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Mutchler AL, Haynes AP, Saleem M, Jamison S, Khan MM, Ertuglu L, Kirabo A. Epigenetic Regulation of Innate and Adaptive Immune Cells in Salt-Sensitive Hypertension. Circ Res 2025; 136:232-254. [PMID: 39819017 PMCID: PMC11750173 DOI: 10.1161/circresaha.124.325439] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/19/2025]
Abstract
Access to excess dietary sodium has heightened the risk of cardiovascular diseases, particularly affecting individuals with salt sensitivity of blood pressure. Our research indicates that innate antigen-presenting immune cells contribute to rapid blood pressure increases in response to excess sodium intake. Emerging evidence suggests that epigenetic reprogramming, with subsequent transcriptional and metabolic changes, of innate immune cells allows these cells to have a sustained response to repetitive stimuli. Epigenetic mechanisms also steer T-cell differentiation in response to innate immune signaling. Immune cells respond to environmental and nutritional cues, such as salt, promoting epigenetic regulation changes. This article aims to identify and discuss the role of epigenetic mechanisms in the immune system contributing to salt-sensitive hypertension.
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Affiliation(s)
- Ashley L. Mutchler
- Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Alexandria Porcia Haynes
- Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Mohammad Saleem
- Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
| | | | - Mohd Mabood Khan
- Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Lale Ertuglu
- Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Annet Kirabo
- Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
- Department of Molecular Physiology & Biophysics, Vanderbilt University, Nashville, TN 37212-8802, USA
- Vanderbilt Center for Immunobiology
- Vanderbilt Institute for Infection, Immunology and Inflammation
- Vanderbilt Institute for Global Health
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Wang Y, Chu D, Li H, Fan J, Zhu X, Ma Y, Gu Z, Xie N, Jing P. A comprehensive investigation of PRMT5 in the prognosis and ion channel features of lung cancer. Front Oncol 2024; 14:1478672. [PMID: 39678513 PMCID: PMC11638061 DOI: 10.3389/fonc.2024.1478672] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2024] [Accepted: 11/07/2024] [Indexed: 12/17/2024] Open
Abstract
The increasing incidence and mortality associated with lung cancer (LC) is a significant global health challenge. The underlying mechanisms contributing to LC remain inadequately understood. However, emerging evidence suggests that the epigenetic modifier protein arginine methyltransferase 5 (PRMT5) plays a complex role in various cellular processes, including DNA repair, gene transcription, and alternative splicing, through its function in catalyzing the symmetric dimethylation of both histone and non-histone proteins. In this study, we examined the functional role of PRMT5 utilizing LC-related datasets (GSE30219, GSE50081, and TCGA LC cohort) through a series of analyses. Our findings revealed that PRMT5 was significantly overexpressed in LC samples compared to normal tissues and was correlated with overall survival and disease-free survival rates. Additionally, PRDM1 was identified as a key protein exhibiting a strong interaction with PRMT5. The prognostic model that integrated PRMT5 with clinical factors demonstrated robust performance in assessing survival outcomes. Elevated levels of PRMT5 were associated with poor prognosis in LC, as evidenced by analyses of the GSE30219, GSE50081, and TCGA-LC datasets. Furthermore, we identified 27 ion channel (IC) genes exhibited a correlation with PRMT5 in lung adenocarcinoma (LUAD), of which 9 genes were identified as statistically significant with KM survival analysis. Strikingly, all of the 9 genes, including LRRC8A, the same as PRMT5, were associated with poor prognosis in LUAD. Our research highlights the potential of PRMT5 as a novel prognostic biomarker and its relationship with IC genes in LC.
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Affiliation(s)
- Yan Wang
- Department of Thoracic Surgery, The Second Affiliated Hospital, Air Force Medical University, Xi’an, China
| | - Daifang Chu
- Department of Pulmonary and Critical Care Medicine, The Second Affiliated Hospital, Air Force Medical University, Xi’an, China
| | - Haichao Li
- Department of Thoracic Surgery, The Second Affiliated Hospital, Air Force Medical University, Xi’an, China
| | - Jiangjiang Fan
- Department of Thoracic Surgery, The Second Affiliated Hospital, Air Force Medical University, Xi’an, China
| | - Ximing Zhu
- Department of Thoracic Surgery, The Second Affiliated Hospital, Air Force Medical University, Xi’an, China
| | - Yulong Ma
- Department of Thoracic Surgery, Yicheng County People’s Hospital, Linfen, Shanxi, China
| | - Zhongping Gu
- Department of Thoracic Surgery, The Second Affiliated Hospital, Air Force Medical University, Xi’an, China
| | - Nianlin Xie
- Department of Thoracic Surgery, The Second Affiliated Hospital, Air Force Medical University, Xi’an, China
| | - Pengyu Jing
- Department of Thoracic Surgery, The Second Affiliated Hospital, Air Force Medical University, Xi’an, China
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Johnston JG, Welch AK, Cain BD, Sayeski PP, Gumz ML, Wingo CS. Aldosterone: Renal Action and Physiological Effects. Compr Physiol 2023; 13:4409-4491. [PMID: 36994769 PMCID: PMC11472823 DOI: 10.1002/cphy.c190043] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/31/2023]
Abstract
Aldosterone exerts profound effects on renal and cardiovascular physiology. In the kidney, aldosterone acts to preserve electrolyte and acid-base balance in response to changes in dietary sodium (Na+ ) or potassium (K+ ) intake. These physiological actions, principally through activation of mineralocorticoid receptors (MRs), have important effects particularly in patients with renal and cardiovascular disease as demonstrated by multiple clinical trials. Multiple factors, be they genetic, humoral, dietary, or otherwise, can play a role in influencing the rate of aldosterone synthesis and secretion from the adrenal cortex. Normally, aldosterone secretion and action respond to dietary Na+ intake. In the kidney, the distal nephron and collecting duct are the main targets of aldosterone and MR action, which stimulates Na+ absorption in part via the epithelial Na+ channel (ENaC), the principal channel responsible for the fine-tuning of Na+ balance. Our understanding of the regulatory factors that allow aldosterone, via multiple signaling pathways, to function properly clearly implicates this hormone as central to many pathophysiological effects that become dysfunctional in disease states. Numerous pathologies that affect blood pressure (BP), electrolyte balance, and overall cardiovascular health are due to abnormal secretion of aldosterone, mutations in MR, ENaC, or effectors and modulators of their action. Study of the mechanisms of these pathologies has allowed researchers and clinicians to create novel dietary and pharmacological targets to improve human health. This article covers the regulation of aldosterone synthesis and secretion, receptors, effector molecules, and signaling pathways that modulate its action in the kidney. We also consider the role of aldosterone in disease and the benefit of mineralocorticoid antagonists. © 2023 American Physiological Society. Compr Physiol 13:4409-4491, 2023.
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Affiliation(s)
- Jermaine G Johnston
- Division of Nephrology, Hypertension and Renal Transplantation, Department of Medicine, University of Florida, Gainesville, Florida, USA
- Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, USA
- Nephrology Section, Veteran Administration Medical Center, North Florida/South Georgia Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida, USA
| | - Amanda K Welch
- Division of Nephrology, Hypertension and Renal Transplantation, Department of Medicine, University of Florida, Gainesville, Florida, USA
- Nephrology Section, Veteran Administration Medical Center, North Florida/South Georgia Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida, USA
| | - Brian D Cain
- Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA
| | - Peter P Sayeski
- Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, USA
| | - Michelle L Gumz
- Division of Nephrology, Hypertension and Renal Transplantation, Department of Medicine, University of Florida, Gainesville, Florida, USA
- Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, USA
- Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA
- Nephrology Section, Veteran Administration Medical Center, North Florida/South Georgia Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida, USA
| | - Charles S Wingo
- Division of Nephrology, Hypertension and Renal Transplantation, Department of Medicine, University of Florida, Gainesville, Florida, USA
- Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, USA
- Nephrology Section, Veteran Administration Medical Center, North Florida/South Georgia Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida, USA
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Zhong S, Zhang B, Qin L, Wang Q, Luo X. Aldosterone inhibits Dot1l expression in guinea pig cochlea. Eur J Med Res 2023; 28:26. [PMID: 36639782 PMCID: PMC9838020 DOI: 10.1186/s40001-023-00994-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2022] [Accepted: 01/04/2023] [Indexed: 01/15/2023] Open
Abstract
BACKGROUND Aldosterone relieves transcriptional repression of epithelial sodium channel (ENaC) by inhibiting Dot1a and Af9 expression and their interaction with ENaC promoter in various tissues. Expressions of ENaC and Af9 in inner ear have been identified. However, it is not known how Dot1l is regulated by aldosterone in inner ear. METHODS Twenty-eight adult guinea pigs were randomly divided into the control group and treatment group. Aldosterone 1 mg/kg/d was injected intraperitoneally in the treatment group and saline in the control group for 7 days. Animals were killed 1 month later following auditory brainstem response examination. Histomorphology of cochlea was detected with hematoxylin-eosin staining, and Dot1l expression was examined with immunohistochemistry and Western blot. RESULTS There was no significant difference in ABR thresholds before and after injection of aldosterone or saline in either group. Endolymphatic hydrops was found in 75% of animals in the treatment group. Dot1l was found in both groups in the stria vascularis, Reissner's membrane, spiral limbus, organ of Corti and spiral ligament. Dot1l expression in the treatment group was decreased by aldosterone. CONCLUSIONS Dot1l in guinea pig cochlea is inhibited by aldosterone with induction of endolymphatic hydrops. Dot1l may be closely related to endolymph regulation by aldosterone and to pathogenesis of Meniere's disease.
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Affiliation(s)
- Shixun Zhong
- grid.452206.70000 0004 1758 417XDepartment of Otolaryngology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing, 400016 China
| | - Biyun Zhang
- grid.452206.70000 0004 1758 417XDepartment of Otolaryngology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing, 400016 China
| | - Li Qin
- grid.490255.f0000 0004 7594 4364 Department of Otolaryngology, Mianyang Central Hospital, Mianyang, China
| | - Qianying Wang
- grid.452206.70000 0004 1758 417XDepartment of Otolaryngology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing, 400016 China
| | - Xiaoli Luo
- Department of Otolaryngology, Chongqing Traditional Chinese Medicine Hospital, Chongqing, China
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5
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Zhou X, Chen H, Li J, Shi Y, Zhuang S, Liu N. The Role and Mechanism of Lysine Methyltransferase and Arginine Methyltransferase in Kidney Diseases. Front Pharmacol 2022; 13:885527. [PMID: 35559246 PMCID: PMC9086358 DOI: 10.3389/fphar.2022.885527] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Accepted: 04/08/2022] [Indexed: 11/13/2022] Open
Abstract
Methylation can occur in both histones and non-histones. Key lysine and arginine methyltransferases under investigation for renal disease treatment include enhancer of zeste homolog 2 (EZH2), G9a, disruptor of telomeric silencing 1-like protein (DOT1L), and protein arginine methyltransferases (PRMT) 1 and 5. Recent studies have shown that methyltransferases expression and activity are also increased in several animal models of kidney injury, such as acute kidney injury(AKI), obstructive nephropathy, diabetic nephropathy and lupus nephritis. The inhibition of most methyltransferases can attenuate kidney injury, while the role of methyltransferase in different animal models remains controversial. In this article, we summarize the role and mechanism of lysine methyltransferase and arginine methyltransferase in various kidney diseases and highlight methyltransferase as a potential therapeutic target for kidney diseases.
