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Schallmayer E, Morozov V, Duensing-Kropp S, Schallmayer L, Schüffner L, Schubert-Zsilavecz M, Pabel J, Höfner G, Heering J, Marschner JA, Merk D. A First-in-Class Hepatocyte Nuclear Factor 4 Agonist. J Med Chem 2025; 68:10410-10424. [PMID: 40336482 DOI: 10.1021/acs.jmedchem.5c00595] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/09/2025]
Abstract
Hepatocyte nuclear factor 4 (HNF4) is an orphan nuclear receptor implicated, for example, in pancreatic islet gene expression and hepatic regulation of glucose and lipid metabolism. Mutations in the HNF4α gene are responsible for the inheritable maturity-onset diabetes of the young 1 (MODY-1), supporting the therapeutic potential of HNF4 activation in metabolic diseases. However, exploration and validation of HNF4 as a therapeutic target is hindered by the lack of suitable ligands. Here, we report the development of the first high-affinity HNF4 agonists by extension of a fragment screening hit and systematic SAR elucidation. Structural modification allowed tuning of the chemotype for both HNF4 agonism and inverse agonism. X-ray structure analysis demonstrated orthosteric site occupation by the new ligand scaffold mimicking the natural fatty acid ligand binding. The most active descendant displayed low nanomolar HNF4 agonist potency and binding affinity and favorable selectivity, enabling unprecedented studies on HNF4 biology as a chemical tool.
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Affiliation(s)
- Espen Schallmayer
- Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, 60438 Frankfurt, Germany
| | - Vasily Morozov
- Ludwig-Maximilians-Universität (LMU) München, Department of Pharmacy, 81377 Munich, Germany
| | - Silke Duensing-Kropp
- Ludwig-Maximilians-Universität (LMU) München, Department of Pharmacy, 81377 Munich, Germany
| | - Lasse Schallmayer
- Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, 60438 Frankfurt, Germany
| | - Leann Schüffner
- Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, 60438 Frankfurt, Germany
| | | | - Jörg Pabel
- Ludwig-Maximilians-Universität (LMU) München, Department of Pharmacy, 81377 Munich, Germany
| | - Georg Höfner
- Ludwig-Maximilians-Universität (LMU) München, Department of Pharmacy, 81377 Munich, Germany
| | - Jan Heering
- Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, 60596 Frankfurt, Germany
| | - Julian A Marschner
- Ludwig-Maximilians-Universität (LMU) München, Department of Pharmacy, 81377 Munich, Germany
| | - Daniel Merk
- Ludwig-Maximilians-Universität (LMU) München, Department of Pharmacy, 81377 Munich, Germany
- Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, 60438 Frankfurt, Germany
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2
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Das S, Ravi H, Devi Rajeswari V, Venkatraman G, Ramasamy M, Dhanasekaran S, Ramanathan G. Therapeutic insight into the role of nuclear protein HNF4α in liver carcinogenesis. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2024; 143:1-37. [PMID: 39843133 DOI: 10.1016/bs.apcsb.2024.05.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
Abstract
Hepatocyte nuclear factor 4-alpha (HNF4α), a well-preserved member of the nuclear receptor superfamily of transcription factors, is found in the liver. It is recognized as a central controller of gene expression specific to the liver and plays a key role in preserving the liver's homeostasis. Irregular expression of HNF4α is increasingly recognized as a crucial factor in the proliferation, cell death, invasiveness, loss of specialized functions, and metastasis of cancer cells. An increasing number of studies are pointing to abnormal HNF4α expression as a key component of cancer cell invasion, apoptosis, proliferation, dedifferentiation, and metastasis. Understanding HNF4α's intricate involvement in liver carcinogenesis provides a promising avenue for therapeutic intervention. This chapter attempts to shed light on the diverse aspects of HNF4's role in liver carcinogenesis and demonstrate how this knowledge can be harnessed for approaches to prevent and treat liver cancer. This comprehensive chapter will offer an elaborate perspective on HNF4's function in liver cancer, delineating its molecular mechanisms that aid in the emergence of liver cancer. Furthermore, it will highlight the potential to help create more effective and precisely targeted therapeutic strategies, rekindling fresh optimism in the fight against this formidable condition.
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Affiliation(s)
- Soumik Das
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India
| | - Harini Ravi
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India
| | - V Devi Rajeswari
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India
| | - Ganesh Venkatraman
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India
| | - Magesh Ramasamy
- Department of Biotechnology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
| | - Sivaraman Dhanasekaran
- School of Energy Technology, Pandit Deendayal Energy University, Knowledge Corridor, Gandhinagar, Gujarat, India
| | - Gnanasambandan Ramanathan
- Department of Bio-Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India.
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Kaci A, Solheim MH, Silgjerd T, Hjaltadottir J, Hornnes LH, Molnes J, Madsen A, Sjøholt G, Bellanné-Chantelot C, Caswell R, Sagen JV, Njølstad PR, Aukrust I, Bjørkhaug L. Functional characterization of HNF4A gene variants identify promoter and cell line specific transactivation effects. Hum Mol Genet 2024; 33:894-904. [PMID: 38433330 PMCID: PMC11070132 DOI: 10.1093/hmg/ddae027] [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: 10/27/2023] [Revised: 01/26/2024] [Accepted: 02/11/2024] [Indexed: 03/05/2024] Open
Abstract
Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and β-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.
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Affiliation(s)
- Alba Kaci
- Mohn Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Haukelandsbakken 1, Bergen 5020, Norway
- Center for Laboratory Medicine, Østfold Hospital Trust, Kalnesveien 300, Grålum 1714, Norway
| | - Marie Holm Solheim
- Mohn Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Haukelandsbakken 1, Bergen 5020, Norway
| | - Trine Silgjerd
- Department of Safety, Chemistry, and Biomedical Laboratory Sciences, Western Norway University of Applied Sciences, Inndalsveien 28, Bergen 5020, Norway
| | - Jorunn Hjaltadottir
- Mohn Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Haukelandsbakken 1, Bergen 5020, Norway
- Department of Safety, Chemistry, and Biomedical Laboratory Sciences, Western Norway University of Applied Sciences, Inndalsveien 28, Bergen 5020, Norway
| | - Lorentze Hope Hornnes
- Department of Medical Biochemistry and Pharmacology, Haukeland University Hospital, Jonas Lies veg 87, Bergen 5021, Norway
| | - Janne Molnes
- Mohn Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Haukelandsbakken 1, Bergen 5020, Norway
- Department of Medical Genetics, Haukeland University Hospital, Jonas Lies veg 87, Bergen 5021, Norway
| | - Andre Madsen
- Department of Clinical Science, University of Bergen, Jonas Lies veg 87, Bergen 5020, Norway
| | - Gry Sjøholt
- Department of Safety, Chemistry, and Biomedical Laboratory Sciences, Western Norway University of Applied Sciences, Inndalsveien 28, Bergen 5020, Norway
| | - Christine Bellanné-Chantelot
- Départment of Medical Genetics, Sorbonne University, AP-HP, Hôpital Pitié-Salpêtriére, 21 rue de l'école de médecine, 75006 Paris, France
| | - Richard Caswell
- Exeter Genomics Laboratory, Royal Devon University Healthcare NHS Foundation Trust, Barrack Rd, Exeter EX2 5DW, United Kingdom
| | - Jørn V Sagen
- Mohn Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Haukelandsbakken 1, Bergen 5020, Norway
- Department of Medical Biochemistry and Pharmacology, Haukeland University Hospital, Jonas Lies veg 87, Bergen 5021, Norway
| | - Pål R Njølstad
- Mohn Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Haukelandsbakken 1, Bergen 5020, Norway
- Children and Youth Clinic, Haukeland University Hospital, Haukelandsbakken 1, Bergen 5021, Norway
| | - Ingvild Aukrust
- Mohn Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Haukelandsbakken 1, Bergen 5020, Norway
- Department of Medical Genetics, Haukeland University Hospital, Jonas Lies veg 87, Bergen 5021, Norway
| | - Lise Bjørkhaug
- Department of Safety, Chemistry, and Biomedical Laboratory Sciences, Western Norway University of Applied Sciences, Inndalsveien 28, Bergen 5020, Norway
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Rastinejad F. The protein architecture and allosteric landscape of HNF4α. Front Endocrinol (Lausanne) 2023; 14:1219092. [PMID: 37732120 PMCID: PMC10507258 DOI: 10.3389/fendo.2023.1219092] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Accepted: 08/01/2023] [Indexed: 09/22/2023] Open
Abstract
Hepatocyte nuclear factor 4 alpha (HNF4α) is a multi-faceted nuclear receptor responsible for governing the development and proper functioning of liver and pancreatic islet cells. Its transcriptional functions encompass the regulation of vital metabolic processes including cholesterol and fatty acid metabolism, and glucose sensing and control. Various genetic mutations and alterations in HNF4α are associated with diabetes, metabolic disorders, and cancers. From a structural perspective, HNF4α is one of the most comprehensively understood nuclear receptors due to its crystallographically observed architecture revealing interconnected DNA binding domains (DBDs) and ligand binding domains (LBDs). This review discusses key properties of HNF4α, including its mode of homodimerization, its binding to fatty acid ligands, the importance of post-translational modifications, and the mechanistic basis for allosteric functions. The surfaces linking HNF4α's DBDs and LBDs create a convergence zone that allows signals originating from any one domain to influence distant domains. The HNF4α-DNA complex serves as a prime illustration of how nuclear receptors utilize individual domains for specific functions, while also integrating these domains to create cohesive higher-order architectures that allow signal responsive functions.
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Affiliation(s)
- Fraydoon Rastinejad
- Nuffield Department of Medicine, Target Discovery Institute (NDMRB), University of Oxford, Oxford, United Kingdom
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5
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Choi WJ, Haratipour Z, Blind RD. Full-length nuclear receptor allosteric regulation. J Lipid Res 2023; 64:100406. [PMID: 37356665 PMCID: PMC10388211 DOI: 10.1016/j.jlr.2023.100406] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2021] [Revised: 06/14/2023] [Accepted: 06/15/2023] [Indexed: 06/27/2023] Open
Abstract
Nuclear receptors are a superfamily of transcription factors regulated by a wide range of lipids that include phospholipids, fatty acids, heme-based metabolites, and cholesterol-based steroids. Encoded as classic two-domain modular transcription factors, nuclear receptors possess a DNA-binding domain (DBD) and a lipid ligand-binding domain (LBD) containing a transcriptional activation function. Decades of structural studies on the isolated LBDs of nuclear receptors established that lipid-ligand binding allosterically regulates the conformation of the LBD, regulating transcriptional coregulator recruitment and thus nuclear receptor function. These structural studies have aided the development of several FDA-approved drugs, highlighting the importance of understanding the structure-function relationships between lipids and nuclear receptors. However, there are few published descriptions of full-length nuclear receptor structure and even fewer descriptions of how lipids might allosterically regulate full-length structure. Here, we examine multidomain interactions based on the published full-length nuclear receptor structures, evaluating the potential of interdomain interfaces within these nuclear receptors to act as inducible sites of allosteric regulation by lipids.
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Affiliation(s)
- Woong Jae Choi
- Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Zeinab Haratipour
- Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA; Vanderbilt University Center for Structural Biology, Nashville, TN, USA; Program in Precision Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Raymond D Blind
- Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA; Vanderbilt University Center for Structural Biology, Nashville, TN, USA; Program in Precision Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA; Diabetes Research and Training Center, Vanderbilt University Medical Center, Nashville, TN, USA.
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Role of Actionable Genes in Pursuing a True Approach of Precision Medicine in Monogenic Diabetes. Genes (Basel) 2022; 13:genes13010117. [PMID: 35052457 PMCID: PMC8774614 DOI: 10.3390/genes13010117] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 01/04/2022] [Accepted: 01/05/2022] [Indexed: 12/16/2022] Open
Abstract
Monogenic diabetes is a genetic disorder caused by one or more variations in a single gene. It encompasses a broad spectrum of heterogeneous conditions, including neonatal diabetes, maturity onset diabetes of the young (MODY) and syndromic diabetes, affecting 1-5% of patients with diabetes. Some of these variants are harbored by genes whose altered function can be tackled by specific actions ("actionable genes"). In suspected patients, molecular diagnosis allows the implementation of effective approaches of precision medicine so as to allow individual interventions aimed to prevent, mitigate or delay clinical outcomes. This review will almost exclusively concentrate on the clinical strategy that can be specifically pursued in carriers of mutations in "actionable genes", including ABCC8, KCNJ11, GCK, HNF1A, HNF4A, HNF1B, PPARG, GATA4 and GATA6. For each of them we will provide a short background on what is known about gene function and dysfunction. Then, we will discuss how the identification of their mutations in individuals with this form of diabetes, can be used in daily clinical practice to implement specific monitoring and treatments. We hope this article will help clinical diabetologists carefully consider who of their patients deserves timely genetic testing for monogenic diabetes.
