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Yutoku M, Fujita K, Chiba N, Tada R, Ohnishi T, Sugimura M, Matsuguchi T. Early Growth Response 1 Plays an Essential Role in Proinflammatory and Osteoclastogenic Activities of Lipopolysaccharide-Stimulated Osteoblasts. FASEB J 2025; 39:e70532. [PMID: 40193242 DOI: 10.1096/fj.202402623r] [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: 10/28/2024] [Revised: 03/13/2025] [Accepted: 03/31/2025] [Indexed: 04/09/2025]
Abstract
Lipopolysaccharide (LPS) of Gram-negative bacteria in oral plaque is the major cause of periodontal disease. It is involved in the induction of inflammation and alveolar bone resorption at least partly by directly reacting to Toll-like receptor (TLR) 4 on osteoblasts. LPS induces osteoblasts to express proinflammatory cytokines, chemokines, and prostaglandins, as well as macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), which directly activate adjacent osteoclasts toward bone resorption. However, the regulator mechanisms have not been fully revealed at the molecular level. Here, we have demonstrated that LPS rapidly induces expression of early growth response 1 (EGR1), a zinc-finger transcription factor, and analyzed its physiological functions in osteoblasts. In both primary osteoblasts and an osteoblast cell line, LPS induced expression of EGR1 mRNA and protein within 30 min and 60 min, respectively, which were relatively slower than in macrophages. Inhibition of EGR1 by siRNA significantly inhibited LPS-induced mRNA expression of the tumor necrosis factor (TNF), interleukin-6 (IL-6), chemokines, cyclooxygenase-2 (COX2), matrix metalloproteinase-13 (MMP13), M-CSF, and RANKL in osteoblasts. Moreover, forced overexpression of EGR1 by the inducible expression system was sufficient to increase mRNA expression levels of TNF, IL-6, COX2, MMP13, and RANKL without LPS stimulation. As for the intracellular signal transduction, LPS-induced EGR1 expression in osteoblasts was dependent on the unique c-Jun N-terminal kinase (JNK)-extracellular signal-regulated kinase (ERK) activation pathway. Our data suggest an essential role of EGR1 in osteoblast responses to LPS-inducing tissue inflammation and osteolysis, providing new insights into the pathogenesis of periodontal disease.
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Affiliation(s)
- Miyoko Yutoku
- Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
- Department of Dental Anesthesiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Kosuke Fujita
- Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Norika Chiba
- Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Ryohei Tada
- Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Tomokazu Ohnishi
- Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Mitsutaka Sugimura
- Department of Dental Anesthesiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Tetsuya Matsuguchi
- Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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2
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van Oostrum M, Schuman EM. Understanding the molecular diversity of synapses. Nat Rev Neurosci 2025; 26:65-81. [PMID: 39638892 DOI: 10.1038/s41583-024-00888-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/08/2024] [Indexed: 12/07/2024]
Abstract
Synapses are composed of thousands of proteins, providing the potential for extensive molecular diversity to shape synapse type-specific functional specializations. In this Review, we explore the landscape of synaptic diversity and describe the mechanisms that expand the molecular complexity of synapses, from the genotype to the regulation of gene expression to the production of specific proteoforms and the formation of localized protein complexes. We emphasize the importance of examining every molecular layer and adopting a systems perspective to understand how these interconnected mechanisms shape the diverse functional and structural properties of synapses. We explore current frameworks for classifying synapses and methodologies for investigating different synapse types at varying scales, from synapse-type-specific proteomics to advanced imaging techniques with single-synapse resolution. We highlight the potential of synapse-type-specific approaches for integrating molecular data with cellular functions, circuit organization and organismal phenotypes to enable a more holistic exploration of neuronal phenomena across different scales.
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Affiliation(s)
- Marc van Oostrum
- Max Planck Institute for Brain Research, Frankfurt am Main, Germany
- Biozentrum, University of Basel, Basel, Switzerland
| | - Erin M Schuman
- Max Planck Institute for Brain Research, Frankfurt am Main, Germany.
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Mukhametzyanova L, Schmitt LT, Torres-Rivera J, Rojo-Romanos T, Lansing F, Paszkowski-Rogacz M, Hollak H, Brux M, Augsburg M, Schneider PM, Buchholz F. Activation of recombinases at specific DNA loci by zinc-finger domain insertions. Nat Biotechnol 2024; 42:1844-1854. [PMID: 38297187 PMCID: PMC11631766 DOI: 10.1038/s41587-023-02121-y] [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: 04/18/2023] [Accepted: 12/22/2023] [Indexed: 02/02/2024]
Abstract
Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site. The developed fusion improved targeted editing efficiencies of recombinases by four-fold and abolished measurable off-target activity in mammalian cells. The ZFD-dependent activity is transferable to a recombinase with relaxed specificity, providing the means for developing fully programmable recombinases. Our engineered recombinases provide improved genome editing tools with increased precision and efficiency.
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Affiliation(s)
- Liliya Mukhametzyanova
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
| | - Lukas Theo Schmitt
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | - Julia Torres-Rivera
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
| | - Teresa Rojo-Romanos
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | - Felix Lansing
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | | | - Heike Hollak
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | - Melanie Brux
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
| | - Martina Augsburg
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
| | - Paul Martin Schneider
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | - Frank Buchholz
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany.
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4
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Li X, Huang L, Liu B, Zhang Z, Zhou G, Guo Z, Song J, Wang X. Cyclic negative pressure promotes chondrocyte growth: Association of IGF-2 with EGR-1. BIOMOLECULES & BIOMEDICINE 2024; 24:1761-1775. [PMID: 38912889 PMCID: PMC11496872 DOI: 10.17305/bb.2024.10487] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/16/2024] [Revised: 06/14/2024] [Accepted: 06/14/2024] [Indexed: 06/25/2024]
Abstract
Knee osteoarthritis (KOA) is one of the most common degenerative joint diseases in the elderly worldwide. The primary lesion in patients with KOA is the degeneration of articular cartilage. This study aimed to observe the biological effects of cyclic negative pressure on C28/I2 chondrocytes and to elucidate the underlying molecular mechanisms. We designed a bi-directional intelligent micro-pressure control device for cyclic negative pressure intervention on C28/I2 chondrocytes. Chondrocyte vitality and proliferation were assessed using Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays. The extracellular matrix was analyzed using real-time fluorescence quantitative polymerase chain reaction (PCR) and western blot, while the molecular mechanism of the chondrocyte response to cyclic negative pressure was explored through mRNA sequencing. Experimental data demonstrated that cyclic negative pressure promoted chondrocyte proliferation and upregulated the expression of chondrocyte-specific protein, namely the collagen type II alpha 1 chain (COL2A1) protein, and the transcription factor SRY-box transcription factor 9 (SOX9). Additionally, RNA sequencing analysis revealed that the gene levels of insulin-like growth factor 2 (IGF-2) and early growth response 1 (EGR-1) were significantly elevated in the cyclic negative pressure group. This study demonstrates that cyclic negative pressure stimulates the proliferation of C28/I2 chondrocytes by promoting the expression of EGR-1 and IGF-2. This new discovery may provide novel insights into cartilage health and KOA prevention.
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Affiliation(s)
- Xiaoyu Li
- School of Medicine, Jianghan University, Wuhan, China
| | - Lixia Huang
- Tianyuan Translational Medicine Research and Development Team, School of Medicine, Jianghan University, Wuhan, China
- Institute of Translational Medicine, Wuhan College of Arts and Science, Wuhan, China
| | - Bingxue Liu
- School of Medicine, Jianghan University, Wuhan, China
| | - Zehua Zhang
- School of Medicine, Jianghan University, Wuhan, China
| | - Guoyong Zhou
- Tianyuan Translational Medicine Research and Development Team, School of Medicine, Jianghan University, Wuhan, China
| | - Zirui Guo
- Department of Materials, Swiss Federal Institute of Technology, Zurich, Switzerland
| | - Jiuhong Song
- Wuhan FL Medical Science and Technology Ltd., Wuhan, China
| | - Xiang Wang
- School of Medicine, Jianghan University, Wuhan, China
- Tianyuan Translational Medicine Research and Development Team, School of Medicine, Jianghan University, Wuhan, China
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Hu X, Zhang X, Sun W, Liu C, Deng P, Cao Y, Zhang C, Xu N, Zhang T, Zhang Y, Liu JJ, Wang H. Systematic discovery of DNA-binding tandem repeat proteins. Nucleic Acids Res 2024; 52:10464-10489. [PMID: 39189466 PMCID: PMC11417379 DOI: 10.1093/nar/gkae710] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Revised: 07/30/2024] [Accepted: 08/07/2024] [Indexed: 08/28/2024] Open
Abstract
Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools.
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Affiliation(s)
- Xiaoxuan Hu
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Xuechun Zhang
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Wen Sun
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Chunhong Liu
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Pujuan Deng
- State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China
- Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China
| | - Yuanwei Cao
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Chenze Zhang
- National Key Laboratory of Efficacy and Mechanism on Chinese Medicine for Metabolic Diseases, Beijing University of Chinese Medicine, Beijing 100029, China
| | - Ning Xu
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Tongtong Zhang
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Yong E Zhang
- University of Chinese Academy of Sciences, Beijing 100049, China
- Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jun-Jie Gogo Liu
- State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China
- Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China
| | - Haoyi Wang
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
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6
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Chen SY, Fang CY, Su BH, Chen HM, Huang SC, Wu PT, Shiau AL, Wu CL. Early Growth Response Protein 1 Exacerbates Murine Inflammatory Bowel Disease by Transcriptional Activation of Matrix Metalloproteinase 12. Biomedicines 2024; 12:780. [PMID: 38672136 PMCID: PMC11047900 DOI: 10.3390/biomedicines12040780] [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: 02/19/2024] [Revised: 03/19/2024] [Accepted: 03/26/2024] [Indexed: 04/28/2024] Open
Abstract
Inflammatory bowel disease (IBD) is an inflammatory condition affecting the colon and small intestine, with Crohn's disease and ulcerative colitis being the major types. Individuals with long-term IBD are at an increased risk of developing colorectal cancer. Early growth response protein 1 (Egr1) is a nuclear protein that functions as a transcriptional regulator. Egr1 is known to control the expression of numerous genes and play a role in cell growth, proliferation, and differentiation. While IBD has been associated with severe inflammation, the precise mechanisms underlying its pathogenesis remain unclear. This study aimed to investigate the role of Egr1 in the development of IBD. High levels of Egr1 expression were observed in a mouse model of colitis induced by dextran sulfate sodium (DSS), as determined by immunohistochemical (IHC) staining. Chronic DSS treatment showed that Egr1 knockout (KO) mice exhibited resistance to the development of IBD, as determined by changes in their body weight and disease scores. Additionally, enzyme-linked immunosorbent assay (ELISA) and IHC staining demonstrated decreased expression levels of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, as well as matrix metalloproteinase 12 (MMP12). Putative Egr1 binding sites were identified within the MMP12 promoter region. Through reporter assays and chromatin immunoprecipitation (ChIP) analysis, it was shown that Egr1 binds to the MMP12 promoter and regulates MMP12 expression. In conclusion, we found that Egr1 plays a role in the inflammation process of IBD through transcriptionally activating MMP12.