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Affiliation(s)
- Xun Zhou
- Department of Nephrology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
| | - Hui Chen
- Department of Nephrology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
| | - Jinqing Li
- Department of Nephrology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
| | - Yingfeng Shi
- Department of Nephrology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
| | - Shougang Zhuang
- Department of Nephrology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
- Department of Medicine, Rhode Island Hospital and Alpert Medical School, Brown University, Providence, RI, United States
| | - Na Liu
- Department of Nephrology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
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Xiao Y, Zhou H, Jiang L, Liu R, Chen Q. Epigenetic regulation of ion channels in the sense of taste. Pharmacol Res 2021; 172:105760. [PMID: 34450315 DOI: 10.1016/j.phrs.2021.105760] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/06/2021] [Revised: 07/02/2021] [Accepted: 07/05/2021] [Indexed: 02/05/2023]
Abstract
There are five fundamental tastes discovered so far: sweet, bitter, umami, sour and salty. Taste is mediated by the specialized neuroepithelial cells mainly located at the tongue papillae, namely taste receptor cells, which can be classified into type I, type II, type III and type IV. Ion channels are necessary for diverse cell physiological activities including taste sensing, smell experience and temperature perception. Existing evidences have demonstrated distinct structures and working models of ion channels. Epigenetic modifications regulate gene expression mainly through histone modifications, DNA methylation and non-coding RNA-mediated regulation, without altering DNA sequence. This review summarizes how ion channels work during the transduction of multiple tastes, as well as the recent progressions in the epigenetic regulation of ion channels.
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Affiliation(s)
- Yanxuan Xiao
- State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Chinese Academy of Medical Sciences Research Unit of Oral Carcinogenesis and Management, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China
| | - Hangfan Zhou
- State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Chinese Academy of Medical Sciences Research Unit of Oral Carcinogenesis and Management, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China
| | - Lu Jiang
- State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Chinese Academy of Medical Sciences Research Unit of Oral Carcinogenesis and Management, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China
| | - Rui Liu
- State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Chinese Academy of Medical Sciences Research Unit of Oral Carcinogenesis and Management, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China.
| | - Qianming Chen
- State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Chinese Academy of Medical Sciences Research Unit of Oral Carcinogenesis and Management, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China.
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7
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Cildir G, Tumes DJ. DOT1L leaves its mark on adaptive immunity. Immunol Cell Biol 2021; 99:348-350. [PMID: 33604914 DOI: 10.1111/imcb.12440] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Accepted: 01/25/2021] [Indexed: 11/26/2022]
Affiliation(s)
- Gökhan Cildir
- Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, 5000, Australia
| | - Damon J Tumes
- Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, 5000, Australia
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8
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Aslam MA, Alemdehy MF, Kwesi-Maliepaard EM, Muhaimin FI, Caganova M, Pardieck IN, van den Brand T, van Welsem T, de Rink I, Song JY, de Wit E, Arens R, Jacobs H, van Leeuwen F. Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation. EMBO Rep 2021; 22:e51184. [PMID: 33410591 PMCID: PMC7857439 DOI: 10.15252/embr.202051184] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 12/01/2020] [Accepted: 12/08/2020] [Indexed: 12/13/2022] Open
Abstract
Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B‐cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B‐cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro‐proliferative, pro‐GC program. In addition, DOT1L indirectly supports the repression of an anti‐proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B‐cell naivety and GC B‐cell differentiation.
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Affiliation(s)
- Muhammad Assad Aslam
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands.,Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan
| | - Mir Farshid Alemdehy
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands
| | | | | | | | - Iris N Pardieck
- Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands
| | - Teun van den Brand
- Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands.,Division of Gene Regulation, Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Tibor van Welsem
- Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Iris de Rink
- Genome Core Facility, Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Ji-Ying Song
- Division of Experimental Animal Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Elzo de Wit
- Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands.,Division of Gene Regulation, Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Ramon Arens
- Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands
| | - Heinz Jacobs
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Fred van Leeuwen
- Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands.,Department of Medical Biology, Amsterdam UMC, Location AMC, University of Amsterdam, Amsterdam, The Netherlands
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Scheer S, Runting J, Bramhall M, Russ B, Zaini A, Ellemor J, Rodrigues G, Ng J, Zaph C. The Methyltransferase DOT1L Controls Activation and Lineage Integrity in CD4 + T Cells during Infection and Inflammation. Cell Rep 2020; 33:108505. [PMID: 33326781 DOI: 10.1016/j.celrep.2020.108505] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Revised: 10/05/2020] [Accepted: 11/17/2020] [Indexed: 12/12/2022] Open
Abstract
CD4+ T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-γ under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-γ response, mice with a T-cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and are resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th2 cell lineage commitment and stability and suggest that inhibition of DOT1L may provide a therapeutic strategy to limit type 2 immune responses.
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Affiliation(s)
- Sebastian Scheer
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia.
| | - Jessica Runting
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia
| | - Michael Bramhall
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia
| | - Brendan Russ
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Microbiology, Monash University, Clayton VIC 3800, Australia
| | - Aidil Zaini
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia
| | - Jessie Ellemor
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia
| | - Grace Rodrigues
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia
| | - Judy Ng
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia
| | - Colby Zaph
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia.
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Assmus AM, Mullins JJ, Brown CM, Mullins LJ. Cellular plasticity: A mechanism for homeostasis in the kidney. Acta Physiol (Oxf) 2020; 229:e13447. [PMID: 31991057 DOI: 10.1111/apha.13447] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Revised: 01/15/2020] [Accepted: 01/24/2020] [Indexed: 12/30/2022]
Abstract
Cellular plasticity is a topical subject with interest spanning a wide range of fields from developmental biology to regenerative medicine. Even the nomenclature is a subject of debate, and the underlying mechanisms are still under investigation. On top of injury repair, cell plasticity is a constant physiological process in adult organisms and tissues, in response to homeostatic challenges. In this review we discuss two examples of plasticity for the maintenance of homeostasis in the renal system-namely the renin-producing juxtaglomerular cells (JG cells) and cortical collecting duct (CCD) cells. JG cells show plasticity through recruitment mechanisms, answering the demand for an increase in renin production. In the CCD, cells appear to have the ability to transdifferentiate between principal and intercalated cells to help maintain the highly regulated solute transport levels of that segment. These two cases highlight the complexity of plasticity processes and the role they can play in the kidney.
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Affiliation(s)
- Adrienne M. Assmus
- The University of Edinburgh ‐ Cardiovascular Science (CVS) Queen's Medical Research Institute Edinburgh Scotland UK
| | - John J. Mullins
- The University of Edinburgh ‐ Cardiovascular Science (CVS) Queen's Medical Research Institute Edinburgh Scotland UK
| | - Cara M. Brown
- The University of Edinburgh ‐ Cardiovascular Science (CVS) Queen's Medical Research Institute Edinburgh Scotland UK
| | - Linda J. Mullins
- The University of Edinburgh ‐ Cardiovascular Science (CVS) Queen's Medical Research Institute Edinburgh Scotland UK
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11
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Zhang L, Chen L, Gao C, Chen E, Lightle AR, Foulke L, Zhao B, Higgins PJ, Zhang W. Loss of Histone H3 K79 Methyltransferase Dot1l Facilitates Kidney Fibrosis by Upregulating Endothelin 1 through Histone Deacetylase 2. J Am Soc Nephrol 2019; 31:337-349. [PMID: 31843983 DOI: 10.1681/asn.2019070739] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2019] [Accepted: 11/01/2019] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND The progression rate of CKD varies substantially among patients. The genetic and epigenetic contributions that modify how individual patients respond to kidney injury are largely unknown. Emerging evidence has suggested that histone H3 K79 methyltransferase Dot1l has an antifibrotic effect by repressing Edn1, which encodes endothelin 1 in the connecting tubule/collecting duct. METHODS To determine if deletion of the Dot1l gene is a genetic and epigenetic risk factor through regulating Edn1, we studied four groups of mice: wild-type mice, connecting tubule/collecting duct-specific Dot1l conditional knockout mice (Dot1lAC ), Dot1l and Edn1 double-knockout mice (DEAC ), and Edn1 connecting tubule/collecting duct-specific conditional knockout mice (Edn1AC ), under three experimental conditions (streptozotocin-induced diabetes, during normal aging, and after unilateral ureteral obstruction). We used several approaches (colocalization, glutathione S-transferase pulldown, coimmunoprecipitation, yeast two-hybrid, gel shift, and chromatin immunoprecipitation assays) to identify and confirm interaction of Dot1a (the major Dot1l splicing variant in the mouse kidney) with histone deacetylase 2 (HDAC2), as well as the function of the Dot1a-HDAC2 complex in regulating Edn1 transcription. RESULTS In each case, Dot1lAC mice developed more pronounced kidney fibrosis and kidney malfunction compared with wild-type mice. These Dot1lAC phenotypes were ameliorated in the double-knockout DEAC mice. The interaction between Dot1a and HDAC2 prevents the Dot1a-HDAC2 complex from association with DNA, providing a counterbalancing mechanism governing Edn1 transcription by modulating H3 K79 dimethylation and H3 acetylation at the Edn1 promoter. CONCLUSIONS Our study confirms Dot1l to be a genetic and epigenetic modifier of kidney fibrosis, reveals a new mechanism regulating Edn1 transcription by Dot1a and HDAC2, and reinforces endothelin 1 as a therapeutic target of kidney fibrosis.