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7
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Guo Y, Zhao J, Huang R, Xu T, Zhou K, Zheng L. Scalable Dual-Fluorescence Assay for Functional Interpretation of HNF-4α Missense Variants. Front Endocrinol (Lausanne) 2022; 13:812747. [PMID: 35237236 PMCID: PMC8883583 DOI: 10.3389/fendo.2022.812747] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 01/17/2022] [Indexed: 11/15/2022] Open
Abstract
AIM The study aimed to develop a scalable dual-fluorescence assay in cells to enable the functional interpretation of HNF-4α missense variants identified in exome sequencing, which can be used to guide clinical diagnosis. METHODS Using mOrange2 and GFP fluorescence proteins to track the expression of HNF-4α (HNF-4α-mOrange2) and reporter activity under the control of the HNF-1α promoter (pHNF1A-GFP), respectively, we designed a dual-fluorescence assay to evaluate the expression level, cellular localization, and transcriptional function of HNF-4α simultaneously in live cells. To assess the scalable characteristic of the assay, a small library containing five previously reported mutations and wild-type HNF-4α was constructed. Cells infected with this library were sorted into different populations through fluorescence-activated cell sorting (FACS) according to the transcription activity and expression abundance. Cloning and Sanger sequencing were used to detect the mutations of the different groups. High content screening (HCS) assay was used for the validation of individual mutants in the function and expression point of view. RESULTS HNF-4α-mOrange2 exhibited nuclear localization and transactivation capability on the HNF-1α promoter as physical HNF-4α does. The expression of HNF-4α-mOrange2 shows a 6-fold induction of GFP expression compared to the control without HNF-4α-mOrange2, which was significantly abolished by the known loss-of-function mutant M373R. The different performances of wild-type and mutant M373R made them distinguishable in the FACS system, empowering the scalable capability of this assay for classifying large numbers of variants combining functional stratification and sequencing. Further application of the assay in the small library showed that three cell populations were seen grouped as Normal (same transactivation as wild type), Reducedexp_nor (reduced transactivation with normal or higher expression), and Reducedexp_low (reduced transactivation with lower expression). Subsequently, Sanger sequencing showed that wild-type HNF-4α was in the Normal group, two mutations (M373R and G79C) were enriched in the Reducedexp_nor group, and three mutations (C115S, L272P, and F83C) belonged to the Reducedexp_low group. These results were validated by further imaging data using HCS assay for individual mutation. CONCLUSIONS Our study proposes a scalable and informative approach for the characterization of the variants in HNF-4α genes in a quantitative and high-throughput manner.
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Affiliation(s)
- Yiming Guo
- College of Life Sciences, the University of Chinese Academy of Sciences, Beijing, China
| | - Jing Zhao
- College of Life Sciences, the University of Chinese Academy of Sciences, Beijing, China
| | | | - Tao Xu
- College of Life Sciences, the University of Chinese Academy of Sciences, Beijing, China
- Guangzhou Laboratory, Guangzhou, China
- Key Laboratory of RNA Biology, Center for Big Data Research in Health, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, China
- *Correspondence: Li Zheng, ; Kaixin Zhou, ; Tao Xu,
| | - Kaixin Zhou
- College of Life Sciences, the University of Chinese Academy of Sciences, Beijing, China
- Guangzhou Laboratory, Guangzhou, China
- *Correspondence: Li Zheng, ; Kaixin Zhou, ; Tao Xu,
| | - Li Zheng
- Key Laboratory of RNA Biology, Center for Big Data Research in Health, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- *Correspondence: Li Zheng, ; Kaixin Zhou, ; Tao Xu,
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8
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Vijayakumar P, Raut AA, Chingtham S, Murugkar HV, Kulkarni DD, Sood R, Singh VP, Mishra A. Proteomic analysis of differential expression of lung proteins in response to highly pathogenic avian influenza virus infection in chickens. Arch Virol 2021; 167:141-152. [PMID: 34786609 DOI: 10.1007/s00705-021-05287-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2021] [Accepted: 09/16/2021] [Indexed: 12/21/2022]
Abstract
Elucidation of the molecular pathogenesis underlying virus-host interactions is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) virus infection in chickens. However, the pathogenesis of HPAI virus in chickens is not completely understood. To identify the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we analyzed the lung proteome of a chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). Mass spectrometry data sets were searched against the chicken UniProt reference database. At the local false discovery rate level of 5%, a total of 3313 proteins with the presence of at least one unique peptide were identified in the chicken lung proteome datasets. Differential expression analysis of these proteins showed that 247 and 1754 proteins were downregulated at 12 h and 48 h postinfection, respectively. We observed expression of proteins of the predominant signaling pathways, including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and JAK-STAT signaling. Activation of these pathways is associated with the cytokine storm effect and thus may be the cause of the severity of HPAI H5N1 infection in chickens. We also observed the expression of myeloid differentiation primary response protein (MyD88), inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), interleukin 1 receptor associated kinase 4 (IRAK4), RELA proto-oncogene NF-κB subunit (RELA), and mitochondrial antiviral signaling protein (MAVS), which are involved in critical signaling pathways, as well as other, less-commonly identified proteins such as hepatocyte nuclear factor 4 alpha (HNF4A), ELAV-like RNA binding protein 1 (ELAVL1), fibronectin 1 (FN1), COP9 signalosome subunit 5 (COPS5), cullin 1 (CUL1), breast cancer type 1 susceptibility protein (BRCA1), and the FYN proto-oncogene Src family tyrosine kinase (FYN) as main hub proteins that might play important roles in influenza pathogenesis in chickens. In summary, we identified the signaling pathways and the proteomic determinants associated with disease pathogenesis in chickens infected with HPAI H5N1 virus.
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Affiliation(s)
- Periyasamy Vijayakumar
- Pathogenomics Laboratory, ICAR-National Institute of High-Security Animal Diseases, OIE Reference lab for Avian Influenza, Bhopal, 462021, Madhya Pradesh, India.,Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Orathanadu, 614625, Tamil Nadu, India
| | - Ashwin Ashok Raut
- Pathogenomics Laboratory, ICAR-National Institute of High-Security Animal Diseases, OIE Reference lab for Avian Influenza, Bhopal, 462021, Madhya Pradesh, India
| | - Santhalembi Chingtham
- Pathogenomics Laboratory, ICAR-National Institute of High-Security Animal Diseases, OIE Reference lab for Avian Influenza, Bhopal, 462021, Madhya Pradesh, India
| | - Harshad V Murugkar
- ICAR -National Institute of High-Security Animal Diseases, OIE Reference lab for Avian Influenza, Bhopal, 462021, Madhya Pradesh, India
| | - Diwakar D Kulkarni
- ICAR -National Institute of High-Security Animal Diseases, OIE Reference lab for Avian Influenza, Bhopal, 462021, Madhya Pradesh, India
| | - Richa Sood
- ICAR -National Institute of High-Security Animal Diseases, OIE Reference lab for Avian Influenza, Bhopal, 462021, Madhya Pradesh, India
| | - Vijendra Pal Singh
- ICAR -National Institute of High-Security Animal Diseases, OIE Reference lab for Avian Influenza, Bhopal, 462021, Madhya Pradesh, India
| | - Anamika Mishra
- Pathogenomics Laboratory, ICAR-National Institute of High-Security Animal Diseases, OIE Reference lab for Avian Influenza, Bhopal, 462021, Madhya Pradesh, India.
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9
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Jiang S, Tanaka T, Yagami R, Hasegawa G, Umezu H, Fujiyoshi Y, Kodama T, Naito M, Ajioka Y. Immunohistochemical detection of hepatocyte nuclear factor-4α in vertebrates. Microsc Res Tech 2021; 84:2906-2914. [PMID: 34196449 DOI: 10.1002/jemt.23848] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 05/16/2021] [Accepted: 05/23/2021] [Indexed: 11/06/2022]
Abstract
Hepatocyte nuclear factor-4α (HNF4α) presents in multiple isoforms generated using alternative promoter (P1 and P2) and splicing. Neither conservation of tissue distribution of HNF4α isoforms, nor presence of alternative promoter usage is known. In this study, to detect the expression of HNF4α in some species of animals, we have applied monoclonal antibodies against P1 (K9218) and P2 (H6939) promoter-driven and P1/P2 promoter-driven H1415 HNF4α for immunohistochemistry and western blot analysis. Antibody K9218 was observed in the hepatocytes, proximal tubules of the kidney, and epithelial cells in the mucosa of the small intestine and colon of rats, chicken, and tortoise, whereas antibody H6939 signal were detected in the stomach, pancreas, bile duct, and pancreatic duct of human and rats. The signal for antibody K9218 was recognized in tissues of a wide range of mammals, bird, reptile, amphibian, and fish as well. Antibody H1415 displayed a positive reaction in hepatocytes and intestinal epithelial cells in chicken and tortoise, whereas the bile duct, mucosal epithelial cells in the stomach, or pancreas in these animals were negative. Western blotting showed the binding of the antibody with HNF4α protein from each animal. The sequence of human HNF4α was 100% identical to murine and rat HNF4α, 88.9% to chicken, 77.8% to Xenopus HNF4α, and 81.5% to medaka. However, the specific part of human and invertebrate Drosophila HNF4 shares only 14.8% sequence identity. This antibody is useful for detecting HNF4α isoforms in a wide range of vertebrates, and suggests many insights into animal evolution.
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Affiliation(s)
- Shuying Jiang
- Niigata College of Medical Technology, Niigata, Japan.,Division of Molecular and Diagnostic Pathology, Graduate Scholl of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Toshiya Tanaka
- Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
| | - Ren Yagami
- Division of International Health (Public Health) Graduate Scholl of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Go Hasegawa
- Division of Pathology, Niigata University Medical and Dental Hospital, Niigata, Japan
| | - Hajime Umezu
- Uonuma Institute of Community Medicine, Niigata University Medical and Dental Hospital, Niigata, Japan
| | - Yukio Fujiyoshi
- Department of Pathology and Molecular Diagnostics, Nagoya City Graduate School of Medical Sciences, Nagoya, Japan
| | - Tatsuhiko Kodama
- Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
| | - Makoto Naito
- Department of Pathology, Niigata Medical Center, Nishi-ku, Niigata, Japan
| | - Yoichi Ajioka
- Division of Molecular and Diagnostic Pathology, Graduate Scholl of Medical and Dental Sciences, Niigata University, Niigata, Japan
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A Review of Functional Characterization of Single Amino Acid Change Mutations in HNF Transcription Factors in MODY Pathogenesis. Protein J 2021; 40:348-360. [PMID: 33950347 DOI: 10.1007/s10930-021-09991-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/22/2021] [Indexed: 12/15/2022]
Abstract
Mutations in HNF transcription factor genes cause the most common subtypes of maturity-onset of diabetes of youth (MODY), a monogenic form of diabetes mellitus. Mutations in the HNF1-α, HNF4-α, and HNF1-β genes are primarily considered as the cause of MODY3, MODY1, and MODY5 subtypes, respectively. Although patients with different subtypes display similar symptoms, they may develop distinct diabetes-related complications and require different treatments depending on the type of the mutation. Genetic analysis of MODY patients revealed more than 400 missense/nonsense mutations in HNF1-α, HNF4-α, and HNF1-β genes, however only a small portion of them are functionally characterized. Evaluation of nonsense mutations are more direct as they lead to premature stop codons and mostly in mRNA decay or nonfunctional truncated proteins. However, interpretation of the single amino acid change (missense) mutation is not such definite, as effect of the variant may vary depending on the location and also the substituted amino acid. Mutations with benign effect on the protein function may not be the pathologic variant and further genetic testing may be required. Here, we discuss the functional characterization analysis of single amino acid change mutations identified in HNF1-α, HNF4-α, and HNF1-β genes and evaluate their roles in MODY pathogenesis. This review will contribute to comprehend HNF nuclear family-related molecular mechanisms and to develop more accurate diagnosis and treatment based on correct evaluation of pathologic effects of the variants.
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11
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Sanchez Caballero L, Gorgogietas V, Arroyo MN, Igoillo-Esteve M. Molecular mechanisms of β-cell dysfunction and death in monogenic forms of diabetes. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2021; 359:139-256. [PMID: 33832649 DOI: 10.1016/bs.ircmb.2021.02.005] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Monogenetic forms of diabetes represent 1%-5% of all diabetes cases and are caused by mutations in a single gene. These mutations, that affect genes involved in pancreatic β-cell development, function and survival, or insulin regulation, may be dominant or recessive, inherited or de novo. Most patients with monogenic diabetes are very commonly misdiagnosed as having type 1 or type 2 diabetes. The severity of their symptoms depends on the nature of the mutation, the function of the affected gene and, in some cases, the influence of additional genetic or environmental factors that modulate severity and penetrance. In some patients, diabetes is accompanied by other syndromic features such as deafness, blindness, microcephaly, liver and intestinal defects, among others. The age of diabetes onset may also vary from neonatal until early adulthood manifestations. Since the different mutations result in diverse clinical presentations, patients usually need different treatments that range from just diet and exercise, to the requirement of exogenous insulin or other hypoglycemic drugs, e.g., sulfonylureas or glucagon-like peptide 1 analogs to control their glycemia. As a consequence, awareness and correct diagnosis are crucial for the proper management and treatment of monogenic diabetes patients. In this chapter, we describe mutations causing different monogenic forms of diabetes associated with inadequate pancreas development or impaired β-cell function and survival, and discuss the molecular mechanisms involved in β-cell demise.
