Modulation of plasmid DNA methylation and expression in zebrafish embryos
Modulation of plasmid DNA methylation and expression in zebrafish embryos
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DOI：
10.1093/nar/26.19.4454
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发表时间：
1998-10-01
影响因子：
14.9
通讯作者：
Collas, P
中科院分区：
文献类型：
--
作者：
Collas, P
基因表达受DNA甲基化和染色质结构组装的影响。本文报道了用5-氮杂胞苷改变DNA甲基化和用组蛋白去乙酰化抑制剂丁酸钠改变异染色质的形成来调控转基因在斑马鱼胚胎中的表达。组蛋白去乙酰化抑制剂CMV启动子-荧光素酶融合基因构建物(PCMVL)显微注射到斑马鱼卵中，在发育过程中逐渐甲基化，大约在注射后12h开始。当在注射前被HpalI甲基酶体外甲基化时，该构建体在被从头甲基化之前在体内被快速去甲基化。去甲基化与DNA复制无关，表明它是一个活跃的DNA修复过程。斑马鱼胚胎核提取物的脱甲基化活性是不稳定的，对蛋白酶和核糖核酸酶敏感，需要ATP水解酶，体外去甲基化活性取决于制备提取物的胚胎的发育阶段。在体内，荧光素酶转录本在从头开始的质粒甲基化之前被检测到。此外，注射pCMVL的胚胎在注射后立即与5-氮杂胞苷或丁酸盐孵育可抑制质粒甲基化并延长荧光素酶的表达时间，当在从头开始甲基化发生后应用时，这两种抑制物都能阻止新复制的DNA的甲基化并促进转基因的表达。这些数据表明，在早期发育过程中，注射的结构物的甲基化可能通过将结构物转化为抑制性染色质结构来诱导转基因的抑制。
Gene expression is under the influence of DNA methylation and assembly of chromatin structure. This paper reports the modulation of transgene expression in zebrafish embryos by altering DNA methylation with 5-azacytidine and heterochromatin formation with sodium butyrate, an inhibitor of histone deacetylation, A CMV promoter-luciferase fusion gene construct (pCMVL) microinjected into zebrafish eggs becomes gradually methylated during development, starting at similar to 12 h post-injection. When methylated in vitro by HpalI methylase prior to injection, the construct is rapidly demethylated in vivo before being de novo methylated. Demethylation is independent of DNA replication, indicating that it is an active DNA repair process. Demethylating activity has been characterized in zebrafish embryo nuclear extracts, in which this activity is heat-labile, sensitive to protease and RNase and requires ATP hydrolysis, Demethylating activity in vitro is dependent on the developmental stage of the embryo from which extracts are prepared. In vivo, luciferase transcripts are detected prior to de novo plasmid methylation. Furthermore, incubation of pCMVL-injected embryos with 5-azacytidine or butyrate immediately after injection inhibits plasmid methylation and extends the period of luciferase expression, When applied after de novo methylation has occurred, both inhibitors prevent methylation of newly replicated DNA and promote transgene expression. These data suggest that methylation of the injected construct during early development induces repression of the transgene, perhaps by converting the construct to a repressive chromatin structure.