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

Xiaohui Liu,Jun Zhou,Yi Jin,Hao Yuan,Hugues De The,Zhu Chen,Min Deng,Yi Zhou,Xiaoe Jia,Saijuan Chen,Weijun Pan,Leonard I Zon,Ke Ma,Yi Chen,Jun Zhu

doi:10.1186/s13045-015-0115-7

Abstract

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 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.ResultsA 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.ConclusionsDnmt1 is required for HSPC maintenance via cebpa regulation during definitive hematopoiesis in zebrafish.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0115-7) contains supplementary material, which is available to authorized users.

Highlights

DNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes through maintaining parental DNA methylation patterns on daughter DNA strands during mitosis
Definitive hematopoietic stem cells (HSCs) arise from the ventral wall of the dorsal aorta (VDA), the zebrafish equivalent of the aorta/gonad/mesonephros (AGM) of mammals [10,11], HSCs migrate through the caudal hematopoietic tissue (CHT) to the thymus and kidney marrow [12], where adult hematopoiesis occurs, similar to HSC migration through fetal liver and home to bone marrow in mammals
Zebrafish mutant line ldd794 displays impaired definitive hematopoiesis To search for novel genes involved in regulating definitive hematopoiesis, we established an ENU-based genetic screening strategy to identify putative mutants with defects in definitive hematopoietic stem and progenitor cells (HSPCs) in zebrafish

Summary

Introduction

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). Known as embryonic hematopoiesis, predominantly produces erythroid and myeloid cells [3,4]; while definitive hematopoiesis, called adult hematopoietic wave, generates hematopoietic stem cells (HSCs) that are capable of producing all lineages of blood [5]. DNA methylation is an important epigenetic regulation mechanism that regulates normal development through influencing gene transcription, genomic imprinting, and genome stability in mammal cells [14,15,16]. Dysregulation of DNA methylation may result in global shifts in gene expression, which frequently leads to increased self-renewal in malignant blood cells at the expense of normal differentiation [17]

Methods

Results

Conclusion