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Affiliation(s)
- Long Zhang
- Departments of Regenerative and Cancer Cell Biology and
| | - Lihe Chen
- Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, Bethesda, Maryland; and
| | - Chao Gao
- Departments of Regenerative and Cancer Cell Biology and
| | - Enuo Chen
- Departments of Regenerative and Cancer Cell Biology and
| | - Andrea R Lightle
- Pathology and Laboratory Medicine, Albany Medical College, Albany, New York
| | - Llewellyn Foulke
- Pathology and Laboratory Medicine, Albany Medical College, Albany, New York
| | - Bihong Zhao
- Department of Pathology and Laboratory Medicine, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas
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Franz H, Villarreal A, Heidrich S, Videm P, Kilpert F, Mestres I, Calegari F, Backofen R, Manke T, Vogel T. DOT1L promotes progenitor proliferation and primes neuronal layer identity in the developing cerebral cortex. Nucleic Acids Res 2019; 47:168-183. [PMID: 30329130 PMCID: PMC6326801 DOI: 10.1093/nar/gky953] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2018] [Accepted: 10/04/2018] [Indexed: 01/01/2023] Open
Abstract
Cortical development is controlled by transcriptional programs, which are orchestrated by transcription factors. Yet, stable inheritance of spatio-temporal activity of factors influencing cell fate and localization in different layers is only partly understood. Here we find that deletion of Dot1l in the murine telencephalon leads to cortical layering defects, indicating DOT1L activity and chromatin methylation at H3K79 impact on the cell cycle, and influence transcriptional programs conferring upper layer identity in early progenitors. Specifically, DOT1L prevents premature differentiation by increasing expression of genes that regulate asymmetric cell division (Vangl2, Cenpj). Loss of DOT1L results in reduced numbers of progenitors expressing genes including SoxB1 gene family members. Loss of DOT1L also leads to altered cortical distribution of deep layer neurons that express either TBR1, CTIP2 or SOX5, and less activation of transcriptional programs that are characteristic for upper layer neurons (Satb2, Pou3f3, Cux2, SoxC family members). Data from three different mouse models suggest that DOT1L balances transcriptional programs necessary for proper neuronal composition and distribution in the six cortical layers. Furthermore, because loss of DOT1L in the pre-neurogenic phase of development impairs specifically generation of SATB2-expressing upper layer neurons, our data suggest that DOT1L primes upper layer identity in cortical progenitors.
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Affiliation(s)
- Henriette Franz
- Institute of Anatomy and Cell Biology, Department of Molecular Embryology, Medical Faculty, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany
| | - Alejandro Villarreal
- Institute of Anatomy and Cell Biology, Department of Molecular Embryology, Medical Faculty, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany
| | - Stefanie Heidrich
- Institute of Anatomy and Cell Biology, Department of Molecular Embryology, Medical Faculty, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany
| | - Pavankumar Videm
- Bioinformatics Group, Department of Computer Science, Albert-Ludwigs-University Freiburg, 79110 Freiburg, Germany
| | - Fabian Kilpert
- Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany
| | - Ivan Mestres
- DFG-Research Center and Cluster of Excellence for Regenerative Therapies (CRTD), School of Medicine, Technical University Dresden, 01307 Dresden, Germany
| | - Federico Calegari
- DFG-Research Center and Cluster of Excellence for Regenerative Therapies (CRTD), School of Medicine, Technical University Dresden, 01307 Dresden, Germany
| | - Rolf Backofen
- Bioinformatics Group, Department of Computer Science, Albert-Ludwigs-University Freiburg, 79110 Freiburg, Germany.,Centre for Biological Signalling Studies (BIOSS), Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany.,Centre for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany.,Center for non-coding RNA in Technology and Health, University of Copenhagen, DK-1870 Frederiksberg C, Denmark
| | - Thomas Manke
- Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany
| | - Tanja Vogel
- Institute of Anatomy and Cell Biology, Department of Molecular Embryology, Medical Faculty, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany
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13
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Chen L, Higgins PJ, Zhang W. Development and Diseases of the Collecting Duct System. Results Probl Cell Differ 2017; 60:165-203. [PMID: 28409346 DOI: 10.1007/978-3-319-51436-9_7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The collecting duct of the mammalian kidney is important for the regulation of extracellular volume, osmolarity, and pH. There are two major structurally and functionally distinct cell types: principal cells and intercalated cells. The former regulates Na+ and water homeostasis, while the latter participates in acid-base homeostasis. In vivo lineage tracing using Cre recombinase or its derivatives such as CreGFP and CreERT2 is a powerful new technique to identify stem/progenitor cells in their native environment and to decipher the origins of the tissue that they give rise to. Recent studies using this technique in mice have revealed multiple renal progenitor cell populations that differentiate into various nephron segments and collecting duct. In particular, emerging evidence suggests that like principal cells, most of intercalated cells originate from the progenitor cells expressing water channel Aquaporin 2. Mutations or malfunctions of the channels, pumps, and transporters expressed in the collecting duct system cause various human diseases. For example, gain-of-function mutations in ENaC cause Liddle's syndrome, while loss-of-function mutations in ENaC lead to Pseudohypoaldosteronism type 1. Mutations in either AE1 or V-ATPase B1 result in distal renal tubular acidosis. Patients with disrupted AQP2 or AVPR2 develop nephrogenic diabetes insipidus. A better understanding of the function and development of the collecting duct system may facilitate the discovery of new therapeutic strategies for treating kidney disease.
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Affiliation(s)
- Lihe Chen
- Epithelial Systems Biology Laboratory, Systems Biology Center, NHLBI, Bethesda, MD, 20892-1603, USA
| | - Paul J Higgins
- Department of Regenerative and Cancer Cell Biology, Albany Medical College, MC-165, 47 New Scotland Avenue, Albany, NY, 12208, USA
| | - Wenzheng Zhang
- Department of Regenerative and Cancer Cell Biology, Albany Medical College, MC-165, 47 New Scotland Avenue, Albany, NY, 12208, USA.
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Expression of a Diverse Array of Ca2+-Activated K+ Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney. PLoS One 2016; 11:e0155006. [PMID: 27159616 PMCID: PMC4861333 DOI: 10.1371/journal.pone.0155006] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2015] [Accepted: 04/22/2016] [Indexed: 12/02/2022] Open
Abstract
The voltage- and Ca2+-activated, large conductance K+ channel (BK, maxi-K) is expressed in the collecting duct system of kidney where it underlies flow- and Ca2+-dependent K+ excretion. To determine if other Ca2+-activated K+ channels (KCa) may participate in this process, mouse kidney and the K+-secreting mouse cortical collecting duct (CCD) cell line, mCCDcl1, were assessed for TRPV4 and KCa channel expression and cross-talk. qPCR mRNA analysis and immunocytochemical staining demonstrated TRPV4 and KCa expression in mCCDcl1 cells and kidney connecting tubule (CNT) and CCD. Three subfamilies of KCa channels were revealed: the high Ca2+-binding affinity small-conductance SK channels, SK1and SK3, the intermediate conductance channel, IK1, and the low Ca2+-binding affinity, BK channel (BKα subunit). Apparent expression levels varied in CNT/CCD where analysis of CCD principal cells (PC) and intercalated cells (IC) demonstrated differential staining: SK1:PC<IC, and SK3:PC>IC, IK1:PC>IC, BKα:PC = IC, and TRPV4:PC>IC. Patch clamp analysis and fluorescence Ca2+ imaging of mCCDcl1 cells demonstrated potent TRPV4-mediated Ca2+ entry and strong functional cross-talk between TRPV4 and KCa channels. TRPV4-mediated Ca2+ influx activated each KCa channel, as evidenced by selective inhibition of KCa channels, with each active KCa channel enhancing Ca2+ entry (due to membrane hyperpolarization). Transepithelial electrical resistance (TEER) analysis of confluent mCCDcl1 cells grown on permeable supports further demonstrated this cross-talk where TRPV4 activation induce a decrease in TEER which was partially restored upon selective inhibition of each KCa channel. It is concluded that SK1/SK3 and IK1 are highly expressed along with BKα in CNT and CCD and are closely coupled to TRPV4 activation as observed in mCCDcl1 cells. The data support a model in CNT/CCD segments where strong cross talk between TRPV4-mediated Ca2+ influx and each KCa channel leads to enhance Ca2+ entry which will support activation of the low Ca2+-binding affinity BK channel to promote BK-mediated K+ secretion.
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15
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Vlaming H, van Leeuwen F. The upstreams and downstreams of H3K79 methylation by DOT1L. Chromosoma 2016; 125:593-605. [PMID: 26728620 DOI: 10.1007/s00412-015-0570-5] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Revised: 12/16/2015] [Accepted: 12/21/2015] [Indexed: 12/14/2022]
Abstract
Histone modifications regulate key processes of eukaryotic genomes. Misregulation of the enzymes that place these modifications can lead to disease. An example of this is DOT1L, the enzyme that can mono-, di-, and trimethylate the nucleosome core on lysine 79 of histone H3 (H3K79). DOT1L plays a role in development and its misregulation has been implicated in several cancers, most notably leukemias caused by a rearrangement of the MLL gene. A DOT1L inhibitor is in clinical trials for these leukemias and shows promising results, yet we are only beginning to understand DOT1L's function and regulation in the cell. Here, we review what happens upstream and downstream of H3K79 methylation. H3K79 methylation levels are highest in transcribed genes, where H2B ubiquitination can promote DOT1L activity. In addition, DOT1L can be targeted to transcribed regions of the genome by several of its interaction partners. Although methylation levels strongly correlate with transcription, the mechanistic link between the two is unclear and probably context-dependent. Methylation of H3K79 may act through recruiting or repelling effector proteins, but we do not yet know which effectors mediate DOT1L's functions. Understanding DOT1L biology better will help us to understand the effects of DOT1L inhibitors and may allow the development of alternative strategies to target the DOT1L pathway.
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Affiliation(s)
- Hanneke Vlaming
- Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, 1066 CX, The Netherlands
| | - Fred van Leeuwen
- Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, 1066 CX, The Netherlands.
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16
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Xiao Z, Chen L, Zhou Q, Zhang W. Dot1l deficiency leads to increased intercalated cells and upregulation of V-ATPase B1 in mice. Exp Cell Res 2015; 344:167-75. [PMID: 26404731 DOI: 10.1016/j.yexcr.2015.09.014] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2015] [Revised: 09/16/2015] [Accepted: 09/19/2015] [Indexed: 01/19/2023]
Abstract
The collecting duct in the mammalian kidney consists of principal cells (PCs) and intercalated cells (ICs), which regulate electrolyte/fluid and acid/base balance, respectively. The epigenetic regulators of PC and IC differentiation remain obscure. We previously used Aqp2 and V-ATPase B1B2 to label PCs and ICs, respectively. We found that mice with histone H3 K79 methyltransferase Dot1l disrupted in Aqp2-expressing cells (Dot1l(AC)) vs. Dot1l(f/f) possessed ~20% more ICs coupled with a similar decrease in PCs. Here, we performed multiple double immunofluorescence staining using various PC and IC markers and confirmed that this finding. Both α-IC and β-IC populations were significantly expanded in Dot1l(AC) vs. Dot1l(f/f). These changes are associated with significantly upregulated V-ATPase B1 and B2, but not Aqp2, AE1, and Pendrin. Chromatin immunoprecipitation assay unveiled a significant reduction of Dot1l and H3K79 di-methylation bound at the Atp6v1b1 5' flanking region. Overexpression of Dot1a significantly downregulated a stably-transfected luciferase reporter driven by the Atp6v1b1 promoter in IMCD3 cells. This downregulation was impaired, but not completely abolished when a methyltransferase-dead mutant was overexpressed. Taken together, our data suggest that Dot1l is a new epigenetic regulator of PC and IC differentiation and Atp6v1b1 is a new transcriptional target of Dot1l.
