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Wang JB, Du MW, Zheng Y. Effect of ginsenoside Rg1 on hematopoietic stem cells in treating aplastic anemia in mice via MAPK pathway. World J Stem Cells 2024; 16:591-603. [PMID: 38817329 PMCID: PMC11135254 DOI: 10.4252/wjsc.v16.i5.591] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2024] [Revised: 02/20/2024] [Accepted: 04/02/2024] [Indexed: 05/24/2024] Open
Abstract
BACKGROUND Aplastic anemia (AA) presents a significant clinical challenge as a life-threatening condition due to failure to produce essential blood cells, with the current therapeutic options being notably limited. AIM To assess the therapeutic potential of ginsenoside Rg1 on AA, specifically its protective effects, while elucidating the mechanism at play. METHODS We employed a model of myelosuppression induced by cyclophosphamide (CTX) in C57 mice, followed by administration of ginsenoside Rg1 over 13 d. The investigation included examining the bone marrow, thymus and spleen for pathological changes via hematoxylin-eosin staining. Moreover, orbital blood of mice was collected for blood routine examinations. Flow cytometry was employed to identify the impact of ginsenoside Rg1 on cell apoptosis and cycle in the bone marrow of AA mice. Additionally, the study further evaluated cytokine levels with enzyme-linked immunosorbent assay and analyzed the expression of key proteins in the MAPK signaling pathway via western blot. RESULTS Administration of CTX led to significant damage to the bone marrow's structural integrity and a reduction in hematopoietic cells, establishing a model of AA. Ginsenoside Rg1 successfully reversed hematopoietic dysfunction in AA mice. In comparison to the AA group, ginsenoside Rg1 provided relief by reducing the induction of cell apoptosis and inflammation factors caused by CTX. Furthermore, it helped alleviate the blockade in the cell cycle. Treatment with ginsenoside Rg1 significantly alleviated myelosuppression in mice by inhibiting the MAPK signaling pathway. CONCLUSION This study suggested that ginsenoside Rg1 addresses AA by alleviating myelosuppression, primarily through modulating the MAPK signaling pathway, which paves the way for a novel therapeutic strategy in treating AA, highlighting the potential of ginsenoside Rg1 as a beneficial intervention.
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Affiliation(s)
- Jin-Bo Wang
- Internal Medicine of Traditional Chinese Medicine, Tongde Hospital of Zhejiang Province, Hangzhou 310012, Zhejiang Province, China
| | - Ming-Wei Du
- Institute of Cardiovascular Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
- Shanghai Key Laboratory of Molecular Medical Mycology, Second Affiliated Hospital of Naval Medical University, Shanghai 200003, China
- Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China
| | - Yan Zheng
- Department of Hepatic, The Xixi Hospital of Hangzhou Affiliated to Zhejiang University of Traditional Chinese Medicine, Hangzhou 310023, Zhejiang Province, China.
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2
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Martell DJ, Merens HE, Caulier A, Fiorini C, Ulirsch JC, Ietswaart R, Choquet K, Graziadei G, Brancaleoni V, Cappellini MD, Scott C, Roberts N, Proven M, Roy NBA, Babbs C, Higgs DR, Sankaran VG, Churchman LS. RNA polymerase II pausing temporally coordinates cell cycle progression and erythroid differentiation. Dev Cell 2023; 58:2112-2127.e4. [PMID: 37586368 PMCID: PMC10615711 DOI: 10.1016/j.devcel.2023.07.018] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2023] [Revised: 05/23/2023] [Accepted: 07/25/2023] [Indexed: 08/18/2023]
Abstract
Controlled release of promoter-proximal paused RNA polymerase II (RNA Pol II) is crucial for gene regulation. However, studying RNA Pol II pausing is challenging, as pause-release factors are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H, which encodes SPT5, in individuals with β-thalassemia. During erythropoiesis in healthy human cells, cell cycle genes were highly paused as cells transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, RNA Pol II pause release was globally disrupted, and as cells began transitioning from progenitors to precursors, differentiation was delayed, accompanied by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, identifying a role for RNA Pol II pausing in temporally coordinating the cell cycle and erythroid differentiation.
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Affiliation(s)
- Danya J Martell
- Department of Genetics, Harvard University, Boston, MA, USA; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Hope E Merens
- Department of Genetics, Harvard University, Boston, MA, USA
| | - Alexis Caulier
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Claudia Fiorini
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Jacob C Ulirsch
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | | | - Karine Choquet
- Department of Genetics, Harvard University, Boston, MA, USA
| | - Giovanna Graziadei
- Department of Clinical Sciences and Community, University of Milan, IRCCS Ca'Granda Foundation Maggiore Policlinico Hospital, Milan, Italy
| | - Valentina Brancaleoni
- Department of Clinical Sciences and Community, University of Milan, IRCCS Ca'Granda Foundation Maggiore Policlinico Hospital, Milan, Italy
| | - Maria Domenica Cappellini
- Department of Clinical Sciences and Community, University of Milan, IRCCS Ca'Granda Foundation Maggiore Policlinico Hospital, Milan, Italy
| | - Caroline Scott
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
| | - Nigel Roberts
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
| | - Melanie Proven
- Oxford Genetics Laboratories, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
| | - Noémi B A Roy
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK; NIHR Oxford Biomedical Research Centre and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK; Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
| | - Christian Babbs
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
| | - Douglas R Higgs
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
| | - Vijay G Sankaran
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
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3
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Martell DJ, Merens HE, Fiorini C, Caulier A, Ulirsch JC, Ietswaart R, Choquet K, Graziadei G, Brancaleoni V, Cappellini MD, Scott C, Roberts N, Proven M, Roy NB, Babbs C, Higgs DR, Sankaran VG, Churchman LS. RNA Polymerase II pausing temporally coordinates cell cycle progression and erythroid differentiation. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2023:2023.03.03.23286760. [PMID: 36945604 PMCID: PMC10029049 DOI: 10.1101/2023.03.03.23286760] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 06/18/2023]
Abstract
The controlled release of promoter-proximal paused RNA polymerase II (Pol II) into productive elongation is a major step in gene regulation. However, functional analysis of Pol II pausing is difficult because factors that regulate pause release are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H , which encodes SPT5, in individuals with β-thalassemia unlinked to HBB mutations. During erythropoiesis in healthy human cells, cell cycle genes were highly paused at the transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, Pol II pause release was globally disrupted, and the transition from progenitors to precursors was delayed, marked by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.
