jing Lu
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China.
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8
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Pal S, Yadav D, Biswas D. ATM-mediated ELL phosphorylation enhances its self-association through increased EAF1 interaction and inhibits global transcription during genotoxic stress. Nucleic Acids Res 2022; 50:10995-11012. [PMID: 36305813 PMCID: PMC9638944 DOI: 10.1093/nar/gkac943] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Revised: 09/16/2022] [Accepted: 10/15/2022] [Indexed: 12/05/2022] Open
Abstract
Mammalian cells immediately inhibit transcription upon exposure to genotoxic stress to avoid fatal collision between ongoing transcription and newly recruited DNA repair machineries to protect genomic integrity. However, mechanisms of this early transcriptional inhibition are poorly understood. In this study, we decipher a novel role of human EAF1, a positive regulator of ELL-dependent RNA Polymerase II-mediated transcription in vitro, in regulation of temporal inhibition of transcription during genotoxic stress. Our results show that, besides Super Elongation Complex (SEC) and Little Elongation Complex (LEC), human ELL (aka ELL1) also forms a complex with EAF1 alone. Interestingly, contrary to the in vitro studies, EAF1 inhibits ELL-dependent RNA polymerase II-mediated transcription of diverse target genes. Mechanistically, we show that intrinsic self-association property of ELL leads to its reduced interaction with other SEC components. EAF1 enhances ELL self-association and thus reduces its interaction with other SEC components leading to transcriptional inhibition. Physiologically, we show that upon exposure to genotoxic stress, ATM-mediated ELL phosphorylation-dependent enhanced EAF1 association results in reduced ELL interaction with other SEC components that lead to global transcriptional inhibition. Thus, we describe an important mechanism of dynamic transcriptional regulation during genotoxic stress involving post-translational modification of a key elongation factor.
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Affiliation(s)
- Sujay Pal
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata - 32, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Dipika Yadav
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata - 32, India
| | - Debabrata Biswas
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata - 32, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
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Basu S, Nandy A, Barad MK, Pal S, Biswas D. Negative Feedback Loop Mechanism between EAF1/2 and DBC1 in Regulating ELL Stability and Functions. Mol Cell Biol 2022; 42:e0015122. [PMID: 36036574 PMCID: PMC9590304 DOI: 10.1128/mcb.00151-22] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2022] [Revised: 06/05/2022] [Accepted: 08/08/2022] [Indexed: 12/25/2022] Open
Abstract
Although ELL-associated factors 1 and 2 (EAF1/2) have been shown to enhance RNA polymerase II-mediated transcription in vitro, their functional roles in vivo are poorly known. In this report, we show functions of these proteins in regulating ELL stability through their competitive binding with HDAC3 at the N terminus of ELL. Reduced HDAC3 binding to ELL causes increased acetylation leading to reduced ubiquitylation-mediated degradation. Similar functional roles played by DBC1 in regulating ELL stability further prompted in-depth analyses that demonstrated presence of negative feedback loop mechanisms between DBC1 and EAF1/2 in maintaining overall ELL level. Mechanistically, increased DBC1 reduces EAF1/2 level through increased ubiquitylation involving E3 ubiquitin ligase TRIM28, whereas increased EAF1/2 reduces DBC1 level through reduced transcription. Physiologically, after a few passages, ELL levels in either DBC1 or EAF1 knockdown cells are restored through enhanced expression of EAF1 and DBC1, respectively. Interestingly, for maintenance of ELL level, mammalian cells prefer the EAF1-dependent pathway during exposure to genotoxic stress, and the DBC1-dependent pathway during exposure to growth factors. Thus, we describe coordinated functions of multiple factors, including EAF1/2, HDAC3, DBC1, and TRIM28 in regulating ELL protein level for optimal target gene expression in a context-dependent manner within mammalian cells.
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Affiliation(s)
- Subham Basu
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India
| | - Arijit Nandy
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India
| | - Mahesh K. Barad
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India
| | - Sujay Pal
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India
| | - Debabrata Biswas
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India
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Feng ML, Wu C, Zhang HJ, Zhou H, Jiao TW, Liu MY, Sun MJ. Overexpression of ELL-associated factor 2 suppresses invasion, migration, and angiogenesis in colorectal cancer. World J Gastrointest Oncol 2022; 14:1949-1967. [PMID: 36310706 PMCID: PMC9611430 DOI: 10.4251/wjgo.v14.i10.1949] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Revised: 06/20/2022] [Accepted: 09/21/2022] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND The androgen responsive gene, ELL-associated factor 2 (EAF2), expressed in benign prostate tissues, has been shown to play an important role in tumor suppression in a variety of malignant tumors. In addition, some scholars found that EAF2 frameshift mutations are associated with intratumor heterogeneity in colorectal cancer (CRC) and inactivation of EAF2 in microsatellite instability-high CRC. However, the molecular mechanism by which EAF2 is involved in CRC invasion and metastasis remains unclear.
AIM To determine the clinical value of expression of EAF2 protein in CRC, and to study the effects of EAF2 on the invasion, migration, and angiogenesis of CRC cells in vitro.
METHODS In this study, we collected colorectal adenocarcinoma and corresponding adjacent tissues to investigate the clinical expression of EAF2 protein in patients with advanced CRC. Subsequently, we investigated the effect of EAF2 on the invasion, migration, and angiogenesis of CRC cells in vitro using plasmid transfection.
RESULTS EAF2 protein was lowly expressed in cancer tissues of patients with advanced CRC. Kaplan-Meier survival analysis showed that the survival rate of the high EAF2 level group was higher than that of the low EAF2 level group.
CONCLUSION Our results demonstrated that EAF2, as a tumor suppressor, may inhibit the invasion, metastasis, and angiogenesis of CRC cells by regulating the signal transducer and activator of transcription 3/transforming growth factor-β1 crosstalk pathway, and play a cancer suppressive and protective role in the occurrence and development of CRC. Our findings are of great significance to provide a new idea and theoretical basis for the targeted diagnosis and treatment of CRC.
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Affiliation(s)
- Ming-Liang Feng
- Department of Endoscopy, The First Hospital Affiliated to China Medical University, Shenyang 110001, Liaoning Province, China
| | - Can Wu
- Department of Endoscopy, The First Hospital Affiliated to China Medical University, Shenyang 110001, Liaoning Province, China
| | - Hui-Jing Zhang
- Department of Endoscopy, The First Hospital Affiliated to China Medical University, Shenyang 110001, Liaoning Province, China
| | - Huan Zhou
- Department of Endoscopy, The First Hospital Affiliated to China Medical University, Shenyang 110001, Liaoning Province, China
| | - Tai-Wei Jiao
- Department of Endoscopy, The First Hospital Affiliated to China Medical University, Shenyang 110001, Liaoning Province, China
| | - Meng-Yuan Liu
- Department of Endoscopy, The First Hospital Affiliated to China Medical University, Shenyang 110001, Liaoning Province, China
| | - Ming-Jun Sun
- Department of Endoscopy, The First Hospital Affiliated to China Medical University, Shenyang 110001, Liaoning Province, China
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Sequence, structural and functional conservation among the human and fission yeast ELL and EAF transcription elongation factors. Mol Biol Rep 2021; 49:1303-1320. [PMID: 34807377 DOI: 10.1007/s11033-021-06958-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2021] [Accepted: 11/11/2021] [Indexed: 10/19/2022]
Abstract
BACKGROUND Transcription elongation is a dynamic and tightly regulated step of gene expression in eukaryotic cells. Eleven nineteen Lysine rich Leukemia (ELL) and ELL Associated Factors (EAF) family of conserved proteins are required for efficient RNA polymerase II-mediated transcription elongation. Orthologs of these proteins have been identified in different organisms, including fission yeast and humans. METHODS AND RESULTS In the present study, we have examined the sequence, structural and functional conservation between the fission yeast and human ELL and EAF orthologs. Our computational analysis revealed that these proteins share some sequence characteristics, and were predominantly disordered in both organisms. Our functional complementation assays revealed that both human ELL and EAF proteins could complement the lack of ell1+ or eaf1+ in Schizosaccharomyces pombe respectively. Furthermore, our domain mapping experiments demonstrated that both the amino and carboxyl terminal domains of human EAF proteins could functionally complement the S. pombe eaf1 deletion phenotypes. However, only the carboxyl-terminus domain of human ELL was able to partially rescue the phenotypes associated with lack of ell1+ in S. pombe. CONCLUSIONS Collectively, our work adds ELL-EAF to the increasing list of human-yeast complementation gene pairs, wherein the simpler fission yeast can be used to further enhance our understanding of the role of these proteins in transcription elongation and human disease.