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Affiliation(s)
- Laura Sanchez Caballero
- ULB Center for Diabetes Research (UCDR), Université Libre de Bruxelles, Brussels, Belgium. http://www.ucdr.be/
| | - Vyron Gorgogietas
- ULB Center for Diabetes Research (UCDR), Université Libre de Bruxelles, Brussels, Belgium. http://www.ucdr.be/
| | - Maria Nicol Arroyo
- ULB Center for Diabetes Research (UCDR), Université Libre de Bruxelles, Brussels, Belgium. http://www.ucdr.be/
| | - Mariana Igoillo-Esteve
- ULB Center for Diabetes Research (UCDR), Université Libre de Bruxelles, Brussels, Belgium. http://www.ucdr.be/.
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12
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Zhan X, Zhao A. Transcription factor FOXA3 promotes the development of Hepatoblastoma via regulating HNF1A, AFP, and ZFHX3 expression. J Clin Lab Anal 2020; 35:e23686. [PMID: 33368532 PMCID: PMC7957975 DOI: 10.1002/jcla.23686] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 11/19/2020] [Accepted: 12/01/2020] [Indexed: 12/15/2022] Open
Abstract
Objective In this research paper, we aimed to study the role of FOXA3 in hepatoblastoma (HB) and the molecular mechanism. Methods Immunohistochemistry was applied to determine the expression situation of FOXA3 and AFP in HB tissues and the adjacent normal tissues. FOXA3, HNF1A, and ZFHX3 expressions in HB tissues and the normal tissues were measured by Western blot. HB cell lines were randomly divided into 4 groups: Model, si‐NC, si‐FOXA3‐1, and si‐FOXA3‐2 group. The HB cell viability and colony formation characteristics in the 4 groups were explored by CCK‐8 and cell cloning formation assay, respectively. The expression of FOXA3, AFP, HNF1A, ZFHX3, and MYC in HB cells after knockdown of FOXA3 was measured. Results FOXA3, AFP, and HNF1A expressions were significantly up‐regulated in HB tissues, while ZFHX3 expression was down‐regulated. Knockdown of FOXA3 markedly inhibited HB cell viability and cloning formation ability. Knockdown of FOXA3 decreased FOXA3, AFP, and HNF1A/MYC expression, while increased ZFHX3 expression. Conclusion FOXA3 promotes the occurrence and development of HB by up‐regulating AFP and HNF1A/MYC expression, and down‐regulating ZFHX3 expression.
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Affiliation(s)
- Xiaoyun Zhan
- Department of Childhood Hematology, Maternal and Child Hospital of Hubei province, Tongji Medical College Huazhong University of Science and Technology, Wuhan, China
| | - Alan Zhao
- Department of Childhood Hematology, Maternal and Child Hospital of Hubei province, Tongji Medical College Huazhong University of Science and Technology, Wuhan, China
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13
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Hu M, Cherkaoui I, Misra S, Rutter GA. Functional Genomics in Pancreatic β Cells: Recent Advances in Gene Deletion and Genome Editing Technologies for Diabetes Research. Front Endocrinol (Lausanne) 2020; 11:576632. [PMID: 33162936 PMCID: PMC7580382 DOI: 10.3389/fendo.2020.576632] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Accepted: 09/17/2020] [Indexed: 12/13/2022] Open
Abstract
The inheritance of variants that lead to coding changes in, or the mis-expression of, genes critical to pancreatic beta cell function can lead to alterations in insulin secretion and increase the risk of both type 1 and type 2 diabetes. Recently developed clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene editing tools provide a powerful means of understanding the impact of identified variants on cell function, growth, and survival and might ultimately provide a means, most likely after the transplantation of genetically "corrected" cells, of treating the disease. Here, we review some of the disease-associated genes and variants whose roles have been probed up to now. Next, we survey recent exciting developments in CRISPR/Cas9 technology and their possible exploitation for β cell functional genomics. Finally, we will provide a perspective as to how CRISPR/Cas9 technology may find clinical application in patients with diabetes.
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Affiliation(s)
- Ming Hu
- Section of Cell Biology and Functional Genomics, Faculty of Medicine, Imperial College London, London, United Kingdom
| | - Ines Cherkaoui
- Section of Cell Biology and Functional Genomics, Faculty of Medicine, Imperial College London, London, United Kingdom
| | - Shivani Misra
- Metabolic Medicine, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, United Kingdom
| | - Guy A. Rutter
- Section of Cell Biology and Functional Genomics, Faculty of Medicine, Imperial College London, London, United Kingdom
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14
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Control of Cell Identity by the Nuclear Receptor HNF4 in Organ Pathophysiology. Cells 2020; 9:cells9102185. [PMID: 32998360 PMCID: PMC7600215 DOI: 10.3390/cells9102185] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Revised: 09/25/2020] [Accepted: 09/26/2020] [Indexed: 12/14/2022] Open
Abstract
Hepatocyte Nuclear Factor 4 (HNF4) is a transcription factor (TF) belonging to the nuclear receptor family whose expression and activities are restricted to a limited number of organs including the liver and gastrointestinal tract. In this review, we present robust evidence pointing to HNF4 as a master regulator of cellular differentiation during development and a safekeeper of acquired cell identity in adult organs. Importantly, we discuss that transient loss of HNF4 may represent a protective mechanism upon acute organ injury, while prolonged impairment of HNF4 activities could contribute to organ dysfunction. In this context, we describe in detail mechanisms involved in the pathophysiological control of cell identity by HNF4, including how HNF4 works as part of cell-specific TF networks and how its expression/activities are disrupted in injured organs.
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15
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Villemain L, Prigent S, Abou-Lovergne A, Pelletier L, Chiral M, Pontoglio M, Foufelle F, Caruso S, Pineau R, Rebouissou S, Chevet E, Zucman-Rossi J, Combettes L. Sigma 1 Receptor is Overexpressed in Hepatocellular Adenoma: Involvement of ERα and HNF1α. Cancers (Basel) 2020; 12:cancers12082213. [PMID: 32784704 PMCID: PMC7464972 DOI: 10.3390/cancers12082213] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Accepted: 08/03/2020] [Indexed: 11/16/2022] Open
Abstract
Sigma receptor 1 (SigR1) is an endoplasmic reticulum resident integral membrane protein whose functions remain unclear. Although the liver shows the highest expression of SigR1, its role in this organ is unknown. SigR1 is overexpressed in many cancers and its expression is correlated to hormonal status in hormone-dependent cancers. To better understand the role of SigR1 in hepatocytes we focused our work on the regulation of its expression in tumoral liver. In this context, hepatocellular adenomas, benign hepatic tumors associated with estrogen intake are of particular interest. The expression of SigR1 mRNA was assessed in hepatocellular adenoma (HCA) patients using qPCR. The impact of estrogen on the expression of SigR1 was studied in vivo (mice) and in vitro (HepG2 and Huh7 cells). The effect of HNF1α on the expression of SigR1 was studied in vivo by comparing wild type mice to HNF1 knockout mice. Estrogen enhanced SigR1 expression through its nuclear receptor ERα. HNF1α mutated HCA (H-HCA) significantly overexpressed SigR1 compared to all other HCA subtypes. HNF1 knockout mice showed an increase in SigR1 expression. Overexpressing SigR1 in cellular models increases proliferation rate and storage of lipid droplets, which phenocopies the H-HCA phenotype. SigR1 is involved in hepatocyte proliferation and steatosis and may play an important role in the control of the H-HCA phenotype.
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Affiliation(s)
- Laure Villemain
- UMRs 1174, University Paris-Saclay, Inserm, 91405 Orsay, France; (L.V.); (S.P.); (A.A.-L.)
| | - Sylvie Prigent
- UMRs 1174, University Paris-Saclay, Inserm, 91405 Orsay, France; (L.V.); (S.P.); (A.A.-L.)
| | - Aurélie Abou-Lovergne
- UMRs 1174, University Paris-Saclay, Inserm, 91405 Orsay, France; (L.V.); (S.P.); (A.A.-L.)
| | - Laura Pelletier
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Université Sorbonne Paris Nord, Functional Genomics of Solid Tumors laboratory, F-75006 Paris, France; (L.P.); (S.C.); (S.R.); (J.Z.-R.)
| | - Magali Chiral
- Institut Necker - Enfants Malades (INEM), 14, rue Maria Héléna Vieira da Silva Bâtiment Leriche, 75014 Paris, France; (M.C.); (M.P.)
| | - Marco Pontoglio
- Institut Necker - Enfants Malades (INEM), 14, rue Maria Héléna Vieira da Silva Bâtiment Leriche, 75014 Paris, France; (M.C.); (M.P.)
| | - Fabienne Foufelle
- Centre de Recherche des Cordeliers, Sorbonne University, Inserm, U1138” Metabolic diseases, diabetes and co-morbidities” F-75006 Paris, France;
| | - Stefano Caruso
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Université Sorbonne Paris Nord, Functional Genomics of Solid Tumors laboratory, F-75006 Paris, France; (L.P.); (S.C.); (S.R.); (J.Z.-R.)
| | - Raphael Pineau
- UMRs 1242 “Chemistry, Oncogenesis, stress, Signaling” (COSS), University de Rennes-1, 35042 Rennes, France; (R.P.); (E.C.)
- Centre de Lutte Contre le Cancer Eugène Marquis, 35042 Rennes, France
| | - Sandra Rebouissou
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Université Sorbonne Paris Nord, Functional Genomics of Solid Tumors laboratory, F-75006 Paris, France; (L.P.); (S.C.); (S.R.); (J.Z.-R.)
| | - Eric Chevet
- UMRs 1242 “Chemistry, Oncogenesis, stress, Signaling” (COSS), University de Rennes-1, 35042 Rennes, France; (R.P.); (E.C.)
- Centre de Lutte Contre le Cancer Eugène Marquis, 35042 Rennes, France
| | - Jessica Zucman-Rossi
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Université Sorbonne Paris Nord, Functional Genomics of Solid Tumors laboratory, F-75006 Paris, France; (L.P.); (S.C.); (S.R.); (J.Z.-R.)
- Hôpital Européen Georges Pompidou, AP-HP, F-75015 Paris, France
| | - Laurent Combettes
- UMRs 1174, University Paris-Saclay, Inserm, 91405 Orsay, France; (L.V.); (S.P.); (A.A.-L.)
- Correspondence: ; Tel.: +01-6915-6396
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16
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Russell R, Carnese PP, Hennings TG, Walker EM, Russ HA, Liu JS, Giacometti S, Stein R, Hebrok M. Loss of the transcription factor MAFB limits β-cell derivation from human PSCs. Nat Commun 2020; 11:2742. [PMID: 32488111 PMCID: PMC7265500 DOI: 10.1038/s41467-020-16550-9] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2019] [Accepted: 05/06/2020] [Indexed: 12/11/2022] Open
Abstract
Next generation sequencing studies have highlighted discrepancies in β-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human β-cells postnatally, while its expression is restricted to embryonic and neo-natal β-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to β-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide–positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology. The MAF bZIP transcription factor B (MAFB) is present in postnatal human beta cells but its role is unclear. Here, the authors show that MAFB regulates endocrine pancreatic cell fate specification.
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Affiliation(s)
- Ronan Russell
- UCSF Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Phichitpol P Carnese
- UCSF Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Thomas G Hennings
- UCSF Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Emily M Walker
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, 37232, USA
| | - Holger A Russ
- UCSF Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA.,Barbara Davis Center for Diabetes, School of Medicine, University of Colorado Denver, Aurora, CO, 80045, USA
| | - Jennifer S Liu
- UCSF Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Simone Giacometti
- UCSF Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Roland Stein
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, 37232, USA
| | - Matthias Hebrok
- UCSF Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA.
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17
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Mariani L, Weinand K, Gisselbrecht SS, Bulyk ML. MEDEA: analysis of transcription factor binding motifs in accessible chromatin. Genome Res 2020; 30:736-748. [PMID: 32424069 PMCID: PMC7263192 DOI: 10.1101/gr.260877.120] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2020] [Accepted: 04/10/2020] [Indexed: 12/15/2022]
Abstract
Deciphering the interplay between chromatin accessibility and transcription factor (TF) binding is fundamental to understanding transcriptional regulation, control of cellular states, and the establishment of new phenotypes. Recent genome-wide chromatin accessibility profiling studies have provided catalogs of putative open regions, where TFs can recognize their motifs and regulate gene expression programs. Here, we present motif enrichment in differential elements of accessibility (MEDEA), a computational tool that analyzes high-throughput chromatin accessibility genomic data to identify cell-type-specific accessible regions and lineage-specific motifs associated with TF binding therein. To benchmark MEDEA, we used a panel of reference cell lines profiled by ENCODE and curated by the ENCODE Project Consortium for the ENCODE-DREAM Challenge. By comparing results with RNA-seq data, ChIP-seq peaks, and DNase-seq footprints, we show that MEDEA improves the detection of motifs associated with known lineage specifiers. We then applied MEDEA to 610 ENCODE DNase-seq data sets, where it revealed significant motifs even when absolute enrichment was low and where it identified novel regulators, such as NRF1 in kidney development. Finally, we show that MEDEA performs well on both bulk and single-cell ATAC-seq data. MEDEA is publicly available as part of our Glossary-GENRE suite for motif enrichment analysis.