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Affiliation(s)
- Danya J Martell
- Harvard University, Department of Genetics, Boston, MA
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
- Broad Institute of MIT and Harvard, Cambridge, MA
| | - Hope E Merens
- Harvard University, Department of Genetics, Boston, MA
| | - Claudia Fiorini
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
- Broad Institute of MIT and Harvard, Cambridge, MA
| | - Alexis Caulier
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
- Broad Institute of MIT and Harvard, Cambridge, MA
| | - Jacob C Ulirsch
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
- Broad Institute of MIT and Harvard, Cambridge, MA
| | | | | | - Giovanna Graziadei
- Department of Clinical Sciences and Community, University of Milan, IRCCS Ca'Granda Foundation Maggiore Policlinico Hospital, Milan, Italy
| | - Valentina Brancaleoni
- Department of Clinical Sciences and Community, University of Milan, IRCCS Ca'Granda Foundation Maggiore Policlinico Hospital, Milan, Italy
| | - Maria Domenica Cappellini
- Department of Clinical Sciences and Community, University of Milan, IRCCS Ca'Granda Foundation Maggiore Policlinico Hospital, Milan, Italy
| | - Caroline Scott
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Nigel Roberts
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
| | - Melanie Proven
- Oxford Genetics Laboratories, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
| | - Noémi Ba Roy
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
- NIHR Oxford Biomedical Research Centre and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK
- Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
| | - Christian Babbs
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Douglas R Higgs
- MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Vijay G Sankaran
- Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
- Broad Institute of MIT and Harvard, Cambridge, MA
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4
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Karaosmanoglu B, Kursunel MA, Uckan Cetinkaya D, Gumruk F, Esendagli G, Unal S, Taskiran EZ. Proerythroblast Cells of Diamond-Blackfan Anemia Patients With RPS19 and CECR1 Mutations Have Similar Transcriptomic Signature. Front Physiol 2021; 12:679919. [PMID: 34177624 PMCID: PMC8226250 DOI: 10.3389/fphys.2021.679919] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2021] [Accepted: 04/27/2021] [Indexed: 11/18/2022] Open
Abstract
Diamond Blackfan Anemia (DBA) is an inherited bone marrow (BM) failure syndrome, characterized by a paucity of erythroid differentiation. DBA is mainly caused by the mutations in ribosomal protein genes, hence classified as ribosomopathy. However, in approximately 30% of patients, the molecular etiology cannot be discovered. RPS19 germline mutations caused 25% of the cases. On the other hand, CECR1 mutations also cause phenotypes similar to DBA but not being a ribosomopathy. Due to the blockade of erythropoiesis in the BM, we investigated the transcriptomic profile of three different cell types of BM resident cells of DBA patients and compared them with healthy donors. From BM aspirates BM mononuclear cells (MNCs) were isolated and hematopoietic stem cells (HSC) [CD71–CD34+ CD38mo/lo], megakaryocyte–erythroid progenitor cells (MEP) [CD71–CD34+ CD38hi] and Proerythroblasts [CD71+ CD117+ CD38+] were sorted and analyzed with a transcriptomic approach. Among all these cells, proerythroblasts had the most different transcriptomic profile. The genes associated with cellular stress/immune responses were increased and some of the transcription factors that play a role in erythroid differentiation had altered expression in DBA proerythroblasts. We also showed that gene expression levels of ribosomal proteins were decreased in DBA proerythroblasts. In addition to these, colony formation assay (CFU-E) provided functional evidence of the failure of erythroid differentiation in DBA patients. According to our findings that all patients resembling both RPS19 and CECR1 mutations have common transcriptomic signatures, it may be possible that inflammatory BM niche may have a role in DBA pathogenesis.
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Affiliation(s)
- Beren Karaosmanoglu
- Department of Medical Genetics, Faculty of Medicine, Hacettepe University, Ankara, Turkey.,Department of Stem Cell Sciences, Institute of Health Sciences, Hacettepe University, Ankara, Turkey
| | - M Alper Kursunel
- Department of Basic Oncology, Cancer Institute, Hacettepe University, Ankara, Turkey
| | - Duygu Uckan Cetinkaya
- Division of Pediatric Hematology, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey.,Center for Stem Cell Research and Development, Hacettepe University, Ankara, Turkey
| | - Fatma Gumruk
- Division of Pediatric Hematology, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey.,Research Center for Fanconi Anemia and Other IBMFS, Hacettepe University, Ankara, Turkey
| | - Gunes Esendagli
- Department of Basic Oncology, Cancer Institute, Hacettepe University, Ankara, Turkey
| | - Sule Unal
- Division of Pediatric Hematology, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey.,Research Center for Fanconi Anemia and Other IBMFS, Hacettepe University, Ankara, Turkey
| | - Ekim Z Taskiran
- Department of Medical Genetics, Faculty of Medicine, Hacettepe University, Ankara, Turkey
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5
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CD86-based analysis enables observation of bona fide hematopoietic responses. Blood 2021; 136:1144-1154. [PMID: 32438398 DOI: 10.1182/blood.2020004923] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Accepted: 05/01/2020] [Indexed: 12/23/2022] Open
Abstract
Hematopoiesis is a system that provides red blood cells (RBCs), leukocytes, and platelets, which are essential for oxygen transport, biodefense, and hemostasis; its balance thus affects the outcome of various disorders. Here, we report that stem cell antigen-1 (Sca-1), a cell surface marker commonly used for the identification of multipotent hematopoietic progenitors (Lin-Sca-1+c-Kit+ cells; LSKs), is not suitable for the analysis of hematopoietic responses under biological stresses with interferon production. Lin-Sca-1-c-Kit+ cells (LKs), downstream progenitors of LSKs, acquire Sca-1 expression upon inflammation, which makes it impossible to distinguish between LSKs and LKs. As an alternative and stable marker even under such stresses, we identified CD86 by screening 180 surface markers. The analysis of infection/inflammation-triggered hematopoiesis on the basis of CD86 expression newly revealed urgent erythropoiesis producing stress-resistant RBCs and intact reconstitution capacity of LSKs, which could not be detected by conventional Sca-1-based analysis.
