Howell C Y, Steptoe A L, Miller M W, Chaillet J R
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Mol Cell Biol. 1998 Jul;18(7):4149-56. doi: 10.1128/MCB.18.7.4149.
The inheritance of gametic methylation patterns is a critical event in the imprinting of genes. In the case of the imprinted RSVIgmyc transgene, the methylation pattern in the unfertilized egg is maintained by the early mouse embryo, whereas the sperm's methylation pattern is lost in the early embryo. To investigate the cis-acting requirements for this preimplantation stage of genomic imprinting, we examined the fate of different RSVIgmyc methylation patterns, preimposed on RSVIgmyc and introduced into the mouse zygote by pronuclear injection. RSVIgmyc methylation patterns with a low percentage of methylated CpG dinucleotides, generated by using bacterial cytosine methylases with four-base recognition sequences, were lost in the early embryo. In contrast, methylation was maintained when all CpG dinucleotides were methylated with the bacterial SssI (CpG) methylase. This singular maintenance of RSVIgmyc methylation preimposed with SssI methylase appears to be specific to the early, undifferentiated embryo; differentiated NIH 3T3 fibroblasts transfected with methylated versions of RSVIgmyc maintained all methylation patterns, independent of the level of preimposed methylation. The methylation pattern of the RSVIgmyc allele in adult founder transgenic mice that was produced by pronuclear injection of an SssI-methylated construct could not be distinguished from the maternal RSVIgmyc methylation pattern. Thus, a highly methylated allele in adult mice, normally generated by transmission of RSVIgmyc through the female germ line, was also produced in founder transgenic mice by bypassing gametogenesis and introducing a highly methylated RSVIgmyc into the mouse zygote. These results suggest that RSVIgmyc methylation itself is a cis-acting signal for the preimplantation maintenance of the oocyte's methylation pattern and, therefore, a cis-acting signal for RSVIgmyc imprinting. Furthermore, our inability to identify a sequence element within RSVIgmyc that was absolutely required for its imprinting suggests that the extent of RSVIgmyc methylation, rather than a particular pattern of methylation, is the principal feature of this imprinting signal.
配子甲基化模式的遗传是基因印记过程中的关键事件。对于印记的RSVIgmyc转基因而言，未受精卵中的甲基化模式由早期小鼠胚胎维持，而精子的甲基化模式在早期胚胎中则会丢失。为了研究基因组印记着床前阶段的顺式作用需求，我们检测了预先施加于RSVIgmyc并通过原核注射导入小鼠受精卵的不同RSVIgmyc甲基化模式的命运。使用具有四碱基识别序列的细菌胞嘧啶甲基化酶产生的低比例甲基化CpG二核苷酸的RSVIgmyc甲基化模式在早期胚胎中丢失。相反，当所有CpG二核苷酸都被细菌SssI（CpG）甲基化酶甲基化时，甲基化得以维持。这种由SssI甲基化酶预先施加的RSVIgmyc甲基化的独特维持似乎是早期未分化胚胎所特有的；用甲基化的RSVIgmyc版本转染的分化的NIH 3T3成纤维细胞维持所有甲基化模式，与预先施加的甲基化水平无关。通过原核注射SssI甲基化构建体产生的成年转基因奠基小鼠中RSVIgmyc等位基因的甲基化模式与母本RSVIgmyc甲基化模式无法区分。因此，成年小鼠中通常通过RSVIgmyc经雌性生殖系传递产生的高度甲基化等位基因，在转基因奠基小鼠中也通过绕过配子发生并将高度甲基化的RSVIgmyc导入小鼠受精卵而产生。这些结果表明，RSVIgmyc甲基化本身是卵母细胞甲基化模式着床前维持的顺式作用信号，因此也是RSVIgmyc印记的顺式作用信号。此外，我们无法在RSVIgmyc中鉴定出其印记绝对必需的序列元件，这表明RSVIgmyc甲基化的程度而非特定的甲基化模式是该印记信号的主要特征。