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Affiliation(s)
- Shih-Yao Chen
- Department of Nursing, College of Nursing, Chung Hwa University of Medical Technology, Tainan 717302, Taiwan;
| | - Chuan-Yin Fang
- Division of Colon and Rectal Surgery, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chiayi 600566, Taiwan
| | - Bing-Hwa Su
- School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei 110301, Taiwan
| | - Hao-Ming Chen
- Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan 701401, Taiwan
| | - Shih-Chi Huang
- Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan 701401, Taiwan
| | - Po-Ting Wu
- Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan 701401, Taiwan
- Department of Orthopedics, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701401, Taiwan
- Department of Orthopaedics, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701401, Taiwan
- Medical Device Innovation Center, National Cheng Kung University, Tainan 701401, Taiwan
- Department of Biomedical Engineering, National Cheng Kung University, Tainan 701401, Taiwan
| | - Ai-Li Shiau
- Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701401, Taiwan
| | - Chao-Liang Wu
- Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan 701401, Taiwan
- Department of Medical Research, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chiayi 600566, Taiwan
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Yang Z, Chen F, Wei D, Chen F, Jiang H, Qin S. EGR1 mediates MDR1 transcriptional activity regulating gemcitabine resistance in pancreatic cancer. BMC Cancer 2024; 24:268. [PMID: 38408959 PMCID: PMC10895816 DOI: 10.1186/s12885-024-12005-2] [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: 11/11/2023] [Accepted: 02/14/2024] [Indexed: 02/28/2024] Open
Abstract
BACKGROUND Gemcitabine is a cornerstone drug for the treatment of all stages of pancreatic cancer and can prolong the survival of patients with pancreatic cancer, but resistance to gemcitabine in pancreatic cancer patients hinders its efficacy. The overexpression of Early growth response 1(EGR1) in pancreatic ductal adenocarcinoma as a mechanism of gemcitabine chemoresistance in pancreatic cancer has not been explored. The major mechanisms of gemcitabine chemoresistance are related to drug uptake, metabolism, and action. One of the common causes of tumor multidrug resistance (MDR) to chemotherapy in cancer cells is that transporter proteins increase intracellular drug efflux and decrease drug concentrations by inducing anti-apoptotic mechanisms. It has been reported that gemcitabine binds to MDR1 with high affinity. The purpose of this research was to investigate the potential mechanisms by which EGR1 associates with MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. METHODS The following in vitro and in vivo techniques were used in this research to explore the potential mechanisms by which EGR1 binds to MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. Cell culture; in vitro and in vivo study of EGR1 function by loss of function analysis. Binding of EGR1 to the MDR1 promoter was detected using the ChIP assay. qRT-PCR, Western blot assays to detect protein and mRNA expression; use of Annexin V apoptosis detection assay to test apoptosis; CCK8, Edu assay to test cell proliferation viability. The animal model of pancreatic cancer subcutaneous allograft was constructed and the tumours were stained with hematoxylin eosin and Ki-67 expression was detected using immunohistochemistry. FINDINGS We revealed that EGR1 expression was increased in different pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells. Moreover, gemcitabine treatment induced upregulation of EGR1 expression in a dose- and time-dependent manner. EGR1 is significantly enriched in the MDR1 promoter sequence.Upon knockdown of EGR1, cell proliferation was impaired in CFPAC-1 and PANC-1 cell lines, apoptosis was enhanced and MDR1 expression was decreased, thereby partially reversing gemcitabine chemoresistance. In animal experiments, knockdown of EGR1 enhanced the inhibitory effect of gemcitabine on tumor growth compared with the sh-NC group. CONCLUSIONS Our study suggests that EGR1 may be involved in the regulation of MDR1 to enhance gemcitabine resistance in pancreatic cancer cells. EGR1 could be a novel therapeutic target to overcome gemcitabine resistance in pancreatic cancer.
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Affiliation(s)
- Zhe Yang
- Department of Gastroenterology, Guangxi Medical University Cancer Hospital, No 71 Hedi Road, Nanning, Guangxi Zhuang Autonomous Region, PR China
| | - Feiran Chen
- Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, No 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region, PR China
| | - Dafu Wei
- Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, No 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region, PR China
| | - Fengping Chen
- Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, No 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region, PR China
| | - Haixing Jiang
- Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, No 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region, PR China.
| | - Shanyu Qin
- Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, No 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region, PR China.
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Yin L, Zhang J, Sun Y. Early growth response-1 is a new substrate of the GSK3β-FBXW7 axis. Neoplasia 2022; 34:100839. [PMID: 36240645 PMCID: PMC9573921 DOI: 10.1016/j.neo.2022.100839] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Revised: 09/25/2022] [Accepted: 09/26/2022] [Indexed: 11/06/2022]
Abstract
EGR1, a short-lived transcription factor, regulates several biological processes, including cell proliferation and tumor progression. Whether and how EGR1 is regulated by Cullin-RING ligases (CRLs) remains elusive. Here, we report that MLN4924, a small molecule inhibitor of neddylation, causes EGR1 accumulation by inactivating SCFFBXW7 (CRL1), which is a new E3 ligase for EGR1. Specifically, FBXW7 binds to EGR1 via its consensus binding motif/degron, whereas cancer-derived FBXW7 mutants showed a much reduced EGR1 binding. SiRNA-mediated FBXW7 knockdown caused EGR1 accumulation, whereas FBXW7 overexpression reduced EGR1 levels. Likewise, FBXW7 knockdown significantly extended EGR1 protein half-life, while FBXW7 overexpression promotes polyubiquitylation of wild-type EGR1, but not EGR1-S2A mutant with the binding site abrogated. GSK3β kinase is required for the FBXW7-EGR1 binding, and for enhanced EGR1 degradation by wild type FBXW7, but not by FBXW7 mutants. Likewise, GSK3β knockdown or treatment with GSK3β inhibitor significantly increased the EGR1 levels and extended EGR1 protein half-life, while reducing EGR1 polyubiquitylation. Hypoxia exposure reduces the EGR1 levels via enhancing the FBXW7-EGR1 binding, and FBXW7-induced EGR1 polyubiquitylation. Biologically, EGR1 knockdown suppressed cancer cell growth, whereas growth stimulation by FBXW7 knockdown is partially rescued by EGR1 knockdown. Thus, EGR1 is a new substrate of the GSK3β-FBXW7 axis, and the FBXW7-EGR1 axis coordinately regulates growth of cancer cells.
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Affiliation(s)
- Lu Yin
- Cancer Institute of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China; Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou 310029, China
| | - Jiagui Zhang
- Cancer Institute of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China; Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou 310029, China
| | - Yi Sun
- Cancer Institute of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China; Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou 310029, China; Cancer Center, Zhejiang University, Hangzhou 310058, China; Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou 310053, China.
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9
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Stoddart A, Fernald AA, Davis EM, McNerney ME, Le Beau MM. EGR1 Haploinsufficiency Confers a Fitness Advantage to Hematopoietic Stem Cells Following Chemotherapy. Exp Hematol 2022; 115:54-67. [PMID: 35995095 PMCID: PMC10617250 DOI: 10.1016/j.exphem.2022.08.003] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Revised: 08/11/2022] [Accepted: 08/12/2022] [Indexed: 11/17/2022]
Abstract
Therapy-related myeloid neoplasms (t-MNs) share many clinical and molecular characteristics with AML de novo in the elderly. One common factor is that they arise in the setting of chronic inflammation, likely because of advanced age or chemotherapy-induced senescence. Here, we examined the effect of haploinsufficient loss of the del(5q) tumor suppressor gene, EGR1, commonly deleted in high-risk MNs. In mice, under the exogenous stress of either serial transplant or successive doses of the alkylating agent N-ethyl-nitrosourea (ENU), Egr1-haploinsufficient hematopoietic stem cells (HSCs) exhibit a clonal advantage. Complete loss of EGR1 function is incompatible with transformation; mutations of EGR1 are rare and are not observed in the remaining allele in del(5q) patients, and complete knockout of Egr1 in mice leads to HSC exhaustion. Using chromatin immunoprecipitation sequencing (ChIP-seq), we identified EGR1 binding sites in human CD34+ cord blood-derived stem and progenitor cells (HSPCs) and found that EGR1 binds genes critical for stem cell differentiation, inflammatory signaling, and the DNA damage response. Notably, in the chromosome 5 sequences frequently deleted in patients, there is a significant enrichment of innate and inflammatory genes, which may confer a fitness advantage in an inflammatory environment. Short hairpin RNA (shRNA)-mediated silencing of EGR1 biases HSPCs toward a self-renewal transcriptional signature. In the absence of EGR1, HSPCs are characterized by upregulated MYC-driven proliferative signals, downregulated CDKN1A (p21), disrupted DNA damage response, and downregulated inflammation-adaptations anticipated to confer a relative fitness advantage for stem cells especially in an environment of chronic inflammation.
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Affiliation(s)
| | | | | | - Megan E McNerney
- Department of Pathology, University of Chicago, Chicago, IL; University of Chicago Medicine Comprehensive Cancer Center, Chicago, IL; Department of Pediatrics, University of Chicago, Chicago IL
| | - Michelle M Le Beau
- Department of Medicine, University of Chicago, Chicago, IL; University of Chicago Medicine Comprehensive Cancer Center, Chicago, IL
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10
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Woodson CM, Kehn-Hall K. Examining the role of EGR1 during viral infections. Front Microbiol 2022; 13:1020220. [PMID: 36338037 PMCID: PMC9634628 DOI: 10.3389/fmicb.2022.1020220] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2022] [Accepted: 09/26/2022] [Indexed: 09/06/2023] Open
Abstract
Early growth response 1 (EGR1) is a multifunctional mammalian transcription factor capable of both enhancing and/or inhibiting gene expression. EGR1 can be activated by a wide array of stimuli such as exposure to growth factors, cytokines, apoptosis, and various cellular stress states including viral infections by both DNA and RNA viruses. Following induction, EGR1 functions as a convergence point for numerous specialized signaling cascades and couples short-term extracellular signals to influence transcriptional regulation of genes required to initiate the appropriate biological response. The role of EGR1 has been extensively studied in both physiological and pathological conditions of the adult nervous system where it is readily expressed in various regions of the brain and is critical for neuronal plasticity and the formation of memories. In addition to its involvement in neuropsychiatric disorders, EGR1 has also been widely examined in the field of cancer where it plays paradoxical roles as a tumor suppressor gene or oncogene. EGR1 is also associated with multiple viral infections such as Venezuelan equine encephalitis virus (VEEV), Kaposi's sarcoma-associated herpesvirus (KSHV), herpes simplex virus 1 (HSV-1), human polyomavirus JC virus (JCV), human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV). In this review, we examine EGR1 and its role(s) during viral infections. First, we provide an overview of EGR1 in terms of its structure, other family members, and a brief overview of its roles in non-viral disease states. We also review upstream regulators of EGR1 and downstream factors impacted by EGR1. Then, we extensively examine EGR1 and its roles, both direct and indirect, in regulating replication of DNA and RNA viruses.
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Affiliation(s)
- Caitlin M. Woodson
- Department of Biomedical Science and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States
- Center for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States
| | - Kylene Kehn-Hall
- Department of Biomedical Science and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States
- Center for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States
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11
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Brue CR, Dukes MW, Masotti M, Holmgren R, Meade TJ. Functional Disruption of Gli1-DNA Recognition via a Cobalt(III) Complex. ChemMedChem 2022; 17:e202200025. [PMID: 35302712 PMCID: PMC10826845 DOI: 10.1002/cmdc.202200025] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Indexed: 12/29/2022]
Abstract
The aberrant activation of the Gli family of zinc finger transcription factors (ZFTFs) is associated with several types of human cancer, including medulloblastoma and basal cell carcinoma. We have reported the use of cobalt(III) Schiff-base complexes (Co(III)-sb) as potent inhibitors of ZFTFs in vivo. These complexes inhibit transcription by displacing the zinc finger domain's structural Zn(II) ion, destabilizing the alpha helix necessary for DNA recognition. Here, we describe the use of Co(III)-sb complexes for the selective inhibition of Gli1. Spectroscopic and computational studies of the Gli1 DNA binding domain found that Co(III)-sb displaced Zn(II) through direct coordination with the His residues of the Cys2 His2 Zn(II) binding site. As a result, there is a dose-dependent degradation of the alpha-helix content in the DNA binding domain of Gli1 and corresponding inhibition of consensus sequence recognition. We conclude that this strategy is well suited for the development of new and potent inhibitors of Gli1.