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6
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Transcriptional and epigenetic control of hematopoietic stem cell fate decisions in vertebrates. Dev Biol 2021; 475:156-164. [PMID: 33689804 DOI: 10.1016/j.ydbio.2021.03.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Revised: 02/24/2021] [Accepted: 03/04/2021] [Indexed: 12/20/2022]
Abstract
Hematopoietic stem cells (HSCs) are the foundation of adult hematopoiesis that produce all types of mature blood lineages. In vertebrates, HSC development is a stepwise process, coordinately regulated by chromatin architectures and a group of transcriptional and epigenetic regulators. A deeper understanding of the molecular mechanisms governing the generation, expansion, and function of HSCs holds great promise in the generation and expansion of engraftable HSCs in vitro for clinical applications. This study reviewed recent advances in transcriptional and epigenetic control of hematopoietic stem cell fate decisions in vertebrates.
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7
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Jiang X, Prabhakar A, Van der Voorn SM, Ghatpande P, Celona B, Venkataramanan S, Calviello L, Lin C, Wang W, Black BL, Floor SN, Lagna G, Hata A. Control of ribosomal protein synthesis by the Microprocessor complex. Sci Signal 2021; 14:eabd2639. [PMID: 33622983 PMCID: PMC8012103 DOI: 10.1126/scisignal.abd2639] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human "ribosomopathies." Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.
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Affiliation(s)
- Xuan Jiang
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Amit Prabhakar
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Stephanie M Van der Voorn
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
- Department of Medical Physiology, University Medical Center Utrecht, Utrecht, 3584 CM, Netherlands
| | - Prajakta Ghatpande
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Barbara Celona
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Srivats Venkataramanan
- Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Lorenzo Calviello
- Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Chuwen Lin
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Wanpeng Wang
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Brian L Black
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
- Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA
| | - Stephen N Floor
- Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Giorgio Lagna
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Akiko Hata
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA.
- Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA
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8
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Suzuki M, Katayama S, Yamamoto M. Two effects of GATA2 enhancer repositioning by 3q chromosomal rearrangements. IUBMB Life 2019; 72:159-169. [PMID: 31820561 DOI: 10.1002/iub.2191] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2019] [Accepted: 10/09/2019] [Indexed: 01/15/2023]
Abstract
Chromosomal inversion and translocation between 3q21 and 3q26 [inv (3)(q21.3q26.2) and t(3;3)(q21.3;q26.2), respectively] give rise to acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), which have poor prognoses. The chromosomal rearrangements reposition a GATA2 distal hematopoietic enhancer from the original 3q21 locus to the EVI1 (also known as MECOM) locus on 3q26. Therefore, the GATA2 enhancer from one of two GATA2 alleles drives EVI1 gene expression in hematopoietic stem and progenitor cells, which promotes the accumulation of abnormal progenitors and induces leukemogenesis. On the other hand, one allele of the GATA2 gene loses its enhancer, which results in reduced GATA2 expression. The GATA2 gene encodes a transcription factor critical for the generation and maintenance of hematopoietic stem and progenitor cells. GATA2 haploinsufficiency has been known to cause immunodeficiency and myeloid leukemia. Notably, reduced GATA2 expression suppresses the differentiation but promotes the proliferation of EVI1-expressing leukemic cells, which accelerates EVI1-driven leukemogenesis. A series of studies have shown that the GATA2 enhancer repositioning caused by the chromosomal rearrangements between 3q21 and 3q26 provokes misexpression of both the EVI1 and GATA2 genes and that these two effects coordinately elicit high-risk leukemia.
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Affiliation(s)
- Mikiko Suzuki
- Center for Radioisotope Sciences, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Saori Katayama
- Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan.,Department of Pediatrics, Tohoku University Graduate School of Medicine, Sendai, Japan.,Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan
| | - Masayuki Yamamoto
- Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan.,Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan
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9
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The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70. Blood Adv 2017; 1:1959-1976. [PMID: 29296843 DOI: 10.1182/bloodadvances.2017008078] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2017] [Accepted: 08/25/2017] [Indexed: 01/02/2023] Open
Abstract
Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome that exhibits an erythroid-specific phenotype. In at least 70% of cases, DBA is related to a haploinsufficient germ line mutation in a ribosomal protein (RP) gene. Additional cases have been associated with mutations in GATA1. We have previously established that the RPL11+/Mut phenotype is more severe than RPS19+/Mut phenotype because of delayed erythroid differentiation and increased apoptosis of RPL11+/Mut erythroid progenitors. The HSP70 protein is known to protect GATA1, the major erythroid transcription factor, from caspase-3 mediated cleavage during normal erythroid differentiation. Here, we show that HSP70 protein expression is dramatically decreased in RPL11+/Mut erythroid cells while being preserved in RPS19+/Mut cells. The decreased expression of HSP70 in RPL11+/Mut cells is related to an enhanced proteasomal degradation of polyubiquitinylated HSP70. Restoration of HSP70 expression level in RPL11+/Mut cells reduces p53 activation and rescues the erythroid defect in DBA. These results suggest that HSP70 plays a key role in determining the severity of the erythroid phenotype in RP-mutation-dependent DBA.
