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.
Project Narrative These studies will facilitate mechanistic understanding of chromatin modifications and cellular reprogramming in stem cell biology. This is also important for efficient and safe clinical applications of stem cells in regenerative medicine.
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|Hirai, Hiroyuki; Kikyo, Nobuaki (2014) Inhibitors of suppressive histone modification promote direct reprogramming of fibroblasts to cardiomyocyte-like cells. Cardiovasc Res 102:188-90|
|Sabin, Keith; Kikyo, Nobuaki (2014) Microvesicles as mediators of tissue regeneration. Transl Res 163:286-95|
|Hirai, Hiroyuki; Katoku-Kikyo, Nobuko; Keirstead, Susan A et al. (2013) Accelerated direct reprogramming of fibroblasts into cardiomyocyte-like cells with the MyoD transactivation domain. Cardiovasc Res 100:105-13|