ZFP57在人类胚胎干细胞DLK1-DIO3印记调控中的作用研究

Previous studies have suggested that normal activation of the DLK1-DIO3 imprinted cluster has been correlated with the pluripotency state and epigenetic integrity in pluripotent stem cells. Our previous results indicated silencing of the DLK1-DIO3 gene cluster occurs frequently in early human embryonic stem cells (hESC) cultures under 20% atmosphere oxygen, however, hESCs under 5% physiological oxygen can maintain persistent expression of the DLK1-DIO3 Cluster, the mechanism are yet to be identified. ZFP57 was one of the significantly differently expressed genes between DLK1-DIO3 activated hESCs and silencing hESCs, which was described to encode a regulator of imprints within the DLK1-DIO3 locus in mouse. In the present study, we intend to evaluate the function of ZFP57in the epigenetic gene regulatory system of the DLK1-DIO3 cluster under 5% and 2% oxygen, respectively. We intend to first identify the parental chromosomal different epigenetic modification and uncover the role of ZFP57 in regulation of DLK1-DIO3 imprinted cluster under 5% oxygen utilizing the difference of normal fertilized and parthenogenetic embryonic stem cells using CHIP-seq, methylation analysis, CO-IP, ZFP57 interference et al. Further, by identified the changes of ZFP57 related epigenetic modifications in the DLK1-DIO3 locus during the DLK1-DIO3 silencing, we hope to reveal the relationship between ZFP57 and the aberrant silencing of DLK1-DIO3 imprinted culster. This study can provide a more complete picture of how DLK1-DIO3 imprinted cluster is regulated in human.

既往研究提示DLK1-DIO3印记区的正常活化对维持胚胎干细胞全能性和表观遗传稳定有重要意义。我们前期研究发现大气氧培养易引起该区域异常沉默，生理氧分离培养能维持正常活化，机制不明。同时发现重要的印记调控因子ZFP57在印记活化和沉默的人胚胎干细胞表达有显著差异。因此，本研究拟将ZFP57纳入印记调控网络探讨在大气氧和生理氧条件ZFP57分别与哪些基因组差异表观修饰相互作用参与调控？本课题拟首先以印记区处于正常活化状态的正常受精胚胎干细胞和孤雌胚胎干细胞为研究对象，利用CHIP-seq，甲基化分析,CO-IP，ZFP57干扰等方法探讨印记区的差异表观修饰及ZFP57在印记调控中的作用机理。进一步，通过比较活化和沉默细胞系中ZFP57与印记区结合的改变，ZFP57相关观修饰的变化探讨ZFP57在印记沉默中发挥的作用，该研究能完善正常及异常环境下我们对人DLK1-DIO3印记区调控机制的了解。

DLK1-DIO3印记区的正常活化对维持胚胎干细胞全能性和表观遗传稳定有重要意义。本研究比较了正常胚胎干细胞和孤雌胚胎干细胞DLK1-DIO3印记区印记基因表达情况和差异表观修饰，结果提示孤雌胚胎干细胞高表达母源印记基因，不表达父源印记基因，且两个关键的甲基化区域呈现未甲基化状态，同时比较了该印记区活化和沉默状态的表观修饰差异。在人胚胎干细胞样本中采用CHIP-qPCR检测未发现ZFP57与其他组蛋白结合共同调控DLK1-DIO3区域。基于此对人鼠早期胚胎不同发育阶段ZFP57的表达情况进行分析，结果显示在母源性效应蛋白ZFP57存在种属特异性，其在小鼠受精卵合子细胞中特异性高表达，而在人类早期胚胎发育过程直至桑葚胚阶段均无表达，进一步我们发现在早期胚胎发育过程DLK1-DIO3印记区代表性基因MEG3，DLK1在早期胚胎中的变化趋势与ZFP57类似，均是从囊胚阶段开始表达，在建系过程有一上调过程，而建系后急剧下调的过程，我们的研究提示ZFP57对印记的调控作用在人鼠中发挥作用的途径可能不同，接下来将进一步围绕建系过程分析DLK1-DIO3印记调控。进而我们利用人纯和孤雌细胞系和杂合孤雌细胞系以及，采用SNP芯片分析杂合分布模式和杂合度的方法，制定了在微量细胞水平鉴定单性生殖胚胎的方法，为临床筛选正常受精且核型正常的1PN胚胎提供了参考。