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Affiliation(s)
- Christopher R Brue
- Departments of Chemistry, Molecular Biosciences, Neurobiology, and Radiology, Northwestern University, Evanston, IL, 60208-3113, USA
| | - Meghan W Dukes
- Departments of Chemistry, Molecular Biosciences, Neurobiology, and Radiology, Northwestern University, Evanston, IL, 60208-3113, USA
| | - Meghan Masotti
- Departments of Chemistry, Molecular Biosciences, Neurobiology, and Radiology, Northwestern University, Evanston, IL, 60208-3113, USA
| | - Robert Holmgren
- Departments of Chemistry, Molecular Biosciences, Neurobiology, and Radiology, Northwestern University, Evanston, IL, 60208-3113, USA
| | - Thomas J Meade
- Departments of Chemistry, Molecular Biosciences, Neurobiology, and Radiology, Northwestern University, Evanston, IL, 60208-3113, USA
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12
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Dreab A, Bayse CA. Molecular Dynamics Simulations of Reduced and Oxidized TFIIIA Zinc Fingers Free and Interacting with 5S RNA. J Chem Inf Model 2022; 62:903-913. [PMID: 35143196 DOI: 10.1021/acs.jcim.1c01272] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Interactions of zinc finger (ZF) proteins with nucleic acids and proteins play an important role in DNA transcription and repair, biochemical recognition, and protein regulation. The release of Zn2+ through oxidation of cysteine thiolates is associated with disruption of gene expression and DNA repair, preventing tumor growth. Multi-microsecond molecular dynamics (MD) simulations were carried out to examine the effect of Cys oxidation on the ZF456 fragment of transcription factor III A (TFIIIA) and its complex with 5S RNA. In the absence of 5S RNA, the reduced ZF456 peptide undergoes conformational changes in the secondary structure due to the reorientation of the intact ZF domains. Upon oxidation, the individual ZF domains unfold to various degrees, yielding a globular ZF456 peptide with ZF4 and ZF6, responsible for base-specific hydrogen bonds with 5S RNA, losing their ββα-folds. ZF5, on the other hand, participates in nonspecific interactions through its α-helix that conditionally unravels early in the simulation. In the presence of RNA, oxidation of the ZF456 peptide disrupts the key hydrogen bonding interactions between ZF5/ZF6 and 5S RNA. However, interactions with ZF4 are dependent on the protonation state of His119.
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Affiliation(s)
- Ana Dreab
- Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529, United States
| | - Craig A Bayse
- Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529, United States
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13
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Zheng L, Liu J, Niu L, Kamran M, Yang AWH, Jolma A, Dai Q, Hughes TR, Patel DJ, Zhang L, Prasanth SG, Yu Y, Ren A, Lai EC. Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins. Genes Dev 2022; 36:225-240. [PMID: 35144965 PMCID: PMC8887127 DOI: 10.1101/gad.348993.121] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Accepted: 01/05/2022] [Indexed: 11/24/2022]
Abstract
The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.
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Affiliation(s)
- Luqian Zheng
- The Eighth Affiliated Hospital, Sun Yat-Sen University, Shenzhen, Guangdong 518033, China
- Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Jingjing Liu
- State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China
| | - Lijie Niu
- State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China
| | - Mohammad Kamran
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
| | - Ally W H Yang
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada
- Donnelly Centre, University of Toronto, Toronto, Ontario M5S 1A1, Canada
| | - Arttu Jolma
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada
- Donnelly Centre, University of Toronto, Toronto, Ontario M5S 1A1, Canada
| | - Qi Dai
- Developmental Biology Program, Sloan Kettering Institute, New York, New York 10065, USA
| | - Timothy R Hughes
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada
- Donnelly Centre, University of Toronto, Toronto, Ontario M5S 1A1, Canada
| | - Dinshaw J Patel
- Structural Biology Program, Sloan Kettering Institute, New York, New York 10065, USA
| | - Long Zhang
- The Eighth Affiliated Hospital, Sun Yat-Sen University, Shenzhen, Guangdong 518033, China
- Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Supriya G Prasanth
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
| | - Yang Yu
- State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China
- Developmental Biology Program, Sloan Kettering Institute, New York, New York 10065, USA
| | - Aiming Ren
- Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Eric C Lai
- Developmental Biology Program, Sloan Kettering Institute, New York, New York 10065, USA
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14
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Khamis H, Rudnizky S, Melamed P, Kaplan A. Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation. Nucleic Acids Res 2021; 49:10975-10987. [PMID: 34606618 PMCID: PMC8565314 DOI: 10.1093/nar/gkab843] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Revised: 08/04/2021] [Accepted: 09/24/2021] [Indexed: 12/14/2022] Open
Abstract
The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein's binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.
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Affiliation(s)
- Hadeel Khamis
- Faculty of Biology, Technion – Israel Institute of Technology, Haifa 32000, Israel
- Faculty of Physics, Technion – Israel Institute of Technology, Haifa 32000, Israel
| | - Sergei Rudnizky
- Faculty of Biology, Technion – Israel Institute of Technology, Haifa 32000, Israel
| | - Philippa Melamed
- Faculty of Biology, Technion – Israel Institute of Technology, Haifa 32000, Israel
| | - Ariel Kaplan
- Faculty of Biology, Technion – Israel Institute of Technology, Haifa 32000, Israel
- Faculty of Biomedical Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel
- Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 32000, Israel
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15
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The role of zinc finger linkers in zinc finger protein binding to DNA. J Comput Aided Mol Des 2021; 35:973-986. [PMID: 34350488 DOI: 10.1007/s10822-021-00413-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Accepted: 07/26/2021] [Indexed: 10/20/2022]
Abstract
Zinc finger proteins (ZFP) play important roles in cellular processes. The DNA binding region of ZFP consists of 3 zinc finger DNA binding domains connected by amino acid linkers, the sequence TGQKP connects ZF1 and ZF2, and TGEKP connects ZF2 with ZF3. Linkers act to tune the zinc finger protein in the right position to bind its DNA target, the type of amino acid residues and length of linkers reflect on ZF1-ZF2-ZF3 interactions and contribute to the search and recognition process of ZF protein to its DNA target. Linker mutations and the affinity of the resulting mutants to specific and nonspecific DNA targets were studied by MD simulations and MM_GB(PB)SA. The affinity of mutants to DNA varied with type and position of amino acid residue. Mutation of K in TGQKP resulted in loss in affinity due to the loss of positive K interaction with phosphates, mutation of G showed loss in affinity to DNA, WT protein and all linker mutants showed loss in affinity to a nonspecific DNA target, this finding confirms previous reports which interpreted this loss in affinity as due to ZF1 having an anchoring role, and ZF3 playing an explorer role in the binding mechanism. The change in ZFP-DNA affinity with linker mutations is discussed in view of protein structure and role of linker residues in binding.
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16
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Teng L, Huang Y, Guo J, Li B, Lin J, Ma L, Wang Y, Ye C, Chen Q. Cardiac fibroblast miR-27a may function as an endogenous anti-fibrotic by negatively regulating Early Growth Response Protein 3 (EGR3). J Cell Mol Med 2020; 25:73-83. [PMID: 33215816 PMCID: PMC7810947 DOI: 10.1111/jcmm.15814] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2020] [Revised: 08/05/2020] [Accepted: 08/08/2020] [Indexed: 02/06/2023] Open
Abstract
Pathological myocardial fibrosis and hypertrophy occur due to chronic cardiac stress. The microRNA‐27a (miR‐27a) regulates collagen production across diverse cell types and organs to inhibit fibrosis and could constitute an important therapeutic avenue. However, its impact on hypertrophy and cardiac remodelling is less well‐known. We employed a transverse aortic constriction (TAC) murine model of left ventricular pressure overload to investigate the in vivo effects of genetic miR‐27a knockout, antisense inhibition of miR‐27a‐5p and fibroblast‐specific miR‐27a knockdown or overexpression. In silico Venn analysis and reporter assays were used to identify miR‐27a‐5p's targeting of Early Growth Response Protein 3 (Egr3). We evaluated the effects of miR‐27a‐5p and Egr3 upon transforming growth factor‐beta (Tgf‐β) signalling and secretome of cardiac fibroblasts in vitro. miR‐27a‐5p attenuated TAC‐induced cardiac fibrosis and myofibroblast activation in vivo, without a discernible effect on cardiac myocytes. Molecularly, miR‐27a‐5p inhibited transforming growth factor‐beta (Tgf‐β) signalling and pro‐fibrotic protein secretion in cardiac fibroblasts in vitro through suppressing the pro‐fibrotic transcription factor Early Growth Response Protein 3 (Egr3). This body of work suggests that cardiac fibroblast miR‐27a may function as an endogenous anti‐fibrotic by negatively regulating Egr3 expression.
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Affiliation(s)
- Lifeng Teng
- Department of Cardiology, Hainan General Hospital, Haikou, China
| | - Yubing Huang
- Department of Cardiology, Hainan General Hospital, Haikou, China
| | - Jun Guo
- Department of Cardiology, The First Affiliated Hospital of Jinan University, GuangZhou, China
| | - Bin Li
- Department of Cardiology, Hainan General Hospital, Haikou, China
| | - Jin Lin
- Department of Cardiology, Hainan General Hospital, Haikou, China
| | - Lining Ma
- Department of Cardiology, Hainan General Hospital, Haikou, China
| | - Yudai Wang
- Department of Cardiology, Hainan General Hospital, Haikou, China
| | - Cong Ye
- Department of Cardiology, Hainan General Hospital, Haikou, China
| | - Qianqian Chen
- Nursing Department, Hainan Maternal and Child Health Hospital, Haikou, China
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17
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LINC00473 as an Immediate Early Gene under the Control of the EGR1 Transcription Factor. Noncoding RNA 2020; 6:ncrna6040046. [PMID: 33198374 PMCID: PMC7712511 DOI: 10.3390/ncrna6040046] [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: 10/28/2020] [Revised: 11/09/2020] [Accepted: 11/11/2020] [Indexed: 11/16/2022] Open
Abstract
Immediate early genes play an essential role in cellular responses to different stimuli. Many of them are transcription factors that regulate the secondary response gene expression. Non-coding RNAs may also be involved in this regulatory cascade. In fact, they are emerging as key actors of gene expression regulation, and evidence suggests that their dysregulation may underly pathological states. We previously took a snapshot of both coding and long non-coding RNAs differentially expressed in neuronal cells after brain-derived neurotrophic factor stimulation. Among these, the transcription factor EGR1 (a well-known immediate early gene) and LINC00473 (a primate-specific long non-coding RNA) that has emerged as an interesting RNA candidate involved in neuronal function and in cancer. In this work, we demonstrated that LINC00473 gene expression kinetics resembled that of immediate early genes in SH-SY5Y and HEK293T cells under different cell stimulation conditions. Moreover, we showed that the expression of LINC00473 is under the control of the transcription factor EGR1, providing evidence for an interesting functional relationship in neuron function.