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10
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Tang Y, Joyner CJ, Cabrera-Mora M, Saney CL, Lapp SA, Nural MV, Pakala SB, DeBarry JD, Soderberg S, Kissinger JC, Lamb TJ, Galinski MR, Styczynski MP. Integrative analysis associates monocytes with insufficient erythropoiesis during acute Plasmodium cynomolgi malaria in rhesus macaques. Malar J 2017; 16:384. [PMID: 28938907 PMCID: PMC5610412 DOI: 10.1186/s12936-017-2029-z] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2017] [Accepted: 09/12/2017] [Indexed: 01/06/2023] Open
Abstract
Background Mild to severe anaemia is a common complication of malaria that is caused in part by insufficient erythropoiesis in the bone marrow. This study used systems biology to evaluate the transcriptional and alterations in cell populations in the bone marrow during Plasmodium cynomolgi infection of rhesus macaques (a model of Plasmodium vivax malaria) that may affect erythropoiesis. Results An appropriate erythropoietic response did not occur to compensate for anaemia during acute cynomolgi malaria despite an increase in erythropoietin levels. During this period, there were significant perturbations in the bone marrow transcriptome. In contrast, relapses did not induce anaemia and minimal changes in the bone marrow transcriptome were detected. The differentially expressed genes during acute infection were primarily related to ongoing inflammatory responses with significant contributions from Type I and Type II Interferon transcriptional signatures. These were associated with increased frequency of intermediate and non-classical monocytes. Recruitment and/or expansion of these populations was correlated with a decrease in the erythroid progenitor population during acute infection, suggesting that monocyte-associated inflammation may have contributed to anaemia. The decrease in erythroid progenitors was associated with downregulation of genes regulated by GATA1 and GATA2, two master regulators of erythropoiesis, providing a potential molecular basis for these findings. Conclusions These data suggest the possibility that malarial anaemia may be driven by monocyte-associated disruption of GATA1/GATA2 function in erythroid progenitors resulting in insufficient erythropoiesis during acute infection. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-2029-z) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Yan Tang
- School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA.,Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Chester J Joyner
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Monica Cabrera-Mora
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Celia L Saney
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Stacey A Lapp
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | - Mustafa V Nural
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.,Institute of Bioinformatics, University of Georgia, Athens, GA, USA.,Department of Computer Science, University of Georgia, Athens, GA, USA
| | - Suman B Pakala
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.,Institute of Bioinformatics, University of Georgia, Athens, GA, USA
| | - Jeremy D DeBarry
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.,Institute of Bioinformatics, University of Georgia, Athens, GA, USA
| | - Stephanie Soderberg
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA
| | | | - Jessica C Kissinger
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.,Institute of Bioinformatics, University of Georgia, Athens, GA, USA.,Department of Genetics, University of Georgia, Athens, GA, USA.,Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA.,Department of Computer Science, University of Georgia, Athens, GA, USA
| | - Tracey J Lamb
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.,Department of Pathology, University of Utah, Salt Lake City, UT, USA
| | - Mary R Galinski
- Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.,Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, GA, USA
| | - Mark P Styczynski
- School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA. .,Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA.
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11
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Derepression of the DNA Methylation Machinery of the Gata1 Gene Triggers the Differentiation Cue for Erythropoiesis. Mol Cell Biol 2017; 37:MCB.00592-16. [PMID: 28069743 DOI: 10.1128/mcb.00592-16] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2016] [Accepted: 01/06/2017] [Indexed: 12/21/2022] Open
Abstract
GATA1 is a critical regulator of erythropoiesis. While the mechanisms underlying the high-level expression of GATA1 in maturing erythroid cells have been studied extensively, the initial activation of the Gata1 gene in early hematopoietic progenitors remains to be elucidated. We previously identified a hematopoietic stem and progenitor cell (HSPC)-specific silencer element (the Gata1 methylation-determining region [G1MDR]) that recruits DNA methyltransferase 1 (Dnmt1) and provokes methylation of the Gata1 gene enhancer. In the present study, we hypothesized that removal of the G1MDR-mediated silencing machinery is the molecular basis of the initial activation of the Gata1 gene and erythropoiesis. To address this hypothesis, we generated transgenic mouse lines harboring a Gata1 bacterial artificial chromosome in which the G1MDR was deleted. The mice exhibited abundant GATA1 expression in HSPCs, in a GATA2-dependent manner. The ectopic GATA1 expression repressed Gata2 transcription and induced erythropoiesis and apoptosis of HSPCs. Furthermore, genetic deletion of Dnmt1 in HSPCs activated Gata1 expression and depleted HSPCs, thus recapitulating the HSC phenotype associated with GATA1 gain of function. These results demonstrate that the G1MDR holds the key to HSPC maintenance and suggest that release from this suppressive mechanism is a fundamental requirement for subsequent initiation of erythroid differentiation.