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Affiliation(s)
- Luca Mariani
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Kathryn Weinand
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.,Bioinformatics and Integrative Genomics PhD Program, Harvard University, Cambridge, Massachusetts 02138, USA
| | - Stephen S Gisselbrecht
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Martha L Bulyk
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.,Bioinformatics and Integrative Genomics PhD Program, Harvard University, Cambridge, Massachusetts 02138, USA.,Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA
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18
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Mattis KK, Gloyn AL. From Genetic Association to Molecular Mechanisms for Islet-cell Dysfunction in Type 2 Diabetes. J Mol Biol 2020; 432:1551-1578. [PMID: 31945378 DOI: 10.1016/j.jmb.2019.12.045] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Revised: 12/15/2019] [Accepted: 12/17/2019] [Indexed: 12/30/2022]
Abstract
Genome-wide association studies (GWAS) have identified over 400 signals robustly associated with risk for type 2 diabetes (T2D). At the vast majority of these loci, the lead single nucleotide polymorphisms (SNPs) reside in noncoding regions of the genome, which hampers biological inference and translation of genetic discoveries into disease mechanisms. The study of these T2D risk variants in normoglycemic individuals has revealed that a significant proportion are exerting their disease risk through islet-cell dysfunction. The central role of the islet is also demonstrated by numerous studies, which have shown an enrichment of these signals in islet-specific epigenomic annotations. In recent years the emergence of authentic human beta-cell lines, and advances in genome-editing technologies coupled with improved protocols differentiating human pluripotent stem cells into beta-like cells has opened up new opportunities for T2D disease modeling. Here we review the current understanding on the genetic basis of T2D focusing on approaches, which have facilitated the identification of causal variants and their effector transcripts in human islets. We will present examples of functional studies based on animal and conventional cellular systems and highlight the potential of novel stem cell-based T2D disease models.
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Affiliation(s)
- Katia K Mattis
- Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, UK
| | - Anna L Gloyn
- Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, UK; Wellcome Trust Centre for Human Genetics, University of Oxford, UK; National Institute of Health Research, Biomedical Research Centre, Churchill Hospital, Headington, Oxford, UK.
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19
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Abstract
MODY (Maturity Onset Diabetes of the Young) is a type of diabetes resulting from a pathogenic effect of gene mutations. Up to date, 13 MODY genes are known. Gene HNF1A is one of the most common causes of MODY diabetes (HNF1A-MODY; MODY3). This gene is polymorphic and more than 1200 pathogenic and non-pathogenic HNF1A variants were described in its UTRs, exons and introns. For HNF1A-MODY, not just gene but also phenotype heterogeneity is typical. Although there are some clinical instructions, HNF1A-MODY patients often do not meet every diagnostic criteria or they are still misdiagnosed as type 1 and type 2 diabetics. There is a constant effort to find suitable biomarkers to help with in distinguishing of MODY3 from Type 1 Diabetes (T1D) and Type 2 Diabetes (T2D). DNA sequencing is still necessary for unambiguous confirmation of clinical suspicion of MODY. NGS (Next Generation Sequencing) methods brought discoveries of multiple new gene variants and new instructions for their pathogenicity classification were required. The most actual problem is classification of variants with uncertain significance (VUS) which is a stumbling-block for clinical interpretation. Since MODY is a hereditary disease, DNA analysis of family members is helpful or even crucial. This review is updated summary about HNF1A-MODY genetics, pathophysiology, clinics functional studies and variant classification.
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Chandran S, Rajadurai VS, Hoi WH, Flanagan SE, Hussain K, Yap F. A Novel HNF4A Mutation Causing Three Phenotypic Forms of Glucose Dysregulation in a Family. Front Pediatr 2020; 8:320. [PMID: 32670997 PMCID: PMC7332776 DOI: 10.3389/fped.2020.00320] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/20/2020] [Accepted: 05/18/2020] [Indexed: 12/12/2022] Open
Abstract
Maturity-onset diabetes of the young (MODY) classically describes dominantly inherited forms of monogenic diabetes diagnosed before 25 years of age due to pancreatic β-cell dysfunction. In contrast, mutations in certain MODY genes can also present with transient or persistent hyperinsulinemic hypoglycemia in newborn infants, reflecting instead β-cell dysregulation. Of the MODY genes described to date, only hepatocyte nuclear factor-4-alpha (HNF4A; MODY1) and hepatocyte nuclear factor-1-alpha (HNF1A; MODY3) mutations may result in a biphasic phenotype of hypoglycemia in early life and hyperglycemia in later life. We report a family with a novel HNF4A mutation with diverse phenotypic presentations of glucose dysregulation. The proband was a term, appropriate-for-gestational age male infant with symptomatic hypoglycemia on day 3 of life needing high glucose infusion rate to maintain normoglycemia. He was born to a non-obese and non-diabetic mother. Glucose regulation was optimized using diazoxide upon confirmation of hyperinsulinism. Cascade genetic screening identified the same mutation in his father and elder sister, but mother was negative. Father was diagnosed with Type 1 diabetes at 15 years of age that required insulin therapy. Proband's elder sister, born at term appropriate for gestational age, presented with transient neonatal hypoglycemia needing parenteral glucose infusion for a week followed by spontaneous resolution. The paternal grandparents were negative for this mutation, confirming a paternal de novo mutation and autosomal dominant inheritance in this family. This pedigree suggests that the presence of early-onset paternal diabetes should prompt molecular testing in infants presenting in the newborn period with diazoxide-responsive hyperinsulinemic hypoglycemia, even in the absence of maternal diabetes and macrosomia.
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Affiliation(s)
- Suresh Chandran
- Division of Medicine, KK Women's and Children's Hospital, Singapore, Singapore.,Duke-NUS Medical School, National University of Singapore, Singapore, Singapore.,Lee Kong Chian School of Medicine, Singapore, Singapore.,Yong Loo Lin School of Medicine, Singapore, Singapore
| | - Victor Samuel Rajadurai
- Division of Medicine, KK Women's and Children's Hospital, Singapore, Singapore.,Duke-NUS Medical School, National University of Singapore, Singapore, Singapore.,Lee Kong Chian School of Medicine, Singapore, Singapore.,Yong Loo Lin School of Medicine, Singapore, Singapore
| | - Wai Han Hoi
- Department of Endocrinology, Tan Tock Seng Hospital, Singapore, Singapore
| | - Sarah E Flanagan
- Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, United Kingdom
| | - Khalid Hussain
- Department of Pediatric Endocrinology, Sidra Medicine, Doha, Qatar
| | - Fabian Yap
- Division of Medicine, KK Women's and Children's Hospital, Singapore, Singapore.,Duke-NUS Medical School, National University of Singapore, Singapore, Singapore.,Lee Kong Chian School of Medicine, Singapore, Singapore
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21
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Yabe SG, Nishida J, Fukuda S, Takeda F, Nasiro K, Yasuda K, Iwasaki N, Okochi H. Expression of mutant mRNA and protein in pancreatic cells derived from MODY3- iPS cells. PLoS One 2019; 14:e0217110. [PMID: 31145732 PMCID: PMC6542550 DOI: 10.1371/journal.pone.0217110] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2019] [Accepted: 05/03/2019] [Indexed: 12/12/2022] Open
Abstract
Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 14 disease genes have been identified. However, the pathogenesis of MODY is not fully understood because the patients' pancreatic beta cells are inaccessible. To elucidate the pathology of MODY, we established MODY3 patient-derived iPS (MODY3-iPS) cells using non-integrating Sendai virus (SeV) vector and examined the mutant mRNA and protein of HNF1A (Hepatocyte Nuclear factor 1A) after pancreatic lineage differentiation. Our patient had a cytosine insertion in the HNF1A gene (P291fsinsC) causing frameshift and making a premature termination codon (PTC). We confirmed these MODY3-iPS cells possessed the characteristics of pluripotent stem cells. After we differentiated them into pancreatic beta cells, transcripts of HNF1A gene were cloned and sequenced. We found that P291fsinsC mutant transcripts were much less frequent than wild ones, but they increased after adding cycloheximide (CHX) to the medium. These results suggested that mutant mRNA was destroyed by nonsense-mediated mRNA decay (NMD). Moreover, we were not able to detect any band of mutant proteins in pancreatic lineage cells which were differentiated from MODY3-iPSCs by western blot (WB) analysis. A scarcity of the truncated form of mutant protein may indicate that MODY3 might be caused by a haplo-insufficiency effect rather than a dominant negative manner.
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Affiliation(s)
- Shigeharu G. Yabe
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Junko Nishida
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Satsuki Fukuda
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Fujie Takeda
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Kiyoko Nasiro
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Kazuki Yasuda
- Department of Metabolic Disorders, Diabetes Research Center, National Center for Global Health and Medicine, Tokyo, Japan
| | - Naoko Iwasaki
- Institute of Geriatrics, Diabetes Center, Institute of Medical Genetics, Tokyo Women’s Medical University, Tokyo, Japan
| | - Hitoshi Okochi
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
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22
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Nagano C, Morisada N, Nozu K, Kamei K, Tanaka R, Kanda S, Shiona S, Araki Y, Ohara S, Matsumura C, Kasahara K, Mori Y, Seo A, Miura K, Washiyama M, Sugimoto K, Harada R, Tazoe S, Kourakata H, Enseki M, Aotani D, Yamada T, Sakakibara N, Yamamura T, Minamikawa S, Ishikura K, Ito S, Hattori M, Iijima K. Clinical characteristics of HNF1B-related disorders in a Japanese population. Clin Exp Nephrol 2019; 23:1119-1129. [PMID: 31131422 DOI: 10.1007/s10157-019-01747-0] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Accepted: 05/07/2019] [Indexed: 12/28/2022]
Abstract
BACKGROUND Hepatocyte nuclear factor 1β (HNF1B), located on chromosome 17q12, causes renal cysts and diabetes syndrome (RCAD). Moreover, various phenotypes related to congenital anomalies of the kidney and urinary tract (CAKUT) or Bartter-like electrolyte abnormalities can be caused by HNF1B variants. In addition, 17q12 deletion syndrome presents with multi-system disorders, as well as RCAD. As HNF1B mutations are associated with different phenotypes and genotype-phenotype relationships remain unclear, here, we extensively studied these mutations in Japan. METHODS We performed genetic screening of RCAD, CAKUT, and Bartter-like syndrome cases. Heterozygous variants or whole-gene deletions in HNF1B were detected in 33 cases (19 and 14, respectively). All deletion cases were diagnosed as 17q12 deletion syndrome, confirmed by multiplex ligation probe amplification and/or array comparative genomic hybridization. A retrospective review of clinical data was also conducted. RESULTS Most cases had morphological abnormalities in the renal-urinary tract system. Diabetes developed in 12 cases (38.7%). Hyperuricemia and hypomagnesemia were associated with six (19.3%) and 13 cases (41.9%), respectively. Pancreatic malformations were detected in seven cases (22.6%). Ten patients (32.3%) had liver abnormalities. Estimated glomerular filtration rates were significantly lower in the patients with heterozygous variants compared to those in patients harboring the deletion (median 37.6 vs 58.8 ml/min/1.73 m2; p = 0.0091). CONCLUSION We present the clinical characteristics of HNF1B-related disorders. To predict renal prognosis and complications, accurate genetic diagnosis is important. Genetic testing for HNF1B mutations should be considered for patients with renal malformations, especially when associated with other organ involvement.
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Affiliation(s)
- China Nagano
- Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan
| | - Naoya Morisada
- Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan. .,Department of Clinical Genetics, Hyogo Prefectural Kobe Children's Hospital, 1-6-7, Minatojimaminami-machi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan.