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18
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MicroRNA 27a Is a Key Modulator of Cholesterol Biosynthesis. Mol Cell Biol 2020; 40:MCB.00470-19. [PMID: 32071155 DOI: 10.1128/mcb.00470-19] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2019] [Accepted: 02/10/2020] [Indexed: 12/25/2022] Open
Abstract
Hypercholesterolemia is a strong predictor of cardiovascular diseases. The 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (Hmgcr) coding for the rate-limiting enzyme in the cholesterol biosynthesis pathway is a crucial regulator of plasma cholesterol levels. However, the posttranscriptional regulation of Hmgcr remains poorly understood. The main objective of this study was to explore the role of microRNAs (miRNAs) in the regulation of Hmgcr expression. Systematic in silico predictions and experimental analyses reveal that miRNA 27a (miR-27a) specifically interacts with the Hmgcr 3' untranslated region in murine and human hepatocytes. Moreover, our data show that Hmgcr expression is inversely correlated with miR-27a levels in various cultured cell lines and in human and rodent tissues. Actinomycin D chase assays and relevant experiments demonstrate that miR-27a regulates Hmgcr by translational attenuation followed by mRNA degradation. Early growth response 1 (Egr1) regulates miR-27a expression under basal and cholesterol-modulated conditions. miR-27a augmentation via tail vein injection of miR-27a mimic in high-cholesterol-diet-fed Apoe -/- mice shows downregulation of hepatic Hmgcr and plasma cholesterol levels. Pathway and gene expression analyses show that miR-27a also targets several other genes (apart from Hmgcr) in the cholesterol biosynthesis pathway. Taken together, miR-27a emerges as a key regulator of cholesterol biosynthesis and has therapeutic potential for the clinical management of hypercholesterolemia.
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19
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Havis E, Duprez D. EGR1 Transcription Factor is a Multifaceted Regulator of Matrix Production in Tendons and Other Connective Tissues. Int J Mol Sci 2020; 21:ijms21051664. [PMID: 32121305 PMCID: PMC7084410 DOI: 10.3390/ijms21051664] [Citation(s) in RCA: 125] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Revised: 02/24/2020] [Accepted: 02/25/2020] [Indexed: 12/22/2022] Open
Abstract
Although the transcription factor EGR1 is known as NGF1-A, TIS8, Krox24, zif/268, and ZENK, it still has many fewer names than biological functions. A broad range of signals induce Egr1 gene expression via numerous regulatory elements identified in the Egr1 promoter. EGR1 is also the target of multiple post-translational modifications, which modulate EGR1 transcriptional activity. Despite the myriad regulators of Egr1 transcription and translation, and the numerous biological functions identified for EGR1, the literature reveals a recurring theme of EGR1 transcriptional activity in connective tissues, regulating genes related to the extracellular matrix. Egr1 is expressed in different connective tissues, such as tendon (a dense connective tissue), cartilage and bone (supportive connective tissues), and adipose tissue (a loose connective tissue). Egr1 is involved in the development, homeostasis, and healing processes of these tissues, mainly via the regulation of extracellular matrix. In addition, Egr1 is often involved in the abnormal production of extracellular matrix in fibrotic conditions, and Egr1 deletion is seen as a target for therapeutic strategies to fight fibrotic conditions. This generic EGR1 function in matrix regulation has little-explored implications but is potentially important for tendon repair.
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20
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Popa SC, Shin JA. The Intrinsically Disordered Loop in the USF1 bHLHZ Domain Modulates Its DNA-Binding Sequence Specificity in Hereditary Asthma. J Phys Chem B 2019; 123:9862-9871. [PMID: 31670516 DOI: 10.1021/acs.jpcb.9b06719] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
USF1, a basic region/helix-loop-helix/leucine zipper (bHLHZ) transcription factor, binds to the E-box in the PAI-1 (plasminogen activator inhibitor) promoter. Two alleles containing the E-box control PAI-1 transcription; these alleles are termed "4G" and "5G" based on the G tract flanking E-box. USF1-governed transcription of PAI-1 is elevated in heritable asthma sufferers: the 4G/4G genotype has the highest plasma levels of PAI-1. While USF1 uses its basic region to bind E-box, we found that it uses its 12 amino-acid loop to recognize the flanking sequence and discern the single-nucleotide difference between the alleles. We used the bacterial one-hybrid and electrophoretic mobility shift assays to assess protein-DNA recognition, and circular dichroism to examine protein secondary structure. We mutated Ser233 and Thr234 in the USF1 bHLHZ loop to Ala to generate S233A and T234A. Interestingly, USF1 bHLHZ, S233A, and T234A prefer the 5G sequence (USF1 bHLHZ Kd values 4.1 ± 0.3 nM and 7.0 ± 0.4 nM for 5G and 4G, respectively), whereas studies in stimulated human mast cells showed a preference for 4G. We replaced the 8 amino-acid loop of transcription factor Max bHLHZ with the 12 amino-acid USF1 loop: this mutant now distinguishes the 4G/5G polymorphism-while Max bHLHZ does not-confirming that USF1 differentiation of the 4G/5G is driven by the loop.
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Affiliation(s)
- Serban C Popa
- Department of Chemistry , University of Toronto , 3359 Mississauga Road , Mississauga , Ontario L5L 1C6 , Canada
| | - Jumi A Shin
- Department of Chemistry , University of Toronto , 3359 Mississauga Road , Mississauga , Ontario L5L 1C6 , Canada
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21
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Alkhamis O, Canoura J, Yu H, Liu Y, Xiao Y. Innovative engineering and sensing strategies for aptamer-based small-molecule detection. Trends Analyt Chem 2019; 121. [PMID: 32863483 DOI: 10.1016/j.trac.2019.115699] [Citation(s) in RCA: 81] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Aptamers are nucleic acid-based affinity reagents that have gained widespread attention as biorecognition elements for the detection of targets such as ions, small molecules, and proteins. Over the past three decades, the field of aptamer-based sensing has grown considerably. However, the advancement of aptamer-based small-molecule detection has fallen short of the high demand for such sensors in applications such as diagnostics, environmental monitoring, and forensics. This is due to two challenges: the complexity of developing generalized sensing platforms and the poor sensitivities of assays targeting small molecules. This paper will review new approaches for the streamlined development of high-performance aptamer-based sensors for small-molecule detection. We here provide historical context, explore the current state-of-the art, and offer future directions-with emphasis placed on new aptamer engineering methods, the use of cooperative binding, and label-free approaches using fully-folded, high-affinity aptamers for small-molecule sensing.
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Affiliation(s)
- Obtin Alkhamis
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, USA, 33199
| | - Juan Canoura
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, USA, 33199
| | - Haixiang Yu
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, USA, 33199
| | - Yingzhu Liu
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, USA, 33199
| | - Yi Xiao
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, USA, 33199
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22
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EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity. Nat Commun 2019; 10:3892. [PMID: 31467272 PMCID: PMC6715719 DOI: 10.1038/s41467-019-11905-3] [Citation(s) in RCA: 89] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2018] [Accepted: 08/12/2019] [Indexed: 02/07/2023] Open
Abstract
Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome. It is unclear why neuronal activity induced methylation changes are limited to specific loci in the genome. Here, authors show that the DNA demethylation enzyme, TET1, gains its specificity via the interaction with EGR1, a sequence specific DNA binding protein.
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23
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Yap EL, Greenberg ME. Activity-Regulated Transcription: Bridging the Gap between Neural Activity and Behavior. Neuron 2019; 100:330-348. [PMID: 30359600 DOI: 10.1016/j.neuron.2018.10.013] [Citation(s) in RCA: 401] [Impact Index Per Article: 66.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2018] [Revised: 10/02/2018] [Accepted: 10/05/2018] [Indexed: 12/21/2022]
Abstract
Gene transcription is the process by which the genetic codes of organisms are read and interpreted as a set of instructions for cells to divide, differentiate, migrate, and mature. As cells function in their respective niches, transcription further allows mature cells to interact dynamically with their external environment while reliably retaining fundamental information about past experiences. In this Review, we provide an overview of the field of activity-dependent transcription in the vertebrate brain and highlight contemporary work that ranges from studies of activity-dependent chromatin modifications to plasticity mechanisms underlying adaptive behaviors. We identify key gaps in knowledge and propose integrated approaches toward a deeper understanding of how activity-dependent transcription promotes the refinement and plasticity of neural circuits for cognitive function.
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Affiliation(s)
- Ee-Lynn Yap
- Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA; Program in Neuroscience, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
| | - Michael E Greenberg
- Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.
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Rudnizky S, Khamis H, Malik O, Squires AH, Meller A, Melamed P, Kaplan A. Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context. Nucleic Acids Res 2019; 46:1513-1524. [PMID: 29253225 PMCID: PMC5815098 DOI: 10.1093/nar/gkx1252] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2017] [Accepted: 12/11/2017] [Indexed: 12/31/2022] Open
Abstract
Most functional transcription factor (TF) binding sites deviate from their ‘consensus’ recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harboring three zinc fingers (ZF1, ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 bp bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein–DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.
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Affiliation(s)
- Sergei Rudnizky
- Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel
| | - Hadeel Khamis
- Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.,Faculty of Physics, Technion-Israel Institute of Technology, Haifa 32000, Israel
| | - Omri Malik
- Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.,Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa 32000, Israel
| | - Allison H Squires
- Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA
| | - Amit Meller
- Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa 32000, Israel.,Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.,Faculty of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel
| | - Philippa Melamed
- Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.,Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa 32000, Israel
| | - Ariel Kaplan
- Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.,Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa 32000, Israel
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Hu YT, Chen XL, Huang SH, Zhu QB, Yu SY, Shen Y, Sluiter A, Verhaagen J, Zhao J, Swaab D, Bao AM. Early growth response-1 regulates acetylcholinesterase and its relation with the course of Alzheimer's disease. Brain Pathol 2019; 29:502-512. [PMID: 30511454 DOI: 10.1111/bpa.12688] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2018] [Accepted: 11/28/2018] [Indexed: 01/15/2023] Open
Abstract
Our previous studies showed that the transcription factor early growth response-1 (EGR1) may play a role in keeping the brain cholinergic function intact in the preclinical stages of Alzheimer's disease (AD). In order to elucidate the mechanisms involved, we first performed data mining on our previous microarray study on postmortem human prefrontal cortex (PFC) for the changes in the expression of EGR1 and acetylcholinesterase (AChE) and the relationship between them during the course of AD. The study contained 49 patients, ranging from non-demented controls (Braak stage 0) to late AD patients (Braak stage VI). We found EGR1-mRNA was high in early AD and decreased in late AD stages, while AChE-mRNA was stable in preclinical AD and slightly decreased in late AD stages. A significant positive correlation was found between the mRNA levels of these two molecules. In addition, we studied the relationship between EGR1 and AChE mRNA levels in the frontal cortex of 3-12-months old triple-transgenic AD (3xTg-AD) mice. EGR1- and AChE-mRNA were lower in 3xTg-AD mice compared with wild-type (WT) mice. A significant positive correlation between these two molecules was present in the entire group and in each age group of either WT or 3xTg-AD mice. Subsequently, AChE expression was determined following up- or down-regulating EGR1 in cell lines and the EGR1 levels were found to regulate AChE at both the mRNA and protein levels. Dual-luciferase assay and electrophoretic mobility shift assay in the EGR1-overexpressing cells were performed to determine the functionally effective binding sites of the EGR1 on the AChE gene promoter. We conclude that the EGR1 can upregulate AChE expression by a direct effect on its gene promoter, which may contribute significantly to the changes in cholinergic function in the course of AD. The 3xTg-AD mouse model only reflects later stage AD.