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12
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Protective vaccination and blood-stage malaria modify DNA methylation of gene promoters in the liver of Balb/c mice. Parasitol Res 2017; 116:1463-1477. [PMID: 28315013 DOI: 10.1007/s00436-017-5423-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Accepted: 03/08/2017] [Indexed: 02/07/2023]
Abstract
Epigenetic mechanisms such as DNA methylation are increasingly recognized to be critical for vaccination efficacy and outcome of different infectious diseases, but corresponding information is scarcely available for host defense against malaria. In the experimental blood-stage malaria Plasmodium chabaudi, we investigate the possible effects of a blood-stage vaccine on DNA methylation of gene promoters in the liver, known as effector against blood-stage malaria, using DNA methylation microarrays. Naturally susceptible Balb/c mice acquire, by protective vaccination, the potency to survive P. chabaudi malaria and, concomitantly, modifications of constitutive DNA methylation of promoters of numerous genes in the liver; specifically, promoters of 256 genes are hyper(=up)- and 345 genes are hypo(=down)-methylated (p < 0.05). Protective vaccination also leads to changes in promoter DNA methylation upon challenge with P. chabaudi at peak parasitemia on day 8 post infection (p.i.), when 571 and 1013 gene promoters are up- and down-methylated, respectively, in relation to constitutive DNA methylation (p < 0.05). Gene set enrichment analyses reveal that both vaccination and P. chabaudi infections mainly modify promoters of those genes which are most statistically enriched with functions relating to regulation of transcription. Genes with down-methylated promoters encompass those encoding CX3CL1, GP130, and GATA2, known to be involved in monocyte recruitment, IL-6 trans-signaling, and onset of erythropoiesis, respectively. Our data suggest that vaccination may epigenetically improve parts of several effector functions of the liver against blood-stage malaria, as, e.g., recruitment of monocyte/macrophage to the liver accelerated liver regeneration and extramedullary hepatic erythropoiesis, thus leading to self-healing of otherwise lethal P. chabaudi blood-stage malaria.
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Youn M, Wang N, LaVasseur C, Bibikova E, Kam S, Glader B, Sakamoto KM, Narla A. Loss of Forkhead box M1 promotes erythropoiesis through increased proliferation of erythroid progenitors. Haematologica 2017; 102:826-834. [PMID: 28154085 PMCID: PMC5477601 DOI: 10.3324/haematol.2016.156257] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2016] [Accepted: 01/24/2017] [Indexed: 01/02/2023] Open
Abstract
Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34+ cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis.
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Affiliation(s)
- Minyoung Youn
- Department of Pediatrics, Stanford University School of Medicine, CA, USA
| | - Nan Wang
- Department of Pediatrics, Stanford University School of Medicine, CA, USA
| | - Corinne LaVasseur
- Department of Pediatrics, Stanford University School of Medicine, CA, USA
| | - Elena Bibikova
- Department of Pediatrics, Stanford University School of Medicine, CA, USA
| | - Sharon Kam
- Department of Pediatrics, Stanford University School of Medicine, CA, USA
| | - Bertil Glader
- Department of Pediatrics, Stanford University School of Medicine, CA, USA
| | | | - Anupama Narla
- Department of Pediatrics, Stanford University School of Medicine, CA, USA
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14
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Sureshchandra S, Rais M, Stull C, Grant K, Messaoudi I. Transcriptome Profiling Reveals Disruption of Innate Immunity in Chronic Heavy Ethanol Consuming Female Rhesus Macaques. PLoS One 2016; 11:e0159295. [PMID: 27427759 PMCID: PMC4948771 DOI: 10.1371/journal.pone.0159295] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2016] [Accepted: 06/30/2016] [Indexed: 12/12/2022] Open
Abstract
It is well established that heavy ethanol consumption interferes with the immune system and inflammatory processes, resulting in increased risk for infectious and chronic diseases. However, these processes have yet to be systematically studied in a dose and sex-dependent manner. In this study, we investigated the impact of chronic heavy ethanol consumption on gene expression using RNA-seq in peripheral blood mononuclear cells isolated from female rhesus macaques with daily consumption of 4% ethanol available 22hr/day for 12 months resulting in average ethanol consumption of 4.3 g/kg/day (considered heavy drinking). Differential gene expression analysis was performed using edgeR and gene enrichment analysis using MetaCore™. We identified 1106 differentially expressed genes, meeting the criterion of ≥ two-fold change and p-value ≤ 0.05 in expression (445 up- and 661 down-regulated). Pathway analysis of the 879 genes with characterized identifiers showed that the most enriched gene ontology processes were "response to wounding", "blood coagulation", "immune system process", and "regulation of signaling". Changes in gene expression were seen despite the lack of differences in the frequency of any major immune cell subtype between ethanol and controls, suggesting that heavy ethanol consumption modulates gene expression at the cellular level rather than altering the distribution of peripheral blood mononuclear cells. Collectively, these observations provide mechanisms to explain the higher incidence of infection, delay in wound healing, and increase in cardiovascular disease seen in subjects with Alcohol use disorder.
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Affiliation(s)
- Suhas Sureshchandra
- Graduate Program in Genetics, Genomics and Bioinformatics, University of California Riverside, Riverside, California, United States of America
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, California, United States of America
| | - Maham Rais
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, California, United States of America
| | - Cara Stull
- Division of Neurosciences, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States of America
| | - Kathleen Grant
- Division of Neurosciences, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States of America
| | - Ilhem Messaoudi
- Graduate Program in Genetics, Genomics and Bioinformatics, University of California Riverside, Riverside, California, United States of America
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, California, United States of America
- Division of Neurosciences, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States of America
- * E-mail:
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15
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Guo Y, Fu X, Jin Y, Sun J, Liu Y, Huo B, Li X, Hu X. Histone demethylase LSD1-mediated repression of GATA-2 is critical for erythroid differentiation. DRUG DESIGN DEVELOPMENT AND THERAPY 2015; 9:3153-62. [PMID: 26124638 PMCID: PMC4482369 DOI: 10.2147/dddt.s81911] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Background The transcription factor GATA-2 is predominantly expressed in hematopoietic stem and progenitor cells and counteracts the erythroid-specific transcription factor GATA-1, to modulate the proliferation and differentiation of hematopoietic cells. During hematopoietic cell differentiation, GATA-2 exhibits dynamic expression patterns, which are regulated by multiple transcription factors. Methods Stable LSD1-knockdown cell lines were established by growing murine erythroleukemia (MEL) or mouse embryonic stem cells together with virus particles, in the presence of Polybrene® at 4 μg/mL, for 24–48 hours followed by puromycin selection (1 μg/mL) for 2 weeks. Real-time polymerase chain reaction (PCR)-based quantitative chromatin immunoprecipitation (ChIP) analysis was used to test whether the TAL1 transcription factor is bound to 1S promoter in the GATA-2 locus or whether LSD1 colocalizes with TAL1 at the 1S promoter. The sequential ChIP assay was utilized to confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation. Western blot analysis was employed to detect the protein expression. The alamarBlue® assay was used to examine the proliferation of the cells, and the absorbance was monitored at optical density (OD) 570 nm and OD 600 nm. Results In this study, we showed that LSD1 regulates the expression of GATA-2 during erythroid differentiation. Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells. Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the GATA-2 locus and suppresses GATA-2 expression, via histone demethylation. Conclusion Our data revealed that LSD1 mediates erythroid differentiation, via epigenetic modification of the GATA-2 locus.