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Affiliation(s)
- Zhou Xiao
- Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR China; Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA
| | - Lihe Chen
- Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Qiaoling Zhou
- Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR China
| | - Wenzheng Zhang
- Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA; Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
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17
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Zhang W. Epigenetics of epithelial Na + channel-dependent sodium uptake and blood pressure regulation. World J Nephrol 2015; 4:363-366. [PMID: 26167459 PMCID: PMC4491926 DOI: 10.5527/wjn.v4.i3.363] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/01/2014] [Revised: 12/08/2014] [Accepted: 05/18/2015] [Indexed: 02/06/2023] Open
Abstract
The epithelial Na+ channel (ENaC) consists of α, β, γ subunits. Its expression and function are regulated by aldosterone at multiple levels including transcription. ENaC plays a key role in Na+ homeostasis and blood pressure. Mutations in ENaC subunit genes result in hypertension or hypotension, depending on the nature of the mutations. Transcription of αENaC is considered as the rate-limiting step in the formation of functional ENaC. As an aldosterone target gene, αENaC is activated upon aldosterone- mineralocorticoid receptor binding to the cis-elements in the αENaC promoter, which is packed into chromatin. However, how aldosterone alters chromatin structure to induce changes in transcription is poorly understood. Studies by others and us suggest that Dot1a-Af9 complex represses αENaC by directly binding and regulating targeted histone H3 K79 hypermethylation at the specific subregions of αENaC promoter. Aldosterone decreases Dot1a-Af9 formation by impairing expression of Dot1a and Af9 and by inducing Sgk1, which, in turn, phosphorylates Af9 at S435 to weaken Dot1a-Af9 interaction. MR attenuates Dot1a-Af9 effect by competing with Dot1a for binding Af9. Af17 relieves repression by interfering with Dot1a-Af9 interaction and promoting Dot1a nuclear export. Af17-/- mice exhibit defects in ENaC expression, renal Na+ retention, and blood pressure control. This review gives a brief summary of these novel findings.
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18
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Abstract
Aldosterone is a major regulator of Na(+) absorption and acts primarily by controlling the epithelial Na(+) channel (ENaC) function at multiple levels including transcription. ENaC consists of α, β, and γ subunits. In the classical model, aldosterone enhances transcription primarily by activating mineralocorticoid receptor (MR). However, how aldosterone induces chromatin alternation and thus leads to gene activation or repression remains largely unknown. Emerging evidence suggests that Dot1a-Af9 complex plays an important role in repression of αENaC by directly binding and modulating targeted histone H3 K79 hypermethylation at the specific subregions of αENaC promoter. Aldosterone impairs Dot1a-Af9 formation by decreasing expression of Dot1a and Af9 and by inducing Sgk1, which, in turn, phosphorylates Af9 at S435 to weaken Dot1a-Af9 interaction. MR counterbalances Dot1a-Af9 action by competing with Dot1a for binding Af9. Af17 derepresses αENaC by competitively interacting with Dot1a and facilitating Dot1a nuclear export. Consistently, MR(-/-) mice have impaired ENaC expression at day 5 after birth, which may contribute to progressive development of pseudohypoaldosteronism type 1 in a later stage. Af17(-/-) mice have decreased ENaC expression, renal Na(+) retention, and blood pressure. In contrast, Dot1l(AC) mice have increased αENaC expression, despite a 20% reduction of the principal cells. This chapter reviews these findings linking aldosterone action to ENaC transcription through chromatin modification. Future direction toward the understanding the role of Dot1a-Af9 complex beyond ENaC regulation, in particular, in renal fibrosis is also briefly discussed.
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Affiliation(s)
- Lihe Chen
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, USA; Division of Renal Diseases and Hypertension, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas, USA
| | - Xi Zhang
- Division of Renal Diseases and Hypertension, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas, USA
| | - Wenzheng Zhang
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, USA; Division of Renal Diseases and Hypertension, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas, USA.
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19
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Epigenetics and arterial hypertension: the challenge of emerging evidence. Transl Res 2015; 165:154-65. [PMID: 25035152 DOI: 10.1016/j.trsl.2014.06.007] [Citation(s) in RCA: 70] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/25/2014] [Revised: 06/16/2014] [Accepted: 06/18/2014] [Indexed: 01/11/2023]
Abstract
Epigenetic phenomena include DNA methylation, post-translational histone modifications, and noncoding RNAs, as major marks. Although similar to genetic features of DNA for their heritability, epigenetic mechanisms differ for their potential reversibility by environmental and nutritional factors, which make them potentially crucial for their role in complex and multifactorial diseases. The function of these mechanisms is indeed gaining interest in relation to arterial hypertension (AH) with emerging evidence from cell culture and animal models as well as human studies showing that epigenetic modifications have major functions within pathways related to AH. Among epigenetic marks, the role of DNA methylation is mostly highlighted given the primary role of this epigenetic feature in mammalian cells. A lower global methylation was observed in DNA of peripheral blood mononuclear cells of hypertensive patients. Moreover, DNA hydroxymethylation appears modifiable by salt intake in a Dahl salt-sensitive rat model. The specific function of DNA methylation in regulating the expression of AH-related genes at promoter site was described for hydroxysteroid (11-beta) dehydrogenase 2 (HSD11B2), somatic angiotensin converting enzyme (sACE), Na+/K+/2Cl- cotransporter 1 (NKCC1), angiotensinogen (AGT), α-adducin (ADD1), and for other crucial genes in endocrine hypertension. Post-translational histone methylation at different histone 3 lysine residues was also observed to control the expression of genes related to AH as lysine-specific demethylase-1(LSD1), HSD11B2, and epithelial sodium channel subunit α (SCNN1A). Noncoding RNAs including several microRNAs influence genes involved in steroidogenesis and the renin-angiotensin-aldosterone pathway. In the present review, the current knowledge on the relationship between the main epigenetic marks and AH will be presented, considering the challenge of epigenetic patterns being modifiable by environmental factors that may lead toward novel implications in AH preventive and therapeutic strategies.
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Crambert G, Ernandez T, Lamouroux C, Roth I, Dizin E, Martin PY, Féraille E, Hasler U. Epithelial sodium channel abundance is decreased by an unfolded protein response induced by hyperosmolality. Physiol Rep 2014; 2:2/11/e12169. [PMID: 25413317 PMCID: PMC4255800 DOI: 10.14814/phy2.12169] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Large shifts of osmolality occur in the kidney medulla as part of the urine concentrating mechanism. Hyperosmotic stress profoundly challenges cellular homeostasis and induces endoplasmic reticulum (ER) stress. Here, we examined the unfolded protein response (UPR) in hyperosmotically-challenged principal cells of the kidney collecting duct (CD) and show its relevance in controlling epithelial sodium channel (ENaC) abundance, responsible for the final adjustment of Na(+) excretion. Dehydration increases medullary but not cortical osmolality. Q-PCR analysis of microdissected CD of water-deprived mice revealed increased aquaporin-2 (AQP2) expression in outer medullary and cortical CD while ENaC abundance decreased in outer medullary but not cortical CD. Immunoblotting, Q-PCR and immunofluorescence revealed that hyperosmolality induced a transient ER stress-like response both ex vivo and in cultured CD principal cells and increased activity of the canonical UPR mediators PERK and ATF6. Both hyperosmolality and chemical induction of ER stress decreased ENaC expression in vitro. ENaC depletion by either stimulus was abolished by transcriptional inhibition and by the chemical chaperone 4-phenylbutyric acid and was partly abrogated by either PERK or ATF6 silencing. Our data suggest that induction of the UPR by hyperosmolality may help preserve body fluid homeostasis under conditions of dehydration by uncoupling AQP2 and ENaC abundance in outer medullary CD.
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Affiliation(s)
- Gilles Crambert
- UPMC/INSERM/Paris Descartes U1138 CNRS ERL 8228, Equipe 3 Métabolisme et Physiologie Rénale, Centre de Recherche des Cordeliers, Paris, France
| | - Thomas Ernandez
- Department of Cellular Physiology and Metabolism and Service of Nephrology, University Medical Center, University of Geneva, Geneva, Switzerland
| | - Christine Lamouroux
- UPMC/INSERM/Paris Descartes U1138 CNRS ERL 8228, Equipe 3 Métabolisme et Physiologie Rénale, Centre de Recherche des Cordeliers, Paris, France
| | - Isabelle Roth
- Department of Cellular Physiology and Metabolism and Service of Nephrology, University Medical Center, University of Geneva, Geneva, Switzerland
| | - Eva Dizin
- Department of Cellular Physiology and Metabolism and Service of Nephrology, University Medical Center, University of Geneva, Geneva, Switzerland
| | - Pierre-Yves Martin
- Department of Cellular Physiology and Metabolism and Service of Nephrology, University Medical Center, University of Geneva, Geneva, Switzerland
| | - Eric Féraille
- Department of Cellular Physiology and Metabolism and Service of Nephrology, University Medical Center, University of Geneva, Geneva, Switzerland
| | - Udo Hasler
- Department of Cellular Physiology and Metabolism and Service of Nephrology, University Medical Center, University of Geneva, Geneva, Switzerland
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Abstract
The primary adrenal cortical steroid hormones, aldosterone, and the glucocorticoids cortisol and corticosterone, act through the structurally similar mineralocorticoid (MR) and glucocorticoid receptors (GRs). Aldosterone is crucial for fluid, electrolyte, and hemodynamic homeostasis and tissue repair; the significantly more abundant glucocorticoids are indispensable for energy homeostasis, appropriate responses to stress, and limiting inflammation. Steroid receptors initiate gene transcription for proteins that effect their actions as well as rapid non-genomic effects through classical cell signaling pathways. GR and MR are expressed in many tissues types, often in the same cells, where they interact at molecular and functional levels, at times in synergy, others in opposition. Thus the appropriate balance of MR and GR activation is crucial for homeostasis. MR has the same binding affinity for aldosterone, cortisol, and corticosterone. Glucocorticoids activate MR in most tissues at basal levels and GR at stress levels. Inactivation of cortisol and corticosterone by 11β-HSD2 allows aldosterone to activate MR within aldosterone target cells and limits activation of the GR. Under most conditions, 11β-HSD1 acts as a reductase and activates cortisol/corticosterone, amplifying circulating levels. 11β-HSD1 and MR antagonists mitigate inappropriate activation of MR under conditions of oxidative stress that contributes to the pathophysiology of the cardiometabolic syndrome; however, MR antagonists decrease normal MR/GR functional interactions, a particular concern for neurons mediating cognition, memory, and affect.