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Dabas P, Dhingra Y, Sweta K, Chakrabarty M, Singhal R, Tyagi P, Behera PM, Dixit A, Bhattacharjee S, Sharma N. Arabidopsis thaliana possesses two novel ELL associated factor homologs. IUBMB Life 2021; 73:1115-1130. [PMID: 34089218 DOI: 10.1002/iub.2513] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 05/24/2021] [Accepted: 05/26/2021] [Indexed: 11/06/2022]
Abstract
Transcription elongation is one of the key steps at which RNA polymerase II-directed expression of protein-coding genes is regulated in eukaryotic cells. Different proteins have been shown to control this process, including the ELL/EAF family. ELL Associated Factors (EAFs) were first discovered in a yeast two-hybrid screen as interaction partners of the human ELL (Eleven nineteen Lysine-rich Leukemia) transcription elongation factor. Subsequently, they have been identified in different organisms, including Schizosaccharomyces pombe. However, no homolog(s) of EAF has as yet been characterized from plants. In the present work, we identified EAF orthologous sequences in different plants and have characterized two novel Arabidopsis thaliana EAF homologs, AtEAF-1 (At1g71080) and AtEAF-2 (At5g38050). Sequence analysis showed that both AtEAF-1 and AtEAF-2 exhibit similarity with its S. pombe EAF counterpart. Moreover, both Arabidopsis thaliana and S. pombe EAF orthologs share conserved sequence characteristic features. Computational tools also predicted a high degree of disorder in regions towards the carboxyl terminus of these EAF proteins. We demonstrate that AtEAF-2, but not AtEAF-1 functionally complements growth deficiencies of Schizosaccharomyces pombe eaf mutant. We also show that only AtEAF-1 displays transactivation potential resembling the S. pombe EAF ortholog. Subsequent expression analysis in A. thaliana showed that both homologs were expressed at varying levels during different developmental stages and in different tissues tested in the study. Individual null-mutants of either AtEAF-1 or AtEAF-2 are developmentally normal implying their functional redundancy. Taken together, our results provide first evidence that A. thaliana also possesses functional EAF proteins, suggesting an evolutionary conservation of these proteins across organisms.
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Affiliation(s)
- Preeti Dabas
- University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India
| | - Yukti Dhingra
- University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India
| | - Kumari Sweta
- University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India
| | - Mohima Chakrabarty
- University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India
| | - Ritwik Singhal
- University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India
| | - Prasidhi Tyagi
- University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India
| | | | | | - Saikat Bhattacharjee
- Laboratory of Signal Transduction and plant resistance, Regional Center of Biotechnology, NCR-Biotech Science Cluster, Gurgaon-Faridabad Expressway, Faridabad, Haryana, India
| | - Nimisha Sharma
- University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India
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13
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Takahashi S, Kanai A, Okuda H, Miyamoto R, Komata Y, Kawamura T, Matsui H, Inaba T, Takaori-Kondo A, Yokoyama A. HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis. eLife 2021; 10:e65872. [PMID: 34431785 PMCID: PMC8387021 DOI: 10.7554/elife.65872] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Accepted: 08/12/2021] [Indexed: 12/27/2022] Open
Abstract
Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.
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Affiliation(s)
- Satoshi Takahashi
- Tsuruoka Metabolomics Laboratory, National Cancer CenterTsuruokaJapan
- Department of Hematology and Oncology, Kyoto University Graduate School of MedicineKyotoJapan
| | - Akinori Kanai
- Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima UniversityHiroshimaJapan
| | - Hiroshi Okuda
- Tsuruoka Metabolomics Laboratory, National Cancer CenterTsuruokaJapan
| | - Ryo Miyamoto
- Tsuruoka Metabolomics Laboratory, National Cancer CenterTsuruokaJapan
| | - Yosuke Komata
- Tsuruoka Metabolomics Laboratory, National Cancer CenterTsuruokaJapan
| | | | - Hirotaka Matsui
- Department of Molecular Laboratory Medicine, Faculty of Life Sciences, Kumamoto UniversityKumamotoJapan
| | - Toshiya Inaba
- Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima UniversityHiroshimaJapan
| | - Akifumi Takaori-Kondo
- Department of Hematology and Oncology, Kyoto University Graduate School of MedicineKyotoJapan
| | - Akihiko Yokoyama
- Tsuruoka Metabolomics Laboratory, National Cancer CenterTsuruokaJapan
- Department of Hematology and Oncology, Kyoto University Graduate School of MedicineKyotoJapan
- Division of Hematological Malignancy, National Cancer Center Research InstituteTokyoJapan
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14
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Chen Y, Vos SM, Dienemann C, Ninov M, Urlaub H, Cramer P. Allosteric transcription stimulation by RNA polymerase II super elongation complex. Mol Cell 2021; 81:3386-3399.e10. [PMID: 34265249 DOI: 10.1016/j.molcel.2021.06.019] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2021] [Revised: 05/19/2021] [Accepted: 06/17/2021] [Indexed: 01/11/2023]
Abstract
The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.
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Affiliation(s)
- Ying Chen
- Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany
| | - Seychelle M Vos
- Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany
| | - Christian Dienemann
- Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany
| | - Momchil Ninov
- Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass Spectrometry, Am Fassberg 11, 37077 Göttingen, Germany; University Medical Center Göttingen, Institute of Clinical Chemistry, Bioanalytics Group, Robert-Koch-Straße 40, 37075 Göttingen, Germany
| | - Henning Urlaub
- Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass Spectrometry, Am Fassberg 11, 37077 Göttingen, Germany; University Medical Center Göttingen, Institute of Clinical Chemistry, Bioanalytics Group, Robert-Koch-Straße 40, 37075 Göttingen, Germany
| | - Patrick Cramer
- Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany.
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15
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Panagopoulos I, Andersen K, Eilert-Olsen M, Rognlien AG, Munthe-Kaas MC, Micci F, Heim S. Rare KMT2A-ELL and Novel ZNF56-KMT2A Fusion Genes in Pediatric T-cell Acute Lymphoblastic Leukemia. Cancer Genomics Proteomics 2021; 18:121-131. [PMID: 33608309 DOI: 10.21873/cgp.20247] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2020] [Revised: 01/20/2021] [Accepted: 01/25/2021] [Indexed: 12/28/2022] Open
Abstract
BACKGROUND/AIM Previous reports have associated the KMT2A-ELL fusion gene, generated by t(11;19)(q23;p13.1), with acute myeloid leukemia (AML). We herein report a KMT2A-ELL and a novel ZNF56-KMT2A fusion genes in a pediatric T-lineage acute lymphoblastic leukemia (T-ALL). MATERIALS AND METHODS Genetic investigations were performed on bone marrow of a 13-year-old boy diagnosed with T-ALL. RESULTS A KMT2A-ELL and a novel ZNF56-KMT2A fusion genes were generated on der(11)t(11;19)(q23;p13.1) and der(19)t(11;19)(q23;p13.1), respectively. Exon 20 of KMT2A fused to exon 2 of ELL in KMT2A-ELL chimeric transcript whereas exon 1 of ZNF56 fused to exon 21 of KMT2A in ZNF56-KMT2A transcript. A literature search revealed four more T-ALL patients carrying a KMT2A-ELL fusion. All of them were males aged 11, 11, 17, and 20 years. CONCLUSION KMT2A-ELL fusion is a rare recurrent genetic event in T-ALL with uncertain prognostic implications. The frequency and impact of ZNF56-KMT2A in T-ALL are unknown.
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Affiliation(s)
- Ioannis Panagopoulos
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway;
| | - Kristin Andersen
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Martine Eilert-Olsen
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Anne Gro Rognlien
- Department of Pediatric Hematology and Oncology, Oslo University Hospital Rikshospitalet, Oslo, Norway
| | - Monica Cheng Munthe-Kaas
- Department of Pediatric Hematology and Oncology, Oslo University Hospital Rikshospitalet, Oslo, Norway
| | - Francesca Micci
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Sverre Heim
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.,Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
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16
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Basu S, Nandy A, Biswas D. Keeping RNA polymerase II on the run: Functions of MLL fusion partners in transcriptional regulation. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2020; 1863:194563. [PMID: 32348849 DOI: 10.1016/j.bbagrm.2020.194563] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Revised: 01/13/2020] [Accepted: 04/13/2020] [Indexed: 12/21/2022]
Abstract
Since the identification of key MLL fusion partners as transcription elongation factors regulating expression of HOX cluster genes during hematopoiesis, extensive work from the last decade has resulted in significant progress in our overall mechanistic understanding of role of MLL fusion partner proteins in transcriptional regulation of diverse set of genes beyond just the HOX cluster. In this review, we are going to detail overall understanding of role of MLL fusion partner proteins in transcriptional regulation and thus provide mechanistic insights into possible MLL fusion protein-mediated transcriptional misregulation leading to aberrant hematopoiesis and leukemogenesis.
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Affiliation(s)
- Subham Basu
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 32, India
| | - Arijit Nandy
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India
| | - Debabrata Biswas
- Laboratory of Transcription Biology, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 32, India.
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17
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Multivalent Role of Human TFIID in Recruiting Elongation Components at the Promoter-Proximal Region for Transcriptional Control. Cell Rep 2020; 26:1303-1317.e7. [PMID: 30699356 DOI: 10.1016/j.celrep.2019.01.012] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2018] [Revised: 12/05/2018] [Accepted: 01/02/2019] [Indexed: 01/08/2023] Open
Abstract
Despite substantial progress in our understanding of the players involved and the regulatory mechanisms controlling the initiation and elongation steps of transcription, little is known about the recruitment of elongation factors at promoter-proximal regions for the initiation-to-elongation transition. Here, we show evidence that human TFIID, which initiates pre-initiation complex (PIC) assembly, contributes to regulating the recruitment of super-elongation complex (SEC) components at the promoter-proximal region through interactions among selective TAF and SEC components. In vitro direct interactions, coupled with cell-based assays, identified an important poly-Ser domain within SEC components that are involved in their interaction with TFIID. DNA template-based recruitment assays, using purified components, further show a direct role for poly-Ser domain-dependent TFIID interaction in recruiting SEC components on target DNA. Consistently, ChIP and RNA analyses have shown the importance of this mechanism in TFIID-dependent SEC recruitment and target gene expression within mammalian cells.
