Histone proline isomerization and gene regulation

描述（由申请人提供）： 利用终末分化细胞核作为供体成功克隆体细胞核，已经明确证明卵细胞质中存在强大的核重编程活性。这些重编程活动可以诱导供体细胞核的去分化，导致其获得全能状态。尽管这项研究的历史很长，最近的研究特别密集，但是，目前对克隆过程中核重编程背后的分子机制知之甚少。本项目的长期目标是鉴定这些卵源性核重编程因子，并应用这些因子重编程活细胞的分化状态。该项目可能导致有效制备用于自体移植的患者来源的新组织，补充最近报道的iPS细胞技术。整体染色质去凝聚是在供体细胞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，卵蛋白质核质溶酶和肽基异构酶PPIA负责染色质解凝聚。这两种蛋白质触发特定组蛋白上的乙酰化、磷酸化和甲基化，并促进基因活化。基于这些观察，假设核质蛋白和PPIA在靶基因上组织了一组特定的表观遗传修饰，这导致染色质松弛和核重编程。为了验证这一假设，提出了以下具体目标。在具体目标1中，将通过使用肽片段、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。在具体目标2中，核质蛋白和PPIA如何参与胚胎干细胞的核重编程和多能性将通过基因敲除、DNA微阵列和荧光光漂白技术进行研究。此外，将结合干细胞特异性转录因子研究这些因子对细胞去分化的促进作用。总之，这些研究将提供一个新的见解通过染色质重塑细胞分化的重编程。公共卫生相关性：这些研究将大大有助于我们了解基因活性的调控，这对包括癌症在内的许多疾病的治疗都很重要。此外，这些结果将促进核重编程和再生医学的进展，特别是创造用于移植目的的患者来源的无排斥组织。

DESCRIPTION (provided by applicant): Existence of powerful nuclear reprogramming activities in egg cytoplasm has been unequivocally demonstrated by successful somatic cell nuclear cloning using terminally differentiated cell nuclei as donors. These reprogramming activities can induce dedifferentiation of the donor nuclei, leading to their acquisition of a topipotent state. Despite the long history of the study and the particularly intensive recent research, however, currently little is known about the molecular mechanism behind nuclear reprogramming 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 cells. This project potentially leads to efficient preparation of patients-derived new tissues for autologous transplantation, complementing the recently reported 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 PPIA are responsible for the chromatin decondensation. These two proteins trigger acetylation, phosphorylation and methylation on specific histones and promote gene activation. Based on these observations, it was hypothesized that nucleoplasmin and PPIA 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. In Specific Aim 1 the relationship among phosphorylation, methylation and isomerization of histones will be investigated by using peptide fragments, chromatin and living cells. In Specific Aim 2, how nucleoplasmin and PPIA are involved in nuclear reprogramming and pluripotency in embryonic stem cells will be studied by genetic knockdown, DNA microarray and the fluorescence photobleaching technique. In addition, promotion of cell dedifferentiation by these factors will be investigated in combination with stem cell-specific transcription factors. Together, these studies will provide a novel insight into the reprogramming of cell differentiation through chromatin remodeling. PUBLIC HEALTH RELEVANCE: These studies will substantially contribute to our understanding on the regulation of gene activity, which is important for the therapy of many diseases including cancer. In addition, the results will facilitate the progress of nuclear reprogramming and regenerative medicine, especially creation of patients-derived rejection-free tissues for the purpose of transplantation.