Chongqing Engineering Research Center for Herbivores Resource Protection and Utilization, College of Animal Science and Technology, Southwest University, Chongqing 400715, PR China.
Epigenetic Research Laboratory, Institute for Interdisciplinary Research, Jianghan University, Wuhan, Hubei 430056, PR China.
Gene. 2020 May 20;739:144512. doi: 10.1016/j.gene.2020.144512. Epub 2020 Feb 26.
Pleckstrin homology-like domain family A member 2 (PHLDA2) is essential for placental development in mammals. This study was conducted to investigate transcriptional regulation of goat PHLDA2 in the placenta. Real-time PCR and Western blot analyses showed different expression of the PHLDA2 in goat placentas during gestation with highest expression at 30 and 45 days post coitus (P < 0.05). Luciferase reporter assays demonstrated the highest promoter activity in the region of -1023/+20 (P < 0.05). A CpG island was defined within -631/+379 region, where lower level of CpG-methylation was detected with bisulfite sequencing PCR in the placenta than that in the spleen and liver (P < 0.05). Meanwhile, in vitro experiments showed that 5-AzaC enhanced the gene expression in a dose-dependent manner. Site-directed mutation in vitro demonstrated that transcription factor Ying-yang 1 (YY1) had an inhibitory effect on the PHLDA2 expression, and the inhibition was further confirmed with overexpression and siRNA constructs of YY1. ChIP and RE-ChIP analyses further identified the binding of YY1 to the PHLDA2 promoter by interaction with histone deacetylase 1 (HDAC1) and HDAC3. This study uncovers the negative regulation of the CpG-methylation and YY1 on goat PHLDA2 expression. YY1 prefers binding to CpG-methylation sequences, and inhibits goat PHLDA2 expression via recruiting HDAC1 and 3.
类 PH 结构域家族 A 成员 2（PHLDA2）对于哺乳动物胎盘的发育至关重要。本研究旨在研究山羊 PHLDA2 在胎盘中的转录调控。实时 PCR 和 Western blot 分析显示，在妊娠期间山羊胎盘中的 PHLDA2 表达不同，在交配后 30 和 45 天表达最高（P<0.05）。荧光素酶报告基因分析表明-1023/+20 区域的启动子活性最高（P<0.05）。在-631/+379 区域内定义了一个 CpG 岛，在胎盘中检测到比脾脏和肝脏中更低水平的 CpG 甲基化（P<0.05）。同时，体外实验表明 5-AzaC 以剂量依赖的方式增强基因表达。体外定点突变表明转录因子 Ying-yang 1（YY1）对 PHLDA2 表达具有抑制作用，并且通过 YY1 的过表达和 siRNA 构建进一步证实了这种抑制作用。ChIP 和 RE-ChIP 分析进一步确定了 YY1 通过与组蛋白去乙酰化酶 1（HDAC1）和 HDAC3 的相互作用与 PHLDA2 启动子结合。本研究揭示了 CpG 甲基化和 YY1 对山羊 PHLDA2 表达的负调控。YY1 优先与 CpG 甲基化序列结合，并通过招募 HDAC1 和 3 抑制山羊 PHLDA2 表达。
