Histone isomerization and pluripotency

描述(由申请人提供)： 成功的体细胞核克隆表明，卵子细胞质中含有强大的核重编程活动。这些活性可以诱导终末分化的供体核去分化，导致它们获得全能状态。然而，尽管克隆研究有很长的历史，最近的研究也特别密集，但人们对克隆过程中对供体核重新编程的分子机制知之甚少。该项目的长期目标是确定这些卵子来源的核重新编程因子，并将这些因子应用于重新编程活的哺乳动物细胞的分化状态。该项目希望能有效地为自体移植准备患者来源的新组织，补充诱导多能干细胞(IPS)技术。全球染色质解缩是在供体核中观察到的最显著的细胞生物学变化之一。研究小组最近发现，鸡蛋蛋白核纤溶酶和多肽异构酶p16负责染色质的解缩。这两种蛋白质在特定的组蛋白上启动乙酰化、磷酸化和甲基化，促进基因激活。P16和核纤溶酶的转导显著促进了小鼠成纤维细胞产生iPS细胞。基于这些观察，假设核纤溶酶和p16在靶基因上组织了一组特定的表观遗传修饰，从而导致染色质松弛和核重新编程。为了验证这一假设，提出了以下具体目标。具体目的1旨在了解核纤溶酶和p16如何在诱导性多能性细胞形成过程中对关键多能性基因的表观遗传重新编程做出贡献。在具体目标2中，将通过基因敲除、DNA芯片分析和染色质免疫沉淀来研究p16在ES细胞多能性中的作用。在具体目标3中，将利用多肽片段、染色质和活细胞来研究组蛋白的磷酸化、甲基化和异构化之间的关系。总之，这些研究有望为通过表观遗传重塑重新编程细胞分化提供新的见解。

DESCRIPTION (provided by applicant): Project Summary Successful somatic cell nuclear cloning has demonstrated that egg cytoplasm contains powerful nuclear reprogramming activities. These activities can induce dedifferentiation of the terminally differentiated donor nuclei, leading to their acquisition of a totipotent state. Despite the long history of the cloning study and particularly intensive recent research, however, little is known about the molecular mechanisms that reprogram donor nuclei during cloning. The long-term goal of this project is to identify these egg-derived nuclear reprogramming factors and apply the factors to reprogram the differentiation status of living mammalian cells. This project i expected to lead to efficient preparation of patients-derived new tissues for autologous transplantation, complementing the induced pluripotent stem (iPS) cell technology. Global chromatin decondensation is one of the most striking cell biological changes observed in the donor nuclei. The investigator's group has recently found that the egg protein nucleoplasmin and the peptidyl isomerase p16 are responsible for the chromatin decondensation. These two proteins trigger acetylation, phosphorylation and methylation on specific histones and promote gene activation. Transduction of p16 and nucleoplasmin significantly facilitate the production of iPS cells from mouse fibroblasts. Based on these observations, it was hypothesized that nucleoplasmin and p16 organize a specific set of epigenetic modifications on the target genes, which leads to chromatin relaxation and nuclear reprogramming. To test this hypothesis, following specific aims are proposed. Specific Aim 1 is designed to understand how nucleoplasmin and p16 contributes to epigenetic reprogramming of key pluripotency genes during iPS cell formation. In Specific Aim 2, the role of p16 in pluripotency of ES cells will be studied by gene knockdown, DNA microarray analysis and chromatin immunoprecipitation. In Specific Aim 3, the relationship among phosphorylation, methylation and isomerization of histones will be investigated by using peptide fragments, chromatin and living cells. Together, these studies are expected to provide a novel insight into the reprogramming of cell differentiation through epigenetic remodeling.