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18
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DBC1, p300, HDAC3, and Siah1 coordinately regulate ELL stability and function for expression of its target genes. Proc Natl Acad Sci U S A 2020; 117:6509-6520. [PMID: 32152128 PMCID: PMC7104407 DOI: 10.1073/pnas.1912375117] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Among all of the Super Elongation Complex (SEC) components, ELL1 (also known as ELL) is the only bona fide elongation factor that directly stimulates transcription elongation by RNA polymerase II. However, the mechanism(s) of functional regulation of ELL1 (referred to as ELL hereafter), through its stabilization, is completely unknown. Here, we report a function of human DBC1 in regulating ELL stability involving HDAC3, p300, and Siah1. Mechanistically, we show that p300-mediated site-specific acetylation increases, whereas HDAC3-mediated deacetylation decreases, ELL stability through polyubiquitylation by the E3 ubiquitin ligase Siah1. DBC1 competes with HDAC3 for the same binding sites on ELL and thus increases its acetylation and stability. Knockdown of DBC1 reduces ELL levels and expression of a significant number of genes, including those involved in glucose metabolism. Consistently, Type 2 diabetes patient-derived peripheral blood mononuclear cells show reduced expression of DBC1 and ELL and associated key target genes required for glucose homeostasis. Thus, we describe a pathway of regulating stability and functions of key elongation factor ELL for expression of diverse sets of genes, including ones that are linked to Type 2 diabetes pathogenesis.
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19
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ELL-associated factors EAF1/2 negatively regulate HIV-1 transcription through inhibition of Super Elongation Complex formation. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2020; 1863:194508. [PMID: 32087315 DOI: 10.1016/j.bbagrm.2020.194508] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/13/2019] [Revised: 02/16/2020] [Accepted: 02/16/2020] [Indexed: 01/03/2023]
Abstract
The ELL (ELL1 and ELL2)-containing Super Elongation Complex (SEC) is required for efficient HIV-1 transactivation by the viral-encoded Tat protein. EAF1 and EAF2 are ELL-associated factors and considered as positive regulators of ELL. However, their role in HIV-1 transcriptional control is unknown. In this study, we show that EAF1/2 inhibit the SEC-dependent and Tat-activated HIV-1 transcription. EAF1/2 are found to interact with the SEC components in an ELL1/2-dependent manner. Surprisingly, the depletion of EAF1/2 increases the SEC formation and occupancy on the HIV-1 proviral DNA, thereby stimulating Tat transactivation of HIV-1. Although EAF1/2 interact with members of the SEC in a ELL-dependent manner, this interaction competes with the binding of the scaffolding subunit AFF1 with ELL, thus reducing the SEC formation. Together, these data reveal how EAF1/2 regulate the SEC formation to control HIV-1 transcription.
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20
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The hunt for RNA polymerase II elongation factors: a historical perspective. Nat Struct Mol Biol 2019; 26:771-776. [PMID: 31439940 DOI: 10.1038/s41594-019-0283-1] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2019] [Accepted: 07/18/2019] [Indexed: 02/07/2023]
Abstract
The discovery of the three eukaryotic nuclear RNA polymerases paved the way for serious biochemical investigations of eukaryotic transcription and the identification of eukaryotic transcription factors. Here we describe this adventure from our vantage point, with a focus on the hunt for factors that regulate elongation by RNA polymerase II.
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21
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Pascal LE, Su F, Wang D, Ai J, Song Q, Wang Y, O'Malley KJ, Cross B, Rigatti LH, Green A, Dhir R, Wang Z. Conditional Deletion of Eaf1 Induces Murine Prostatic Intraepithelial Neoplasia in Mice. Neoplasia 2019; 21:752-764. [PMID: 31229879 PMCID: PMC6593215 DOI: 10.1016/j.neo.2019.05.005] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2019] [Revised: 05/15/2019] [Accepted: 05/20/2019] [Indexed: 01/03/2023]
Abstract
ELL-associated factor 1 is a transcription elongation factor that shares significant homology and functional similarity to the androgen-responsive prostate tumor suppressor ELL-associated factor 2. EAF2 is frequently down-regulated in advanced prostate cancer and Eaf2 deletion in the mouse induced the development of murine prostatic intraepithelial neoplasia. Here we show that similar to EAF2, EAF1 is frequently down-regulated in advanced prostate cancer. Co-downregulation of EAF1 and EAF2 occurred in 40% of clinical specimens with Gleason score >7. We developed and characterized a murine model of prostate-epithelial specific deletion of Eaf1 in the prostate and crossed it with our previously generated mouse with conventional deletion of Eaf2. The prostates of Eaf1 deletion mice displayed murine prostatic intraepithelial neoplasia lesions with increased proliferation and inflammation. Combined deletion of Eaf1 and Eaf2 in the murine model induced an increased incidence in mPIN lesions characterized by increased proliferation and CD3+ T cells and CD19+ B cells infiltration compared to individual deletion of either Eaf1 or Eaf2 in the murine prostate. These results suggest that EAF1 may play a tumor suppressive role in the prostate. Cooperation between EAF1 and EAF2 may be important for prostate maintaining prostate epithelial homeostasis, and concurrent loss of these two tumor suppressors may promote prostate tumorigenesis and progression.
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Affiliation(s)
- Laura E Pascal
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA
| | - Fei Su
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA; The Center for Metabolic and Degenerative Diseases, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030
| | - Dan Wang
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA
| | - Junkui Ai
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA
| | - Qiong Song
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA; Key Laboratory of Longevity and Aging-related Diseases of Chinese Ministry of Education, Center for Translational Medicine & School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi 530021, China
| | - Yujuan Wang
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA
| | - Katherine J O'Malley
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA
| | - Brian Cross
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA
| | - Lora H Rigatti
- Division of Laboratory Animal Resources, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Anthony Green
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Rajiv Dhir
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Zhou Wang
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA; Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
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22
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Gopalan S, Gibbon DM, Banks CA, Zhang Y, Florens LA, Washburn MP, Dabas P, Sharma N, Seidel CW, Conaway RC, Conaway JW. Schizosaccharomyces pombe Pol II transcription elongation factor ELL functions as part of a rudimentary super elongation complex. Nucleic Acids Res 2019; 46:10095-10105. [PMID: 30102332 PMCID: PMC6212713 DOI: 10.1093/nar/gky713] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2018] [Accepted: 07/26/2018] [Indexed: 12/21/2022] Open
Abstract
ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including Schizosaccharomyces pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.
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Affiliation(s)
- Sneha Gopalan
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA.,The Open University, Milton Keynes, UK
| | - Dana M Gibbon
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Charles As Banks
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Ying Zhang
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | | | - Michael P Washburn
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA.,Department of Pathology and Laboratory Med icine, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Preeti Dabas
- University School of Biotechnology, G.G.S.Indraprastha University, New Delhi 110078, India
| | - Nimisha Sharma
- University School of Biotechnology, G.G.S.Indraprastha University, New Delhi 110078, India
| | | | - Ronald C Conaway
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA.,Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Joan W Conaway
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA.,Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA
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23
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Candidate Tumor Suppressor Gene EAF2 is Mutated in Colorectal and Gastric Cancers. Pathol Oncol Res 2018; 25:823-824. [DOI: 10.1007/s12253-018-0461-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2016] [Accepted: 08/01/2018] [Indexed: 12/29/2022]
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24
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Liu JX, Xu QH, Yu X, Zhang T, Xie X, Ouyang G. Eaf1 and Eaf2 mediate zebrafish dorsal-ventral axis patterning via suppressing Wnt/β-Catenin activity. Int J Biol Sci 2018; 14:705-716. [PMID: 29910681 PMCID: PMC6001683 DOI: 10.7150/ijbs.18997] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2016] [Accepted: 03/17/2018] [Indexed: 01/02/2023] Open
Abstract
During early vertebrate embryogenesis, maternal Wnt/β-catenin signaling is thought to locally initiate expression of dorsal-specific genes. Here, eaf1 and eaf2 were identified as important maternal and zygotic modulators of Wnt signaling to initiate and specify ventral genes. Expression of ventral ved, vent, and vox was all obviously enhanced in either maternal or zygotic eaf1/2 morphants, and in both eaf1 heterozygous and homozygous mutants, but their expression was suppressed in embryos with over-expression of eaf1/2. Additionally, eaf1/2 were revealed to suppress ventral fates in embryos via Wnt/β-catenin1/Tcf signaling, complimentary to their roles in suppressing dorsal fates via Wnt/β-catenin2 signaling. Moreover, eaf1/2 were also revealed to obviously suppress the expression of axin2 induced by β-catenin2 rather than by β-catenin1, and the dorsal expression of axin2 in embryos was obviously suppressed by ectopic expression of eaf1/2. This study uncovers a novel dorsal-ventral patterning pathway, with eaf1 and eaf2 inhibiting ventral cells via suppressing Wnt/β-catenin1/Tcf signaling and inducing dorsal cells indirectly via suppressing β-catenin2-induced-axin2 on the dorsal side of embryos.
