DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish.
DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish.
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DNA甲基转移酶1通过C/ebpa发挥作用，维持斑马鱼的造血干细胞和祖细胞
DOI：
10.1186/s13045-015-0115-7
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发表时间：
2015-02-22
影响因子：
28.5
通讯作者：
Zhu J
中科院分区：
文献类型：
--
作者：
Liu X;Jia X;Yuan H;Ma K;Chen Y;Jin Y;Deng M;Pan W;Chen S;Chen Z;de The H;Zon LI;Zhou Y;Zhou J;Zhu J
背景DNA甲基转移酶1(Dnmt1)在有丝分裂过程中通过维持亲本DNA甲基化模式来调节许多关键基因的表达。它对胚胎发育和不同的生物学过程是必不可少的，包括维持造血干细胞和祖细胞(HSPC)。然而，Dnmt1参与HSPC维持的确切分子机制尚不清楚。方法基于N-乙基-N-亚硝脲(ENU)的遗传筛选方法，在斑马鱼的造血过程中，鉴定具有特定HSPC缺陷的假定突变体。采用整体挂载原位杂交法(WISH)分析造血标记物的表达。采用定位克隆的方法来鉴定突变体中导致最终造血缺陷的基因。通过吗啉介导的基因敲除、mRNA注射挽救试验、组蛋白H3(PH3)抗磷酸化免疫染色和TUNEL分析、实时定量聚合酶链式反应和亚硫酸氢盐测序分析等方法对其发生机制进行分析。定位克隆表明，在dnmt1中引入了终止密码子突变，导致预测的截短的dnmt1缺少DNA甲基化催化域。分子分析显示，在Dnmt1缺乏的HSPC中，CCAAT/增强子结合蛋白α(C/EBPA)的表达上调，这与头孢菌烯调控区CpG岛的低甲基化有关。过表达转录抑制性SUMO-C/EBPA融合蛋白可以挽救dnmt1突变体的血液学缺陷。Dnmt1和cebpadouble空胚胎未发现明显的造血异常，提示dnmt1突变所引发的HSPC缺陷是C/EBPA依赖的。结论在斑马鱼明确的造血过程中，Dnmt1是HSPC维持所必需的。
BackgroundDNA 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.MethodsAn 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 mountin situhybridization 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.ResultsA heritable mutant line with impaired HSPCs of definitive hematopoiesis was identified. Positional cloning demonstrated that a stop codon mutation was introduced indnmt1which 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 ofcebpagene in Dnmt1 deficient HSPCs. Overexpression of a transcriptional repressive SUMO-C/ebpa fusion protein could rescue hematological defects in thednmt1mutants. Finally,dnmt1andcebpadouble null embryos exhibited no obvious abnormal hematopoiesis indicated that the HSPC defects triggered bydnmt1mutation were C/ebpa dependent.ConclusionsDnmt1 is required for HSPC maintenance viacebparegulation during definitive hematopoiesis in zebrafish.
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影响因子：
64.8
作者：
通讯作者：
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影响因子：
2.7
作者：
Martin, CC;Laforest, L;Ekker, M
通讯作者：
Ekker, M
影响因子：
2.9
作者：
Meijer, Annemarie H.;van der Sar, Astrid M.;Spaink, Herman P.
通讯作者：
Spaink, Herman P.
影响因子：
2.5
作者：
KIMMEL, CB;BALLARD, WW;SCHILLING, TF
通讯作者：
SCHILLING, TF
影响因子：
2.7
作者：
Anderson, Ryan M.;Bosch, Justin A.;Goll, Mary G.;Hesselson, Daniel;Dong, P. Duc Si;Shin, Donghun;Chi, Neil C.;Shin, Chong Hyun;Schlegel, Amnon;Halpern, Marnie;Stainier, Didier Y. R.
通讯作者：
Stainier, Didier Y. R.