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DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish

BACKGROUND: DNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis. It is essential for embryonic development and diverse biological processes, including maintenance of hematopoietic ste...

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Autores principales: Liu, Xiaohui, Jia, Xiaoe, Yuan, Hao, Ma, Ke, Chen, Yi, Jin, Yi, Deng, Min, Pan, Weijun, Chen, Saijuan, Chen, Zhu, de The, Hugues, Zon, Leonard I, Zhou, Yi, Zhou, Jun, Zhu, Jun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372312/
https://www.ncbi.nlm.nih.gov/pubmed/25886310
http://dx.doi.org/10.1186/s13045-015-0115-7
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author Liu, Xiaohui
Jia, Xiaoe
Yuan, Hao
Ma, Ke
Chen, Yi
Jin, Yi
Deng, Min
Pan, Weijun
Chen, Saijuan
Chen, Zhu
de The, Hugues
Zon, Leonard I
Zhou, Yi
Zhou, Jun
Zhu, Jun
author_facet Liu, Xiaohui
Jia, Xiaoe
Yuan, Hao
Ma, Ke
Chen, Yi
Jin, Yi
Deng, Min
Pan, Weijun
Chen, Saijuan
Chen, Zhu
de The, Hugues
Zon, Leonard I
Zhou, Yi
Zhou, Jun
Zhu, Jun
author_sort Liu, Xiaohui
collection PubMed
description BACKGROUND: DNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis. It is essential for embryonic development and diverse biological processes, including maintenance of hematopoietic stem and progenitor cells (HSPCs). However, the precise molecular mechanism of how Dnmt1 is involved in HSPC maintenance remains unexplored. METHODS: An N-ethyl-N-nitrosourea (ENU)-based genetic screening was performed to identify putative mutants with defects in definitive HSPCs during hematopoiesis in zebrafish. The expression of hematopoietic markers was analyzed via whole mount in situ hybridization assay (WISH). Positional cloning approach was carried out to identify the gene responsible for the defective definitive hematopoiesis in the mutants. Analyses of the mechanism were conducted by morpholino-mediated gene knockdown, mRNA injection rescue assays, anti-phosphorylated histone H3 (pH3) immunostaining and TUNEL assay, quantitative real-time PCR, and bisulfite sequencing analysis. RESULTS: A heritable mutant line with impaired HSPCs of definitive hematopoiesis was identified. Positional cloning demonstrated that a stop codon mutation was introduced in dnmt1 which resulted in a predicted truncated Dnmt1 lacking the DNA methylation catalytic domain. Molecular analysis revealed that expression of CCAAT/enhancer-binding protein alpha (C/ebpa) was upregulated, which correlated with hypomethylation of CpG islands in the regulation regions of cebpa gene in Dnmt1 deficient HSPCs. Overexpression of a transcriptional repressive SUMO-C/ebpa fusion protein could rescue hematological defects in the dnmt1 mutants. Finally, dnmt1 and cebpa double null embryos exhibited no obvious abnormal hematopoiesis indicated that the HSPC defects triggered by dnmt1 mutation were C/ebpa dependent. CONCLUSIONS: Dnmt1 is required for HSPC maintenance via cebpa regulation during definitive hematopoiesis in zebrafish. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13045-015-0115-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-43723122015-03-25 DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish Liu, Xiaohui Jia, Xiaoe Yuan, Hao Ma, Ke Chen, Yi Jin, Yi Deng, Min Pan, Weijun Chen, Saijuan Chen, Zhu de The, Hugues Zon, Leonard I Zhou, Yi Zhou, Jun Zhu, Jun J Hematol Oncol Research BACKGROUND: DNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis. It is essential for embryonic development and diverse biological processes, including maintenance of hematopoietic stem and progenitor cells (HSPCs). However, the precise molecular mechanism of how Dnmt1 is involved in HSPC maintenance remains unexplored. METHODS: An N-ethyl-N-nitrosourea (ENU)-based genetic screening was performed to identify putative mutants with defects in definitive HSPCs during hematopoiesis in zebrafish. The expression of hematopoietic markers was analyzed via whole mount in situ hybridization assay (WISH). Positional cloning approach was carried out to identify the gene responsible for the defective definitive hematopoiesis in the mutants. Analyses of the mechanism were conducted by morpholino-mediated gene knockdown, mRNA injection rescue assays, anti-phosphorylated histone H3 (pH3) immunostaining and TUNEL assay, quantitative real-time PCR, and bisulfite sequencing analysis. RESULTS: A heritable mutant line with impaired HSPCs of definitive hematopoiesis was identified. Positional cloning demonstrated that a stop codon mutation was introduced in dnmt1 which resulted in a predicted truncated Dnmt1 lacking the DNA methylation catalytic domain. Molecular analysis revealed that expression of CCAAT/enhancer-binding protein alpha (C/ebpa) was upregulated, which correlated with hypomethylation of CpG islands in the regulation regions of cebpa gene in Dnmt1 deficient HSPCs. Overexpression of a transcriptional repressive SUMO-C/ebpa fusion protein could rescue hematological defects in the dnmt1 mutants. Finally, dnmt1 and cebpa double null embryos exhibited no obvious abnormal hematopoiesis indicated that the HSPC defects triggered by dnmt1 mutation were C/ebpa dependent. CONCLUSIONS: Dnmt1 is required for HSPC maintenance via cebpa regulation during definitive hematopoiesis in zebrafish. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13045-015-0115-7) contains supplementary material, which is available to authorized users. BioMed Central 2015-02-22 /pmc/articles/PMC4372312/ /pubmed/25886310 http://dx.doi.org/10.1186/s13045-015-0115-7 Text en © Liu et al.; licensee BioMed Central. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Liu, Xiaohui
Jia, Xiaoe
Yuan, Hao
Ma, Ke
Chen, Yi
Jin, Yi
Deng, Min
Pan, Weijun
Chen, Saijuan
Chen, Zhu
de The, Hugues
Zon, Leonard I
Zhou, Yi
Zhou, Jun
Zhu, Jun
DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish
title DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish
title_full DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish
title_fullStr DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish
title_full_unstemmed DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish
title_short DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish
title_sort dna methyltransferase 1 functions through c/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372312/
https://www.ncbi.nlm.nih.gov/pubmed/25886310
http://dx.doi.org/10.1186/s13045-015-0115-7
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