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Affiliation(s)
- Jing-Xia Liu
- College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, P. R. China.,Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China
| | - Qin-Han Xu
- College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, P. R. China
| | - XueDong Yu
- College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, P. R. China
| | - Ting Zhang
- College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, P. R. China
| | - XunWei Xie
- Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China
| | - Gang Ouyang
- Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China
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25
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EZH2 promotes metabolic reprogramming in glioblastomas through epigenetic repression of EAF2-HIF1α signaling. Oncotarget 2018; 7:45134-45143. [PMID: 27259264 PMCID: PMC5216711 DOI: 10.18632/oncotarget.9761] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2015] [Accepted: 05/13/2016] [Indexed: 12/12/2022] Open
Abstract
Cancer cells prefer glycolysis for energy metabolism, even when there is sufficient oxygen to make it unnecessary. This is called the Warburg effect, and it promotes tumorigenesis and malignant progression. In this study, we demonstrated that EZH2, a multifaceted oncogenic protein involved in tumor proliferation, invasion and metastasis, promotes glioblastoma tumorigenesis and malignant progression through activation of the Warburg effect. We observed that HIF1α is a target of EZH2 whose activation is necessary for EZH2-mediated metabolic adaption, and that HIF1α is activated upon EZH2 overexpression. EZH2 suppressed expression of EAF2, which in turn upregulated HIF1α levels. We conclude from these results that EZH2 promotes tumorigenesis and malignant progression in part by activating glycolysis through an EAF2-HIF1α signaling axis.
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26
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Feng K, Guo HK. Eaf2 protects human lens epithelial cells against oxidative stress‑induced apoptosis by Wnt signaling. Mol Med Rep 2017; 17:2795-2802. [PMID: 29257273 PMCID: PMC5783493 DOI: 10.3892/mmr.2017.8246] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2016] [Accepted: 04/24/2017] [Indexed: 01/11/2023] Open
Abstract
The tumor suppressor protein ELL-associated factor 2 (Eaf2) serves an important role in lens development and maturation; however, its role in oxidative stress-induced cataract formation remains unclear. In the present study, an in vitro apoptosis model was constructed by treating HLE-B3 cells with 50 µM hydrogen peroxide (H2O2), and was confirmed by flow cytometry. Subsequently, overexpression of Eaf2 was induced in H2O2-induced HLE-B3 cells by ligating Eaf2 cDNA to a pcDNA3.0 plasmid and the role of Wnt3a in the function of Eaf2 was also assessed by inhibiting the expression of the gene in Eaf2-overexpression cells. The expression levels of glycogen synthase kinase 3β, β-catenin, Eaf2, caspase 3, Wnt3a, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein were examined using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Immunocytochemistry was used to locate Eaf2 and Wnt3 protein expression in the H2O2-induced HLE-B3 cells. The results indicated that Eaf2 was able to effectively suppress H2O2-induced apoptosis of HLE cells via inhibition of caspase 3 production and activation of Wnt3a signaling. In addition, knockdown of Wnt3a in Eaf2-overexpression cells evidently counteracted the effect of Eaf2 in antagonizing H2O2-induced apoptosis. Taken together, these findings suggested that Eaf2 may suppress oxidative stress-induced apoptosis of HLE-B3 cells exerted through the activation of Wnt3a signaling.
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Affiliation(s)
- Ke Feng
- Department of Ophthalmology, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China
| | - Hai-Ke Guo
- Department of Ophthalmology, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China
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Wang Y, Pascal LE, Zhong M, Ai J, Wang D, Jing Y, Pilch J, Song Q, Rigatti LH, Graham LE, Nelson JB, Parwani AV, Wang Z. Combined Loss of EAF2 and p53 Induces Prostate Carcinogenesis in Male Mice. Endocrinology 2017; 158:4189-4205. [PMID: 29029019 PMCID: PMC5711381 DOI: 10.1210/en.2017-00409] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2017] [Accepted: 09/13/2017] [Indexed: 01/03/2023]
Abstract
Mutations in the p53 tumor suppressor are frequent in patients with castration-resistant prostate cancer but less so in patients with localized disease, and patients who have Li-Fraumeni with germline p53 mutations do not have an increased incidence of prostate cancer, suggesting that additional molecular and/or genetic changes are required for p53 to promote prostate carcinogenesis. ELL-associated factor 2 (EAF2) is a tumor suppressor that is frequently downregulated in advanced prostate cancer. Previous studies have suggested that p53 binds to EAF2, providing a potential mechanism for their functional interactions. In this study, we tested whether p53 and EAF2 could functionally interact in prostate cancer cells and whether concurrent inactivation of p53 and EAF2 could promote prostate carcinogenesis in a murine knockout model. Endogenous p53 coprecipitated with EAF2 in prostate cancer cells, and deletion mutagenesis indicated that this interaction was mediated through the C terminus of EAF2 and the DNA binding domain of p53. Concurrent knockdown of p53 and EAF2 induced an increase in proliferation and migration in cultured prostate cancer cells, and conventional p53 and EAF2 knockout mice developed prostate cancer. In human prostate cancer specimens, concurrent p53 nuclear staining and EAF2 downregulation was associated with high Gleason score. These findings suggest that EAF2 and p53 functionally interact in prostate tumor suppression and that simultaneous inactivation of EAF2 and p53 can drive prostate carcinogenesis.
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Affiliation(s)
- Yao Wang
- Department of Urology, China-Japan Union Hospital of Jilin University, China
- Department of Urology, University of Pittsburgh School of Medicine
| | - Laura E Pascal
- Department of Urology, University of Pittsburgh School of Medicine
| | - Mingming Zhong
- Department of Urology, University of Pittsburgh School of Medicine
| | - Junkui Ai
- Department of Urology, University of Pittsburgh School of Medicine
| | - Dan Wang
- Department of Urology, University of Pittsburgh School of Medicine
| | - Yifeng Jing
- Department of Urology, University of Pittsburgh School of Medicine
- Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, China
| | - Jan Pilch
- Saarland University Medical Center, Institute of Clinical Hemostaseology and Transfusion Medicine, Germany
| | - Qiong Song
- Department of Urology, University of Pittsburgh School of Medicine
- Center for Translational Medicine, Guangxi Medical University, China
| | - Lora H Rigatti
- Division of Laboratory Animal Resources, University of Pittsburgh School of Medicine
| | - Lara E Graham
- Department of Urology, University of Pittsburgh School of Medicine
| | - Joel B Nelson
- Department of Urology, University of Pittsburgh School of Medicine
| | - Anil V Parwani
- Department of Pathology, University of Pittsburgh School of Medicine
| | - Zhou Wang
- Department of Urology, University of Pittsburgh School of Medicine
- Department of Pathology, University of Pittsburgh School of Medicine
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine
- University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine
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Zang Y, Pascal LE, Zhou Y, Qiu X, Wei L, Ai J, Nelson JB, Zhong M, Xue B, Wang S, Yang D, Lan L, Shan Y, Wang Z. ELL2 regulates DNA non-homologous end joining (NHEJ) repair in prostate cancer cells. Cancer Lett 2017; 415:198-207. [PMID: 29179998 DOI: 10.1016/j.canlet.2017.11.028] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2017] [Revised: 11/21/2017] [Accepted: 11/22/2017] [Indexed: 12/12/2022]
Abstract
ELL2 is an androgen-responsive gene that is expressed by prostate epithelial cells and is frequently down-regulated in prostate cancer. Deletion of Ell2 in the murine prostate induced murine prostatic intraepithelial neoplasia and ELL2 knockdown enhanced proliferation and migration in C4-2 prostate cancer cells. Here, knockdown of ELL2 sensitized prostate cancer cells to DNA damage and overexpression of ELL2 protected prostate cancer cells from DNA damage. Knockdown of ELL2 impaired non-homologous end joining repair but not homologous recombination repair. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. ELL2 could bind to and co-accumulate with Ku70/Ku80 proteins at sites of DNA damage. Knockdown of ELL2 dramatically inhibited Ku70 and Ku80 recruitment and retention at DNA double-strand break sites in prostate cancer cells. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2. This study suggests that ELL2 is required for efficient NHEJ repair via Ku70/Ku80 in prostate cancer cells.
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Affiliation(s)
- Yachen Zang
- Department of Urology, The Second Affiliated Hospital of Soochow University, Suzhou, China; Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA.
| | - Laura E Pascal
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA.
| | - Yibin Zhou
- Department of Urology, The Second Affiliated Hospital of Soochow University, Suzhou, China; Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA.
| | - Xiaonan Qiu
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA.
| | - Leizhen Wei
- University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213, USA; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
| | - Junkui Ai
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA.
| | - Joel B Nelson
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA; University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213, USA.
| | - Mingming Zhong
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA.
| | - Boxin Xue
- Department of Urology, The Second Affiliated Hospital of Soochow University, Suzhou, China.
| | - Shaoxiong Wang
- Department of Urology, The Second Affiliated Hospital of Soochow University, Suzhou, China.
| | - Dongrong Yang
- Department of Urology, The Second Affiliated Hospital of Soochow University, Suzhou, China.
| | - Li Lan
- University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213, USA; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
| | - Yuxi Shan
- Department of Urology, The Second Affiliated Hospital of Soochow University, Suzhou, China.
| | - Zhou Wang
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, USA; University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15216, USA.