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Affiliation(s)
- Yu-Ting Hu
- Department of Neurobiology, and Department of Neurology of the Second Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, P.R. China
| | - Xin-Lu Chen
- Department of Neurobiology, and Department of Neurology of the Second Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, P.R. China
| | - Shu-Han Huang
- Department of Neurobiology, and Department of Neurology of the Second Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, P.R. China
| | - Qiong-Bin Zhu
- Department of Neurobiology, and Department of Neurology of the Second Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, P.R. China
| | - Si-Yang Yu
- Department of Neurobiology, and Department of Neurology of the Second Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, P.R. China
| | - Yi Shen
- Department of Neurobiology, and Department of Neurology of the Second Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, P.R. China
| | - Arja Sluiter
- Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences, University of Amsterdam, Amsterdam, the Netherlands
| | - Joost Verhaagen
- Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences, University of Amsterdam, Amsterdam, the Netherlands
| | - Juan Zhao
- Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences, University of Amsterdam, Amsterdam, the Netherlands
| | - Dick Swaab
- Department of Neurobiology, and Department of Neurology of the Second Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, P.R. China.,Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences, University of Amsterdam, Amsterdam, the Netherlands
| | - Ai-Min Bao
- Department of Neurobiology, and Department of Neurology of the Second Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, P.R. China
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Li L, Chen J, Ge C, Zhao F, Chen T, Tian H, Li J, Li H. CD24 isoform a promotes cell proliferation, migration and invasion and is downregulated by EGR1 in hepatocellular carcinoma. Onco Targets Ther 2019; 12:1705-1716. [PMID: 30881025 PMCID: PMC6400134 DOI: 10.2147/ott.s196506] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
INTRODUCTION CD24 is known as a heavily glycosylated cell surface molecule that is highly expressed in a wide variety of human malignancies. Previous studies have shown that CD24 plays an important role in self-renewal, proliferation, migration, invasion and drug resistance of hepatocellular carcinoma (HCC). However, little is known about the expression and function of CD24 isoform a (CD24A) and CD24 isoform b (CD24B) in HCC. MATERIALS AND METHODS Quantitative real-time polymerase chain reaction (qPCR) and Western blotting were performed to detect CD24 and EGR1 expression in HCC cells and tissue. The function of CD24 in cell proliferation was verified with MTT assays, colony formation assays and tumor xenograft models. Wound healing assays and invasion assays were performed to clarify the function of CD24 in the regulation of cell migration and invasion in HCC. A dual luciferase reporter assay and chromatin immunoprecipitation assay were used to analyze the regulation mechanism of CD24A. RESULTS CD24A but not CD24B, which was barely detected by qPCR and Western blotting, is significantly upregulated in HCC tissue. Both CD24A and CD24B contribute to HCC cell proliferation, migration and invasion, but CD24A is more effective than CD24B. EGR1 downregulates CD24A and exerts transcription-promoting activity on the CD24A promoter. Furthermore, EGR1 represses HCC cell proliferation via downregulation of CD24A. CONCLUSION CD24A is the predominant CD24 isoform in HCC and plays a major role in cell proliferation, migration, and invasion. EGR1 can exert its antitumor effect through transcriptional downregulation of CD24A in HCC.
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Affiliation(s)
- Liangyu Li
- Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China
| | - Jing Chen
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China,
| | - Chao Ge
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China,
| | - Fangyu Zhao
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China,
| | - Taoyang Chen
- Qi Dong Liver Cancer Institute, Qi Dong, Jiangsu Province, People's Republic of China
| | - Hua Tian
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China,
| | - Jinjun Li
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China,
| | - Hong Li
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China,
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Baltaci SB, Mogulkoc R, Baltaci AK. Molecular Mechanisms of Early and Late LTP. Neurochem Res 2019; 44:281-296. [PMID: 30523578 DOI: 10.1007/s11064-018-2695-4] [Citation(s) in RCA: 53] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2018] [Revised: 10/31/2018] [Accepted: 12/04/2018] [Indexed: 12/01/2022]
Abstract
LTP is the most intensively studied cellular model of the memory and generally divided at least two distinct phases as early and late. E-LTP requires activation of CaMKII that initiates biochemical events and trafficking of proteins, which eventually potentiate synaptic transmission, and is independent of de novo protein synthesis. In contrast, L-LTP requires gene expression and local protein synthesis regulated via TrkB receptor- and functional prions CPEB2-3-mediated translation. Maintenance of LTP for longer periods depends on constitutively active PKMζ. Throughout this review, current knowledge about early and late phases of LTP will be reviewed.
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Affiliation(s)
- Saltuk Bugra Baltaci
- Faculty of Medicine, Department of Physiology, Selcuk University, 42031, Konya, Turkey
| | - Rasim Mogulkoc
- Faculty of Medicine, Department of Physiology, Selcuk University, 42031, Konya, Turkey
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28
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EGR-mediated control of STIM expression and function. Cell Calcium 2018; 77:58-67. [PMID: 30553973 DOI: 10.1016/j.ceca.2018.12.003] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2018] [Revised: 12/03/2018] [Accepted: 12/04/2018] [Indexed: 12/22/2022]
Abstract
Ca2+ is a ubiquitous, dynamic and pluripotent second messenger with highly context-dependent roles in complex cellular processes such as differentiation, proliferation, and cell death. These Ca2+ signals are generated by Ca2+-permeable channels located on the plasma membrane (PM) and endoplasmic reticulum (ER) and shaped by PM- and ER-localized pumps and transporters. Differences in the expression of these Ca2+ homeostasis proteins contribute to cell and context-dependent differences in the spatiotemporal organization of Ca2+ signals and, ultimately, cell fate. This review focuses on the Early Growth Response (EGR) family of zinc finger transcription factors and their role in the transcriptional regulation of Stromal Interaction Molecule (STIM1), a critical regulator of Ca2+ entry in both excitable and non-excitable cells.
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Inoue K, Fry EA. Tumor suppression by the EGR1, DMP1, ARF, p53, and PTEN Network. Cancer Invest 2018; 36:520-536. [PMID: 30396285 PMCID: PMC6500763 DOI: 10.1080/07357907.2018.1533965] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2017] [Revised: 02/25/2018] [Accepted: 10/05/2018] [Indexed: 01/08/2023]
Abstract
Recent studies have indicated that EGR1 is a direct regulator of tumor suppressors including TGFβ1, PTEN, and p53. The Myb-like transcription factor Dmp1 is a physiological regulator of the Arf-p53 pathway through transactivation of the Arf promoter and physical interaction of p53. The Dmp1 promoter has binding sites for Egr proteins, and Egr1 is a target for Dmp1. Crosstalks between p53 and PTEN have been reported. The Egr1-Dmp1-Arf-p53-Pten pathway displays multiple modes of interaction with each other, suggesting the existence of a functional network of tumor suppressors that maintain normal cell growth and prevent the emergence of incipient cancer cells.
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Affiliation(s)
- Kazushi Inoue
- The Department of Pathology, Wake Forest University Health Sciences,
Medical Center Boulevard, Winston-Salem, NC 27157 USA
| | - Elizabeth A. Fry
- The Department of Pathology, Wake Forest University Health Sciences,
Medical Center Boulevard, Winston-Salem, NC 27157 USA
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30
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Wang R, Hao W, Pan L, Boldogh I, Ba X. The roles of base excision repair enzyme OGG1 in gene expression. Cell Mol Life Sci 2018; 75:3741-3750. [PMID: 30043138 PMCID: PMC6154017 DOI: 10.1007/s00018-018-2887-8] [Citation(s) in RCA: 66] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2018] [Revised: 07/11/2018] [Accepted: 07/19/2018] [Indexed: 12/13/2022]
Abstract
Modifications of DNA strands and nucleobases-both induced and accidental-are associated with unfavorable consequences including loss or gain in genetic information and mutations. Therefore, DNA repair proteins have essential roles in keeping genome fidelity. Recently, mounting evidence supports that 8-oxoguanine (8-oxoG), one of the most abundant genomic base modifications generated by reactive oxygen and nitrogen species, along with its cognate repair protein 8-oxoguanine DNA glycosylase1 (OGG1), has distinct roles in gene expression through transcription modulation or signal transduction. Binding to 8-oxoG located in gene regulatory regions, OGG1 acts as a transcription modulator, which can control transcription factor homing, induce allosteric transition of G-quadruplex structure, or recruit chromatin remodelers. In addition, post-repair complex formed between OGG1 and its repair product-free 8-oxoG increases the levels of active small GTPases and induces downstream signaling cascades to trigger gene expressions. The present review discusses how cells exploit damaged guanine base(s) and the authentic repair protein to orchestrate a profile of various transcriptomes in redox-regulated biological processes.
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Affiliation(s)
- Ruoxi Wang
- Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Genetics and Cytology, Northeast Normal University, 5268 Renmin Street, Changchun, 130024, Jilin, China
- School of Life Science, Northeast Normal University, Changchun, 130024, Jilin, China
| | - Wenjing Hao
- Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Genetics and Cytology, Northeast Normal University, 5268 Renmin Street, Changchun, 130024, Jilin, China
- School of Life Science, Northeast Normal University, Changchun, 130024, Jilin, China
| | - Lang Pan
- Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Genetics and Cytology, Northeast Normal University, 5268 Renmin Street, Changchun, 130024, Jilin, China
- Department of Physiology, Xiangya Medicine School in Central South University, Changsha, 410078, Hunan, China
| | - Istvan Boldogh
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX, 77555, USA
- Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, TX, 77555, USA
| | - Xueqing Ba
- Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Genetics and Cytology, Northeast Normal University, 5268 Renmin Street, Changchun, 130024, Jilin, China.
- School of Life Science, Northeast Normal University, Changchun, 130024, Jilin, China.
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Rogers JM, Bulyk ML. Diversification of transcription factor-DNA interactions and the evolution of gene regulatory networks. WILEY INTERDISCIPLINARY REVIEWS. SYSTEMS BIOLOGY AND MEDICINE 2018; 10:e1423. [PMID: 29694718 PMCID: PMC6202284 DOI: 10.1002/wsbm.1423] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/08/2017] [Revised: 02/23/2018] [Accepted: 03/11/2018] [Indexed: 01/17/2023]
Abstract
Sequence-specific transcription factors (TFs) bind short DNA sequences in the genome to regulate the expression of target genes. In the last decade, numerous technical advances have enabled the determination of the DNA-binding specificities of many of these factors. Large-scale screens of many TFs enabled the creation of databases of TF DNA-binding specificities, typically represented as position weight matrices (PWMs). Although great progress has been made in determining and predicting binding specificities systematically, there are still many surprises to be found when studying a particular TF's interactions with DNA in detail. Paralogous TFs' binding specificities can differ in subtle ways, in a manner that is not immediately apparent from looking at their PWMs. These differences affect gene regulatory outputs and enable TFs to rewire transcriptional networks over evolutionary time. This review discusses recent observations made in the study of TF-DNA interactions that highlight the importance of continued in-depth analysis of TF-DNA interactions and their inherent complexity. This article is categorized under: Biological Mechanisms > Regulatory Biology.