| | - Kandai Nozu
- Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan
| | - Koichi Kamei
- Division of Nephrology and Rheumatology, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan
| | - Ryojiro Tanaka
- Department of Nephrology, Hyogo Prefectural Kobe Children's Hospital, 1-6-7 Minatojima Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan
| | - Shoichiro Kanda
- Department of Pediatrics, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Shinichi Shiona
- Department of Pediatrics, Oita Prefectural Hospital, 476, Oaza-Bujyo, Oita, Oita, 870-8511, Japan
| | - Yoshinori Araki
- Department of Pediatrics, Hokkaido Medical Center, 5-7-1-1 Yamanote Nishi-ku, Sapporo, Hokkaido, 063-0005, Japan
| | - Shinichiro Ohara
- Department of Pediatrics, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima-shi, Fukushima, 960-1295, Japan
| | - Chieko Matsumura
- Department of Pediatrics, National Hospital Organization Chibahigashi National Hospital, 673 Nitonacho, Chuo-ku, Chiba, Chiba, 260-8712, Japan
| | - Katsuaki Kasahara
- Department of Pediatric Nephrology, Japanese Red Cross Nagoya Daini Hospital, 2-9 Myokencho, Syowa-ku, Nagoya, 4668-650, Japan
| | - Yukiko Mori
- Department of Pediatrics, Japanese Red Cross Fukui Hospital, 2-4-1, Tsukimi, Fukui, 918-8501, Japan
| | - Akane Seo
- Department of Diabetes and Endocrinology, Kobe University Hospital, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan
| | - Kenichiro Miura
- Department of Pediatric Nephrology, Tokyo Women's Medical University, School of Medicine, 8-1, Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Miki Washiyama
- Department of Diabetes and Endocrinology, Kusatsu General Hospital, 1660 Yabase-cho, Kusatsu, Siga, 525-8585, Japan
| | - Keisuke Sugimoto
- Department of Pediatrics, Faculty of Medicine, Kindai University, 377-2, Ohno-Higashi, Osakasayama, Osaka, 589-8511, Japan
| | - Ryoko Harada
- Department of Nephrology, Tokyo Metropolitan Children's Medical Center, 2-8-29, Musashidai, Fuchu, Tokyo, 183-8561, Japan
| | - Satoshi Tazoe
- Department of Metabolism, Osaka City General Hospital, 2-13-22 Miyakojimahondori, Miyakojima-ku, Osaka, 534-0021, Japan
| | - Hiroyo Kourakata
- Department of Respiratory Medicine, Niigata Saiseikai Sanjo Hospital, 6-18 Oonohata, Sanjyo-shi, Niigata, 955-8511, Japan
| | - Mayumi Enseki
- Department of Pediatrics, Tokai University Hospital, 143, Shimokasuya, Isehara-shi, Tokyo, 259-1193, Japan
| | - Daisuke Aotani
- Department of Gastroenterology and Metabolism, Graduate School of Medical Sciences and Medical School, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan
| | - Takeshi Yamada
- Department of Pediatrics, Niigata University School of Medicine, 1-757, Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan
| | - Nana Sakakibara
- Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan
| | - Tomohiko Yamamura
- Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan
| | - Shogo Minamikawa
- Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan
| | - Kenji Ishikura
- Division of Nephrology and Rheumatology, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan.,Department of Pediatrics, Kitasato University School of Medicine, 1-15-1, Kitasato, Minami-ku, Sagamihara, 252-0375, Japan
| | - Shuichi Ito
- Department of Pediatrics, Yokohama City University, 3-9, Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan
| | - Motoshi Hattori
- Department of Pediatric Nephrology, Tokyo Women's Medical University, School of Medicine, 8-1, Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Kazumoto Iijima
- Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan
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23
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Ng NHJ, Jasmen JB, Lim CS, Lau HH, Krishnan VG, Kadiwala J, Kulkarni RN, Ræder H, Vallier L, Hoon S, Teo AKK. HNF4A Haploinsufficiency in MODY1 Abrogates Liver and Pancreas Differentiation from Patient-Derived Induced Pluripotent Stem Cells. iScience 2019; 16:192-205. [PMID: 31195238 PMCID: PMC6562146 DOI: 10.1016/j.isci.2019.05.032] [Citation(s) in RCA: 43] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2018] [Revised: 11/20/2018] [Accepted: 05/22/2019] [Indexed: 01/01/2023] Open
Abstract
Maturity-onset diabetes of the young 1 (MODY1) is a monogenic diabetes condition caused by heterozygous HNF4A mutations. We investigate how HNF4A haploinsufficiency from a MODY1/HNF4A mutation influences the development of foregut-derived liver and pancreatic cells through differentiation of human induced pluripotent stem cells from a MODY1 family down the foregut lineage. In MODY1-derived hepatopancreatic progenitors, which expressed reduced HNF4A levels and mislocalized HNF4A, foregut genes were downregulated, whereas hindgut-specifying HOX genes were upregulated. MODY1-derived hepatocyte-like cells were found to exhibit altered morphology. Hepatic and β cell gene signatures were also perturbed in MODY1-derived hepatocyte-like and β-like cells, respectively. As mutant HNF4A (p.Ile271fs) did not undergo complete nonsense-mediated decay or exert dominant negativity, HNF4A-mediated loss of function is likely due to impaired transcriptional activation of target genes. Our results suggest that in MODY1, liver and pancreas development is perturbed early on, contributing to altered hepatic proteins and β cell defects in patients.
HNF4A is downregulated and predominantly mislocalized in the cytoplasm in MODY1 Foregut markers, pancreatic and hepatic genes, were downregulated in MODY1-HPPs A reciprocal upregulation of hindgut HOX genes was observed in MODY1-HPPs Mutant HNF4A resulted in loss of transcriptional activation of target genes
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Affiliation(s)
- Natasha Hui Jin Ng
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
| | - Joanita Binte Jasmen
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
| | - Chang Siang Lim
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore
| | - Hwee Hui Lau
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore
| | | | - Juned Kadiwala
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, University of Cambridge, Cambridge CB2 0SZ, UK
| | - Rohit N Kulkarni
- Section of Islet Cell and Regenerative Biology, Joslin Diabetes Center, Harvard Stem Cell Institute, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02215, USA
| | - Helge Ræder
- Department of Pediatrics, Haukeland University Hospital, 5021 Bergen, Norway; KG Jebsen Center for Diabetes Research, Department of Clinical Science, Faculty of Medicine, University of Bergen, 5020 Bergen, Norway
| | - Ludovic Vallier
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, University of Cambridge, Cambridge CB2 0SZ, UK; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK
| | - Shawn Hoon
- Molecular Engineering Lab, A*STAR, Singapore 138673, Singapore
| | - Adrian Kee Keong Teo
- Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore; Department of Biochemistry and Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore.
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24
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Singh P, Tung SP, Han EH, Lee IK, Chi YI. Dimerization defective MODY mutations of hepatocyte nuclear factor 4α. Mutat Res 2019; 814:1-6. [PMID: 30648609 DOI: 10.1016/j.mrfmmm.2019.01.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Revised: 11/13/2018] [Accepted: 01/08/2019] [Indexed: 10/27/2022]
Abstract
HNF4α is a culprit gene product for a monogenic and dominantly-inherited form of diabetes, referred to as MODY1 (Maturity Onset Diabetes of the Young type 1). Reduced HNF4α activities have been linked to impaired insulin secretion and β-cell function. Numerous mutations have been identified from the patients and they have been instructive as to the individual residue's role in protein structure-function and dysfunction. As a member of the nuclear receptor (NR) superfamily, HNF4α is made of characteristic modular domains and it functions exclusively as a homodimer despite its sequence homology to RXR, a common heterodimer partner of non-steroidal NRs. Transcription factors commonly dimerize to enhance their molecular functions mainly by facilitating the recognition of double helix target DNAs that display an intrinsic pseudo-2-fold symmetry and the recruitment of the remainder of the main transcriptional machinery. HNF4α is no exception and its dimerization is maintained by the ligand binding domain (LBD) mainly through the leucine-zipper-like interactions at the stalk of two interacting helices. Although many MODY1 mutations have been previously characterized, including DNA binding disruptors, ligand binding disruptors, coactivator binding disruptors, and protein stability disruptors, protein dimerization disruptors have not been formally reported. In this report, we present a set of data for the two MODY1 mutations found right at the dimerization interface (L332 P and L328del mutations) which clearly exhibit the disruptive effects of directly affecting dimerization, protein stability, and transcriptional activities. These data reinforced the fact that MODY mutations are loss-of-function mutations and HNF4α dimerization is essential for its optimal function and normal physiology.
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Affiliation(s)
- Puja Singh
- Section of Structural Biology, Hormel Institute, University of Minnesota, Austin, MN, United States
| | - Shu-Ping Tung
- Section of Structural Biology, Hormel Institute, University of Minnesota, Austin, MN, United States
| | - Eun Hee Han
- Drug & Disease Target Group, Division of Life Science, Korea Basic Science Institute, Cheongju, Republic of Korea
| | - In-Kyu Lee
- Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu, Republic of Korea
| | - Young-In Chi
- Section of Structural Biology, Hormel Institute, University of Minnesota, Austin, MN, United States.
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25
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Abstract
In addition to the common types of diabetes mellitus, two major monogenic diabetes forms exist. Maturity-onset diabetes of the young (MODY) represents a heterogenous group of monogenic, autosomal dominant diseases. MODY accounts for 1-2% of all diabetes cases, and it is not just underdiagnosed but often misdiagnosed to type 1 or type 2 diabetes. More than a dozen MODY genes have been identified to date, and their molecular classification is of great importance in the correct treatment decision and in the judgment of the prognosis. The most prevalent subtypes are HNF1A, GCK, and HNF4A. Genetic testing for MODY has changed recently due to the technological advancements, as contrary to the sequential testing performed in the past, nowadays all MODY genes can be tested simultaneously by next-generation sequencing. The other major group of monogenic diabetes is neonatal diabetes mellitus which can be transient or permanent, and often the diabetes is a part of a syndrome. It is a severe monogenic disease appearing in the first 6 months of life. The hyperglycemia usually requires insulin. There are two forms, permanent neonatal diabetes mellitus (PNDM) and transient neonatal diabetes mellitus (TNDM). In TNDM, the diabetes usually reverts within several months but might relapse later in life. The incidence of NDM is 1:100,000-1:400,000 live births, and PNDM accounts for half of the cases. Most commonly, neonatal diabetes is caused by mutations in KCNJ11 and ABCC8 genes encoding the ATP-dependent potassium channel of the β cell. Neonatal diabetes has experienced a quick and successful transition into the clinical practice since the discovery of the molecular background. In case of both genetic diabetes groups, recent guidelines recommend genetic testing.
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Affiliation(s)
- Zsolt Gaál
- 4th Department of Medicine, Jósa András Teaching Hospital, Nyíregyháza, Hungary
| | - István Balogh
- Division of Clinical Genetics, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
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26
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Up-regulation of PCSK9 gene expression and diminished level of LDL-receptor in rat liver as a potential cause of post-lipectomy hypercholesterolemia. Mol Cell Biochem 2018; 455:207-217. [PMID: 30483910 PMCID: PMC6445806 DOI: 10.1007/s11010-018-3484-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2018] [Accepted: 11/23/2018] [Indexed: 12/14/2022]
Abstract
Studies designed to examine effects of fat mass reduction (including lipodystrophy and lipectomy) on human serum total and LDL-cholesterol concentrations are inconsistent. The purpose of this study was to examine effect of partial lipectomy in rats (as an experimental model of fat mass reduction in humans) on (1) circulating total cholesterol, LDL-cholesterol + VLDL-cholesterol and HDL-cholesterol concentrations, and (2) factors which may affect serum cholesterol concentrations such as: (a) liver LDL-receptor level, (b) expression of liver PCSK9 and (c) circulating PCSK9 concentration. Reduction of rat adipose tissue mass resulted in an increase in circulating total and LDL + VLDL—cholesterol concentrations, which was associated with (a) decrease in liver LDL-R level, (b) increase in liver PCSK9 expression, and (c) increase in circulating PCSK9 concentration as compared with sham controls. These changes were accompanied by elevated liver HNF1α (and HNF4α) mRNA levels. Silencing HNF1α in HepG2 cells by siRNA led to decrease in PCSK9 mRNA levels. This suggests that overexpression of HNF1α gene in liver of lipectomized rats can lead to overproduction of PCSK9. In conclusion, up-regulation of PCSK9, due to overexpression of HNF1α gene in liver of lipectomized rats and subsequently increase in circulating PCSK9 concentration lead to decrease in liver LDL-R level. This may contribute, at least in part, to an increase in the concentration of circulating cholesterol in rats with reduced fat mass. These findings provide a possible explanation for the molecular mechanism of hypercholesterolemia observed sometimes after reduction of fat mass in human.
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27
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Astro V, Adamo A. Epigenetic Control of Endocrine Pancreas Differentiation in vitro: Current Knowledge and Future Perspectives. Front Cell Dev Biol 2018; 6:141. [PMID: 30410880 PMCID: PMC6209628 DOI: 10.3389/fcell.2018.00141] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2018] [Accepted: 10/04/2018] [Indexed: 12/11/2022] Open
Abstract
The raising worldwide prevalence of Type 1 and Type 2 diabetes mellitus (T1DM and T2DM) solicits the derivation of in vitro methods yielding mature and fully functional β-cells to be used in regenerative medicine. Several protocols to differentiate human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into human pancreatic β-like cells have recently been developed. These methods, coupled with a bioengineering approach using biocompatible encapsulating devices, have recently led to experimental clinical trials showing great promises to ultimately end the battle of diabetic patients for managing hyperglycemia. However, in vitro differentiation protocols face the challenge of achieving homogenous population of mono-hormonal insulin-secreting mature β-cells. Major epigenetic events such as DNA methylation, post-translational modification of histones and non-coding RNAs expression, orchestrate physiological endocrine pancreas specification into α-, β-, γ-, and δ-cells, both in vivo and in vitro. The dysregulation of such epigenetic processes is associated to multiple pancreatic disorders including diabetes. Understanding the epigenomic and transcriptomic landscape underlying endocrine pancreas development could, therefore, improve in vitro differentiation methods. In this review, we summarize the most effective protocols for in vitro differentiation of hESCs/hiPSCs toward pancreatic β-cells and we discuss the current limitations in the derivation of functional glucose-responsive, insulin-releasing β-cells. Moreover, we focus on the main transcriptional and epigenetic events leading to pancreatic specification and on the applicative potential of novel epigenetic drugs for the establishment of innovative pharmacological therapeutic approaches.