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Pascal LE, Masoodi KZ, Liu J, Qiu X, Song Q, Wang Y, Zang Y, Yang T, Wang Y, Rigatti LH, Chandran U, Colli LM, Vencio RZN, Lu Y, Zhang J, Wang Z. Conditional deletion of ELL2 induces murine prostate intraepithelial neoplasia. J Endocrinol 2017; 235:123-136. [PMID: 28870994 PMCID: PMC5679084 DOI: 10.1530/joe-17-0112] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/18/2017] [Accepted: 08/04/2017] [Indexed: 12/19/2022]
Abstract
Elongation factor, RNA polymerase II, 2 (ELL2) is an RNA Pol II elongation factor with functional properties similar to ELL that can interact with the prostate tumor suppressor EAF2. In the prostate, ELL2 is an androgen response gene that is upregulated in benign prostatic hyperplasia (BPH). We recently showed that ELL2 loss could enhance prostate cancer cell proliferation and migration, and that ELL2 gene expression was downregulated in high Gleason score prostate cancer specimens. Here, prostate-specific deletion of ELL2 in a mouse model revealed a potential role for ELL2 as a prostate tumor suppressor in vivoEll2-knockout mice exhibited prostatic defects including increased epithelial proliferation, vascularity and PIN lesions similar to the previously determined prostate phenotype in Eaf2-knockout mice. Microarray analysis of prostates from Ell2-knockout and wild-type mice on a C57BL/6J background at age 3 months and qPCR validation at 17 months of age revealed a number of differentially expressed genes associated with proliferation, cellular motility and epithelial and neural differentiation. OncoPrint analysis identified combined downregulation or deletion in prostate adenocarcinoma cases from the Cancer Genome Atlas (TCGA) data portal. These results suggest that ELL2 and its pathway genes likely play an important role in the development and progression of prostate cancer.
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Affiliation(s)
- Laura E Pascal
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Khalid Z Masoodi
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Transcriptomics LabDivision of Plant Biotechnology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, Jammu and Kashmir, India
| | - June Liu
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Xiaonan Qiu
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- School of MedicineTsinghua University, Beijing, China
| | - Qiong Song
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Center for Translational MedicineGuangxi Medical University, Nanning, Guangxi, China
| | - Yujuan Wang
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Yachen Zang
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of UrologyThe Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Tiejun Yang
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of UrologyHenan Cancer Hospital, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, China
| | - Yao Wang
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of UrologyChina-Japan Hospital of Jilin University, Changchun, Jilin, China
| | - Lora H Rigatti
- Division of Laboratory Animal ResourcesUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Uma Chandran
- Department of Biomedical InformaticsUniversity of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Leandro M Colli
- Ribeirao Preto Medical SchoolUniversity of São Paulo, Ribeirão Preto-SP, Brazil
| | - Ricardo Z N Vencio
- Department of Computing and Mathematics FFCLRP-USPUniversity of São Paulo, Ribeirão Preto, Brazil
| | - Yi Lu
- Key Laboratory of Longevity and Aging-related DiseasesMinistry of Education, China and Center for Translational Medicine Guangxi Medical University, Nanning, Guangxi, China
- Department of BiologySouthern University of Science and Technology School of Medicine, Shenzhen, Guangdong, China
| | - Jian Zhang
- Key Laboratory of Longevity and Aging-related DiseasesMinistry of Education, China and Center for Translational Medicine Guangxi Medical University, Nanning, Guangxi, China
- Department of BiologySouthern University of Science and Technology School of Medicine, Shenzhen, Guangdong, China
| | - Zhou Wang
- Department of UrologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- University of Pittsburgh Cancer InstituteUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Pharmacology and Chemical BiologyUniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
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30
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Sweta K, Dabas P, Jain K, Sharma N. The amino-terminal domain of ELL transcription elongation factor is essential for ELL function in Schizosaccharomyces pombe. MICROBIOLOGY-SGM 2017; 163:1641-1653. [PMID: 29043956 DOI: 10.1099/mic.0.000554] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Transcriptional elongation is a critical step for regulating expression of protein-coding genes. Multiple transcription elongation factors have been identified in vitro, but the physiological roles of many of them are still not clearly understood. The ELL (Eleven nineteen Lysine rich Leukemia) family of transcription elongation factors are conserved from fission yeast to humans. Schizosaccharomyces pombe contains a single ELL homolog (SpELL) that is not essential for its survival. Therefore to gain insights into the in vivo cellular functions of SpELL, we identified phenotypes associated with deletion of ell1 in S. pombe. Our results demonstrate that SpELL is required for normal growth of S. pombe cells. Furthermore, cells lacking ell1+ exhibit a decrease in survival when exposed to DNA-damaging conditions, but their growth is not affected under environmental stress conditions. ELL orthologs in different organisms contain three conserved domains, an amino-terminal domain, a middle domain and a carboxyl-terminal domain. We also carried out an in vivo functional mapping of these conserved domains within S. pombe ELL and uncovered a critical role for its amino-terminus in regulating all its cellular functions, including growth under different conditions, transcriptional elongation potential and interaction with S. pombe EAF. Taken together our results suggest that the domain organization of ELL proteins is conserved across species, but the in vivo functions as well as the relationship between the various domains and roles of ELL show species-specific differences.
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Affiliation(s)
- Kumari Sweta
- University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi-110078, India
| | - Preeti Dabas
- University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi-110078, India
| | - Kamal Jain
- University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi-110078, India
| | - Nimisha Sharma
- University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi-110078, India
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31
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Dabas P, Sweta K, Ekka M, Sharma N. Structure function characterization of the ELL Associated Factor (EAF) from Schizosaccharomyces pombe. Gene 2017; 641:117-128. [PMID: 29032152 DOI: 10.1016/j.gene.2017.10.031] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2017] [Revised: 10/07/2017] [Accepted: 10/11/2017] [Indexed: 01/03/2023]
Abstract
EAF (ELL Associated Factor) proteins interact with the transcription elongation factor, ELL (Eleven nineteen Lysine rich Leukemia) and enhance its ability to stimulate RNA polymerase II-mediated transcriptional elongation in vitro. Schizosaccharomyces pombe contains a single homolog of EAF (SpEAF), which is not essential for survival of S. pombe in contrast to its essential higher eukaryotic homologs. The physiological role of SpEAF is not well understood. In this study, we show that S. pombe EAF is important in regulating growth of S. pombe cells during normal growth conditions. Moreover, SpEAF is also essential for survival under conditions of DNA damage, while its deletion does not affect growth under environmental stress conditions. Our in vivo structure-function studies further demonstrate that while both the amino and carboxyl terminal domains of SpEAF possess the potential to activate transcription, only the amino terminal domain of SpEAF is involved in interaction with the S. pombe ELL protein. The carboxyl-terminus of SpEAF is required for rescue of the growth defect under normal and DNA damaging conditions that is associated with the absence of SpEAF. Using bioinformatics and circular dichroism spectroscopy, we show that the carboxyl-terminus of SpEAF has a disordered conformation. Furthermore, addition of trifluoroethanol triggered its transition from a disordered to α-helical conformation. Taken together, the results presented here identify novel structural and functional features of SpEAF protein, providing insights into how EAF proteins may enforce transcriptional control of gene expression.
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Affiliation(s)
- Preeti Dabas
- University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi 110078, India
| | - Kumari Sweta
- University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi 110078, India
| | - Mary Ekka
- CSIR-Institute of Genomics and Integrative Biology, Mathura Road, Opp. Sukhdev Vihar Bus Depot, New Delhi, Delhi 110025, India
| | - Nimisha Sharma
- University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi 110078, India.