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Affiliation(s)
- Julia M. Rogers
- Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115, USA
- Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, MA, 02138, USA
| | - Martha L. Bulyk
- Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115, USA
- Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, MA, 02138, USA
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115, USA
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Ernø H, Monard D. Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter. Gene Expr 2018; 3:163-74. [PMID: 8268720 PMCID: PMC6081634] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
The first three exons and the promoter of rat glia-derived nexin, also called protease nexin-1 (GDN/PN-1), have been identified through analysis of rat genomic clones. A 1.6 kilobase (kb) fragment containing 105 base pairs of the first exon and 5'-flanking sequences was sequenced. The 5'-flanking sequence and the first exon were found to be GC-rich, indicating that the 5' region of the rat GDN/PN-1 gene resides within a CpG island. A TATA box-like sequence, but no CAAT box, was found. The rat GDN/PN-1 promoter contains five SP1 consensus sites, four consensus sites for the MyoD1 transcription factor, and one binding site for the transcription factors NGFI-A, NGFI-C, Krox-20, and Wilms tumor factor. The presence of these consensus sequences is consistent with the known expression pattern of GDN/PN-1. Primer extension and RNase protection assays identified one transcriptional start site. The 1.6 kb promoter fragment cloned in a reporter plasmid was found to induce firefly luciferase expression in a cell-specific manner. A positive regulatory element is localized in the region -1545 to -389. In vitro CpG methylation blocked transcription from the GDN/PN-1 promoter in rat hepatoma cells but not in C6 rat glioma cells.
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Affiliation(s)
- H Ernø
- Friedrich Miescher Institute, Basel, Switzerland
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Hankey W, Chen Z, Bergman MJ, Fernandez MO, Hancioglu B, Lan X, Jegga AG, Zhang J, Jin VX, Aronow BJ, Wang Q, Groden J. Chromatin-associated APC regulates gene expression in collaboration with canonical WNT signaling and AP-1. Oncotarget 2018; 9:31214-31230. [PMID: 30131849 PMCID: PMC6101278 DOI: 10.18632/oncotarget.25781] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2018] [Accepted: 07/05/2018] [Indexed: 11/25/2022] Open
Abstract
Mutation of the APC gene occurs in a high percentage of colorectal tumors and is a central event driving tumor initiation in the large intestine. The APC protein performs multiple tumor suppressor functions including negative regulation of the canonical WNT signaling pathway by both cytoplasmic and nuclear mechanisms. Published reports that APC interacts with β-catenin in the chromatin fraction to repress WNT-activated targets have raised the possibility that chromatin-associated APC participates more broadly in mechanisms of transcriptional control. This screening study has used chromatin immunoprecipitation and next-generation sequencing to identify APC-associated genomic regions in colon cancer cell lines. Initial target selection was performed by comparison and statistical analysis of 3,985 genomic regions associated with the APC protein to whole transcriptome sequencing data from APC-deficient and APC-wild-type colon cancer cells, and two types of murine colon adenomas characterized by activated Wnt signaling. 289 transcripts altered in expression following APC loss in human cells were linked to APC-associated genomic regions. High-confidence targets additionally validated in mouse adenomas included 16 increased and 9 decreased in expression following APC loss, indicating that chromatin-associated APC may antagonize canonical WNT signaling at both WNT-activated and WNT-repressed targets. Motif analysis and comparison to ChIP-seq datasets for other transcription factors identified a prevalence of binding sites for the TCF7L2 and AP-1 transcription factors in APC-associated genomic regions. Our results indicate that canonical WNT signaling can collaborate with or antagonize the AP-1 transcription factor to fine-tune the expression of shared target genes in the colorectal epithelium. Future therapeutic strategies for APC-deficient colorectal cancers might be expanded to include agents targeting the AP-1 pathway.
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Affiliation(s)
- William Hankey
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, Ohio, United States of America
| | - Zhong Chen
- Department of Pathology, Duke University School of Medicine, Durham, North Carolina, United States of America
| | - Maxwell J Bergman
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, Ohio, United States of America
| | - Max O Fernandez
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, Ohio, United States of America
| | - Baris Hancioglu
- Biomedical Informatics Shared Resource, The Ohio State University, Columbus, Ohio, United States of America
| | - Xun Lan
- Department of Basic Medical Sciences, Tsinghua University School of Medicine, Beijing, China
| | - Anil G Jegga
- Division of Bioinformatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America
| | - Jie Zhang
- Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Victor X Jin
- Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America
| | - Bruce J Aronow
- Division of Bioinformatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America
| | - Qianben Wang
- Department of Pathology, Duke University School of Medicine, Durham, North Carolina, United States of America
| | - Joanna Groden
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, Ohio, United States of America
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Li X, Wang Z, Tong H, Yan Y, Li S. Effects of COL8A1 on the proliferation of muscle-derived satellite cells. Cell Biol Int 2018; 42:1132-1140. [PMID: 29696735 DOI: 10.1002/cbin.10979] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2018] [Accepted: 04/21/2018] [Indexed: 11/10/2022]
Abstract
Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix. Our previous studies suggested that COL8A1 is associated with the proliferation of muscle-derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells. Therefore, we predicted that COL8A1 is associated with the proliferation of bovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 in bovine MDSCs using the CRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, Western blotting, and dual-luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU-positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P-AKT. The expression of P-Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3-kinase). In contrast, repression of COL8A1 reduced the number of EdU-positive cells and expression of CCNB1 and P-AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual-luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation of bovine MDSCs via the PI3 K/AKT signaling pathway.
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Affiliation(s)
- Xiaofan Li
- Laboratory of Cellular and Developmental Biology, Life Science College, North-east Agricultural University, Harbin, 150030, China
| | - Zhao Wang
- Laboratory of Cellular and Developmental Biology, Life Science College, North-east Agricultural University, Harbin, 150030, China
| | - Huili Tong
- Laboratory of Cellular and Developmental Biology, Life Science College, North-east Agricultural University, Harbin, 150030, China
| | - Yunqin Yan
- Laboratory of Cellular and Developmental Biology, Life Science College, North-east Agricultural University, Harbin, 150030, China
| | - Shufeng Li
- Laboratory of Cellular and Developmental Biology, Life Science College, North-east Agricultural University, Harbin, 150030, China
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Lampen J, McAuley JD, Chang SE, Wade J. Neural activity associated with rhythmicity of song in juvenile male and female zebra finches. Behav Processes 2017; 163:45-52. [PMID: 29247695 DOI: 10.1016/j.beproc.2017.12.003] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2017] [Revised: 11/09/2017] [Accepted: 12/05/2017] [Indexed: 12/28/2022]
Abstract
Rhythm is an important aspect of both human speech and birdsong. Adult zebra finches show increased neural activity following exposure to arrhythmic compared to rhythmic song in regions similar to the mammalian auditory association cortex and amygdala. This pattern may indicate that birds are detecting errors in the arrhythmic song relative to their learned song template or to more general expectations of song structure. Here we exposed juvenile zebra finches to natural conspecific song (rhythmic) or song with altered inter-syllable intervals (arrhythmic) prior to or during template formation, or afterward as males are matching vocal production to a memorized song template (sensorimotor integration). Before template formation, expression of the immediate early gene ZENK was increased in the caudomedial nidopallium (NCM) of birds exposed to rhythmic relative to arrhythmic song. During template formation, ZENK expression was increased in the caudomedial mesopallium (CMM) of birds exposed to arrhythmic relative to rhythmic song. These results suggest that the youngest birds may be predisposed to respond to a more natural stimulus, and a template may be required for arrhythmic song to elicit increased neural activity. It also appears that functional development across the brain regions investigated continues to maturity.
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Affiliation(s)
- Jennifer Lampen
- Neuroscience Program, Michigan State University, East Lansing, MI 48824-1101, USA.
| | - J Devin McAuley
- Neuroscience Program, Michigan State University, East Lansing, MI 48824-1101, USA; Department of Psychology, Michigan State University, East Lansing, MI 48824-1101, USA
| | - Soo-Eun Chang
- Department of Psychiatry, University of Michigan, Ann Arbor, MI 48109, USA
| | - Juli Wade
- Neuroscience Program, Michigan State University, East Lansing, MI 48824-1101, USA; Department of Psychology, Michigan State University, East Lansing, MI 48824-1101, USA
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Abstract
The liver is an essential organ for nutrient and drug metabolism - possessing the remarkable ability to sense environmental and metabolic stimuli and provide an optimally adaptive response. Early growth response 1 (Egr1), an immediate early transcriptional factor which acts as a coordinator of the complex response to stress, is induced during liver injury and controls the expression of a wide range of genes involved in metabolism, cell proliferation, and role of Egr1 in liver injury and repair, deficiency of Egr1 delays liver regeneration process. The known upstream regulators of Egr1 include, but are not limited to, growth factors (e.g. transforming growth factor β1, platelet-derived growth factor, epidermal growth factor, hepatocyte growth factor), nuclear receptors (e.g. hepatocyte nuclear factor 4α, small heterodimer partner, peroxisome proliferator-activated receptor-γ), and other transcription factors (e.g. Sp1, E2F transcription factor 1). Research efforts using various animal models such as fatty liver, liver injury, and liver fibrosis contribute greatly to the elucidation of Egr1 function in the liver. Hepatocellular carcinoma (HCC) represents the second leading cause of cancer mortality worldwide due to the heterogeneity and the late stage at which cancer is generally diagnosed. Recent studies highlight the involvement of Egr1 in HCC development. The purpose of this review is to summarize current studies pertaining to the role of Egr1 in liver metabolism and liver diseases including liver cancer.
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Affiliation(s)
- Nancy Magee
- Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Yuxia Zhang
- Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA
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Ba X, Boldogh I. 8-Oxoguanine DNA glycosylase 1: Beyond repair of the oxidatively modified base lesions. Redox Biol 2017; 14:669-678. [PMID: 29175754 PMCID: PMC5975208 DOI: 10.1016/j.redox.2017.11.008] [Citation(s) in RCA: 167] [Impact Index Per Article: 20.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Revised: 10/08/2017] [Accepted: 11/08/2017] [Indexed: 12/11/2022] Open
Abstract
Oxidative stress and the resulting damage to genomic DNA are inevitable consequences of endogenous physiological processes, and they are amplified by cellular responses to environmental exposures. One of the most frequent reactions of reactive oxygen species with DNA is the oxidation of guanine to pre-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG). Despite the vulnerability of guanine to oxidation, vertebrate genes are primarily embedded in GC-rich genomic regions, and over 72% of the promoters of human genes belong to a class with a high GC content. In the promoter, 8-oxoG may serve as an epigenetic mark, and when complexed with the oxidatively inactivated repair enzyme 8-oxoguanine DNA glycosylase 1, provide a platform for the coordination of the initial steps of DNA repair and the assembly of the transcriptional machinery to launch the prompt and preferential expression of redox-regulated genes. Deviations/variations from this artful coordination may be the etiological links between guanine oxidation and various cellular pathologies and diseases during ageing processes.
