Developmentally regulated DNA methylation in Dicyostelium discoideum
Developmentally regulated DNA methylation in Dicyostelium discoideum
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DOI：
10.1128/ec.5.1.18-25.2006
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发表时间：
2006-01-01
期刊：
影响因子：
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通讯作者：
Shaulsky, G
中科院分区：
文献类型：
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作者：
Katoh, M;Curk, T;Shaulsky, G
DNA中胞嘧啶残基的甲基化在真核生物基因表达的沉默、染色质结构的组织和细胞分化中起着至关重要的作用。以往的研究未能检测到碟形骨菌基因组DNA中的5-甲基胞嘧啶，但最近对碟形骨菌基因组的测序发现了一个候选DNA甲基转移酶基因（dnmA）。基因组序列还揭示了整个基因组中潜在甲基化位点CpG岛的不寻常分布。DnmA属于Dnmt2亚家族，包含胞嘧啶甲基转移酶所需的所有催化基序。Dnmt2在果蝇、小鼠和人类细胞中的活性通常较弱，这些系统中的基因功能尚不清楚。我们研究了Dictyostelium基因组DNA的甲基化状态，用抗体培养5-甲基胞嘧啶，检测到低水平的修饰核苷酸。我们还发现DNA甲基化在发育过程中增加。我们在基因组中寻找潜在的甲基化位点，并在逆转录转座元件和其他几个基因中发现了它们。使用甲基化敏感和不敏感的限制性内切酶进行Southern blot分析，我们发现DIRS反转录转座子和guaB基因确实被甲基化了。然后我们突变了dnmA基因，发现DNA甲基化降低到野生型水平的50%左右。突变细胞在发育后期表现出形态缺陷，表明DNA甲基化在盘基ostelum发育中具有调节作用。我们的发现确定了Dnmt2甲基转移酶在真核生物发育中的作用。
Methylation of cytosine residues in DNA plays a critical role in the silencing of gene expression, organization of chromatin structure, and cellular differentiation of eukaryotes. Previous studies failed to detect 5-methyl-cytosine in Dictyostelium genomic DNA, but the recent sequencing of the Dictyostelium genome revealed a candidate DNA methyltransferase gene (dnmA). The genome sequence also uncovered an unusual distribution of potential methylation sites, CpG islands, throughout the genome. DnmA belongs to the Dnmt2 subfamily and contains all the catalytic motifs necessary for cytosine methyltransferases. Dnmt2 activity is typically weak in Drosophila melanogaster, mouse, and human cells and the gene function in these systems is unknown. We have investigated the methylation status of Dictyostelium genomic DNA with antibodies raised against 5-methylcytosine and detected low levels of the modified nucleotide. We also found that DNA methylation increased during development. We searched the genome for potential methylation sites and found them in retrotransposable elements and in several other genes. Using Southern blot analysis with methylation-sensitive and -insensitive restriction endonucleases, we found that the DIRS retrotransposon and the guaB gene were indeed methylated. We then mutated the dnmA gene and found that DNA methylation was reduced to about 50% of the wild-type level. The mutant cells exhibited morphological defects in late development, indicating that DNA methylation has a regulatory role in Dictyostelium development. Our findings establish a role for a Dnmt2 methyltransferase in eukaryotic development.