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Affiliation(s)
- Veronica Astro
- Biological and Environmental Science and Engineering Division, KAUST Environmental Epigenetics Program, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
| | - Antonio Adamo
- Biological and Environmental Science and Engineering Division, KAUST Environmental Epigenetics Program, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
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28
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Hepatocyte nuclear factors as possible C-reactive protein transcriptional inducer in the liver and white adipose tissue of rats with experimental chronic renal failure. Mol Cell Biochem 2018; 446:11-23. [PMID: 29330688 PMCID: PMC6096500 DOI: 10.1007/s11010-018-3268-1] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2017] [Accepted: 01/04/2018] [Indexed: 12/11/2022]
Abstract
Inflammation related to chronic kidney disease (CKD) is an important clinical problem. We recently determined that hepatocyte nuclear factor 1α (HNF1α) was upregulated in the livers of chronic renal failure (CRF) rats—experimental model of CKD. Considering that the promoter region of gene encoding C-reactive protein (CRP) contains binding sites for HNF1α and that the loss-of-function mutation in the Hnfs1α leads to significant reduction in circulating CRP levels, we hypothesized that HNF1α can activate the Crp in CRF rats. Here, we found coordinated upregulation of genes encoding CRP, interleukin-6 (IL-6), HNF1α, and HNF4α in the livers and white adipose tissue (WAT) of CRF rats, as compared to the pair-fed and control animals. This was accompanied by elevated serum levels of CRP and IL-6. CRP and HNFs’ mRNA levels correlated positively with CRP and HNFs’ protein levels in the liver and WAT. Similar upregulation of the Crp, Il-6, and Hnfs in the liver and WAT and increased serum CRP and IL-6 concentrations were found in lipopolysaccharide (LPS)-induced systemic inflammation in rats. Moreover, silencing HNF1α in HepG2 cells by small interfering RNA led to decrease in CRP mRNA levels. Our results suggests that (a) HNFs act in concert with IL-6 in the upregulation of CRP production by the liver and WAT, leading to an increase in circulating CRP concentration in CRF rats and (b) CRF-related inflammation plays an important role in the upregulation of genes that encode HNFs and CRP in the liver and WAT of CRF rats.
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29
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Sun M, Tong P, Kong W, Dong B, Huang Y, Park IY, Zhou L, Liu XD, Ding Z, Zhang X, Bai S, German P, Powell R, Wang Q, Tong X, Tannir NM, Matin SF, Rathmell WK, Fuller GN, McCutcheon IE, Walker CL, Wang J, Jonasch E. HNF1B Loss Exacerbates the Development of Chromophobe Renal Cell Carcinomas. Cancer Res 2017; 77:5313-5326. [PMID: 28807937 DOI: 10.1158/0008-5472.can-17-0986] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2017] [Revised: 06/16/2017] [Accepted: 07/25/2017] [Indexed: 12/25/2022]
Abstract
Chromophobe renal cell carcinoma (ChRCC) is characterized by major changes in chromosomal copy number (CN). No model is available to precisely elucidate the molecular drivers of this tumor type. HNF1B is a master regulator of gene expression. Here, we report that the transcription factor HNF1B is downregulated in the majority of ChRCC and that the magnitude of HNF1B loss is unique to ChRCC. We also observed a strong correlation between reduced HNF1B expression and aneuploidy in ChRCC patients. In murine embryonic fibroblasts or ACHN cells, HNF1B deficiency reduced expression of the spindle checkpoint proteins MAD2L1 and BUB1B, and the cell-cycle checkpoint proteins RB1 and p27. Furthermore, it altered the chromatin accessibility of Mad2l1, Bub1b, and Rb1 genes and triggered aneuploidy development. Analysis of The Cancer Genome Atlas database revealed TP53 mutations in 33% of ChRCC where HNF1B expression was repressed. In clinical specimens, combining HNF1B loss with TP53 mutation produced an association with poor patient prognosis. In cells, combining HNF1B loss and TP53 mutation increased cell proliferation and aneuploidy. Our results show how HNF1B loss leads to abnormal mitotic protein regulation and induction of aneuploidy. We propose that coordinate loss of HNF1B and TP53 may enhance cellular survival and confer an aggressive phenotype in ChRCC. Cancer Res; 77(19); 5313-26. ©2017 AACR.
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Affiliation(s)
- Mianen Sun
- Department of Genitourinary Medical Oncology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Pan Tong
- Department of Bioinformatics and Computational Biology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Wen Kong
- Department of Urology, Shanghai Jiaotong University School of Medicine, Shanghai, China
| | - Baijun Dong
- Department of Urology, Shanghai Jiaotong University School of Medicine, Shanghai, China
| | - Yiran Huang
- Department of Urology, Shanghai Jiaotong University School of Medicine, Shanghai, China
| | - In Young Park
- Institute of Biosciences and Technology, Texas A&M University Health Science Center, Bryan, Texas
| | - Lijun Zhou
- Department of Genitourinary Medical Oncology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Xian-De Liu
- Department of Genitourinary Medical Oncology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Zhiyong Ding
- Department of System Biology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Xuesong Zhang
- Department of Genitourinary Medical Oncology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Shanshan Bai
- Department of Genitourinary Medical Oncology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Peter German
- Department of Genitourinary Medical Oncology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Reid Powell
- Institute of Biosciences and Technology, Texas A&M University Health Science Center, Bryan, Texas
| | - Quan Wang
- Institute of Biosciences and Technology, Texas A&M University Health Science Center, Bryan, Texas
| | - Xuefei Tong
- Institute of Biosciences and Technology, Texas A&M University Health Science Center, Bryan, Texas
| | - Nizar M Tannir
- Institute of Biosciences and Technology, Texas A&M University Health Science Center, Bryan, Texas
| | - Surena F Matin
- Department of Urology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - W Kimryn Rathmell
- Department of Urology, University of North Carolina, Chapel Hill, North Carolina
| | - Gregory N Fuller
- Department of Pathology, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Ian E McCutcheon
- Department of Neurosurgery, University of Texas at MD Anderson Cancer Center, Houston, Texas
| | - Cheryl L Walker
- Institute of Biosciences and Technology, Texas A&M University Health Science Center, Bryan, Texas
| | - Jing Wang
- Department of Bioinformatics and Computational Biology, University of Texas at MD Anderson Cancer Center, Houston, Texas
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30
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Al-Khawaga S, Memon B, Butler AE, Taheri S, Abou-Samra AB, Abdelalim EM. Pathways governing development of stem cell-derived pancreatic β cells: lessons from embryogenesis. Biol Rev Camb Philos Soc 2017. [DOI: 10.1111/brv.12349] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Affiliation(s)
- Sara Al-Khawaga
- Diabetes Research Center, Qatar Biomedical Research Institute; Hamad Bin Khalifa University, Qatar Foundation, Education City; Doha Qatar
| | - Bushra Memon
- Diabetes Research Center, Qatar Biomedical Research Institute; Hamad Bin Khalifa University, Qatar Foundation, Education City; Doha Qatar
| | - Alexandra E. Butler
- Larry L. Hillblom Islet Research Center, David Geffen School of Medicine; University of California; Los Angeles CA 90095 U.S.A
| | - Shahrad Taheri
- Department of Medicine; Weill Cornell Medicine in Qatar, Qatar Foundation, Education City, PO BOX 24144; Doha Qatar
- Department of Medicine; Qatar Metabolic Institute, Hamad Medical Corporation; Doha Qatar
| | - Abdul B. Abou-Samra
- Department of Medicine; Weill Cornell Medicine in Qatar, Qatar Foundation, Education City, PO BOX 24144; Doha Qatar
- Department of Medicine; Qatar Metabolic Institute, Hamad Medical Corporation; Doha Qatar
| | - Essam M. Abdelalim
- Diabetes Research Center, Qatar Biomedical Research Institute; Hamad Bin Khalifa University, Qatar Foundation, Education City; Doha Qatar
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Genome Editing in hPSCs Reveals GATA6 Haploinsufficiency and a Genetic Interaction with GATA4 in Human Pancreatic Development. Cell Stem Cell 2017; 20:675-688.e6. [PMID: 28196600 DOI: 10.1016/j.stem.2017.01.001] [Citation(s) in RCA: 129] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2015] [Revised: 11/08/2016] [Accepted: 01/03/2017] [Indexed: 01/19/2023]
Abstract
Human disease phenotypes associated with haploinsufficient gene requirements are often not recapitulated well in animal models. Here, we have investigated the association between human GATA6 haploinsufficiency and a wide range of clinical phenotypes that include neonatal and adult-onset diabetes using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome editing coupled with human pluripotent stem cell (hPSC) directed differentiation. We found that loss of one GATA6 allele specifically affects the differentiation of human pancreatic progenitors from the early PDX1+ stage to the more mature PDX1+NKX6.1+ stage, leading to impaired formation of glucose-responsive β-like cells. In addition to this GATA6 haploinsufficiency, we also identified dosage-sensitive requirements for GATA6 and GATA4 in the formation of both definitive endoderm and pancreatic progenitor cells. Our work expands the application of hPSCs from studying the impact of individual gene loci to investigation of multigenic human traits, and it establishes an approach for identifying genetic modifiers of human disease.
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Lu H, Lei X, Liu J, Klaassen C. Regulation of hepatic microRNA expression by hepatocyte nuclear factor 4 alpha. World J Hepatol 2017; 9:191-208. [PMID: 28217257 PMCID: PMC5295159 DOI: 10.4254/wjh.v9.i4.191] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2016] [Revised: 10/02/2016] [Accepted: 12/02/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To uncover the role of hepatocyte nuclear factor 4 alpha (HNF4α) in regulating hepatic expression of microRNAs.
METHODS Microarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4α expression in liver (Hnf4α-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4α, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4α on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3’UTR) of two genes in human hepatoma cells.
RESULTS Microarray data indicated that most microRNAs remained unaltered by Hnf4α deficiency in Hnf4α-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4α-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4α-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4α directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4α markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3’UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1.
CONCLUSION HNF4α is essential for hepatic basal expression of a group of liver-enriched microRNAs, including miR-101, miR-192, miR-193a, miR-194 and miR-802, through which HNF4α may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.
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Magaña‐Cerino JM, Luna‐Arias JP, Labra‐Barrios ML, Avendaño‐Borromeo B, Boldo‐León XM, Martínez‐López MC. Identification and functional analysis of c.422_423InsT, a novel mutation of the HNF1A gene in a patient with diabetes. Mol Genet Genomic Med 2017; 5:50-65. [PMID: 28116330 PMCID: PMC5241209 DOI: 10.1002/mgg3.261] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2016] [Revised: 10/07/2016] [Accepted: 10/11/2016] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND HNF1A gene regulates liver-specific genes, and genes that have a role in glucose metabolism, transport, and secretion of insulin. HNF1A gene mutations are frequently associated with type 2 diabetes. HNF1A protein has three domains: the dimerization domain, the DNA-binding domain, and the trans-activation domain. Some mutations in the dimerization or DNA-binding domains have no influence on the normal allele, while others have dominant negative effects. The I27L, A98V, and S487N polymorphisms are common variants of the HNF1A gene; they have been found in T2D and non-diabetic subjects. METHODS AND RESULTS We searched for mutations in the first three exons of the HNF1A gen in an Amerindian population of 71 diabetic patients. DNA sequencing revealed the previously reported I27L polymorphism (c.79A>C) in 53% of diabetic patients and in 67% of the control group. Thus, the I27L/L27L polymorphism might be a marker of Amerindians. In addition, we found the c.422_423InsT mutation in the HNF1A gene of one patient, which had not been previously reported. This mutation resulted in a frame shift of the open reading frame and a new translation stop in codon 187, leading to a truncated polypeptide of 186 amino acids (Q141Hfs*47). This novel mutation affects the DNA-binding capacity of the mutant HNF1A protein by EMSA; its intracellular localization by fluorescence and confocal microscopy, and a dominant-negative effect affecting the DNA-binding capacity of the normal HNF1A by EMSA. We also studied the homology modeling structure to understand the effect of this mutation on its DNA-binding capacity and its dominant negative effect. CONCLUSION The HNF1A Q141Hfs*47 mutant polypeptide has no DNA-binding capacity and exerts a dominant negative effect on the HNF1A protein. Therefore, it might produce severe phenotypic effects on the expression levels of a set of β-cell genes. Consequently, its screening should be included in the genetic analysis of diabetic patients after more functional studies are performed.