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Affiliation(s)
- Elise Gomez-Sanchez
- G.V.(Sonny) Montgomery V.A. Medical Center and Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi
| | - Celso E. Gomez-Sanchez
- G.V.(Sonny) Montgomery V.A. Medical Center and Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi
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Abstract
The apical membrane epithelial Na(+) channel subunit (ENaC) in series with the basolateral Na(+)/K(+)-adenosine triphosphatase mediates collecting duct Na(+) reabsorption. Aldosterone induces αENaC gene transcription, which appears to be rate limiting for ENaC activity in this segment. Although this response has long been assumed to be solely the result of liganded nuclear hormone receptors trans-activating αENaC, epigenetic controls of basal and aldosterone-induced transcription of αENaC in the collecting duct recently were described. These epigenetic pathways involve dynamic nuclear repressor complexes targeted to specific subregions of the αENaC promoter and consisting of the histone methyltransferase disrupter of telomeric silencing (Dot)1a together with the transcriptional factor Af9 or the nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase Sirt1, key co-regulatory proteins, including serum- and glucocorticoid-induced kinase-1 and the putative transcription factor Af17, and targeted chromatin modifications. The complexes, through the action of Dot1a, maintain chromatin associated with the αENaC promoter in a stable hypermethylated state, constraining αENaC transcription under basal conditions. Aldosterone and serum- and glucocorticoid-induced kinase-1, itself, activate αENaC transcription in large part by disrupting or diminishing the Dot1a-Af9 and Dot1a-Sirt1 complexes and their effects on chromatin. Mouse models indicate potential roles of the Dot1a pathways in renal salt excretion and hypertension.
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Affiliation(s)
- Bruce C Kone
- Division of Renal Diseases and Hypertension, Department of Internal Medicine, The University of Texas Medical School, Houston, TX.
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23
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The emerging roles of DOT1L in leukemia and normal development. Leukemia 2014; 28:2131-8. [DOI: 10.1038/leu.2014.169] [Citation(s) in RCA: 95] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2014] [Revised: 05/06/2014] [Accepted: 05/15/2014] [Indexed: 01/30/2023]
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Berrout J, Mamenko M, Zaika OL, Chen L, Zang W, Pochynyuk O, O'Neil RG. Emerging role of the calcium-activated, small conductance, SK3 K+ channel in distal tubule function: regulation by TRPV4. PLoS One 2014; 9:e95149. [PMID: 24762817 PMCID: PMC3999037 DOI: 10.1371/journal.pone.0095149] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2013] [Accepted: 03/24/2014] [Indexed: 12/24/2022] Open
Abstract
The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K+ channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+-dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+-affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion.
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Affiliation(s)
- Jonathan Berrout
- Department of Integrative Biology, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America
| | - Mykola Mamenko
- Department of Integrative Biology, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America
| | - Oleg L. Zaika
- Department of Integrative Biology, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America
| | - Lihe Chen
- Department of Internal Medicine-Division of Renal Diseases and Hypertension, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America
| | - Wenzheng Zang
- Department of Internal Medicine-Division of Renal Diseases and Hypertension, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America
| | - Oleh Pochynyuk
- Department of Integrative Biology, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America
| | - Roger G. O'Neil
- Department of Integrative Biology, The University of Texas Health Science Center Medical School, Houston, Texas, United States of America
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25
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Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the α-ENaC gene in a renal collecting duct cell line. Biosci Rep 2013; 33:BSR20130086. [PMID: 24070375 PMCID: PMC3979232 DOI: 10.1042/bsr20130086] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
The physical and functional interaction of Rnf2 (RING finger protein 2), a central component of the PRC (Polycomb repressive complex) 1 and Af9 (ALL1-fused gene from chromosome 9 protein), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the α-ENaC (epithelial Na+ channel α-subunit) gene was explored in mIMCD3 CD (collecting duct) cells. Since Rnf2 lacks DNA-specific binding activity, other factors must mediate its site-specific chromatin recruitment. Rnf2 and Af9 co-localized in the nucleus and co-immunoprecipitated. A GST (glutathione transferase)-Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knock down enhanced basal and aldosterone-stimulated α-ENaC mRNA levels and α-ENaC promoter activity. ChIP/QPCR (chromatin immunoprecipitation/quantitative PCR) assays demonstrated enrichment of Rnf2, H2AK119 (mono-ubiquitinated histone H2A lysine 119), and H3K27me3 (histone H3 lysine 27 trimethylated), a PRC2 chromatin mark, at multiple α-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2-Af9 co-occupancy of the α-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the α-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the α-ENaC promoter to constrain basal α-ENaC transcription in collecting duct cells, and aldosterone reverses this process.
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Yu Z, Kong Q, Kone BC. Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting duct. Am J Physiol Renal Physiol 2013; 305:F1006-13. [PMID: 23926181 PMCID: PMC3798741 DOI: 10.1152/ajprenal.00407.2013] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2013] [Accepted: 07/30/2013] [Indexed: 02/06/2023] Open
Abstract
Aldosterone increases tubular Na(+) absorption largely by increasing α-epithelial Na(+) channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.
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Affiliation(s)
- Zhiyuan Yu
- Div. of Renal Diseases and Hypertension, The Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 5.124, Houston, TX 77030.
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Zhang X, Zhou Q, Chen L, Berger S, Wu H, Xiao Z, Pearce D, Zhou X, Zhang W. Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increase αENaC transcription. Am J Physiol Renal Physiol 2013; 305:F1436-44. [PMID: 24026182 DOI: 10.1152/ajprenal.00202.2013] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
Aldosterone is a major regulator of Na(+) absorption and acts by activating the mineralocorticoid receptor (MR) to stimulate the epithelial Na(+) channel (ENaC). MR(-/-) mice exhibited pseudohypoaldosteronism type 1 (hyponatremia, hyperkalemia, salt wasting, and high levels of aldosterone) and died around postnatal day 10. However, if and how MR regulates ENaC transcription remain incompletely understood. Our earlier work demonstrated that aldosterone activates αENaC transcription by reducing expression of Dot1a and Af9 and by impairing Dot1a-Af9 interaction. Most recently, we reported identification of a major Af9 binding site in the αENaC promoter and upregulation of αENaC mRNA expression in mouse kidneys lacking Dot1a. Despite these findings, the putative antagonism between the MR/aldosterone and Dot1a-Af9 complexes has never been addressed. The molecular defects leading to PHA-1 in MR(-/-) mice remain elusive. Here, we report that MR competes with Dot1a to bind Af9. MR/aldosterone and Dot1a-Af9 complexes mutually counterbalance ENaC mRNA expression in inner medullary collecting duct 3 (IMCD3) cells. Real-time RT-quantitative PCR revealed that 5-day-old MR(-/-) vs. MR(+/+) mice had significantly lower αENaC mRNA levels. This change was associated with an increased Af9 binding and H3 K79 hypermethylation in the αENaC promoter. Therefore, this study identified MR as a novel binding partner and regulator of Af9 and a novel mechanism coupling MR-mediated activation with relief of Dot1a-Af9-mediated repression via MR-Af9 interaction. Impaired ENaC expression due to failure to inhibit Dot1a-Af9 may play an important role in the early stages of PHA-1 (before postnatal day 8) in MR(-/-) mice.
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Affiliation(s)
- Xi Zhang
- Dept. of Internal Medicine, Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 5.135, Houston, TX 77030.
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Staruschenko A. Aldosterone-dependent trans-activation and epigenetic derepression of ENaC: where is the balance? Am J Physiol Renal Physiol 2013; 305:F968-9. [PMID: 23842778 DOI: 10.1152/ajprenal.00386.2013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Affiliation(s)
- Alexander Staruschenko
- Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.
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Yu Z, Kong Q, Kone BC. Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells. Am J Physiol Renal Physiol 2013; 305:F653-62. [PMID: 23804453 DOI: 10.1152/ajprenal.00177.2013] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC.
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Affiliation(s)
- Zhiyuan Yu
- Div. of Renal Diseases and Hypertension, The Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 5.124, Houston, TX 77030, USA
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Zhang W, Yu Z, Wu H, Chen L, Kong Q, Kone BC. An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the αENaC gene. Am J Physiol Renal Physiol 2013; 304:F367-75. [PMID: 23152297 PMCID: PMC3566494 DOI: 10.1152/ajprenal.00537.2011] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2011] [Accepted: 11/11/2012] [Indexed: 01/17/2023] Open
Abstract
The epithelial Na(+) channel subunit-α (αENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress αENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to αENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the αENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the -57/+439 "R3" subregion of αENaC, the principal site for Dot1a-Af9 interaction, in mIMCD3 cells. We also exploited connecting tubule/collecting duct-specific Dot1l-deficient mice (Dot1l(AC)) to determine the impact of Dot1l inactivation on renal αENaC expression in vivo. mIMCD3 cell lines expressing αENaC promoter-reporter constructs harboring deletion of +74/+107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the αENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing +78/+92 αENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the +78/+92 element resulted in higher basal αENaC promoter activity and impaired Dot1a-mediated inhibition in trans-repression assays. In agreement, mice with connecting tubule/collecting duct-specific knockout of Dot1l exhibited greater αENaC mRNA levels in kidney compared with control. Thus, we conclude that +78/+92 of αENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive αENaC transcription and that Dot1l inactivation promotes αENaC mRNA expression by eliminating Dot1a-mediated repression.
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Affiliation(s)
- Wenzheng Zhang
- Division of Renal Diseases and Hypertension, The University of Texas Medical School at Houston, Houston, TX 77030, USA
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Wu H, Chen L, Zhou Q, Zhang X, Berger S, Bi J, Lewis DE, Xia Y, Zhang W. Aqp2-expressing cells give rise to renal intercalated cells. J Am Soc Nephrol 2013; 24:243-52. [PMID: 23308014 DOI: 10.1681/asn.2012080866] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
Abstract
The mammalian collecting duct comprises principal and intercalated cells, which maintain sodium/water and acid/base balance, respectively, but the epigenetic contributors to the differentiation of these cell types remain unknown. Here, we investigated whether the histone H3 K79 methyltransferase Dot1l, which is highly expressed in principal cells, participates in this process. Taking advantage of the distribution of aquaporin 2 (Aqp2), which localizes to principal cells of the collecting duct, we developed mice lacking Dot1l in Aqp2-expressing cells (Dot1l(AC)) and found that these mice had approximately 20% fewer principal cells and 13%-16% more intercalated cells than control mice. This deletion of Dot1l in principal cells abolished histone H3 K79 methylation in these cells, but unexpectedly, most intercalated cells also had undetectable di-methyl K79, suggesting that Aqp2(+) cells give rise to intercalated cells. These Aqp2(+) cell-derived intercalated cells were present in both developing and mature kidneys. Furthermore, compared with control mice, Dot1l(AC) mice had 40% higher urine volume and 18% lower urine osmolarity with relatively normal electrolyte and acid-base homeostasis. In conclusion, these data suggest that Dot1l deletion facilitates the differentiation of some α- and β-intercalated cells from Aqp2-expressing progenitor cells or mature principal cells.
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Affiliation(s)
- Hongyu Wu
- Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas, USA
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Wu H, Chen L, Zhang X, Zhou Q, Li JM, Berger S, Borok Z, Zhou B, Xiao Z, Yin H, Liu M, Wang Y, Jin J, Blackburn MR, Xia Y, Zhang W. Aqp5 is a new transcriptional target of Dot1a and a regulator of Aqp2. PLoS One 2013; 8:e53342. [PMID: 23326416 PMCID: PMC3542343 DOI: 10.1371/journal.pone.0053342] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2012] [Accepted: 11/27/2012] [Indexed: 12/02/2022] Open
Abstract
Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1l(AC)) develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1l(AC) vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1l(AC) kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5' flanking region in Dot1l(AC) vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1l(AC) mice and in patients with diabetic nephropathy.