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Affiliation(s)
- Yidi Guo
- School of Life Sciences, Jilin University, Changchun, People's Republic of China
| | - Xueqi Fu
- School of Life Sciences, Jilin University, Changchun, People's Republic of China ; Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, People's Republic of China
| | - Yue Jin
- School of Life Sciences, Jilin University, Changchun, People's Republic of China
| | - Jing Sun
- School of Life Sciences, Jilin University, Changchun, People's Republic of China
| | - Ye Liu
- School of Life Sciences, Jilin University, Changchun, People's Republic of China
| | - Bo Huo
- School of Life Sciences, Jilin University, Changchun, People's Republic of China
| | - Xiang Li
- School of Life Sciences, Jilin University, Changchun, People's Republic of China
| | - Xin Hu
- School of Life Sciences, Jilin University, Changchun, People's Republic of China ; Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, People's Republic of China ; National Engineering Laboratory of AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, People's Republic of China
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16
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TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors. Blood 2014; 124:3791-8. [PMID: 25270909 DOI: 10.1182/blood-2014-06-584656] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-α (TNF-α) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.
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17
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Yamazaki H, Suzuki M, Otsuki A, Shimizu R, Bresnick EH, Engel JD, Yamamoto M. A remote GATA2 hematopoietic enhancer drives leukemogenesis in inv(3)(q21;q26) by activating EVI1 expression. Cancer Cell 2014; 25:415-27. [PMID: 24703906 PMCID: PMC4012341 DOI: 10.1016/j.ccr.2014.02.008] [Citation(s) in RCA: 177] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/13/2013] [Revised: 12/20/2013] [Accepted: 02/18/2014] [Indexed: 10/25/2022]
Abstract
Chromosomal inversion between 3q21 and 3q26 results in high-risk acute myeloid leukemia (AML). In this study, we identified a mechanism whereby a GATA2 distal hematopoietic enhancer (G2DHE or -77-kb enhancer) is brought into close proximity to the EVI1 gene in inv(3)(q21;q26) inversions, leading to leukemogenesis. We examined the contribution of G2DHE to leukemogenesis by creating a bacterial artificial chromosome (BAC) transgenic model that recapitulates the inv(3)(q21;q26) allele. Transgenic mice harboring a linked BAC developed leukemia accompanied by EVI1 overexpression-neoplasia that was not detected in mice bearing the same transgene but that was missing the GATA2 enhancer. These results establish the mechanistic basis underlying the pathogenesis of a severe form of leukemia through aberrant expression of the EVI1 proto-oncogene.
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MESH Headings
- Animals
- Base Sequence
- Chromosome Inversion
- Chromosomes, Human, Pair 3
- DNA-Binding Proteins/biosynthesis
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/metabolism
- GATA2 Transcription Factor/genetics
- GATA2 Transcription Factor/metabolism
- Hematopoiesis/genetics
- Humans
- Leukemia, Myeloid, Acute/genetics
- Leukemia, Myeloid, Acute/metabolism
- Leukemia, Myeloid, Acute/pathology
- MDS1 and EVI1 Complex Locus Protein
- Mice
- Mice, Transgenic
- Proto-Oncogene Mas
- Proto-Oncogenes/genetics
- Transcription Factors/biosynthesis
- Transcription Factors/genetics
- Transcription Factors/metabolism
- Transfection
- Transgenes
- Translocation, Genetic
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Affiliation(s)
- Hiromi Yamazaki
- Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
| | - Mikiko Suzuki
- Center for Radioisotope Sciences, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan; Department of Molecular Hematology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
| | - Akihito Otsuki
- Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
| | - Ritsuko Shimizu
- Department of Molecular Hematology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
| | - Emery H Bresnick
- UW-Madison Blood Research Program, Carbone Cancer Center, Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA
| | - James Douglas Engel
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Masayuki Yamamoto
- Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan; Tohoku Medical Megabank, Tohoku University, Sendai 980-8573, Japan.
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18
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Liu Z, Sun W, Zhao Y, Xu C, Fu Y, Li Y, Chen J. The effect of variants in the promoter of BMPER on the intramuscular fat deposition in longissimus dorsi muscle of pigs. Gene 2014; 542:168-72. [PMID: 24667095 DOI: 10.1016/j.gene.2014.03.038] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2014] [Revised: 03/12/2014] [Accepted: 03/21/2014] [Indexed: 11/19/2022]
Abstract
The aim of the study was to evaluate the contribution of BMPER promoter SNPs to the gene expression and intramuscular fat content in longissimus dorsi muscle. Firstly the promoter region of BMPER was comparatively scanned by direct sequencing with pool DNA of two groups (n=15 for each group) with high (H) or low (L) IMF content. Two SNPs, c.-1423A>G and c.-1344A>C, were found to have reverse allele distribution in the two groups. Genotyping by PCR-SSCP in a larger population revealed that the two SNPs interlock completely to form only A-A or G-C haplotype. The IMF content and BMPER expression level of A-A/A-A genotype were higher than G-C/G-C genotype, and luciferase assay revealed that A-A haplotype promoter activity was also higher than G-C haplotype. Putative transcription factor prediction suggested that c.-1344 A>C mutation might shift the promoter binding affinity with GATAs. We concluded that BMPER promoter polymorphisms have an effect on IMF content, and A-A haplotype could be used as a candidate genetic marker for preferable IMF deposition.
