Use Forward Genetics Methods To Study The Function Of DNA Methyltransferase 1(Dnmt1) In Definitive Hematopoiesis Of Zebrafish

Hematopoietic stem cell(HSC) can self-renew and give rise to all lineages of mature blood cells. Abnormal proliferation and differentiation can lead to serious blood diseases. Some genes which regulate self-renewal and differentiation of HSCs had been reported, such as bmi1, hoxB4, wnt, pten, hedgehog. But most of the molecular regulation networks are still unknown. To discover novel genes which play critical roles in hematopoiesis, ENU mediated-forward genetics screen was carried out in zebrafish.The hematopoietic regulation network of zebrafish was conserved that of human. N-Ethyl-N-Nitrosourea(ENU) was used as the chemical mutagen to treat male zebrafish. One thousand mutant families were raised and screened. Definitive hematopoietic stem cell marker cmyb was used to screen mutant families with HSC defects. After several generation screening, two hundred mutant families were obtained.Ldd794 mutant family was chosen for the further molecular research. According positional cloning, we found that in dnmt1 there was a T to A transversion, which introduced a stop codon at 743 th amino acid, resulting in a predicted truncated Dnmt1 lacking the DNA methylation catalytic domain. 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.In the mutant line ldd794, HSCs was reduced from 36 hours post-fertilization(hpf) and was almost absent at 5 days post-fertilization(dpf). TUNEL and PH3 assays suggested that the defects of HSPCs in dnmt1 deficient embroys were caused by decreased proliferation. Molecular analysis revealed that expression of CCAAT/enhancer-binding protein alpha(C/ebpa) was up-regulated, which correlated with hypo-methylation of Cp G islands in the regulation regions of cebpa gene in dnmt1 mutant/knock-down zebrafish embryos. 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.