Department of Cancer Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, California, USA.
Mol Carcinog. 2010 Dec;49(12):999-1006. doi: 10.1002/mc.20683.
Methylated cytosines within CpG dinucleotides (mCpGs) along the DNA-binding domain of the TP53 tumor suppressor gene (exons ~5-8) are the single most significant mutational target in human cancers. The human p53 knock-in (Hupki) mouse model was constructed using gene-targeting technology to create a mouse strain that harbors human wild-type TP53 DNA sequences spanning exons 4-9 in both copies of the mouse p53 gene. To date, however, the methylation status of cytosines within CpGs in the substituted segment of the Hupki mouse genome has not been determined. This lack of information deserves special attention because DNA methylation in mammals, which occurs almost exclusively within CpG dinucleotides, is a dynamic process throughout developmental stages and may vary among different species. Here, we have investigated the status of CpG methylation in the substituted segment of the Hupki mouse genome, and compared it to the methylation profile of the corresponding segment in the human genome using the combined bisulfite-restriction analysis and sodium bisulfite genomic sequencing. We found that all cytosines within CpGs of the TP53 DNA-binding domain, on both the coding and noncoding strands, were heavily methylated in Hupki fibroblasts, as they were in human fibroblasts. This is in keeping with the fully methylated status of TP53 CpGs that is known to prevail in adult human tissues. The remarkably similar patterns of cytosine methylation within CpG dinucleotides in Hupki cells and human cells further validates the suitability of mutagenesis assays in Hupki cells for experimental induction of TP53 mutations that have been observed in human tumors.
CpG 二核苷酸中的甲基化胞嘧啶（mCpG）位于 TP53 肿瘤抑制基因的 DNA 结合域内（外显子~5-8），是人类癌症中突变的单一最重要靶标。人类 p53 敲入（Hupki）小鼠模型是使用基因靶向技术构建的，用于创建一种携带人类野生型 TP53 DNA 序列的小鼠品系，该序列跨越小鼠 p53 基因的两个拷贝的外显子 4-9。然而，迄今为止，尚未确定 Hupki 小鼠基因组中取代片段内 CpG 中胞嘧啶的甲基化状态。这种信息的缺失值得特别关注，因为哺乳动物中的 DNA 甲基化几乎仅在 CpG 二核苷酸内发生，是一个贯穿发育阶段的动态过程，并且可能因不同物种而异。在这里，我们研究了 Hupki 小鼠基因组中取代片段内 CpG 甲基化的状态，并使用联合亚硫酸氢盐限制性分析和亚硫酸氢盐基因组测序，将其与人类基因组中相应片段的甲基化谱进行了比较。我们发现，Hupki 成纤维细胞中 TP53 DNA 结合域的两条链上的 CpG 中的所有胞嘧啶都高度甲基化，就像人类成纤维细胞一样。这与已知在成人组织中普遍存在的 TP53 CpG 完全甲基化状态一致。Hupki 细胞和人类细胞中 CpG 二核苷酸内的胞嘧啶甲基化模式非常相似，进一步验证了在 Hupki 细胞中进行诱变实验以诱导人类肿瘤中观察到的 TP53 突变的适用性。
