FAM20C could be targeted by TET1 to promote odontoblastic differentiation potential of human dental pulp cells
FAM20C could be targeted by TET1 to promote odontoblastic differentiation potential of human dental pulp cells
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FAM20C可被TET1靶向促进人牙髓细胞的成牙本质细胞分化潜能
DOI：
10.1111/cpr.12426
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发表时间：
2018-04-01
影响因子：
8.5
通讯作者：
Xu, Qiong
中科院分区：
文献类型：
--
作者：
Li, Qimeng;Yi, Baicheng;Xu, Qiong
目的十十一易位1（TET1）是最近发现的一种DNA甲基胞嘧啶（mC）双加氧酶，可以将5-mC转化为5-羟甲基胞嘧啶（5hmC）。我们之前报道过 TET1 促进人牙髓细胞 (hDPC) 的成牙本质细胞分化。编码具有序列相似性20的家族成员C (FAM20C)蛋白的基因是潜在的TET1靶标，并且在我们之前的研究中在hDPC的成牙本质细胞分化过程中表现出去甲基化。本研究旨在探讨TET1介导的羟甲基化是否可以激活FAM20C基因，从而调节hDPC分化。材料与方法通过Western blotting评估FAM20C的表达模式及其在成牙本质细胞诱导hDPCs过程中的潜在变化。使用慢病毒介导的短发夹 RNA (shRNA) 转导来敲低 hDPC 中的 FAM20C 和 TET1 表达。通过 ALPase 活性测定和观察矿化基质沉积以及成牙本质细胞相关标记 DSPP 和 DMP1 的表达来评估 hDPC 的矿化潜力。在救援实验中，重组人 FAM20C 蛋白 (rhFAM20C) 被重新引入 shTET1 细胞中。使用羟甲基化 DNA 免疫沉淀 (IP)-PCR 检查 FAM20C 基因启动子的动态羟甲基化状态。利用染色质 IP-PCR 和琼脂糖凝胶电泳验证 TET1 招募到 FAM20C 启动子中的目标位点。结果 hDPC 成牙本质细胞诱导后，FAM20C 蛋白水平上调。 shRNA介导的FAM20C抑制在成牙本质细胞诱导7天和14天后降低了DSPP和DMP1的表达。在 FAM20C 抑制的 hDPC 中成牙本质细胞诱导后，ALPase 活性在第 7 天降低，矿化结节的形成在第 14 天减弱。在 TET1 缺陷的 hDPC 中，基因组 5hmC 水平显着降低，总 5mC 水平升高。此外，FAM20C也出现大幅减少。 shTET1 细胞的 rhFAM20C 处理减轻了 TET1 耗竭引起的矿化异常。 TET1 缺失导致 FAM20C 启动子上多个区域的 5hmC 水平下降。在成牙本质细胞诱导过程中，在 FAM20C 启动子的相应位点检测到 TET1 募集增强。结论 TET1 敲低通过抑制其与 FAM20C 启动子的直接结合来抑制成牙本质细胞分化，从而抑制 FAM20C 羟甲基化和随后的转录。这些结果表明，TET1 通过其 DNA 去甲基化机制和上调 FAM20C 蛋白表达，可能促进 hDPC 的细胞分化潜力。
ObjectivesTen-eleven translocation 1 (TET1) is a DNA methylcytosine (mC) dioxygenase discovered recently that can convert 5-mC into 5-hydroxymethylcytosine (5hmC). We previously reported that TET1 promotes odontoblastic differentiation of human dental pulp cells (hDPCs). The gene encoding the family with sequence similarity 20, member C (FAM20C) protein, is a potential TET1 target and showed demethylation during odontoblastic differentiation of hDPCs in our previous study. This study aimed to explore whether TET1-mediated hydroxymethylation could activate the FAM20C gene, thereby regulating hDPC differentiation.Materials and methodsThe expression pattern of FAM20C and its potential changes during odontoblastic induction of hDPCs were assessed by Western blotting. Lentivirus-mediated transduction with short hairpin RNA (shRNA) was used to knock down FAM20C and TET1 expression in hDPCs. The mineralization potential of hDPCs was evaluated with an ALPase activity assay and by observing the mineralized matrix deposition and the expression of odontoblast-related markers DSPP and DMP1. Recombinant human FAM20C protein (rhFAM20C) was reintroduced into shTET1 cells in a rescue experiment. The dynamic hydroxymethylation status of the FAM20C gene promoter was examined using hydroxymethylated DNA immunoprecipitation (IP)-PCR. Chromatin IP-PCR and agarose gel electrophoresis were utilized to validate the recruitment of TET1 to its target loci in the FAM20C promoter.ResultsFAM20C protein level was upregulated after the odontoblastic induction of hDPCs. shRNA-mediated FAM20C suppression reduced the expression of DSPP and DMP1 after odontoblastic induction for 7 and 14days. ALPase activity was reduced on day 7, and the formation of mineralized nodules was attenuated on day 14 after odontoblastic induction in FAM20C-inhibited hDPCs. Genomic 5hmC levels significantly decreased, and total 5mC levels increased in TET1-deficient hDPCs. In addition, a significant reduction in FAM20C also emerged. The rhFAM20C treatment of shTET1 cells attenuated the mineralization abnormalities caused by TET1 depletion. TET1 depletion prompted a decline in 5hmC levels in several regions on the FAM20C promoter. Enhanced TET1 recruitment was detected at the corresponding loci in the FAM20C promoter during odontoblastic induction.ConclusionTET1 knockdown suppressed odontoblastic differentiation by restraining its direct binding to FAM20C promoter, and hence inhibiting FAM20C hydroxymethylation and subsequent transcription. These results suggest that TET1 potentially promotes the cytodifferentiation potential of hDPCs through its DNA demethylation machinery and upregulation of FAM20C protein expression.