Histone proline isomerization and gene regulation

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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.