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Affiliation(s)
- Zhi Liu
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Wenxing Sun
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Yongyan Zhao
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Chunying Xu
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Yingying Fu
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Yan Li
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Jie Chen
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China.
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19
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Moriguchi T, Yamamoto M. A regulatory network governing Gata1 and Gata2 gene transcription orchestrates erythroid lineage differentiation. Int J Hematol 2014; 100:417-24. [PMID: 24638828 DOI: 10.1007/s12185-014-1568-0] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2014] [Revised: 03/04/2014] [Accepted: 03/04/2014] [Indexed: 12/17/2022]
Abstract
GATA transcription factor family members GATA1 and GATA2 play crucial roles in the regulation of lineage-restricted genes during erythroid differentiation. GATA1 is indispensable for survival and terminal differentiation of erythroid, megakaryocytic and eosinophilic progenitors, whereas GATA2 regulates proliferation and maintenance of hematopoietic stem and progenitor cells. Expression levels of GATA1 and GATA2 are primarily regulated at the transcriptional level through auto- and reciprocal regulatory networks formed by these GATA factors. The dynamic and strictly controlled change of expression from GATA2 to GATA1 during erythropoiesis has been referred to as GATA factor switching, which plays a crucial role in erythropoiesis. The regulatory network comprising GATA1 and GATA2 gives rise to the stage-specific changes in Gata1 and Gata2 gene expression during erythroid differentiation, which ensures specific expression of early and late erythroid genes at each stage. Recent studies have also shed light on the genome-wide binding profiles of GATA1 and GATA2, and the significance of epigenetic modification of Gata1 gene during erythroid differentiation. This review summarizes the current understanding of network regulation underlying stage-dependent Gata1 and Gata2 gene expressions and the functional contribution of these GATA factors in erythroid differentiation.
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Affiliation(s)
- Takashi Moriguchi
- Department of Medical Biochemistry, Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan
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20
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Cooperative interaction of Etv2 and Gata2 regulates the development of endothelial and hematopoietic lineages. Dev Biol 2014; 389:208-18. [PMID: 24583263 DOI: 10.1016/j.ydbio.2014.02.018] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2013] [Revised: 02/07/2014] [Accepted: 02/19/2014] [Indexed: 12/31/2022]
Abstract
Regulatory mechanisms that govern lineage specification of the mesodermal progenitors to become endothelial and hematopoietic cells remain an area of intense interest. Both Ets and Gata factors have been shown to have important roles in the transcriptional regulation in endothelial and hematopoietic cells. We previously reported Etv2 as an essential regulator of vasculogenesis and hematopoiesis. In the present study, we demonstrate that Gata2 is co-expressed and interacts with Etv2 in the endothelial and hematopoietic cells in the early stages of embryogenesis. Our studies reveal that Etv2 interacts with Gata2 in vitro and in vivo. The protein-protein interaction between Etv2 and Gata2 is mediated by the Ets and Gata domains. Using the embryoid body differentiation system, we demonstrate that co-expression of Gata2 augments the activity of Etv2 in promoting endothelial and hematopoietic lineage differentiation. We also identify Spi1 as a common downstream target gene of Etv2 and Gata2. We provide evidence that Etv2 and Gata2 bind to the Spi1 promoter in vitro and in vivo. In summary, we propose that Gata2 functions as a cofactor of Etv2 in the transcriptional regulation of mesodermal progenitors during embryogenesis.
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21
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May G, Soneji S, Tipping A, Teles J, McGowan S, Wu M, Guo Y, Fugazza C, Brown J, Karlsson G, Pina C, Olariu V, Taylor S, Tenen D, Peterson C, Enver T. Dynamic analysis of gene expression and genome-wide transcription factor binding during lineage specification of multipotent progenitors. Cell Stem Cell 2013; 13:754-68. [PMID: 24120743 PMCID: PMC3878573 DOI: 10.1016/j.stem.2013.09.003] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2012] [Revised: 08/06/2013] [Accepted: 09/12/2013] [Indexed: 12/30/2022]
Abstract
We used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.
Cross-platform resource for TF-network regulation of multipotent blood cell fate DNA motif dependence and changing specificity of GATA factors in lineage choice Modeling-based inference identifies GATA2 repression of PU.1 in multipotent cells Priming, recruitment, and switching modes of GATA interplay during differentiation
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Affiliation(s)
- Gillian May
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - Shamit Soneji
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - Alex J. Tipping
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - Jose Teles
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Computational Biology and Biological Physics, Department of Theoretical Physics, Lund University, 223 62 Lund, Sweden
| | - Simon J. McGowan
- Computational Biology Research Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - Mengchu Wu
- Cancer Science Institute, National University of Singapore, Singapore 117599
| | - Yanping Guo
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - Cristina Fugazza
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - John Brown
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - Göran Karlsson
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
| | - Cristina Pina
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - Victor Olariu
- Computational Biology and Biological Physics, Department of Theoretical Physics, Lund University, 223 62 Lund, Sweden
| | - Stephen Taylor
- Computational Biology Research Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - Daniel G. Tenen
- Cancer Science Institute, National University of Singapore, Singapore 117599
- Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Carsten Peterson
- Computational Biology and Biological Physics, Department of Theoretical Physics, Lund University, 223 62 Lund, Sweden
| | - Tariq Enver
- Stem Cell Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
- Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
- Corresponding author
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Shimizu R, Hasegawa A, Ottolenghi S, Ronchi A, Yamamoto M. Verification of the in vivo activity of three distinct cis-acting elements within the Gata1 gene promoter-proximal enhancer in mice. Genes Cells 2013; 18:1032-41. [PMID: 24118212 DOI: 10.1111/gtc.12096] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2013] [Accepted: 08/13/2013] [Indexed: 12/27/2022]
Abstract
The transcription factor GATA1 is essential for erythroid and megakaryocytic cell differentiation. Gata1 hematopoietic regulatory domain (G1HRD) has been shown to recapitulate endogenous Gata1 gene expression in transgenic mouse assays in vivo. G1HRD contains a promoter-proximal enhancer composed of a GATA-palindrome motif, four CP2-binding sites and two CACCC boxes. We prepared transgenic reporter mouse lines in which green fluorescent protein and β-galactosidase expression are driven by wild-type G1HRD (as a positive control) and the G1HRD harboring mutations within these cis-acting elements (as the experimental conditions), respectively. Exploiting this transgenic dual reporter (TDR) assay, we show here that in definitive erythropoiesis, G1HRD activity was markedly affected by individual mutations in the GATA-palindrome motif and the CACCC boxes. Mutation of CP2-binding sites also moderately decreased G1HRD activity. The combined mutation of the CP2-binding sites and the GATA-palindrome motif resulted in complete loss of G1HRD activity. In contrast, in primitive erythroid cells, individual mutations of each element did not affect G1HRD activity; G1HRD activity was abolished only when these three mutations were combined. These results thus show that all three elements independently and cooperatively contribute to G1HRD activity in vivo in definitive erythropoiesis, although these are contributing redundantly to primitive erythropoiesis.