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Affiliation(s)
- Jesús Miguel Magaña‐Cerino
- Centro de Investigación y PosgradoLaboratorio de Diagnóstico MolecularDivisión Académica de Ciencias de la Salud (DACS)Universidad Juárez Autónoma de Tabasco (UJAT)Ave. Gregorio Méndez Magaña. No 2838‐A, Col. Tamulté de las BarrancasVillahermosaC.P. 86150México
| | - Juan P. Luna‐Arias
- Departamento de Biología CelularCentro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV‐IPN)Ave. Instituto Politécnico Nacional 2508, Col. San Pedro ZacatencoCiudad de MéxicoC.P. 07360México
| | - María Luisa Labra‐Barrios
- Departamento de Biología CelularCentro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV‐IPN)Ave. Instituto Politécnico Nacional 2508, Col. San Pedro ZacatencoCiudad de MéxicoC.P. 07360México
| | - Bartolo Avendaño‐Borromeo
- Departamento de Biología CelularCentro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV‐IPN)Ave. Instituto Politécnico Nacional 2508, Col. San Pedro ZacatencoCiudad de MéxicoC.P. 07360México
| | - Xavier Miguel Boldo‐León
- Centro de Investigación y PosgradoLaboratorio de Diagnóstico MolecularDivisión Académica de Ciencias de la Salud (DACS)Universidad Juárez Autónoma de Tabasco (UJAT)Ave. Gregorio Méndez Magaña. No 2838‐A, Col. Tamulté de las BarrancasVillahermosaC.P. 86150México
| | - Mirian Carolina Martínez‐López
- Centro de Investigación y PosgradoLaboratorio de Diagnóstico MolecularDivisión Académica de Ciencias de la Salud (DACS)Universidad Juárez Autónoma de Tabasco (UJAT)Ave. Gregorio Méndez Magaña. No 2838‐A, Col. Tamulté de las BarrancasVillahermosaC.P. 86150México
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Zhao Y, Hou J, Mi P, Mao L, Xu L, Zhang Y, Xiao L, Cao H, Zhang W, Zhang B, Song G, Hu T, Zhan YY. Exo70 is transcriptionally up-regulated by hepatic nuclear factor 4α and contributes to cell cycle control in hepatoma cells. Oncotarget 2016; 7:9150-62. [PMID: 26848864 PMCID: PMC4891032 DOI: 10.18632/oncotarget.7133] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2015] [Accepted: 01/19/2016] [Indexed: 11/25/2022] Open
Abstract
Exo70, a member of the exocyst complex, is involved in cell exocytosis, migration, invasion and autophagy. However, the expression regulation and function of Exo70 in hepatocellular carcinoma are still poorly understood. In this study, we found Exo70 expression in human hepatoma cells was greatly reduced after knocking down hepatic nuclear factor 4α (HNF4α), the most important and abundant transcription factor in liver. This regulation occurred at the transcriptional level but not post-translational level. HNF4α transactivated Exo70 promoter through directly binding to the HNF4α-response element in this promoter. Cell cycle analysis further revealed that down-regulation of HNF4α and Exo70 was essential to berberine-stimulated G2/M cell cycle arrest in hepatoma cells. Moreover, knocking down either Exo70 or HNF4α induced G2/M phase arrest of hepatoma cells. Exo70 acted downstream of HNF4α to stimulate G2/M transition via increasing Cdc2 expression. Together, our results identify Exo70 as a novel transcriptional target of HNF4α to promote cell cycle progression in hepatoma, thus provide a basis for the development of therapeutic strategies for hepatocellular carcinoma.
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Affiliation(s)
- Yujie Zhao
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Jihuan Hou
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Panying Mi
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Liyuan Mao
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Liang Xu
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Youyu Zhang
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Li Xiao
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China.,Department of Oncology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen 361004, Fujian Province, PR China
| | - Hanwei Cao
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Wenqing Zhang
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Bing Zhang
- Department of Basic Medicine, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Gang Song
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Tianhui Hu
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
| | - Yan-yan Zhan
- Cancer Research Center, Xiamen University Medical College, Xiamen 361102, Fujian Province, PR China
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Lee K, Goh GYS, Wong MA, Klassen TL, Taubert S. Gain-of-Function Alleles in Caenorhabditis elegans Nuclear Hormone Receptor nhr-49 Are Functionally Distinct. PLoS One 2016; 11:e0162708. [PMID: 27618178 PMCID: PMC5019492 DOI: 10.1371/journal.pone.0162708] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Accepted: 08/27/2016] [Indexed: 02/07/2023] Open
Abstract
Nuclear hormone receptors (NHRs) are transcription factors that regulate numerous physiological and developmental processes and represent important drug targets. NHR-49, an ortholog of Hepatocyte Nuclear Factor 4 (HNF4), has emerged as a key regulator of lipid metabolism and life span in the nematode worm Caenorhabditis elegans. However, many aspects of NHR-49 function remain poorly understood, including whether and how it regulates individual sets of target genes and whether its activity is modulated by a ligand. A recent study identified three gain-of-function (gof) missense mutations in nhr-49 (nhr-49(et7), nhr-49(et8), and nhr-49(et13), respectively). These substitutions all affect the ligand-binding domain (LBD), which is critical for ligand binding and protein interactions. Thus, these alleles provide an opportunity to test how three specific residues contribute to NHR-49 dependent gene regulation. We used computational and molecular methods to delineate how these mutations alter NHR-49 activity. We find that despite originating from a screen favoring the activation of specific NHR-49 targets, all three gof alleles cause broad upregulation of NHR-49 regulated genes. Interestingly, nhr-49(et7) and nhr-49(et8) exclusively affect nhr-49 dependent activation, whereas the nhr-49(et13) surprisingly affects both nhr-49 mediated activation and repression, implicating the affected residue as dually important. We also observed phenotypic non-equivalence of these alleles, as they unexpectedly caused a long, short, and normal life span, respectively. Mechanistically, the gof substitutions altered neither protein interactions with the repressive partner NHR-66 and the coactivator MDT-15 nor the subcellular localization or expression of NHR-49. However, in silico structural modeling revealed that NHR-49 likely interacts with small molecule ligands and that the missense mutations might alter ligand binding, providing a possible explanation for increased NHR-49 activity. In sum, our findings indicate that the three nhr-49 gof alleles are non-equivalent, and highlight the conserved V411 residue affected by et13 as critical for gene activation and repression alike.
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Affiliation(s)
- Kayoung Lee
- Graduate Program in Medical Genetics, University of British Columbia, Vancouver, BC, Canada
- Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada
| | - Grace Ying Shyen Goh
- Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada
- Graduate Program in Cell and Developmental Biology, University of British Columbia, Vancouver, BC, Canada
| | - Marcus Andrew Wong
- Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada
| | - Tara Leah Klassen
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada
| | - Stefan Taubert
- Graduate Program in Medical Genetics, University of British Columbia, Vancouver, BC, Canada
- Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada
- Graduate Program in Cell and Developmental Biology, University of British Columbia, Vancouver, BC, Canada
- Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
- * E-mail:
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Sucajtys-Szulc E, Szolkiewicz M, Swierczynski J, Rutkowski B. Up-regulation of Hnf1α gene expression in the liver of rats with experimentally induced chronic renal failure – A possible link between circulating PCSK9 and triacylglycerol concentrations. Atherosclerosis 2016; 248:17-26. [DOI: 10.1016/j.atherosclerosis.2016.02.027] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/10/2015] [Revised: 02/04/2016] [Accepted: 02/23/2016] [Indexed: 12/12/2022]
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Bockenhauer D, Jaureguiberry G. HNF1B-associated clinical phenotypes: the kidney and beyond. Pediatr Nephrol 2016; 31:707-14. [PMID: 26160100 DOI: 10.1007/s00467-015-3142-2] [Citation(s) in RCA: 99] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2015] [Revised: 05/19/2015] [Accepted: 06/04/2015] [Indexed: 12/17/2022]
Abstract
Mutations in HNF1B, the gene encoding hepatocyte nuclear factor 1β are the most commonly identified genetic cause of renal malformations. HNF1B was first identified as a disease gene for diabetes (MODY5) in 1997, and its involvement in renal disease was subsequently noted through clinical observations in pedigrees affected by MODY5. Since then, a whole spectrum of associated phenotypes have been reported, including genital malformations, autism, epilepsy, gout, hypomagnesaemia, primary hyperparathyroidism, liver and intestinal abnormalities and a rare form of kidney cancer. The most commonly identified mutation, in approximately 50 % of patients, is an entire gene deletion occurring in the context of a 17q12 chromosomal microdeletion that also includes several other genes. Some of the associated phenotypes, especially the neurologic ones, appear to occur only in the context of this microdeletion and thus may not be directly linked to HNF1B. Here we review the spectrum of associated phenotypes and discuss potential implications for clinical management.
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Affiliation(s)
- Detlef Bockenhauer
- UCL Institute of Child Health, 30 Guilford Street, London, WC1N 3EH, UK. .,Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK.
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Voevoda MI, Ivanova AA, Shakhtshneider EV, Ovsyannikova AK, Mikhailova SV, Astrakova KS, Voevoda SM, Rymar OD. Molecular genetics of maturity-onset diabetes of the young. TERAPEVT ARKH 2016. [DOI: 10.17116/terarkh2016884117-124] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
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Kim YI, Lee J, Choi YJ, Seo J, Park J, Lee SY, Cho JY. Proteogenomic Study beyond Chromosome 9: New Insight into Expressed Variant Proteome and Transcriptome in Human Lung Adenocarcinoma Tissues. J Proteome Res 2015; 14:5007-5016. [PMID: 26584007 DOI: 10.1021/acs.jproteome.5b00544] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
This is a report of a human proteome project (HPP) related to chromosome 9 (Chr 9). To reveal missing proteins and undiscovered features in proteogenomes, both LC-MS/MS analysis and next-generation RNA sequencing (RNA-seq)-based identification and characterization were conducted on five pairs of lung adenocarcinoma tumors and adjacent nontumor tissues. Before our previous Chromosome-Centric Human Proteome Project (C-HPP) special issue, there were 170 remaining missing proteins on Chr 9 (neXtProt 2013.09.26 rel.); 133 remain at present (neXtProt 2015.04.28 rel.). In the proteomics study, we found two missing protein candidates that require follow-up work and one unrevealed protein across all chromosomes. RNA-seq analysis detected RNA expression for four nonsynonymous (NS) single nucleotide polymorphisms (SNPs) (in CDH17, HIST1H1T, SAPCD2, and ZNF695) and three synonymous SNPs (in CDH17, CST1, and HNF1A) in all five tumor tissues but not in any of the adjacent normal tissues. By constructing a cancer patient sample-specific protein database based on individual RNA-seq data and by searching the proteomics data from the same sample, we identified four missense mutations in four genes (LTF, HDLBP, TF, and HBD). Two of these mutations were found in tumor samples but not in paired normal tissues. In summary, our proteogenomic study of human primary lung tumor tissues detected additional and revealed novel missense mutations and synonymous SNP signatures, some of which are specific to lung cancers. Data from mass spectrometry have been deposited in the ProteomeXchange with the identifier PXD002523.
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Affiliation(s)
- Yong-In Kim
- Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University , Seoul 08826, South Korea
| | - Jongan Lee
- Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine , Seoul 06351, South Korea
| | - Young-Jin Choi
- Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University , Seoul 08826, South Korea
- ProtAnBio , Seoul 08826, South Korea
| | - Jawon Seo
- Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University , Seoul 08826, South Korea
| | - Jisook Park
- Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine , Seoul 06351, South Korea
| | - Soo-Youn Lee
- Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine , Seoul 06351, South Korea
- Department of Clinical Pharmacology & Therapeutics, Samsung Medical Center, Sungkyunkwan University School of Medicine , Seoul 06351, South Korea
| | - Je-Yoel Cho
- Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University , Seoul 08826, South Korea
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Li J, Inoue J, Choi JM, Nakamura S, Yan Z, Fushinobu S, Kamada H, Kato H, Hashidume T, Shimizu M, Sato R. Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α. J Biol Chem 2015; 290:24021-35. [PMID: 26272613 DOI: 10.1074/jbc.m115.645200] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2015] [Indexed: 01/14/2023] Open
Abstract
Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.
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Affiliation(s)
- Juan Li
- From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 1-1-1 Yayoi, 113-8657, Japan
| | - Jun Inoue
- From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 1-1-1 Yayoi, 113-8657, Japan,
| | - Jung-Min Choi
- From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 1-1-1 Yayoi, 113-8657, Japan
| | - Shugo Nakamura
- the Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
| | - Zhen Yan
- the Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
| | - Shinya Fushinobu
- the Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
| | - Haruhiko Kamada
- the Laboratory of Biopharmaceutical Research, National Institute of Biomedical Innovation, Osaka 567-0085, Japan
| | - Hisanori Kato
- From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 1-1-1 Yayoi, 113-8657, Japan, the Corporate Sponsored Research Program "Food for Life," Organization for Interdisciplinary Research Projects, University of Tokyo, Tokyo, 113-8657, Japan, and
| | - Tsutomu Hashidume
- the Institute of Gerontology, University of Tokyo, Tokyo 113-8656, Japan
| | - Makoto Shimizu
- From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 1-1-1 Yayoi, 113-8657, Japan
| | - Ryuichiro Sato
- From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 1-1-1 Yayoi, 113-8657, Japan,
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Genetic Variants Associated with Lipid Profiles in Chinese Patients with Type 2 Diabetes. PLoS One 2015; 10:e0135145. [PMID: 26252223 PMCID: PMC4529182 DOI: 10.1371/journal.pone.0135145] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2015] [Accepted: 07/17/2015] [Indexed: 11/30/2022] Open
Abstract
Dyslipidemia is a strong risk factor for cardiovascular disease among patients with type 2 diabetes (T2D). The aim of this study was to identify lipid-related genetic variants in T2D patients of Han Chinese ancestry. Among 4,908 Chinese T2D patients who were not taking lipid-lowering medications, single nucleotide polymorphisms (SNPs) in seven genes previously found to be associated with lipid traits in genome-wide association studies conducted in populations of European ancestry (ABCA1, GCKR, BAZ1B, TOMM40, DOCK7, HNF1A, and HNF4A) were genotyped. After adjusting for multiple covariates, SNPs in ABCA1, GCKR, BAZ1B, TOMM40, and HNF1A were identified as significantly associated with triglyceride levels in T2D patients (P < 0.05). The associations between the SNPs in ABCA1 (rs3890182), GCKR (rs780094), and BAZ1B (rs2240466) remained significant even after correction for multiple testing (P = 8.85×10−3, 7.88×10−7, and 2.03×10−6, respectively). BAZ1B (rs2240466) also was associated with the total cholesterol level (P = 4.75×10−2). In addition, SNP rs157580 in TOMM40 was associated with the low-density lipoprotein cholesterol level (P = 6.94×10−3). Our findings confirm that lipid-related genetic loci are associated with lipid profiles in Chinese patients with type 2 diabetes.
