Normal development involves the interplay of genetic and epigenetic regulatory mechanisms. Pax6 is an eye-selector factor responsible for initiating the regulatory cascade for the development of the eyes. For the olive ridley sea turtle (Lepidochelys olivacea), a threatened species, eye malformations have been reported. In order to study the DNA methylation status of the putative promoter of the Pax6 gene in embryos with ocular malformations, an exploratory study was carried out in which DNA was isolated from embryos with anophthalmia, microphthalmia, and cyclopia, as well as from their normal counterparts. The 5'-flanking region from the Pax6 gene was isolated, showing two CpG islands (CGIs). The methylation status of CGIs in malformed embryos was compared with that of normal embryos by bisulfite sequencing. Putative transcription factor binding sites and regulatory features were identified. Methylation patterns were observed in both CpG and non-CpG contexts, and were unique for each malformed embryo; in the CpG context, an embryo with cyclopia showed a methylated cytosine upstream the CGI-1 not present in other embryos, an embryo with left anophthalmia presented two methylated cytosines in the CGI-1, whereas an embryo with left anophthalmia and right microphthalmia showed two methylated cytosines in the CGI-2. Normal embryos did not show methylated cytosines in the CGI-1, but one of them showed one methylcytosine in the CGI-2. Methylated transcription factor-binding sites may affect Pax6 expression associated to the cellular response to environmental compounds and hypoxia, signal transduction, cell cycle, lens physiology and development, as well as the transcription rate. Although preliminary, these results suggest that embryos with ocular malformations present unique DNA methylation patterns in the putative promoter of the Pax6 gene in L. olivacea, and probably those subtle, random changes in the methylation status can cause (at least in part) the aberrant phenotypes observed in these embryos.

中文翻译：

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具有眼部缺陷的橄榄树海龟胚胎中假定的 Pax6 启动子的甲基化状态：初步方法

正常发育涉及遗传和表观遗传调控机制的相互作用。Pax6 是一种眼睛选择因子，负责启动眼睛发育的调节级联反应。据报道，橄榄树海龟 (Lepidochelys olivacea) 是一种受威胁的物种，其眼部畸形。为了研究眼部畸形胚胎中 Pax6 基因的推定启动子的 DNA 甲基化状态，进行了一项探索性研究，其中从患有无眼症、小眼症和独眼症的胚胎以及它们的正常对应物中分离 DNA . 分离了 Pax6 基因的 5'-侧翼区域，显示了两个 CpG 岛 (CGI)。通过亚硫酸氢盐测序将畸形胚胎中 CGI 的甲基化状态与正常胚胎的甲基化状态进行比较。确定了假定的转录因子结合位点和调节特征。在 CpG 和非 CpG 环境中都观察到甲基化模式，并且每个畸形胚胎都是独一无二的；在 CpG 背景下，一个有独眼的胚胎在 CGI-1 上游显示一个甲基化的胞嘧啶，在其他胚胎中不存在，一个有左眼的胚胎在 CGI-1 中表现出两个甲基化的胞嘧啶，而一个有左眼和右小眼的胚胎在 CGI-1 中表现出两个甲基化胞嘧啶。 CGI-2 中的甲基化胞嘧啶。正常胚胎在 CGI-1 中没有显示甲基化胞嘧啶，但其中一个在 CGI-2 中显示出一种甲基胞嘧啶。甲基化转录因子结合位点可能会影响与细胞对环境化合物和缺氧的反应、信号转导、细胞周期、晶状体生理和发育相关的 Pax6 表达，以及转录率。尽管是初步的，但这些结果表明，具有眼部畸形的胚胎在 L. olivacea 的 Pax6 基因的推定启动子中呈现出独特的 DNA 甲基化模式，并且甲基化状态的那些微妙的、随机的变化可能会导致（至少部分）异常在这些胚胎中观察到的表型。