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Affiliation(s)
- Ritsuko Shimizu
- Department of Molecular Hematology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai, 980-8575, Japan
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Suzuki M, Kobayashi-Osaki M, Tsutsumi S, Pan X, Ohmori S, Takai J, Moriguchi T, Ohneda O, Ohneda K, Shimizu R, Kanki Y, Kodama T, Aburatani H, Yamamoto M. GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation. Genes Cells 2013; 18:921-33. [PMID: 23911012 DOI: 10.1111/gtc.12086] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2013] [Accepted: 06/16/2013] [Indexed: 11/30/2022]
Abstract
Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the 'GATA factor switching'. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1-knockdown embryos that concomitantly express Gata2-GFP reporter, high-level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1-binding sites widely overlap with the sites pre-occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient-phase genes.
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Affiliation(s)
- Mikiko Suzuki
- Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan; Center for Radioisotope Sciences, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan; Department of Molecular Hematology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan
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Shimizu R, Yamamoto M. Contribution of GATA1 dysfunction to multi-step leukemogenesis. Cancer Sci 2012; 103:2039-44. [PMID: 22937757 DOI: 10.1111/cas.12007] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2012] [Revised: 08/19/2012] [Accepted: 08/23/2012] [Indexed: 01/01/2023] Open
Abstract
In mammals, hematopoietic homeostasis is maintained by a fine-tuned balance among the self-renewal, proliferation, differentiation and survival of hematopoietic stem cells and their progenies. Each process is also supported by the delicate balance of the expression of multiple genes specific to each process. GATA1 is a transcription factor that comprehensively regulates the genes that are important for the development of erythroid and megakaryocytic cells. Accumulating evidence supports the notion that defects in GATA1 function are intimately linked to hematopoietic disorders. In particular, the somatic mutation of the GATA1 gene, which leads to the production of N-terminally truncated GATA1, contributes to the genesis of transient myeloproliferative disorder and acute megakaryoblastic leukemia in infants with Down syndrome. Similarly, a mutation in the GATA1 regulatory region that reduces GATA1 expression is involved in the onset of erythroid leukemia in mice. In both cases, the accumulation of immature progenitor cells caused by GATA1 dysregulation underlies the pathogenesis of the leukemia. This review provides a summary of multi-step leukemogenesis with a focus on GATA1 dysfunction.
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Affiliation(s)
- Ritsuko Shimizu
- Department of Molecular Hematology, Tohoku University Graduate School of Medicine, Sendai, Japan
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Whitfield TW, Wang J, Collins PJ, Partridge EC, Aldred SF, Trinklein ND, Myers RM, Weng Z. Functional analysis of transcription factor binding sites in human promoters. Genome Biol 2012; 13:R50. [PMID: 22951020 PMCID: PMC3491394 DOI: 10.1186/gb-2012-13-9-r50] [Citation(s) in RCA: 121] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2011] [Revised: 04/19/2012] [Accepted: 06/18/2012] [Indexed: 12/19/2022] Open
Abstract
Background The binding of transcription factors to specific locations in the genome is integral to the orchestration of transcriptional regulation in cells. To characterize transcription factor binding site function on a large scale, we predicted and mutagenized 455 binding sites in human promoters. We carried out functional tests on these sites in four different immortalized human cell lines using transient transfections with a luciferase reporter assay, primarily for the transcription factors CTCF, GABP, GATA2, E2F, STAT, and YY1. Results In each cell line, between 36% and 49% of binding sites made a functional contribution to the promoter activity; the overall rate for observing function in any of the cell lines was 70%. Transcription factor binding resulted in transcriptional repression in more than a third of functional sites. When compared with predicted binding sites whose function was not experimentally verified, the functional binding sites had higher conservation and were located closer to transcriptional start sites (TSSs). Among functional sites, repressive sites tended to be located further from TSSs than were activating sites. Our data provide significant insight into the functional characteristics of YY1 binding sites, most notably the detection of distinct activating and repressing classes of YY1 binding sites. Repressing sites were located closer to, and often overlapped with, translational start sites and presented a distinctive variation on the canonical YY1 binding motif. Conclusions The genomic properties that we found to associate with functional TF binding sites on promoters -- conservation, TSS proximity, motifs and their variations -- point the way to improved accuracy in future TFBS predictions.
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Affiliation(s)
- Troy W Whitfield
- Program in Bioinformatics and Integrative Biology and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA
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