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Liu JX, Xu QH, Li S, Yu X, Liu W, Ouyang G, Zhang T, Chen LL. Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm formation via suppressing TGF-β signaling. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2017; 1860:1103-1116. [DOI: 10.1016/j.bbagrm.2017.09.001] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/04/2017] [Revised: 09/02/2017] [Accepted: 09/03/2017] [Indexed: 01/11/2023]
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Qiu X, Pascal LE, Song Q, Zang Y, Ai J, O'Malley KJ, Nelson JB, Wang Z. Physical and Functional Interactions between ELL2 and RB in the Suppression of Prostate Cancer Cell Proliferation, Migration, and Invasion. Neoplasia 2017; 19:207-215. [PMID: 28167296 PMCID: PMC5293724 DOI: 10.1016/j.neo.2017.01.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2016] [Revised: 12/22/2016] [Accepted: 01/02/2017] [Indexed: 12/24/2022] Open
Abstract
Elongation factor, RNA polymerase II, 2 (ELL2) is expressed and regulated by androgens in the prostate. ELL2 and ELL-associated factor 2 (EAF2) form a stable complex, and their orthologs in Caenorhabditis elegans appear to be functionally similar. In C. elegans, the EAF2 ortholog eaf-1 was reported to interact with the retinoblastoma (RB) pathway to control development and fertility in worms. Because RB loss is frequent in prostate cancer, ELL2 interaction with RB might be important for prostate homeostasis. The present study explored physical and functional interaction of ELL2 with RB in prostate cancer. ELL2 expression in human prostate cancer specimens was detected using quantitative polymerase chain reaction coupled with laser capture microdissection. Co-immunoprecipitation coupled with deletion mutagenesis was used to determine ELL2 association with RB. Functional interaction between ELL2 and RB was tested using siRNA knockdown, BrdU incorporation, Transwell, and/or invasion assays in LNCaP, C4-2, and 22Rv1 prostate cancer cells. ELL2 expression was downregulated in high-Gleason score prostate cancer specimens. ELL2 could be bound and stabilized by RB, and this interaction was mediated through the N-terminus of ELL2 and the C-terminus of RB. Concurrent siRNA knockdown of ELL2 and RB enhanced cell proliferation, migration, and invasion as compared to knockdown of ELL2 or RB alone in prostate cancer cells. ELL2 and RB can interact physically and functionally to suppress prostate cancer progression.
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Affiliation(s)
- Xiaonan Qiu
- Tsinghua MD Program, School of Medicine, Tsinghua University, Beijing, China; Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
| | - Laura E Pascal
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
| | - Qiong Song
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Center for Translational Medicine, Guangxi Medical University, Nanning, Guangxi, China.
| | - Yachen Zang
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Urology, The Second Affiliate Hospital of Soochow University, Suzhou, China.
| | - Junkui Ai
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
| | - Katherine J O'Malley
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
| | - Joel B Nelson
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
| | - Zhou Wang
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Center for Translational Medicine, Guangxi Medical University, Nanning, Guangxi, China; University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, and University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
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Lin S, Luo RT, Ptasinska A, Kerry J, Assi SA, Wunderlich M, Imamura T, Kaberlein JJ, Rayes A, Althoff MJ, Anastasi J, O'Brien MM, Meetei AR, Milne TA, Bonifer C, Mulloy JC, Thirman MJ. Instructive Role of MLL-Fusion Proteins Revealed by a Model of t(4;11) Pro-B Acute Lymphoblastic Leukemia. Cancer Cell 2016; 30:737-749. [PMID: 27846391 DOI: 10.1016/j.ccell.2016.10.008] [Citation(s) in RCA: 85] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Revised: 04/21/2016] [Accepted: 10/12/2016] [Indexed: 01/11/2023]
Abstract
The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34+ cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.
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Affiliation(s)
- Shan Lin
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Roger T Luo
- Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA
| | - Anetta Ptasinska
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B15 2TT, UK
| | - Jon Kerry
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford OX3 9DS, UK
| | - Salam A Assi
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B15 2TT, UK
| | - Mark Wunderlich
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Toshihiko Imamura
- Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA
| | - Joseph J Kaberlein
- Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA
| | - Ahmad Rayes
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Mark J Althoff
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - John Anastasi
- Department of Pathology, University of Chicago, Chicago, IL 60637, USA
| | - Maureen M O'Brien
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Amom Ruhikanta Meetei
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Thomas A Milne
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford OX3 9DS, UK
| | - Constanze Bonifer
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B15 2TT, UK
| | - James C Mulloy
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
| | - Michael J Thirman
- Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA.
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Sharma N. Regulation of RNA polymerase II-mediated transcriptional elongation: Implications in human disease. IUBMB Life 2016; 68:709-16. [DOI: 10.1002/iub.1538] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2016] [Accepted: 07/14/2016] [Indexed: 01/03/2023]
Affiliation(s)
- Nimisha Sharma
- University School of Biotechnology, G.G.S. Indraprastha University; Dwarka New Delhi 110078 India
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36
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Knutson BA, Smith ML, Walker-Kopp N, Xu X. Super elongation complex contains a TFIIF-related subcomplex. Transcription 2016; 7:133-40. [PMID: 27223670 DOI: 10.1080/21541264.2016.1194027] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Super elongation complex (SEC) belongs to a family of RNA polymerase II (Pol II) elongation factors that has similar properties as TFIIF, a general transcription factor that increases the transcription elongation rate by reducing pausing. Although SEC has TFIIF-like functional properties, it apparently lacks sequence and structural homology. Using HHpred, we find that SEC contains an evolutionarily related TFIIF-like subcomplex. We show that the SEC subunit ELL interacts with the Pol II Rbp2 subunit, as expected for a TFIIF-like factor. These findings suggest a new model for how SEC functions as a Pol II elongation factor and how it suppresses Pol II pausing.
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Affiliation(s)
- Bruce A Knutson
- a Department of Biochemistry and Molecular Biology , SUNY Upstate Medical University , Syracuse , NY , USA
| | - Marissa L Smith
- a Department of Biochemistry and Molecular Biology , SUNY Upstate Medical University , Syracuse , NY , USA
| | - Nancy Walker-Kopp
- a Department of Biochemistry and Molecular Biology , SUNY Upstate Medical University , Syracuse , NY , USA
| | - Xia Xu
- a Department of Biochemistry and Molecular Biology , SUNY Upstate Medical University , Syracuse , NY , USA
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EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity. Nat Commun 2016; 7:10836. [PMID: 26935903 PMCID: PMC4782062 DOI: 10.1038/ncomms10836] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2015] [Accepted: 01/25/2016] [Indexed: 01/03/2023] Open
Abstract
Regulated apoptosis of germinal centre (GC) B cells is critical for normal humoral immune responses. ELL-associated factor 2 (EAF2) regulates transcription elongation and has been shown to be an androgen-responsive potential tumour suppressor in prostate by inducing apoptosis. Here we show that EAF2 is selectively upregulated in GC B cells among various immune cell types and promotes apoptosis of GC B cells both in vitro and in vivo. EAF2 deficiency results in enlarged GCs and elevated antibody production during a T-dependent immune response. After immunization with type II collagen, mice lacking EAF2 produce high levels of collagen-specific autoantibodies and rapidly develop severe arthritis. Moreover, the mutant mice spontaneously produce anti-dsDNA, rheumatoid factor and anti-nuclear antibodies as they age. These results demonstrate that EAF2-mediated apoptosis in GC B cells limits excessive humoral immune responses and is important for maintaining self-tolerance.
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Liu X, Chen Z, Ouyang G, Song T, Liang H, Liu W, Xiao W. ELL Protein-associated Factor 2 (EAF2) Inhibits Transforming Growth Factor β Signaling through a Direct Interaction with Smad3. J Biol Chem 2015; 290:25933-45. [PMID: 26370086 DOI: 10.1074/jbc.m115.663542] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2015] [Indexed: 12/29/2022] Open
Abstract
A series of in vitro and in vivo studies has shown that EAF2 can affect multiple signaling pathways involved in cellular processes. However, the molecular mechanisms underlying its effects have remained elusive. Here we report the discovery of a new functional link between EAF2 and TGF-β signaling. Promoter reporter assays indicated that EAF2 suppresses Smad3 transcriptional activity, resulting in inhibition of TGF-β signaling. Coimmunoprecipitation assays showed that EAF2 specifically interacts with Smad3 in vitro and in vivo but not with other Smad proteins. In addition, we observed that EAF2 binding does not alter Smad3 phosphorylation but causes Smad3 cytoplasmic retention, competes with Smad4 for binding to Smad3, and prevents p300-Smad3 complex formation. Furthermore, we demonstrated that EAF2 suppresses both TGF-β-induced G1 cell cycle arrest and TGF-β-induced cell migration. This study identifies and characterizes a novel repressor of TGF-β signaling.
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Affiliation(s)
- Xing Liu
- From the Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
| | - Zhu Chen
- From the Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China, Department of Reproduction, Maternal and Child Health Hospital of Hubei Province, Wuhan 430070, China
| | - Gang Ouyang
- From the Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
| | - Tieshan Song
- Hubei University of Science and Technology, Xianning 437100, China, and
| | - Huageng Liang
- Department of Urology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
| | - Wei Liu
- From the Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
| | - Wuhan Xiao
- From the Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China,
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Zebrafish eaf1 suppresses foxo3b expression to modulate transcriptional activity of gata1 and spi1 in primitive hematopoiesis. Dev Biol 2014; 388:81-93. [DOI: 10.1016/j.ydbio.2014.01.005] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2013] [Revised: 12/13/2013] [Accepted: 01/11/2014] [Indexed: 12/28/2022]
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Qiao Z, Wang D, Hahn J, Ai J, Wang Z. Pirin down-regulates the EAF2/U19 protein and alleviates its growth inhibition in prostate cancer cells. Prostate 2014; 74:113-20. [PMID: 24272884 PMCID: PMC4827099 DOI: 10.1002/pros.22729] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/04/2013] [Accepted: 08/20/2013] [Indexed: 12/14/2022]
Abstract
BACKGROUND The tumor suppressor ELL associated factor 2 (EAF2/U19) has been reported to induce apoptosis of LNCaP cells and suppress AT6.1 xenograft prostate tumor growth. EAF2/U19 expression level is down-regulated in advanced human prostate cancer. EAF2/U19 is also a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Identification of binding partners and regulators of EAF2/U19 is essential to understand its function in regulating apoptosis/survival of prostate cancer cells. METHODS Through a yeast two-hybrid screening system, we identified Pirin as a binding partner of EAF2. We further determined the interaction between epitope-tagged EAF2/U19 and Pirin by co-immunoprecipitation in mammalian cells. The effect of Pirin on EAF2/U19 inhibition of LNCaP growth was assayed by colony formation. RESULTS Pirin co-immunoprecipitated with EAF2/U19 and the overexpressed Pirin decreased the expression level of EAF2/U19 protein in prostate cancer cell lines LNCaP and PC3. Furthermore, overexpression of EAF2/U19 suppressed LNCaP colony formation, and co-expression of Pirin significantly blocked the growth inhibition induced by EAF2/U19 overexpression. CONCLUSION Pirin is a newly identified binding partner of EAF2/U19 capable of down-regulating EAF2/U19 protein and alleviating its inhibition of prostate cancer cell survival/proliferation. Pirin may play an important role involved in EAF2/U19 function as an androgen-responsive gene and tumor repressor.