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Affiliation(s)
- Xueqing Ba
- The Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, Changchun, Jilin 130024, China; School of Life Science, Northeast Normal University, Changchun, Jilin 130024, China.
| | - Istvan Boldogh
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA; Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
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EGR-1/ASPP1 inter-regulatory loop promotes apoptosis by inhibiting cyto-protective autophagy. Cell Death Dis 2017; 8:e2869. [PMID: 28594407 PMCID: PMC5520923 DOI: 10.1038/cddis.2017.268] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Revised: 05/10/2017] [Accepted: 05/10/2017] [Indexed: 01/07/2023]
Abstract
The decrease of ASPP1 (Apoptosis-Stimulating Protein of p53 1), a known p53 activator, has been linked to carcinogenesis and the cytotoxic resistance in various cancers, yet the underlying mechanisms of ASPP1 expression and its complex functions are not yet clear. Here, we report that ASPP1 forms an inter-regulatory loop with Early Growth Response 1 (EGR-1), and promotes apoptosis via inhibiting cyto-protective autophagy, independent of the well-documented p53-dependent mechanisms. We show that ASPP1 mRNA and protein were remarkably elevated by ectopic EGR-1 expression or endogenous EGR-1 activation, in cells with different tissue origins and p53 status. Conversely, RNAi-mediated EGR-1 knockdown suppressed ASPP1. The further mechanism studies revealed that ASPP1 promoter, mapped to -283/+88, which contained three conserved EGR-1 binding sites, was required for both binding and transactivity of EGR-1. In addition, we demonstrate that ASPP1 promoted EGR-1 in a positive feedback loop by preventing proteasome-mediated EGR-1 degradation or promoting EGR-1 nuclear import in response to anticancer natural compound Quercetin. Furthermore, albeit activating p53 in the nucleus is the well-studied function of ASPP1, we found that ASPP1 was predominately localized in the cytoplasm. Interestingly, the cytoplasmic ASPP1 retained its pro-apoptosis capability. Mechanistically, ASPP1 suppressed Atg5-Atg12 and also bound with Atg5-Atg12 to prevent its further complex formation with Atg16, resulting in the inhibition of cyto-protective autophagy. In conclusion, our results provide new insights into EGR-1/ASPP1 regulatory loop in sensitizing Quercetin-induced apoptosis. EGR-1/ASPP1, therefore, may be potentially used as therapeutic targets to improve cancer's response to pro-apoptosis treatments.
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Sung SR, Song SH, Kang KM, Park JE, Nam YJ, Shin YJ, Cha DH, Seo JT, Yoon TK, Shim SH. Sequence variations of the EGR4 gene in Korean men with spermatogenesis impairment. BMC MEDICAL GENETICS 2017; 18:47. [PMID: 28464846 PMCID: PMC5414287 DOI: 10.1186/s12881-017-0408-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/19/2016] [Accepted: 04/19/2017] [Indexed: 11/17/2022]
Abstract
Background Egr4 is expressed in primary and secondary spermatocytes in adult mouse testes and has a crucial role in regulating germ cell maturation. The functional loss of Egr4 blocks spermatogenesis, significantly reducing the number of spermatozoa that are produced. In this study, we examined whether EGR4 variants are present in Korean men with impaired spermatogenesis. Methods A total 170 Korean men with impaired spermatogenesis and 272 normal controls were screened. The coding regions including exon-intron boundaries of EGR4 were sequenced by PCR-direct sequencing method. Results We identified eight sequence variations in the coding region and 3′-UTR regions of the EGR4 gene. Four were nonsynonymous variants (rs771189047, rs561568849, rs763487015, and rs546250227), three were synonymous variants (rs115948271, rs528939702, and rs7558708), and one variant (rs2229294) was localized in the 3′-UTR. Three nonsynonymous variants [c.65_66InsG (p. Cys23Leufs*37), c.236C > T (p. Pro79Leu), c.1294G > T (p. Val432Leu)] and one synonymous variant [c.1230G > A (p. Thr410)] were not detected in controls. To evaluate the pathogenic effects of nonsynonymous variants, we used seven prediction methods. The c.214C > A (p. Arg72Ser) and c.236C > T (p. Pro79Leu) variants were predicted as “damaging” by SIFT and SNAP2. The c.65_66insG (p. Cys23Leufs*37) variants were predicted as “disease causing” by Mutation Taster, SNPs &GO and SNAP2. The c.867C > G (p. Leu289) variants were predicted as “disease causing” only by Mutation Taster. Conclusion To date, this study is the first to screen the EGR4 gene in relation to male infertility. However, our findings did not clearly explain how nonsynonymous EGR4 variations affect spermatogenesis. Therefore, further studies are required to validate the functional impact of EGR4 variations on spermatogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12881-017-0408-5) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Se Ra Sung
- Genetics Laboratory, Fertility Center of CHA Gangnam Medical Center, Seoul, South Korea
| | - Seung Hun Song
- Department of Urology, CHA Gangnam Medical Center, Seoul, South Korea
| | - Kyung Min Kang
- Genetics Laboratory, Fertility Center of CHA Gangnam Medical Center, Seoul, South Korea
| | - Ji Eun Park
- Genetics Laboratory, Fertility Center of CHA Gangnam Medical Center, Seoul, South Korea
| | - Yeo Jung Nam
- Department of Biomedical Science, College of Life Science, CHA University, Seoul, South Korea
| | - Yun-Jeong Shin
- Genetics Laboratory, Fertility Center of CHA Gangnam Medical Center, Seoul, South Korea
| | - Dong Hyun Cha
- Department of Obstetrics and Gynecology, CHA Gangnam Medical Center, Seoul, South Korea
| | - Ju Tae Seo
- Department of Urology, Cheil General Hospital, Seoul, South Korea
| | - Tae Ki Yoon
- Department of Obstetrics and Gynecology, CHA Gangnam Medical Center, Seoul, South Korea
| | - Sung Han Shim
- Genetics Laboratory, Fertility Center of CHA Gangnam Medical Center, Seoul, South Korea. .,Department of Biomedical Science, College of Life Science, CHA University, Seoul, South Korea.
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Duclot F, Kabbaj M. The Role of Early Growth Response 1 (EGR1) in Brain Plasticity and Neuropsychiatric Disorders. Front Behav Neurosci 2017; 11:35. [PMID: 28321184 PMCID: PMC5337695 DOI: 10.3389/fnbeh.2017.00035] [Citation(s) in RCA: 247] [Impact Index Per Article: 30.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2016] [Accepted: 02/21/2017] [Indexed: 12/11/2022] Open
Abstract
It is now clearly established that complex interactions between genes and environment are involved in multiple aspects of neuropsychiatric disorders, from determining an individual's vulnerability to onset, to influencing its response to therapeutic intervention. In this perspective, it appears crucial to better understand how the organism reacts to environmental stimuli and provide a coordinated and adapted response. In the central nervous system, neuronal plasticity and neurotransmission are among the major processes integrating such complex interactions between genes and environmental stimuli. In particular, immediate early genes (IEGs) are critical components of these interactions as they provide the molecular framework for a rapid and dynamic response to neuronal activity while opening the possibility for a lasting and sustained adaptation through regulation of the expression of a wide range of genes. As a result, IEGs have been tightly associated with neuronal activity as well as a variety of higher order processes within the central nervous system such as learning, memory and sensitivity to reward. The immediate early gene and transcription factor early growth response 1 (EGR1) has thus been revealed as a major mediator and regulator of synaptic plasticity and neuronal activity in both physiological and pathological conditions. In this review article, we will focus on the role of EGR1 in the central nervous system. First, we will summarize the different factors influencing its activity. Then, we will analyze the amount of data, including genome-wide, that has emerged in the recent years describing the wide variety of genes, pathways and biological functions regulated directly or indirectly by EGR1. We will thus be able to gain better insights into the mechanisms underlying EGR1's functions in physiological neuronal activity. Finally, we will discuss and illustrate the role of EGR1 in pathological states with a particular interest in cognitive functions and neuropsychiatric disorders.
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Affiliation(s)
- Florian Duclot
- Department of Biomedical Sciences, Florida State UniversityTallahassee, FL, USA; Program in Neuroscience, Florida State UniversityTallahassee, FL, USA
| | - Mohamed Kabbaj
- Department of Biomedical Sciences, Florida State UniversityTallahassee, FL, USA; Program in Neuroscience, Florida State UniversityTallahassee, FL, USA
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Oben KZ, Gachuki BW, Alhakeem SS, McKenna MK, Liang Y, St. Clair DK, Rangnekar VM, Bondada S. Radiation Induced Apoptosis of Murine Bone Marrow Cells Is Independent of Early Growth Response 1 (EGR1). PLoS One 2017; 12:e0169767. [PMID: 28081176 PMCID: PMC5230770 DOI: 10.1371/journal.pone.0169767] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2016] [Accepted: 11/14/2016] [Indexed: 12/03/2022] Open
Abstract
An understanding of how each individual 5q chromosome critical deleted region (CDR) gene contributes to malignant transformation would foster the development of much needed targeted therapies for the treatment of therapy related myeloid neoplasms (t-MNs). Early Growth Response 1 (EGR1) is a key transcriptional regulator of myeloid differentiation located within the 5q chromosome CDR that has been shown to regulate HSC (hematopoietic stem cell) quiescence as well as the master regulator of apoptosis—p53. Since resistance to apoptosis is a hallmark of malignant transformation, we investigated the role of EGR1 in apoptosis of bone marrow cells; a cell population from which myeloid malignancies arise. We evaluated radiation induced apoptosis of Egr1+/+ and Egr1-/- bone marrow cells in vitro and in vivo. EGR1 is not required for radiation induced apoptosis of murine bone marrow cells. Neither p53 mRNA (messenger RNA) nor protein expression is regulated by EGR1 in these cells. Radiation induced apoptosis of bone marrow cells by double strand DNA breaks induced p53 activation. These results suggest EGR1 dependent signaling mechanisms do not contribute to aberrant apoptosis of malignant cells in myeloid malignancies.
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Affiliation(s)
- Karine Z. Oben
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America
| | - Beth W. Gachuki
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America
| | - Sara S. Alhakeem
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America
| | - Mary K. McKenna
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America
| | - Ying Liang
- Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, Kentucky, United States of America
- Department of Internal Medicine, University of Kentucky, Lexington, Kentucky, United States of America
| | - Daret K. St. Clair
- Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, Kentucky, United States of America
| | - Vivek M. Rangnekar
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America
- Department of Radiation Medicine, University of Kentucky, Lexington, Kentucky, United States of America
- Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America
| | - Subbarao Bondada
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America
- Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America
- * E-mail:
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Adams KW, Kletsov S, Lamm RJ, Elman JS, Mullenbrock S, Cooper GM. Role for Egr1 in the Transcriptional Program Associated with Neuronal Differentiation of PC12 Cells. PLoS One 2017; 12:e0170076. [PMID: 28076410 PMCID: PMC5226839 DOI: 10.1371/journal.pone.0170076] [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: 11/03/2016] [Accepted: 12/28/2016] [Indexed: 11/17/2022] Open
Abstract
PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30–60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4–6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here.
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Affiliation(s)
- Kenneth W Adams
- Department of Biological Sciences, Bridgewater State University, Bridgewater, Massachusetts, United States of America
| | - Sergey Kletsov
- Department of Biological Sciences, Bridgewater State University, Bridgewater, Massachusetts, United States of America
| | - Ryan J Lamm
- Department of Biology, Boston University, Boston, Massachusetts, United States of America
| | - Jessica S Elman
- Department of Biology, Boston University, Boston, Massachusetts, United States of America
| | - Steven Mullenbrock
- Department of Biology, Boston University, Boston, Massachusetts, United States of America
| | - Geoffrey M Cooper
- Department of Biology, Boston University, Boston, Massachusetts, United States of America
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De-novo protein function prediction using DNA binding and RNA binding proteins as a test case. Nat Commun 2016; 7:13424. [PMID: 27869118 PMCID: PMC5121330 DOI: 10.1038/ncomms13424] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2016] [Accepted: 10/03/2016] [Indexed: 12/14/2022] Open
Abstract
Of the currently identified protein sequences, 99.6% have never been observed in the laboratory as proteins and their molecular function has not been established experimentally. Predicting the function of such proteins relies mostly on annotated homologs. However, this has resulted in some erroneous annotations, and many proteins have no annotated homologs. Here we propose a de-novo function prediction approach based on identifying biophysical features that underlie function. Using our approach, we discover DNA and RNA binding proteins that cannot be identified based on homology and validate these predictions experimentally. For example, FGF14, which belongs to a family of secreted growth factors was predicted to bind DNA. We verify this experimentally and also show that FGF14 is localized to the nucleus. Mutating the predicted binding site on FGF14 abrogated DNA binding. These results demonstrate the feasibility of automated de-novo function prediction based on identifying function-related biophysical features. Identification of the function of proteins is difficult when there are no structurally or biochemically characterized homologs. Here, the authors present an approach that allows the prediction of nucleic-acid binding proteins based on sequence alone, and they are able to experimentally validate their method.
