title: DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish.
abstract: 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.
authors.affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. liuxiaohuiuestc@126.com.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, 200031, China. jiaxiaoe@gmail.com.
authors.forename: Xiaoe
authors.initials: X
authors.lastname: Jia
authors.affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. hyuan@sibs.ac.cn.
authors.forename: Hao
authors.initials: H
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, 200031, China. mke2002@163.com.
authors.forename: Ke
authors.initials: K
authors.lastname: Ma
authors.affiliation: Laboratory of Development and Diseases, State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. chenyi1827@gmail.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Laboratory of Development and Diseases, State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. jy88235@126.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, 200031, China. mdeng@sibs.ac.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Deng
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, 200031, China. weijunpan@sibs.ac.cn.
authors.forename: Weijun
authors.initials: W
authors.lastname: Pan
authors.affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. sjchen@stn.sh.cn.
authors.forename: Saijuan
authors.initials: S
authors.lastname: Chen
authors.affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. zchen@stn.sh.cn.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Chen
authors.affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. hugues.dethe@inserm.fr.|Equipe Labellisée No. 11 Ligue Nationale Contre le Cancer, Hôpital St. Louis, Université de Paris 7/INSERM/CNRS UMR 944/7212, 75475, Paris, France. hugues.dethe@inserm.fr.
authors.forename: Hugues
authors.initials: H
authors.lastname: de The
authors.affiliation: Stem Cell Program, Hematology/Oncology Program at Children's Hospital Boston, Harvard Medical School, Boston, MA, 02114, USA. zon@enders.tch.harvard.edu.
authors.forename: Leonard I
authors.initials: LI
authors.lastname: Zon
authors.affiliation: Stem Cell Program, Hematology/Oncology Program at Children's Hospital Boston, Harvard Medical School, Boston, MA, 02114, USA. yzhou@enders.tch.harvard.edu.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. junjun_j@yahoo.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhou
authors.affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. zhuj1966@yahoo.com.|Equipe Labellisée No. 11 Ligue Nationale Contre le Cancer, Hôpital St. Louis, Université de Paris 7/INSERM/CNRS UMR 944/7212, 75475, Paris, France. zhuj1966@yahoo.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006410:Hematopoiesis / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D017403:In Situ Hybridization; D060888:Real-Time Polymerase Chain Reaction; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D029961:Zebrafish Proteins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C514253:Dnmt1 protein, zebrafish