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Affiliation(s)
- Zhongjie Qiao
- Department of Urology, The 3rd Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China
- Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Dan Wang
- Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Junghyun Hahn
- Department of Urology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
| | - Junkui Ai
- Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Zhou Wang
- Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania
- Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania
- University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
- Correspondence to: Zhou Wang, PhD, Department of Urology, Shadyside Medical Center, Suite G40, 5200 Centre Avenue, Pittsburgh, PA 15232.
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EAF2 suppresses hypoxia-induced factor 1α transcriptional activity by disrupting its interaction with coactivator CBP/p300. Mol Cell Biol 2014; 34:1085-99. [PMID: 24421387 DOI: 10.1128/mcb.00718-13] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Previous studies revealed that the potential tumor suppressor EAF2 binds to and stabilizes pVHL, suggesting that EAF2 may function by disturbing the hypoxia signaling pathway. However, the extent to which EAF2 affects hypoxia and the mechanisms underlying this activity remain largely unknown. In this study, we found that EAF2 is a hypoxia response gene harboring the hypoxia response element (HRE) in its promoter. By taking advantage of the pVHL-null cell lines RCC4 and 786-O, we demonstrated that hypoxia-induced factor 1α (HIF-1α), but not HIF-2α, induced EAF2 under hypoxia. Subsequent experiments showed that EAF2 bound to and suppressed HIF-1α but not HIF-2α transactivity. In addition, we observed that EAF2 inhibition of HIF-1α activity resulted from the disruption of p300 recruitment and that this occurred independently of FIH-1 (factor inhibiting HIF-1) and Sirt1. Furthermore, we found that EAF2 protected cells against hypoxia-induced cell death and inhibited cellular uptake of glucose under hypoxic conditions, suggesting that EAF2 indeed may act by modulating the hypoxia-signaling pathway. Our findings not only uncover a unique feedback regulation loop between EAF2 and HIF-1α but also provide a novel insight into the mechanism of EAF2 tumor suppression.
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Ma X, Liu JX. Eafs control erythroid cell fate by regulating c-myb expression through Wnt signaling. PLoS One 2013; 8:e64576. [PMID: 23717633 PMCID: PMC3661582 DOI: 10.1371/journal.pone.0064576] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2012] [Accepted: 04/16/2013] [Indexed: 01/11/2023] Open
Abstract
ELL associated factor 1 and ELL associated factor 2 (EAF1/2 factors) are reported to play important roles in tumor suppression and embryogenesis. Our previous studies showed that eaf factors mediated effective convergence and extension (C&E) movements and modulated mesoderm and neural patterning by regulating both non-canonical and canonical Wnt signaling in the early embryonic process. In this study, through knockdown of both eaf1 and eaf2 in embryos, we found that differentiation of primary erythroid cells was blocked, but hematopoietic precursor cells maintained in eafs morphants. Co-injection of c-myb-MO rescued the erythroid differentiation in eafs morphants, as indicated by the restored expression of the erythroid-specific gene, βe3 globin. In addition, low dosage of c-myb effectively blocked the βe3 globin expression in embryos, and did not affect the expression of markers of hematopoietic progenitor cells and other mesoderm, which was similar to the phenotypes we observed in eafs morphants. We also revealed that knockdown Wnt signaling by transiently inducing dn-Tcf in embryos at the bud stage down-regulated the increased c-myb to normal level and also restored βe3 globin expression in eafs morphants. Our evidence points to a novel role for eaf factors in controlling erythroid cell fate by regulating c-Myb expression through canonic Wnt signaling.
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Affiliation(s)
- Xufa Ma
- College of Fisheries, Huazhong Agricultural University, Wuhan, P. R. China
| | - Jing-Xia Liu
- Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, P. R. China
- * E-mail:
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Su F, Correa BRS, Luo J, Vencio RZN, Pascal LE, Wang Z. Gene Expression Profiling Reveals Regulation of ERK Phosphorylation by Androgen-Induced Tumor Suppressor U19/EAF2 in the Mouse Prostate. CANCER MICROENVIRONMENT 2013; 6:247-61. [PMID: 23440596 DOI: 10.1007/s12307-013-0132-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/26/2012] [Accepted: 02/12/2013] [Indexed: 01/02/2023]
Abstract
U19/EAF2 is regulated by androgens in the prostate and capable of regulating transcriptional elongation of RNA Pol II via interaction with the ELL family proteins. Inactivation of U19/EAF2 induces tumorigenesis in multiple organs; however the mechanism of U19/EAF2 tumor suppression remains unclear. To elucidate potential mechanisms of U19/EAF2 action, we performed cDNA microarray analysis and identified 164 mRNA transcripts regulated by U19/EAF2 in the mouse ventral prostate. Bioinformatics analysis indicated that U19/EAF2 knockout activates the RAS-BRAF-ERK signaling pathway, which is known to play important roles in carcinogenesis. qPCR verified increased expression of BRAF mRNA, and immunostaining and Western blot analysis demonstrated increased expression of p-ERK at the protein level suggested U19/EAF2 knockout activates this important pathway. These findings indicate that loss of EAF2 up-regulates transcription of RAS cascade genes including Grb2, PI3K, and BRAF, leading to elevated p-ERK levels, which may represent a major functional role of U19/EAF2 in the prostate. Furthermore, these observations suggest that U19/EAF2 is a key player in crosstalk between androgen receptor and the RAS-BRAF-ERK signaling pathway.
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Affiliation(s)
- Fei Su
- Department of Urology, University of Pittsburgh, Pittsburgh, PA, 15232, USA
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Liu JX, Zhang D, Xie X, Ouyang G, Liu X, Sun Y, Xiao W. Eaf1 and Eaf2 negatively regulate canonical Wnt/β-catenin signaling. Development 2013; 140:1067-78. [PMID: 23364330 DOI: 10.1242/dev.086157] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Eaf factors play a crucial role in tumor suppression and embryogenesis. To investigate the potential mechanism of Eaf activity, we performed loss- and gain-of-function assays in zebrafish using morpholino and mRNA injections, respectively. We found that eaf1 and eaf2 inhibit Wnt/β-catenin signaling, thereby modulating mesodermal and neural patterning in the embryo. Moreover, ectopic expression of eaf1 and eaf2 in embryos and cultured cells blocked β-catenin reporter activity. By immunoprecipitation, we also observed that Eaf1 and Eaf2 bound to the Armadillo repeat region and C-terminus of β-catenin, as well as to other β-catenin transcription complex proteins, such as c-Jun, Tcf and Axin, suggesting the formation of a novel complex. In addition, the N-terminus of Eaf1 and Eaf2 bound to β-catenin and exhibited dominant-negative activity, whereas the C-terminus appeared to either harbor a suppression domain or to recruit a repressor. Both the N- and C-terminus must be intact for Eaf1 and Eaf2 suppressive activity. Lastly, we demonstrate a conservation of biological activities for Eaf family proteins across species. In summary, our evidence points to a novel role for Eaf1 and Eaf2 in inhibiting canonical Wnt/β-catenin signaling, which might form the mechanistic basis for Eaf1 and Eaf2 tumor suppressor activity.