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Affiliation(s)
- Hongyu Wu
- Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Lihe Chen
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Xi Zhang
- Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Qiaoling Zhou
- Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Ju-Mei Li
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Stefan Berger
- German Cancer Research Center, Division Molecular Biology of the Cell I, Heidelberg, Germany
| | - Zea Borok
- Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Southern California, Los Angeles, California, United States of America
| | - Beiyun Zhou
- Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Southern California, Los Angeles, California, United States of America
| | - Zhou Xiao
- Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Hongling Yin
- Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Mingyao Liu
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
- Institute of Biosciences and Technology and Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, Houston, Texas, United States of America
| | - Ying Wang
- Institute of Biosciences and Technology and Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, Houston, Texas, United States of America
| | - Jianping Jin
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Michael R. Blackburn
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Yang Xia
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Wenzheng Zhang
- Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
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Developmental programming of hypertension and kidney disease. Int J Nephrol 2012; 2012:760580. [PMID: 23251800 PMCID: PMC3516001 DOI: 10.1155/2012/760580] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2012] [Revised: 09/18/2012] [Accepted: 10/21/2012] [Indexed: 02/05/2023] Open
Abstract
A growing body of evidence supports the concept that changes in the intrauterine milieu during "sensitive" periods of embryonic development or in infant diet after birth affect the developing individual, resulting in general health alterations later in life. This phenomenon is referred to as "developmental programming" or "developmental origins of health and disease." The risk of developing late-onset diseases such as hypertension, chronic kidney disease (CKD), obesity or type 2 diabetes is increased in infants born prematurely at <37 weeks of gestation or in low birth weight (LBW) infants weighing <2,500 g at birth. Both genetic and environmental events contribute to the programming of subsequent risks of CKD and hypertension in premature or LBW individuals. A number of observations suggest that susceptibility to subsequent CKD and hypertension in premature or LBW infants is mediated, at least in part, by reduced nephron endowment. The major factors influencing in utero environment that are associated with a low final nephron number include uteroplacental insufficiency, maternal low-protein diet, hyperglycemia, vitamin A deficiency, exposure to or interruption of endogenous glucocorticoids, and ethanol exposure. This paper discusses the effect of premature birth, LBW, intrauterine milieu, and infant feeding on the development of hypertension and renal disease in later life as well as examines the role of the kidney in developmental programming of hypertension and CKD.
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Spironolactone rescues Dot1a-Af9-mediated repression of endothelin-1 and improves kidney injury in streptozotocin-induced diabetic rats. PLoS One 2012; 7:e47360. [PMID: 23077601 PMCID: PMC3471839 DOI: 10.1371/journal.pone.0047360] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2012] [Accepted: 09/11/2012] [Indexed: 01/01/2023] Open
Abstract
The molecular mechanism linking aldosterone and endothelin-1 in the development of diabetic nephropathy has not been completely elucidated. Here, we provide evidence showing that streptozotocin-induced diabetic rats have significantly increased aldosterone and endothelin-1 in the kidney tissue and markedly decreased expression of Dot1a and Af9. Blocking aldosterone with spironolactone significantly reduced proteinuria, glomerulosclerosis, tubulointerstitial injury and endothelin-1 expression, and significantly increased Dot1a and Af9 expression. Increasing Dot1a and Af9 expression by spironolactone or by stable transfection led to impaired endothelin-1 expression in NRK-52 cells. In contrast, downregulation of Dot1a and Af9 by aldosterone in NRK-52E cells caused upregulation of endothelin-1. Genetic inactivation of Dot1l, which encodes Dot1a, in Aqp2-expressing principal cells of mouse kidney impaired association of Dot1a and H3 dimethyl K79 with the specific subregions of endothelin-1 promoter, and upregulates endothelin-1 mRNA and protein expression. Our data suggest that Dot1a and Af9 repress endothelin-1 in vitro and in vivo. Excessive aldosterone induces kidney injury, in part possibly by downregulating Dot1a and Af9, and thus relieving Dot1a-Af9-mediated repression to increase endothelin-1 transcription. Spironolactone ameliorates kidney injury in Streptozotocin-induced diabetic rats, possibly by restoring Dot1a-Af9-mediated repression to reduce endothelin-1 expression. Therefore, Dot1a and Af9 as aldosterone-downregulated targets are negative regulators of endothelin-1 transcription in vitro and in vivo, and may be considered as new potential therapeutic targets of kidney injury in diabetes.
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Duarte JD, Zineh I, Burkley B, Gong Y, Langaee TY, Turner ST, Chapman AB, Boerwinkle E, Gums JG, Cooper-DeHoff RM, Beitelshees AL, Bailey KR, Fillingim RB, Kone BC, Johnson JA. Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure response to hydrochlorothiazide. J Transl Med 2012; 10:56. [PMID: 22440088 PMCID: PMC3320544 DOI: 10.1186/1479-5876-10-56] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2011] [Accepted: 03/22/2012] [Indexed: 11/21/2022] Open
Abstract
BACKGROUND Nearly one-third of the United States adult population suffers from hypertension. Hydrochlorothiazide (HCTZ), one of the most commonly used medications to treat hypertension, has variable efficacy. The renal epithelial sodium channel (ENaC) provides a mechanism for fine-tuning sodium excretion, and is a major regulator of blood pressure homeostasis. DOT1L, MLLT3, SIRT1, and SGK1 encode genes in a pathway that controls methylation of the histone H3 globular domain at lysine 79 (H3K79), thereby modulating expression of the ENaCα subunit. This study aimed to determine the role of variation in these regulatory genes on blood pressure response to HCTZ, and secondarily, untreated blood pressure. METHODS We investigated associations between genetic variations in this candidate pathway and HCTZ blood pressure response in two separate hypertensive cohorts (clinicaltrials.gov NCT00246519 and NCT00005520). In a secondary, exploratory analysis, we measured associations between these same genetic variations and untreated blood pressure. Associations were measured by linear regression, with only associations with P ≤ 0.01 in one cohort and replication by P ≤ 0.05 in the other cohort considered significant. RESULTS In one cohort, a polymorphism in DOT1L (rs2269879) was strongly associated with greater systolic (P = 0.0002) and diastolic (P = 0.0016) blood pressure response to hydrochlorothiazide in Caucasians. However, this association was not replicated in the other cohort. When untreated blood pressure levels were analyzed, we found directionally similar associations between a polymorphism in MLLT3 (rs12350051) and greater untreated systolic (P < 0.01 in both cohorts) and diastolic (P < 0.05 in both cohorts) blood pressure levels in both cohorts. However, when further replication was attempted in a third hypertensive cohort and in smaller, normotensive samples, significant associations were not observed. CONCLUSIONS Our data suggest polymorphisms in DOT1L, MLLT3, SIRT1, and SGK1 are not likely associated with blood pressure response to HCTZ. However, a possibility exists that rs2269879 in DOT1L could be associated with HCTZ response in Caucasians. Additionally, exploratory analyses suggest rs12350051 in MLLT3 may be associated with untreated blood pressure in African-Americans. Replication efforts are needed to verify roles for these polymorphisms in human blood pressure regulation.
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Affiliation(s)
- Julio D Duarte
- Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
- Department of Pharmacy Practice, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Issam Zineh
- Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
- Office of Clinical Pharmacology, Office of Translational Sciences - CDER, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
| | - Ben Burkley
- Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
| | - Yan Gong
- Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
| | - Taimour Y Langaee
- Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
| | - Stephen T Turner
- Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 55905, USA
| | - Arlene B Chapman
- Renal Division, Department of Medicine, Emory University, Atlanta, GA 30322, USA
| | - Eric Boerwinkle
- Human Genetics Center and Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA
| | - John G Gums
- Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
| | - Rhonda M Cooper-DeHoff
- Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
| | - Amber L Beitelshees
- Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland, Baltimore, MD 21201, USA
| | - Kent R Bailey
- Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN 55905, USA
| | - Roger B Fillingim
- Department of Community Dentistry and Behavioral Science, University of Florida, Gainesville, FL 32610, USA
| | - Bruce C Kone
- Division of Nephrology, Hypertension & Renal Transplantation, University of Florida, Gainesville, FL 32610, USA
- Division of Renal Diseases and Hypertension, University of Texas Health Science Center, Houston, TX 77030, USA
| | - Julie A Johnson
- Center for Pharmacogenomics and Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL 32610, USA
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Abstract
The epithelial Na(+) channel (ENaC) and acid-sensitive ion channel (ASIC) branches of the ENaC/degenerin superfamily of cation channels have drawn increasing attention as potential therapeutic targets in a variety of diseases and conditions. Originally thought to be solely expressed in fluid absorptive epithelia and in neurons, it has become apparent that members of this family exhibit nearly ubiquitous expression. Therapeutic opportunities range from hypertension, due to the role of ENaC in maintaining whole body salt and water homeostasis, to anxiety disorders and pain associated with ASIC activity. As a physiologist intrigued by the fundamental mechanics of salt and water transport, it was natural that Dale Benos, to whom this series of reviews is dedicated, should have been at the forefront of research into the amiloride-sensitive sodium channel. The cloning of ENaC and subsequently the ASIC channels has revealed a far wider role for this channel family than was previously imagined. In this review, we will discuss the known and potential roles of ENaC and ASIC subunits in the wide variety of pathologies in which these channels have been implicated. Some of these, such as the role of ENaC in Liddle's syndrome are well established, others less so; however, all are related in that the fundamental defect is due to inappropriate channel activity.
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Affiliation(s)
- Yawar J Qadri
- Department of Physiology and Biophysics, University of Alabama at Birmingham, AL 35294, USA
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Wu H, Chen L, Zhou Q, Zhang W. AF17 facilitates Dot1a nuclear export and upregulates ENaC-mediated Na+ transport in renal collecting duct cells. PLoS One 2011; 6:e27429. [PMID: 22087315 PMCID: PMC3210795 DOI: 10.1371/journal.pone.0027429] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2011] [Accepted: 10/16/2011] [Indexed: 01/22/2023] Open
Abstract
Our previous work in 293T cells and AF17-/- mice suggests that AF17 upregulates expression and activity of the epithelial Na+ channel (ENaC), possibly by relieving Dot1a-AF9-mediated repression. However, whether and how AF17 directly regulates Dot1a cellular distribution and ENaC function in renal collecting duct cells remain unaddressed. Here, we report our findings in mouse cortical collecting duct M-1 cells that overexpression of AF17 led to preferential distribution of Dot1a in the cytoplasm. This effect could be blocked by nuclear export inhibitor leptomycin B. siRNA-mediated depletion of AF17 caused nuclear accumulation of Dot1a. AF17 overexpression elicited multiple effects that are reminiscent of aldosterone action. These effects include 1) increased mRNA and protein expression of the three ENaC subunits (α, β and γ) and serum- and glucocorticoid inducible kinase 1, as revealed by real-time RT-qPCR and immunoblotting analyses; 2) impaired Dot1a-AF9 interaction and H3 K79 methylation at the αENaC promoter without affecting AF9 binding to the promoter, as evidenced by chromatin immunoprecipitation; and 3) elevated ENaC-mediated Na+ transport, as analyzed by measurement of benzamil-sensitive intracellular [Na+] and equivalent short circuit current using single-cell fluorescence imaging and an epithelial Volt-ohmmeter, respectively. Knockdown of AF17 elicited opposite effects. However, combination of AF17 overexpression or depletion with aldosterone treatment did not cause an additive effect on mRNA expression of the ENaC subunits. Taken together, we conclude that AF17 promotes Dot1a nuclear export and upregulates basal, but not aldosterone-stimulated ENaC expression, leading to an increase in ENaC-mediated Na+ transport in renal collecting duct cells.
