Direct induction of DNA hypermethylation in sea urchin embryos by microinjection of 5-methyl dCTP stimulates early histone gene expression and leads to developmental arrest.
Direct induction of DNA hypermethylation in sea urchin embryos by microinjection of 5-methyl dCTP stimulates early histone gene expression and leads to developmental arrest.
复制标题
通过显微注射 5-甲基 dCTP 直接诱导海胆胚胎 DNA 高甲基化，刺激早期组蛋白基因表达并导致发育停滞。
DOI：
10.1006/dbio.1993.1008
复制
发表时间：
1993
影响因子：
2.7
通讯作者：
Jones,PA
中科院分区：
文献类型：
--
作者：
Chen,J;Maxson,R;Jones,PA
DNA甲基化在基因失活中的作用在脊椎动物中已被广泛研究，但尚不清楚它是否在其他生物中具有类似的功能。我们设计了一种新的方法来诱导海胆Lytechinus pictus中内源性和注射DNA的超甲基化，以研究DNA甲基化对这种无脊椎动物基因表达的影响。通过将5-甲基dCTP单独或与克隆的DNA构建体一起注射到受精的海胆卵中，复制DNA在DNA合成期间由于甲基化核苷酸代替胞嘧啶的随机掺入而变得高度甲基化。在随后的几轮复制过程中，注射的5-甲基dCTP被耗尽，但CpG位点的甲基化仍然升高，这可能是由于甲基转移酶的作用。利用这种方法，我们研究了超甲基化对海胆多基因家族的两个成员，早期H2 B和晚期H2 B基因的影响。甲基化对基因活性的影响使用RNase保护测定来探测。甲基化导致早期H2 B组蛋白基因表达增加，但对晚期H2 B组蛋白基因表达没有影响。这些结果表明，甲基化并不一定像以前认为的那样使海胆中的基因失活。有趣的是，用5-甲基dCTP注射的胚胎的发育通常在囊胚期被阻止，并且从注射的胚胎中提取的基因组DNA的分析显示内源性甲基化含量显著增加。这些数据表明，在发育中的海胆胚胎甲基化模式的扰动可能是负责通过改变基因表达模式的发育停滞。
The role of DNA methylation in gene inactivation has been studied extensively in vertebrates but it is not clear whether it serves similar functions in other organisms. We devised a novel approach to induce hypermethylation of both endogenous and injected DNA in the sea urchinLytechinus pictusin order to study the effect of DNA methylation on gene expression in this invertebrate. By injecting 5-methyl dCTP either alone or together with a cloned DNA construct into fertilized sea urchin eggs, replicating DNA became hypermethylated from the random incorporation of the methylated nucleotide in place of cytosine during DNA synthesis. During subsequent rounds of replication, the injected 5-methyl dCTP became depleted but methylation at CpG sites was still elevated presumably due to the action of a methyltransferase enzyme. Using this approach, we studied the effect of hypermethylation on two members of the sea urchin multigene family, the early H2B and the late H2B genes.De novomethylation was shown to occur at knowncis-regulatory regions of the genes. The effect of methylation on gene activity was probed using RNase protection assay. Methylation resulted in increased early H2B histone gene expression but had no effect on late H2B histone gene expression. These results demonstrate that methylation does not necessarily inactivate genes in the sea urchins as previously thought. Interestingly, the development of embryos injected with 5-methyl dCTP typically was arrested at the blastula stage, and analysis of the genomic DNA extracted from injected embryos showed a significant increase in the endogenous methylation content. These data suggest that perturbation of methylation patterns in developing sea urchin embryos may be responsible for the developmental arrest through altering the gene expression pattern.