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Affiliation(s)
- Jing-Xia Liu
- Key Laboratory of Biodiversity and Conservation of Aquatic Organisms, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, PR China
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Arumemi F, Bayles I, Paul J, Milcarek C. Shared and discrete interacting partners of ELL1 and ELL2 by yeast two-hybrid assay. ACTA ACUST UNITED AC 2013. [DOI: 10.4236/abb.2013.47101] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Xiao F, Zhang JS, Zhao JY, Wu D. Regulation of Eaf2 in mouse lens cells apoptosis induced by ultraviolet radiation. Int J Ophthalmol 2012; 5:570-5. [PMID: 23166866 DOI: 10.3980/j.issn.2222-3959.2012.05.05] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2012] [Accepted: 08/30/2012] [Indexed: 01/11/2023] Open
Abstract
AIM To investigate the regulation of Eaf2 protein in mouse lens cells apoptosis induced by ultraviolet (UV) radiation. METHODS An eye of Eaf2 gene knockout mice or normal control mice was exposed to UV radiation, and the other one was non-exposed. All of lenses were analyzed by TUNEL and caspase 3 activity assays to determine the difference of the apoptosis induced by UV radiation. In addition, exposed and non-exposed lenses were analyzed by quantified p53 expression and real-time reverse transcription-polymerase chain reaction (RT-PCR) of Bax, Bid, Apaf-1, Puma and Noxa, to compare Eaf2 gene knockout mice and normal control mice. RESULTS UV radiation caused apoptosis of lens cells in normal control mice and Eaf2 knockout mice. Activity of caspase 3 was significantly higher in normal control mice than Eaf2 knockout mice. Expression of p53 protein was significantly higher in lenses exposed to UV radiation than nonexposed lenses, but was similar between Eaf2 gene knockout mice and normal control mice in the same UV condition. After exposing to UV radiation, the analysis of real-time RT-PCR demonstrated that mRNA levels of Puma and Noxa were significantly higher in lenses of normal control mice than Eaf2 gene knockout mice, and that mRNA levels of Bax, Bid and Apaf-1 were not significantly different between gene knockout mice and normal control mice. CONCLUSION Eaf2 increases lens cells apoptosis induced by ultraviolet radiation. And Eaf2 up-regulates expression of the Puma and the Noxa to act on lens cells apoptosis after UV radiation.
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Affiliation(s)
- Fan Xiao
- Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Shenyang 110005, Liaoning Province, China
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Cai L, Phong BL, Fisher AL, Wang Z. Regulation of fertility, survival, and cuticle collagen function by the Caenorhabditis elegans eaf-1 and ell-1 genes. J Biol Chem 2011; 286:35915-35921. [PMID: 21880729 DOI: 10.1074/jbc.m111.270454] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
EAF2, an androgen-regulated protein, interacts with members of the ELL (eleven-nineteen lysine-rich leukemia) transcription factor family and also acts as a tumor suppressor. Although these proteins control transcriptional elongation and perhaps modulate the effects of other transcription factors, the mechanisms of their actions remain largely unknown. To gain new insights into the biology of the EAF2 and ELL family proteins, we used Caenorhabditis elegans as a model to explore the in vivo roles of their worm orthologs. Through the use of transgenic worms, RNAi, and an eaf-1 mutant, we found that both genes are expressed in multiple cell types throughout the worm life cycle and that they play important roles in fertility, survival, and body size regulation. ELL-1 and EAF-1 likely contribute to these activities in part through modulating cuticle synthesis, given that we observed a disrupted cuticle structure in ell-1 RNAi-treated or eaf-1 mutant worms. Consistent with disruption of cuticle structure, loss of either ELL-1 or EAF-1 suppressed the rol phenotype of specific collagen mutants, possibly through the control of dpy-3, dpy-13, and sqt-3 collagen gene expression. Furthermore, we also noted the regulation of collagen expression by ELL overexpression in PC3 human prostate cancer cells. Together, these results reveal important roles for the eaf-1 and ell-1 genes in the regulation of extracellular matrix components.
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Affiliation(s)
- Liquan Cai
- Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania 15232
| | - Binh L Phong
- Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania 15232
| | - Alfred L Fisher
- Division of Geriatric Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
| | - Zhou Wang
- Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania 15232.
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Liu L, Ai J, Xiao W, Liu J, Wang Y, Xin D, He Z, Guo Y, Wang Z. ELL is an HIF-1alpha partner that regulates and responds to hypoxia response in PC3 cells. Prostate 2010; 70:797-805. [PMID: 20166137 PMCID: PMC2857586 DOI: 10.1002/pros.21113] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
BACKGROUND Eleven-nineteen lysine-rich leukemia (ELL) plays an important role in tumorigenesis and animal development. HIF-1 is a transcriptional factor that functions as a master regulator of O(2) homeostasis. Our previous studies showed that a binding partner of ELL, U19/Eaf2, can modulate HIF-1alpha activity and hypoxia response, suggesting that ELL may also influence HIF-1alpha pathway and hypoxia response. METHODS Co-localization and co-immunoprecipitation were performed to test the interaction between ELL and HIF-1alpha. PC3 cells with stable ELL knockdown and PC3 cells with stable ELL overexpression, along with their controls, were established using lentiviral expression system. Western blot and real-time PCR were performed to test the effect of ELL on HIF-1alpha protein and its down-stream gene transcription. To elucidate potential effect of hypoxia on ELL, cell growth and colony formation assays were performed using PC3 subline with stable ELL overexpression. RESULTS ELL is associated with HIF-1alpha in transfected cells. In PC3 prostate cancer cells, ELL inhibited HIF-1alpha protein level and down-stream gene expression. As expected, ELL inhibited cell growth and colony formation under normoxia. Interestingly, the inhibition was alleviated under hypoxia. CONCLUSIONS Our findings suggest that ELL and HIF-1alpha are binding partners and can modulate the functions of each other in hypoxia.
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Affiliation(s)
- Lingqi Liu
- Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, Beijing, China
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA
| | - Junkui Ai
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA
| | - Wuhan Xiao
- Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
| | - June Liu
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA
| | - Yujuan Wang
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
| | - Dianqi Xin
- Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, Beijing, China
| | - Zhisong He
- Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, Beijing, China
| | - Yinglu Guo
- Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, Beijing, China
| | - Zhou Wang
- Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA
- Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA
- University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA
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Lin C, Smith ER, Takahashi H, Lai KC, Martin-Brown S, Florens L, Washburn MP, Conaway JW, Conaway RC, Shilatifard A. AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Mol Cell 2010; 37:429-37. [PMID: 20159561 PMCID: PMC2872029 DOI: 10.1016/j.molcel.2010.01.026] [Citation(s) in RCA: 466] [Impact Index Per Article: 31.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2009] [Revised: 12/29/2009] [Accepted: 01/25/2010] [Indexed: 02/08/2023]
Abstract
Chromosomal translocations involving the MLL gene are associated with infant acute lymphoblastic and mixed lineage leukemia. There are a large number of translocation partners of MLL that share very little sequence or seemingly functional similarities; however, their translocations into MLL result in the pathogenesis of leukemia. To define the molecular reason why these translocations result in the pathogenesis of leukemia, we purified several of the commonly occurring MLL chimeras. We have identified super elongation complex (SEC) associated with all chimeras purified. SEC includes ELL, P-TEFb, AFF4, and several other factors. AFF4 is required for SEC stability and proper transcription by poised RNA polymerase II in metazoans. Knockdown of AFF4 in leukemic cells shows reduction in MLL chimera target gene expression, suggesting that AFF4/SEC could be a key regulator in the pathogenesis of leukemia through many of the MLL partners.
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Affiliation(s)
- Chengqi Lin
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
| | - Edwin R. Smith
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
| | - Hidehisa Takahashi
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
| | - Ka-Chun Lai
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
| | - Skylar Martin-Brown
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
| | - Laurence Florens
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
| | - Michael P. Washburn
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
| | - Joan W. Conaway
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
- Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103
| | - Ronald C. Conaway
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
- Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103
| | - Ali Shilatifard
- Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110
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Wan X, Ji W, Mei X, Zhou J, Liu JX, Fang C, Xiao W. Negative feedback regulation of Wnt4 signaling by EAF1 and EAF2/U19. PLoS One 2010; 5:e9118. [PMID: 20161747 PMCID: PMC2817739 DOI: 10.1371/journal.pone.0009118] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2010] [Accepted: 01/20/2010] [Indexed: 01/02/2023] Open
Abstract
Previous studies indicated that EAF (ELL-associated factor) family members, EAF1 and EAF2/U19, play a role in cancer and embryogenesis. For example, EAF2/U19 may serve as a tumor suppressor in prostate cancer. At the same time, EAF2/U19 is a downstream factor in the non-canonical Wnt 4 signaling pathway required for eye development in Xenopus laevis, and along with EAF1, contributes to convergence and extension movements in zebrafish embryos through Wnt maintenance. Here, we used zebrafish embryos and mammalian cells to show that both EAF1 and EAF2/U19 were up-regulated by Wnt4 (Wnt4a). Furthermore, we found that EAF1 and EAF2/U19 suppressed Wnt4 expression by directly binding to the Wnt4 promoter as seen in chromatin immunoprecipitation assays. These findings indicate that an auto-regulatory negative feedback loop occurs between Wnt4 and the EAF family, which is conserved between zebrafish and mammalian. The rescue experiments in zebrafish embryos showed that early embryonic development required the maintenance of the appropriate levels of Wnt4a through the feedback loop. Others have demonstrated that the tumor suppressors p63, p73 and WT1 positively regulate Wnt4 expression while p21 has the opposite effect, suggesting that maintenance of appropriate Wnt4 expression may also be critical for adult tissue homeostasis and prevention against tumor initiation. Thus, the auto-regulatory negative feedback loop that controls expression of Wnt4 and EAF proteins may play an important role in both embryonic development and tumor suppression. Our findings provide the first convincing line of evidence that EAF and Wnt4 form an auto-regulatory negative feedback loop in vivo.
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Affiliation(s)
- Xiaoyang Wan
- Key Laboratory of Biodiversity and Conservation of Aquatic Organisms, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, People's Republic of China.
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