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Affiliation(s)
- Hongyu Wu
- Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Lihe Chen
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Qiaoling Zhou
- Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China
| | - Wenzheng Zhang
- Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
- * E-mail:
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38
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Chen L, Wu H, Pochynyuk OM, Reisenauer MR, Zhang Z, Huang L, Zaika OL, Mamenko M, Zhang W, Zhou Q, Liu M, Xia Y, Zhang W. Af17 deficiency increases sodium excretion and decreases blood pressure. J Am Soc Nephrol 2011; 22:1076-86. [PMID: 21546577 DOI: 10.1681/asn.2010121270] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
The putative transcription factor AF17 upregulates the transcription of the epithelial sodium channel (ENaC) genes, but whether AF17 modulates sodium homeostasis and BP is unknown. Here, we generated Af17-deficient mice to determine whether deletion of Af17 leads to sodium wasting and low BP. Compared with wild-type mice, Af17-deficient mice had lower BP (11 mmHg), higher urine volume, and increased sodium excretion despite mildly increased plasma concentrations of aldosterone. Deletion of Af17 led to increased dimethylation of histone H3 K79 and reduced ENaC function. The attenuated function of ENaC resulted from decreased ENaC mRNA and protein expression, fewer active channels, lower open probability, and reduced effective activity. In contrast, inducing high levels of plasma aldosterone by a variety of methods completely compensated for Af17 deficiency with respect to sodium handling and BP. Taken together, these data identify Af17 as a potential locus for the maintenance of sodium and BP homeostasis and suggest that a particular histone modification is directly linked to these processes. Af17-mediated regulation of BP is largely, but not exclusively, the result of modulating ENaC, suggesting it has potential as a therapeutic target for the control of BP.
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Affiliation(s)
- Lihe Chen
- Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas, USA
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39
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Tang BL. Sirt1's systemic protective roles and its promise as a target in antiaging medicine. Transl Res 2011; 157:276-84. [PMID: 21497775 DOI: 10.1016/j.trsl.2010.11.006] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/11/2010] [Revised: 11/24/2010] [Accepted: 11/24/2010] [Indexed: 12/14/2022]
Abstract
Silent information regulator 2 (Sir2/Sirt1), a member of the sirtuin family of class III histone deacetylases, has been implicated extensively in lifespan extension and is a prominent drug target in antiaging medicine. The mammalian Sirt1 has multiple targets, which include histones, transcription factors, and other molecules that collectively modulate energy metabolism, stress response, and cell/tissue survival. Several of Sirt1's substrates regulate key metabolic processes, and Sirt1 activation may underlie the lifespan prolonging effect of caloric restriction. Recent studies have also identified multifaceted protective roles for Sirt1 against cellular senescence and stress in the neural, cardiovascular, and renal systems. Sirt1's activity in multiple tissues may decline with aging, and sustaining or reactivating this activity seems invariably beneficial. Several studies also point towards a general tumor suppressive role for Sirt1, at least in the context of certain human cancers. Development of Sirt1-based therapeutic interventions against systemic aging and aging-associated diseases will benefit from a thorough understanding of underlying pathological mechanisms of diseases as well as metabolic connections between different tissues and organs.
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Affiliation(s)
- Bor Luen Tang
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore 117597.
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40
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FitzGerald J, Moureau S, Drogaris P, O'Connell E, Abshiru N, Verreault A, Thibault P, Grenon M, Lowndes NF. Regulation of the DNA damage response and gene expression by the Dot1L histone methyltransferase and the 53Bp1 tumour suppressor. PLoS One 2011; 6:e14714. [PMID: 21383990 PMCID: PMC3044716 DOI: 10.1371/journal.pone.0014714] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2010] [Accepted: 11/19/2010] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined. METHODOLOGY/PRINCIPAL FINDINGS Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line. CONCLUSIONS/SIGNIFICANCE Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.
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Affiliation(s)
- Jennifer FitzGerald
- Genome Stability Laboratory, School of Natural Sciences, Centre for Chromosome Biology, National University of Ireland Galway, Galway, Ireland
| | - Sylvie Moureau
- Genome Stability Laboratory, School of Natural Sciences, Centre for Chromosome Biology, National University of Ireland Galway, Galway, Ireland
| | - Paul Drogaris
- Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada
| | - Enda O'Connell
- National Centre for Biomedical Engineering Sciences, National University of Ireland Galway, Galway, Ireland
| | - Nebiyu Abshiru
- Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada
| | - Alain Verreault
- Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada
- Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Québec, Canada
| | - Pierre Thibault
- Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada
- Département de Chimie, Université de Montréal, Montréal, Québec, Canada
| | - Muriel Grenon
- Genome Stability Laboratory, School of Natural Sciences, Centre for Chromosome Biology, National University of Ireland Galway, Galway, Ireland
| | - Noel F. Lowndes
- Genome Stability Laboratory, School of Natural Sciences, Centre for Chromosome Biology, National University of Ireland Galway, Galway, Ireland
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Zhang Z, Huang L, Reisenauer MR, Wu H, Chen L, Zhang Y, Xia Y, Zhang W. Widely expressed Af17 is likely not required for embryogenesis, hematopoiesis, and animal survival. Genesis 2010; 48:693-706. [PMID: 21170927 DOI: 10.1002/dvg.20679] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2010] [Revised: 09/13/2010] [Accepted: 09/25/2010] [Indexed: 01/17/2023]
Abstract
As a putative transcription factor, Af17 may play a role in multiple signaling pathways. However, the Af17 expression profile during development and in adult tissues remains largely uncharacterized. The importance of Af17 function in embryogenesis, hematopoiesis, and animal survival has never been addressed before. Here we report the generation of the first Af17 mutant mouse model and characterization of the Af17 temporal and spatial expression profile in various embryonic stages and adult tissues by X-gal staining, in situ hybridization, and RT-PCR. Af17 expression is detected in specific cell populations in all stages and in multiple tissues examined. In situ hybridization yielded a consistent Af17 expression pattern by X-gal staining. Homozygous mutant mice are viable, fertile, normal in size, and do not display any gross physical, behavioral, or hematopoietic abnormalities. Thus, our studies describe the generation of the first Af17 mutant mouse model, provide the first developmental profile of Af17 expression, and reveal that Af17 may be dispensable for normal embryogenesis, hematopoiesis, and animal survival.
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Affiliation(s)
- Zhijing Zhang
- Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USA
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42
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Reisenauer MR, Wang SW, Xia Y, Zhang W. Dot1a contains three nuclear localization signals and regulates the epithelial Na+ channel (ENaC) at multiple levels. Am J Physiol Renal Physiol 2010; 299:F63-76. [PMID: 20427473 DOI: 10.1152/ajprenal.00105.2010] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
We have previously reported that Dot1a is located in the cytoplasm and nucleus (Reisenauer MR, Anderson M, Huang L, Zhang Z, Zhou Q, Kone BC, Morris AP, Lesage GD, Dryer SE, Zhang W. J Biol Chem 284: 35659-35669, 2009), widely expressed in the kidney as detected by its histone H3K79 methyltransferase activity (Zhang W, Hayashizaki Y, Kone BC. Biochem J 377: 641-651, 2004), and involved in transcriptional control of the epithelial Na(+) channel subunit-alpha gene (alphaENaC) (Zhang W, Xia X, Jalal DI, Kuncewicz T, Xu W, Lesage GD, Kone BC. Am J Physiol Cell Physiol 290: C936-C946, 2006). Aldosterone releases repression of alphaENaC by reducing expression of Dot1a and its partner AF9 (Zhang W, Xia X, Reisenauer MR, Hemenway CS, Kone BC. J Biol Chem 281: 18059-18068, 2006) and by impairing Dot1a-AF9 interaction via Sgk1-mediated AF9 phosphorylation (Zhang W, Xia X, Reisenauer MR, Rieg T, Lang F, Kuhl D, Vallon V, Kone BC. J Clin Invest 117: 773-783, 2007). This network also appears to regulate transcription of several other aldosterone target genes. Here, we provide evidence showing that Dot1a contains at least three potential nuclear localization signals (NLSs). Deletion of these NLSs causes green fluorescent protein-fused Dot1a fusions to localize almost exclusively in the cytoplasm of 293T cells as revealed by confocal microscopy. Deletion of NLSs abolished Dot1a-mediated repression of alphaENaC-promoter luciferase construct in M1 cells. AF9 is widely expressed in mouse kidney. Similar to alphaENaC, the mRNA levels of betaENaC, gammaENaC, and Sgk1 are also downregulated by Dot1a and AF9 overexpression. Small interference RNA-mediated knockdown of Dot1a and AF9 or aldosterone treatment leads to an opposite effect. Using single-cell fluorescence imaging or equivalent short-circuit current in IMCD3 and M1 cells, we show that observed transcriptional alterations correspond to changes in ENaC and Sgk1 protein levels as well as benzamil-sensitive Na(+) transport. In brief, Dot1a and AF9 downregulate Na(+) transport, most likely by regulating ENaC mRNA and subsequent protein expression and ENaC activity.
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Affiliation(s)
- Mary Rose Reisenauer
- Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA
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43
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Reisenauer MR, Anderson M, Huang L, Zhang Z, Zhou Q, Kone BC, Morris AP, Lesage GD, Dryer SE, Zhang W. AF17 competes with AF9 for binding to Dot1a to up-regulate transcription of epithelial Na+ channel alpha. J Biol Chem 2010; 284:35659-69. [PMID: 19864429 DOI: 10.1074/jbc.m109.038448] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
We previously reported that Dot1a.AF9 complex represses transcription of the epithelial Na(+) channel subunit alpha (alpha-ENaC) gene in mouse inner medullary collecting duct mIMCD3 cells and mouse kidney. Aldosterone relieves this repression by down-regulating the complex through various mechanisms. Whether these mechanisms are sufficient and conserved in human cells or can be applied to other aldosterone-regulated genes remains largely unknown. Here we demonstrate that human embryonic kidney 293T cells express the three ENaC subunits and all of the ENaC transcriptional regulators examined. These cells respond to aldosterone and display benzamil-sensitive Na(+) currents, as measured by whole-cell patch clamping. We also show that AF17 and AF9 competitively bind to the same domain of Dot1a in multiple assays and have antagonistic effects on expression of an alpha-ENaC promoter-luciferase construct. Overexpression of Dot1a or AF9 decreased mRNA expression of the ENaC subunits and their transcriptional regulators and reduced benzamil-sensitive Na(+) currents. AF17 overexpression caused the opposite effects, accompanied by redirection of Dot1a from the nucleus to the cytoplasm and reduction in histone H3 K79 methylation. The nuclear export inhibitor leptomycin B blocked the effect of AF17 overexpression on H3 K79 hypomethylation. RNAi-mediated knockdown of AF17 yielded nuclear enrichment of Dot1a and histone H3 K79 hypermethylation. As with AF9, AF17 displays nuclear and cytoplasmic co-localization with Sgk1. Therefore, AF17 competes with AF9 to bind Dot1a, decreases Dot1a nuclear expression by possibly facilitating its nuclear export, and relieves Dot1a.AF9-mediated repression of alpha-ENaC and other target genes.
