Miyadai Rio, Hinata Shiori, Amemiya Yuya, Shigematsu Satori, Umeyama Kazuhiro, Nagashima Hiroshi, Yamatoya Kenji, Ohgane Jun
Laboratory of Genomic Function Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, 214-8571, Kanagawa, Japan.
Meiji University International Institute for Bio-Resource Research (MUIIBR), Kawasaki, 214-8571, Kanagawa, Japan.
Biochem Biophys Rep. 2025 Mar 11;42:101973. doi: 10.1016/j.bbrep.2025.101973. eCollection 2025 Jun.
Fibrillin-1, an extracellular matrix protein encoded by the gene, is crucial for maintaining connective tissue integrity. Mutations in result in haploinsufficiency, leading to Marfan syndrome, in which the expression of functional is correlated with disease onset and severity. Recent studies suggest that expression is modulated by DNA methylation, particularly within the CpG island shores of its promoter region. In porcine models, mRNA levels have been found to correlate with the proportion of hypomethylated alleles in the CpG island shore region. In this study, we employed epigenome editing using the dCas9-TET1 system to induce targeted DNA demethylation within the CpG island shore, which became hypermethylated after a prolonged culture of porcine fetal fibroblast cells. This approach effectively reduced methylation in the targeted region, and cells expressing the dCas9-TET1 system maintained hypomethylation across multiple passages. Critically, DNA demethylation of the CpG island shore restored expression in heterozygous knockout fibroblasts, which developed stochastic hypermethylation after extended culture. These findings highlight the potential of DNA methylation manipulation to restore expression in cells with a haploinsufficient genetic background.
原纤蛋白-1是由该基因编码的一种细胞外基质蛋白，对维持结缔组织完整性至关重要。该基因突变会导致单倍剂量不足，进而引发马凡综合征，其中功能性原纤蛋白-1的表达与疾病的发病和严重程度相关。最近的研究表明，原纤蛋白-1的表达受DNA甲基化调控，尤其是在其启动子区域的CpG岛岸内。在猪模型中，已发现原纤蛋白-1的mRNA水平与CpG岛岸区域低甲基化等位基因的比例相关。在本研究中，我们使用dCas9-TET1系统进行表观基因组编辑，以诱导原纤蛋白-1的CpG岛岸内的靶向DNA去甲基化，在猪胎儿成纤维细胞长期培养后该区域变得高度甲基化。这种方法有效降低了靶向区域的甲基化，并且表达dCas9-TET1系统的细胞在多次传代后维持低甲基化状态。至关重要的是，原纤蛋白-1的CpG岛岸的DNA去甲基化恢复了杂合原纤蛋白-1基因敲除成纤维细胞中的原纤蛋白-1表达，这些细胞在长期培养后出现了随机的高甲基化。这些发现凸显了DNA甲基化操纵在具有单倍剂量不足遗传背景的细胞中恢复原纤蛋白-1表达的潜力。