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Hashimoto H, Zhang X, Zheng Y, Wilson GG, Cheng X. Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications. Nucleic Acids Res 2016; 44:10165-10176. [PMID: 27596598 PMCID: PMC5137435 DOI: 10.1093/nar/gkw766] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2016] [Revised: 08/19/2016] [Accepted: 08/23/2016] [Indexed: 01/10/2023] Open
Abstract
Mutations in human zinc-finger transcription factor WT1 result in abnormal development of the kidneys and genitalia and an array of pediatric problems including nephropathy, blastoma, gonadal dysgenesis and genital discordance. Several overlapping phenotypes are associated with WT1 mutations, including Wilms tumors, Denys-Drash syndrome (DDS), Frasier syndrome (FS) and WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). These conditions vary in severity from individual to individual; they can be fatal in early childhood, or relatively benign into adulthood. DDS mutations cluster predominantly in zinc fingers (ZF) 2 and 3 at the C-terminus of WT1, which together with ZF4 determine the sequence-specificity of DNA binding. We examined three DDS associated mutations in ZF2 of human WT1 where the normal glutamine at position 369 is replaced by arginine (Q369R), lysine (Q369K) or histidine (Q369H). These mutations alter the sequence-specificity of ZF2, we find, changing its affinity for certain bases and certain epigenetic forms of cytosine. X-ray crystallography of the DNA binding domains of normal WT1, Q369R and Q369H in complex with preferred sequences revealed the molecular interactions responsible for these affinity changes. DDS is inherited in an autosomal dominant fashion, implying a gain of function by mutant WT1 proteins. This gain, we speculate, might derive from the ability of the mutant proteins to sequester WT1 into unproductive oligomers, or to erroneously bind to variant target sequences.
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Affiliation(s)
- Hideharu Hashimoto
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Xing Zhang
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Yu Zheng
- RGENE, Inc., 953 Indiana Street, San Francisco, CA 94107, USA
| | | | - Xiaodong Cheng
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA
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Khachigian LM. Early growth response-1 in the pathogenesis of cardiovascular disease. J Mol Med (Berl) 2016; 94:747-53. [PMID: 27251707 DOI: 10.1007/s00109-016-1428-x] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2016] [Revised: 05/12/2016] [Accepted: 05/17/2016] [Indexed: 12/20/2022]
Abstract
This article reviews the regulatory roles of the immediate-early gene product and prototypic zinc finger transcription factor, early growth response-1 in models of cardiovascular pathobiology, focusing on insights using microRNA, DNAzymes, small hairpin RNA, small interfering RNA, oligonucleotide decoy strategies and mice deficient in early growth response-1.
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Affiliation(s)
- Levon M Khachigian
- School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia.
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Mikles DC, Bhat V, Schuchardt BJ, McDonald CB, Farooq A. Effect of osmolytes on the binding of EGR1 transcription factor to DNA. Biopolymers 2016; 103:74-87. [PMID: 25269753 DOI: 10.1002/bip.22556] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2013] [Revised: 08/16/2014] [Accepted: 08/19/2014] [Indexed: 11/11/2022]
Abstract
Osmolytes play a key role in maintaining protein stability and mediating macromolecular interactions within the intracellular environment of the cell. Herein, we show that osmolytes such as glycerol, sucrose, and polyethylene glycol 400 (PEG400) mitigate the binding of early growth response (protein) 1 (EGR1) transcription factor to DNA in a differential manner. Thus, while physiological concentrations of glycerol only moderately reduce the binding affinity, addition of sucrose and PEG400 is concomitant with a loss in the binding affinity by an order of magnitude. This salient observation suggests that EGR1 is most likely subject to conformational equilibrium and that the osmolytes exert their effect via favorable interactions with the unliganded conformation. Consistent with this notion, our analysis reveals that while EGR1 displays rather high structural stability in complex with DNA, the unliganded conformation becomes significantly destabilized in solution. In particular, while liganded EGR1 adopts a well-defined arc-like architecture, the unliganded protein samples a comparatively large conformational space between two distinct states that periodically interconvert between an elongated rod-like shape and an arc-like conformation on a submicrosecond time scale. Consequently, the ability of osmolytes to favorably interact with the unliganded conformation so as to stabilize it could account for the negative effect of osmotic stress on EGR1-DNA interaction observed here. Taken together, our study sheds new light on the role of osmolytes in modulating a key protein-DNA interaction.
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Affiliation(s)
- David C Mikles
- Department of Biochemistry and Molecular Biology, Leonard Miller School of Medicine, University of Miami, Miami, FL, 33136
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Abe K, Murakami Y, Tatsumi A, Sumida K, Kezuka A, Fukaya T, Kumagai T, Osawa Y, Sode K, Ikebukuro K. Enzyme linking to DNA aptamers via a zinc finger as a bridge. Chem Commun (Camb) 2016; 51:11467-9. [PMID: 26087673 DOI: 10.1039/c5cc02906f] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
We propose a novel enzyme-labeling method for DNA aptamers using enzyme-fused zinc finger proteins. We achieved thrombin detection and vascular endothelial growth factor detection using zinc finger-fused firefly luciferase.
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Affiliation(s)
- Koichi Abe
- Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
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Udoko AN, Johnson CA, Dykan A, Rachakonda G, Villalta F, Mandape SN, Lima MF, Pratap S, Nde PN. Early Regulation of Profibrotic Genes in Primary Human Cardiac Myocytes by Trypanosoma cruzi. PLoS Negl Trop Dis 2016; 10:e0003747. [PMID: 26771187 PMCID: PMC4714843 DOI: 10.1371/journal.pntd.0003747] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2015] [Accepted: 10/16/2015] [Indexed: 11/18/2022] Open
Abstract
The molecular mechanisms of Trypanosoma cruzi induced cardiac fibrosis remains to be elucidated. Primary human cardiomyoctes (PHCM) exposed to invasive T. cruzi trypomastigotes were used for transcriptome profiling and downstream bioinformatic analysis to determine fibrotic-associated genes regulated early during infection process (0 to 120 minutes). The identification of early molecular host responses to T. cruzi infection can be exploited to delineate important molecular signatures that can be used for the classification of Chagasic patients at risk of developing heart disease. Our results show distinct gene network architecture with multiple gene networks modulated by the parasite with an incline towards progression to a fibrogenic phenotype. Early during infection, T. cruzi significantly upregulated transcription factors including activator protein 1 (AP1) transcription factor network components (including FOSB, FOS and JUNB), early growth response proteins 1 and 3 (EGR1, EGR3), and cytokines/chemokines (IL5, IL6, IL13, CCL11), which have all been implicated in the onset of fibrosis. The changes in our selected genes of interest did not all start at the same time point. The transcriptome microarray data, validated by quantitative Real-Time PCR, was also confirmed by immunoblotting and customized Enzyme Linked Immunosorbent Assays (ELISA) array showing significant increases in the protein expression levels of fibrogenic EGR1, SNAI1 and IL 6. Furthermore, phosphorylated SMAD2/3 which induces a fibrogenic phenotype is also upregulated accompanied by an increased nuclear translocation of JunB. Pathway analysis of the validated genes and phospho-proteins regulated by the parasite provides the very early fibrotic interactome operating when T. cruzi comes in contact with PHCM. The interactome architecture shows that the parasite induces both TGF-β dependent and independent fibrotic pathways, providing an early molecular foundation for Chagasic cardiomyopathy. Examining the very early molecular events of T. cruzi cellular infection may provide disease biomarkers which will aid clinicians in patient assessment and identification of patient subpopulation at risk of developing Chagasic cardiomyopathy.
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Affiliation(s)
- Aniekanabassi N. Udoko
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, United States of America
| | - Candice A. Johnson
- Food and Drug Administration, Silver Spring, Maryland, United States of America
| | - Andrey Dykan
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, United States of America
| | - Girish Rachakonda
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, United States of America
| | - Fernando Villalta
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, United States of America
| | - Sammed N. Mandape
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, United States of America
| | - Maria F. Lima
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, United States of America
- School of Graduate Studies and Research, Bioinformatics and Molecular Biology Core, Meharry Medical College, Nashville, Tennessee, United States of America
| | - Siddharth Pratap
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, United States of America
- School of Graduate Studies and Research, Bioinformatics and Molecular Biology Core, Meharry Medical College, Nashville, Tennessee, United States of America
| | - Pius N. Nde
- Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, United States of America
- * E-mail:
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Characterization of FGF23-Dependent Egr-1 Cistrome in the Mouse Renal Proximal Tubule. PLoS One 2015; 10:e0142924. [PMID: 26588476 PMCID: PMC4654537 DOI: 10.1371/journal.pone.0142924] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2015] [Accepted: 10/28/2015] [Indexed: 11/19/2022] Open
Abstract
Fibroblast growth factor 23 (FGF23) is a potent regulator of phosphate (Pi) and vitamin D homeostasis. The transcription factor, early growth response 1 (egr-1), is a biomarker for FGF23-induced activation of the ERK1/2 signaling pathway. We have shown that ERK1/2 signaling blockade suppresses renal egr-1 gene expression and prevents FGF23-induced hypophosphatemia and 1,25-dihydroxyvitamin D (1,25(OH)2D) suppression in mice. To test whether egr-1 itself mediates these renal actions of FGF23, we administered FGF23 to egr-1-/- and wild-type (WT) mice. In WT mice, FGF23 induced hypophosphatemia and suppressed expression of the renal Na/Pi cotransporters, Npt2a and Npt2c. In FGF23-treated egr-1-/- mice, hypophosphatemic response was greatly blunted and Na/Pi cotransporter expression was not suppressed. In contrast, FGF23 induced equivalent suppression of serum 1,25(OH)2D concentrations by suppressing renal cyp27b1 and stimulating cyp24a1 mRNA expression in both groups of mice. Thus, downstream of receptor binding and ERK1/2 signaling, we can distinguish the effector pathway that mediates FGF23-dependent inhibition of Pi transport from the pathway that mediates inhibition of 1,25(OH)2D synthesis in the kidney. Furthermore, we demonstrate that the hypophosphatemic effect of FGF23 is significantly blunted in Hyp/egr-1-/- mice; specifically, serum Pi concentrations and renal Npt2a and Npt2c mRNA expression are significantly higher in Hyp/egr-1-/- mice than in Hyp mice. We then characterized the egr-1 cistrome in the kidney using ChIP-sequencing and demonstrate recruitment of egr-1 to regulatory DNA elements in proximity to several genes involved in Pi transport. Thus, our data demonstrate that the effect of FGF23 on Pi homeostasis is mediated, at least in part, by activation of egr-1.
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Cookson VJ, Waite SL, Heath PR, Hurd PJ, Gandhi SV, Chapman NR. Binding loci of RelA-containing nuclear factor-kappaB dimers in promoter regions of PHM1-31 myometrial smooth muscle cells. Mol Hum Reprod 2015; 21:865-83. [DOI: 10.1093/molehr/gav051] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2015] [Accepted: 09/03/2015] [Indexed: 12/15/2022] Open
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