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Affiliation(s)
- Mary Rose Reisenauer
- Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA
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44
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Yu Z, Kong Q, Kone BC. CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells. Am J Physiol Renal Physiol 2010; 298:F617-24. [PMID: 20053791 DOI: 10.1152/ajprenal.00636.2009] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.
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Affiliation(s)
- Zhiyuan Yu
- Department of Medicine, Division of Renal Diseases and Hypertension, The University of Texas Medical School at Houston, Houston, Texas 77006, USA
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45
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Gumz ML, Stow LR, Lynch IJ, Greenlee MM, Rudin A, Cain BD, Weaver DR, Wingo CS. The circadian clock protein Period 1 regulates expression of the renal epithelial sodium channel in mice. J Clin Invest 2009; 119:2423-34. [PMID: 19587447 DOI: 10.1172/jci36908] [Citation(s) in RCA: 171] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2008] [Accepted: 05/13/2009] [Indexed: 11/17/2022] Open
Abstract
The mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and contributes to the control of blood pressure and cardiac function. It specifically functions to increase renal absorption of sodium from tubular fluid via regulation of the alpha subunit of the epithelial sodium channel (alphaENaC). We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line and found that the transcript induced to the greatest extent was the circadian clock gene Period 1. Here, we investigated the role of Period 1 in mediating the downstream effects of aldosterone in renal cells. Aldosterone treatment stimulated expression of Period 1 (Per1) mRNA in renal collecting duct cell lines and in the rodent kidney. RNA silencing of Period 1 dramatically decreased expression of mRNA encoding alphaENaC in the presence or absence of aldosterone. Furthermore, expression of alphaENaC-encoding mRNA was attenuated in the renal medulla of mice with disruption of the Per1 gene, and these mice exhibited increased urinary sodium excretion. Renal alphaENaC-encoding mRNA was expressed in an apparent circadian pattern, and this pattern was dramatically altered in mice lacking functional Period genes. These results suggest a role for Period 1 in the regulation of the renal epithelial sodium channel and more broadly implicate the circadian clock in control of sodium balance.
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46
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Zhang D, Li S, Cruz P, Kone BC. Sirtuin 1 functionally and physically interacts with disruptor of telomeric silencing-1 to regulate alpha-ENaC transcription in collecting duct. J Biol Chem 2009; 284:20917-26. [PMID: 19491102 DOI: 10.1074/jbc.m109.020073] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Aldosterone increases renal tubular Na+ absorption in large part by increasing transcription of the epithelial Na(+) channel alpha-subunit (alpha-ENaC) expressed in the apical membrane of collecting duct principal cells. We recently reported that a complex containing the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) associates with and represses the alpha-ENaC promoter in mouse inner medullary collecting duct mIMCD3 cells, and that aldosterone acts to disrupt this complex and its inhibitory effects (Zhang, W., Xia, X., Reisenauer, M. R., Rieg, T., Lang, F., Kuhl, D., Vallon, V., and Kone, B. C. (2007) J. Clin. Invest. 117, 773-783). Here we demonstrate that the NAD(+)-dependent deacetylase sirtuin 1 (Sirt1) functionally and physically interacts with Dot1 to enhance the distributive activity of Dot1 on H3K79 methylation and thereby represses alpha-ENaC transcription in mIMCD3 cells. Sirt1 overexpression inhibited basal alpha-ENaC mRNA expression and alpha-ENaC promoter activity, surprisingly in a deacetylase-independent manner. The ability of Sirt1 to inhibit alpha-ENaC transcription was retained in a truncated Sirt1 construct expressing only its N-terminal domain. Conversely, Sirt1 knockdown enhanced alpha-ENaC mRNA levels and alpha-ENaC promoter activity, and inhibited global H3K79 methylation, particularly H3K79 trimethylation, in chromatin associated with the alpha-ENaC promoter. Sirt1 and Dot1 co-immunoprecipitated from mIMCD3 cells and colocalized in the nucleus. Sirt1 immunoprecipitated from chromatin associated with regions of the alpha-ENaC promoter known to associate with Dot1. Aldosterone inhibited Sirt1 association at two of these regions, as well as Sirt1 mRNA expression, in a coordinate manner with induction of alpha-ENaC transcription. Overexpressed Sirt1 inhibited aldosterone induction of alpha-ENaC transcription independent of effects on mineralocorticoid receptor trans-activation. These data identify Sirt1 as a novel modulator of alpha-ENaC, Dot1, and the aldosterone signaling pathway.
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Affiliation(s)
- Dongyu Zhang
- Departments of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610, USA
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47
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Epigenetics and the control of epithelial sodium channel expression in collecting duct. Kidney Int 2008; 75:260-7. [PMID: 18818687 DOI: 10.1038/ki.2008.475] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
In eukaryotic nuclei, genomic DNA is compacted with histone and nonhistone proteins into a dynamic polymer termed chromatin. Reorganization of chromatin structure through histone modifications, the action of chromatin factors, or DNA methylation, can profoundly change gene expression. These epigenetic modifications allow heritable and potentially reversible changes in gene functioning to occur without altering the DNA sequence, thus extending the information potential of the genetic code. This review provides an introduction to epigenetic concepts for renal investigators and an overview of our work detailing an epigenetic pathway for aldosterone signaling and the control of epithelial Na(+) channel-alpha (ENaCalpha) subunit gene expression in the collecting duct. This new pathway involves a nuclear repressor complex, consisting of histone H3 Lys-79 methyltransferase disruptor of telomeric silencing-1a (Dot1a), ALL1 fused gene from chromosome 9 (Af9), a sequence-specific DNA-binding protein that binds the ENaCalpha promoter, and potentially other nuclear proteins. This complex regulates targeted histone H3 Lys-79 methylation of chromatin associated with the ENaCalpha promoter, thereby suppressing its transcriptional activity. Aldosterone disrupts the Dot1a-Af9 interaction by serum- and glucocorticoid-induced kinase-1 phosphorylation of Af9, and inhibits Dot1a and Af9 expression, resulting in histone H3 Lys-79 hypomethylation at specific subregions, and derepression of the ENaCalpha promoter. The Dot1a-Af9 pathway may also be involved in the control of genes implicated in renal fibrosis and hypertension.
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48
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Ni Z, Abou El Hassan M, Xu Z, Yu T, Bremner R. The chromatin-remodeling enzyme BRG1 coordinates CIITA induction through many interdependent distal enhancers. Nat Immunol 2008; 9:785-93. [PMID: 18500344 DOI: 10.1038/ni.1619] [Citation(s) in RCA: 87] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2007] [Accepted: 04/21/2008] [Indexed: 01/22/2023]
Abstract
The chromatin-remodeling enzyme BRG1 is critical for interferon-gamma (IFN-gamma)-mediated gene induction. Promoter-proximal elements are sufficient to mediate BRG1 dependency at some IFN-gamma targets. In contrast, we show here that at CIITA, which encodes the 'master regulator' of induction of major histocompatibility complex class II, distal elements conferred BRG1 dependency. At the uninduced locus, many sites formed BRG1-independent loops. One loop juxtaposed a far downstream element adjacent to a far upstream site. Notably, BRG1 was recruited to the latter site, which triggered the appearance of a histone 'mark' linked to activation. This subtle change was crucial, as subsequent IFN-gamma-induced recruitment of the transcription factors STAT1, IRF1 and p300, as well as histone modifications, accessibility and additional loops, showed BRG1 dependency. Like BRG1, each remote element was critical for the induction of CIITA expression. Thus, BRG1 regulates CIITA through many interdependent remote enhancers, not through the promoter alone.
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Affiliation(s)
- Zuyao Ni
- Genetics and Development Division, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8, Canada
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49
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Náray-Fejes-Tóth A, Boyd C, Fejes-Tóth G. Regulation of epithelial sodium transport by promyelocytic leukemia zinc finger protein. Am J Physiol Renal Physiol 2008; 295:F18-26. [PMID: 18448589 DOI: 10.1152/ajprenal.00573.2007] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Aldosterone is the principal regulator of Na homeostasis, and thereby blood pressure. One of the main targets of aldosterone is the epithelial Na channel (ENaC) located in the apical membrane of target cells. Previous studies identified several genes involved in the regulation of ENaC such as SGK1; however, SGK1 knockout mice have only a mild salt-losing phenotype, indicating that further genes must be involved in the action of aldosterone. In our search for further aldosterone-regulated genes, we discovered that aldosterone, at physiological concentrations, induces the expression of the promyelocytic leukemia zinc finger protein (PLZF) in renal cortical collecting duct (CCD) cell lines that stably express mineralocorticoid receptors (MRs). This effect is rapid and does not require de novo protein synthesis, suggesting a direct action. Surprisingly, stable overexpression of human or mouse PLZF isoforms significantly decreased transepithelial Na transport in CCD cells while having no effect on the integrity of the monolayers. In parallel with the decline in Na transport, PLZF suppressed the mRNA levels of beta- and gamma-ENaC subunits. These observations suggest that PLZF is a negative regulator of ENaC in renal epithelial cells and might be part of a negative feedback loop that limits aldosterone's stimulatory effects on sodium reabsorption.
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50
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DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Mol Cell Biol 2008; 28:2825-39. [PMID: 18285465 DOI: 10.1128/mcb.02076-07] [Citation(s) in RCA: 369] [Impact Index Per Article: 21.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recruitment is ubiquitously coupled with active transcription in diverse mammalian cell types. DOT1L preferentially occupies the proximal transcribed region of active genes, correlating with enrichment of H3K79 di- and trimethylation. Furthermore, Dot1l mutant fibroblasts lacked H3K79 di- and trimethylation at all sites examined, indicating that DOT1L is the sole enzyme responsible for these marks. Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. Conversion of H3K79 monomethylation into di- and trimethylation correlated with the transition from low- to high-level gene transcription. We also observed enrichment of H3K79 monomethylation at intergenic regions occupied by DNA-binding transcriptional activators. Our findings highlight several similarities between the patterning of H3K4 methylation and that of H3K79 methylation in mammalian chromatin, suggesting a widespread mechanism for parallel or sequential recruitment of DOT1L and MLL to genes in their normal "on" state.
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