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Polymorphisms in the promoter of interleukin-12β2 and interleukin-23 receptor genes influence milk production traits in Chinese Holstein cows. Livest Sci 2015. [DOI: 10.1016/j.livsci.2015.05.007] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
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Wang Y, Chaudhari S, Ren Y, Ma R. Impairment of hepatic nuclear factor-4α binding to the Stim1 promoter contributes to high glucose-induced upregulation of STIM1 expression in glomerular mesangial cells. Am J Physiol Renal Physiol 2015; 308:F1135-45. [PMID: 25786776 PMCID: PMC4437002 DOI: 10.1152/ajprenal.00563.2014] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Accepted: 03/16/2015] [Indexed: 11/22/2022] Open
Abstract
The present study was carried out to investigate if hepatic nuclear factor (HNF)4α contributed to the high glucose-induced increase in stromal interacting molecule (STIM)1 protein abundance in glomerular mesangial cells (MCs). Western blot and immunofluorescence experiments showed HNF4α expression in MCs. Knockdown of HNF4α using a small interfering RNA approach significantly increased mRNA expression levels of both STIM1 and Orai1 and protein expression levels of STIM1 in cultured human MCs. Consistently, overexpression of HNF4α reduced expressed STIM1 protein expression in human embryonic kidney-293 cells. Furthermore, high glucose treatment did not significantly change the abundance of HNF4α protein in MCs but significantly attenuated HNF4α binding activity to the Stim1 promoter. Moreover, knockdown of HNF4α significantly augmented store-operated Ca(2+) entry, which is known to be gated by STIM1 and has recently been found to be antifibrotic in MCs. In agreement with those results, knockdown of HNF4α significantly attenuated the fibrotic response of high glucose. These results suggest that HNF4α negatively regulates STIM1 transcription in MCs. High glucose increases STIM1 expression levels by impairing HNF4α binding activity to the Stim1 promoter, which subsequently releases Stim1 transcription from HNF4α repression. Since the STIM1-gated store-operated Ca(2+) entry pathway in MCs has an antifibrotic effect, inhibition of HNF4α in MCs might be a potential therapeutic option for diabetic kidney disease.
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Affiliation(s)
- Yanxia Wang
- Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas; and
| | - Sarika Chaudhari
- Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas; and
| | - Yuezhong Ren
- Department of Endocrinology, The Second Affiliated Hospital of Zhejiang University College of Medicine, Hangzhou, Zhejiang, China
| | - Rong Ma
- Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas; and
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Hepatocyte nuclear factor 4 alpha polymorphisms and the metabolic syndrome in French-Canadian youth. PLoS One 2015; 10:e0117238. [PMID: 25671620 PMCID: PMC4325000 DOI: 10.1371/journal.pone.0117238] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2014] [Accepted: 12/19/2014] [Indexed: 01/18/2023] Open
Abstract
Objectives Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor involved in the regulation of serum glucose and lipid levels. Several HNF4A gene variants have been associated with the risk of developing type 2 diabetes mellitus. However, no study has yet explored its association with insulin resistance and the cardiometabolic risk in children. We aimed to investigate the relationship between HNF4A genetic variants and the presence of metabolic syndrome (MetS) and metabolic parameters in a pediatric population. Design and Methods Our study included 1,749 French-Canadians aged 9, 13 and 16 years and evaluated 24 HNF4A polymorphisms that were previously identified by sequencing. Results Analyses revealed that, after correction for multiple testing, one SNP (rs736824; P<0.022) and two haplotypes (P1 promoter haplotype rs6130608-rs2425637; P<0.032 and intronic haplotype rs736824-rs745975-rs3212183; P<0.025) were associated with the risk of MetS. Additionally, a significant association was found between rs3212172 and apolipoprotein B levels (coefficient: -0.14 ± 0.05; P<0.022). These polymorphisms are located in HNF4A P1 promoter or in intronic regions. Conclusions Our study demonstrates that HNF4α genetic variants are associated with the MetS and metabolic parameters in French Canadian children and adolescents. This study, the first exploring the relation between HNF4A genetic variants and MetS and metabolic variables in a pediatric cohort, suggests that HNF4α could represent an early marker for the risk of developing type 2 diabetes mellitus.
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Shende VR, Wu M, Singh AB, Dong B, Kan CFK, Liu J. Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice. J Lipid Res 2015; 56:801-9. [PMID: 25652089 DOI: 10.1194/jlr.m052969] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
The transcription factors hepatic nuclear factor (HNF)1α and HNF1β can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1β) to examine the effects of knockdown of HNF1α or HNF1β on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1β, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (∼ 50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (∼ 1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1β did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1β infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1β, is the primary positive regulator of PCSK9 transcription in mouse liver.
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Affiliation(s)
- Vikram Ravindra Shende
- Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304 Department of Medicine, Stanford University, Stanford, CA 94305
| | - Minhao Wu
- Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304
| | - Amar Bahadur Singh
- Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304 Department of Medicine, Stanford University, Stanford, CA 94305
| | - Bin Dong
- Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304
| | | | - Jingwen Liu
- Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304
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Abstract
Hepatocyte nuclear factor 4α (HNF4α) is an orphan nuclear receptor commonly known as the master regulator of hepatic differentiation, owing to the large number of hepatocyte-specific genes it regulates. Whereas the role of HNF4α in hepatocyte differentiation is well recognized and extensively studied, its role in regulation of cell proliferation is relatively less known. Recent studies have revealed that HNF4α inhibits proliferation not only of hepatocytes but also cells in colon and kidney. Further, a growing number of studies have demonstrated that inhibition or loss of HNF4α promotes tumorigenesis in the liver and colon, and reexpression of HNF4α results in decreased cancer growth. Studies using tissue-specific conditional knockout mice, knock-in studies, and combinatorial bioinformatics of RNA/ChIP-sequencing data indicate that the mechanisms of HNF4α-mediated inhibition of cell proliferation are multifold, involving epigenetic repression of promitogenic genes, significant cross talk with other cell cycle regulators including c-Myc and cyclin D1, and regulation of miRNAs. Furthermore, studies indicate that posttranslational modifications of HNF4α may change its activity and may be at the core of its dual role as a differentiation factor and repressor of proliferation. This review summarizes recent findings on the role of HNF4α in cell proliferation and highlights the newly understood function of this old receptor.
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Affiliation(s)
- Chad Walesky
- *Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA
- †Department of Medicine – Genetics Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Udayan Apte
- *Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA
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Kim EK, Lee JS, Cheong HI, Chung SS, Kwak SH, Park KS. Identification and Functional Characterization of P159L Mutation in HNF1B in a Family with Maturity-Onset Diabetes of the Young 5 (MODY5). Genomics Inform 2014; 12:240-6. [PMID: 25705165 PMCID: PMC4330261 DOI: 10.5808/gi.2014.12.4.240] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2014] [Revised: 11/09/2014] [Accepted: 11/10/2014] [Indexed: 12/16/2022] Open
Abstract
Mutation in HNF1B, the hepatocyte nuclear factor-1β (HNF-1β) gene, results in maturity-onset diabetes of the young (MODY) 5, which is characterized by gradual impairment of insulin secretion. However, the functional role of HNF-1β in insulin secretion and glucose metabolism is not fully understood. We identified a family with early-onset diabetes that fulfilled the criteria of MODY. Sanger sequencing revealed that a heterozygous P159L (CCT to CTT in codon 159 in the DNA-binding domain) mutation in HNF1B was segregated according to the affected status. To investigate the functional consequences of this HNF1B mutation, we generated a P159L HNF1B construct. The wild-type and mutant HNF1B constructs were transfected into COS-7 cells in the presence of the promoter sequence of human glucose transporter type 2 (GLUT2). The luciferase reporter assay revealed that P159L HNF1B had decreased transcriptional activity compared to wild-type (p < 0.05). Electrophoretic mobility shift assay showed reduced DNA binding activity of P159L HNF1B. In the MIN6 pancreatic β-cell line, overexpression of the P159L mutant was significantly associated with decreased mRNA levels of GLUT2 compared to wild-type (p < 0.05). However, INS expression was not different between the wild-type and mutant HNF1B constructs. These findings suggests that the impaired insulin secretion in this family with the P159L HNF1B mutation may be related to altered GLUT2 expression in β-cells rather than decreased insulin gene expression. In conclusion, we have identified a Korean family with an HNF1B mutation and characterized its effect on the pathogenesis of diabetes.
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Affiliation(s)
- Eun Ky Kim
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea. ; Department of Internal Medicine, Seoul National University Hospital, Seoul 110-744, Korea
| | - Ji Seon Lee
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea
| | - Hae Il Cheong
- Department of Pediatrics, Seoul National University Children's Hospital, Seoul 110-744, Korea. ; Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul 110-744, Korea. ; Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul 110-744, Korea
| | - Sung Soo Chung
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea
| | - Soo Heon Kwak
- Department of Internal Medicine, Seoul National University Hospital, Seoul 110-744, Korea
| | - Kyong Soo Park
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea. ; Department of Internal Medicine, Seoul National University Hospital, Seoul 110-744, Korea. ; Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 110-744, Korea
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Effect of interactions between genetic polymorphisms and cigarette smoking on plasma triglyceride levels in elderly Koreans: the Hallym Aging Study. Genes Genomics 2014. [DOI: 10.1007/s13258-014-0234-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
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Delvecchio M, Di Paola R, Mangiacotti D, Sacco M, Menzaghi C, Trischitta V. Clinical heterogeneity of abnormal glucose homeostasis associated with the HNF4A R311H mutation. Ital J Pediatr 2014; 40:58. [PMID: 24947580 PMCID: PMC4100025 DOI: 10.1186/1824-7288-40-58] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/24/2014] [Accepted: 06/12/2014] [Indexed: 11/10/2022] Open
Affiliation(s)
- Maurizio Delvecchio
- Dipartimento di Scienze e Chirurgia Pediatriche, U,O, "B Trambusti", A,O,U, Consorziale Policlinico Giovanni XXIII, Bari 70100, Italy.
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Ogata M, Awaji T, Iwasaki N, Fujimaki R, Takizawa M, Maruyama K, Bell GI, Iwamoto Y, Uchigata Y. Localization of hepatocyte nuclear factor-4α in the nucleolus and nucleus is regulated by its C-terminus. J Diabetes Investig 2014; 3:449-56. [PMID: 24843605 PMCID: PMC4019245 DOI: 10.1111/j.2040-1124.2012.00210.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Aims/Introduction: Mutations in hepatocyte nuclear factor‐4α (HNF4α) lead to various diseases, among which C‐terminal deletions of HNF4α are exclusively responsible for maturity onset diabetes of the young 1 (MODY1). MODY is an autosomal dominant disease characterized by a primary defect in insulin response to glucose, suggesting that the C‐terminus of HNF4α is important for pancreatic β‐cell function. To clarify the role of the C‐terminus of HNF4α, changes in cellular localization and the binding ability to its regulator were examined, specifically in the region containing Q268, which deletion causes MODY1. Materials and Methods: Cellular localization of mutant HNF4α were examined in monkey kidney 7 (COS7), Chinese hamster ovary, rat insulinoma and mouse insulinoma cells, and their binding activity to other proteins were examined by fluorescence resonance energy transfer (FRET) in COS7 cells. Results: Although wild‐type HNF4α was localized in the nucleoplasm in transfected cultured cells, Q268X‐HNF4α was located predominantly in the nucleolus. Deletion analysis of the C‐terminus of HNF4α showed that the S337X‐HNF4α mutant, and other mutants with shorter amino acid sequences (S337‐K194), were mostly localized in the nucleolus. HNF4α mutants with amino acid sequences shorter than the W192X‐HNF4α mutant gradually spread to the nucleoplasm in accordance with their lengths. The A250X‐HNF4α mutant was capable of causing the accumulation of HNF4α or the small heterodimer partner (SHP), one of the HNF4α regulators, in the nucleolus. However, the R154X‐HNF4α mutant did not have binding ability to wild‐type HNF4α or SHP, and thus was seen in the nucleus. Conclusions: The C‐terminus sites might play a key role in facilitating the nucleolar and subnucleolar localization of HNF4α. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2012.00210.x, 2012)
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Affiliation(s)
- Makiko Ogata
- Department of Medicine III and Diabetes Center, Tokyo Women's Medical University School of Medicine, Tokyo
| | - Takeo Awaji
- Department of Pharmacology, Saitama Medical University School of Medicine, Saitama, Japan
| | - Naoko Iwasaki
- Department of Medicine III and Diabetes Center, Tokyo Women's Medical University School of Medicine, Tokyo
| | - Risa Fujimaki
- Department of Medicine III and Diabetes Center, Tokyo Women's Medical University School of Medicine, Tokyo
| | - Miho Takizawa
- Department of Medicine III and Diabetes Center, Tokyo Women's Medical University School of Medicine, Tokyo
| | - Kei Maruyama
- Department of Pharmacology, Saitama Medical University School of Medicine, Saitama, Japan
| | - Graeme I Bell
- Department of Medicine and Human Genetics, University of Chicago, Chicago, IL, USA
| | - Yasuhiko Iwamoto
- Department of Medicine III and Diabetes Center, Tokyo Women's Medical University School of Medicine, Tokyo
| | - Yasuko Uchigata
- Department of Medicine III and Diabetes Center, Tokyo Women's Medical University School of Medicine, Tokyo
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