Global Changes Of H2A.Z Incorporation And Transcriptional Analysis In Mouse Somatic Cell Rerogramming

Stem cells can be applied to treat some diseases that conventional medicines failed, due to the superior features such as the pluripotency and function of repairing the damaged tissue. However, immune rejection of allogeneic cell therapy and ethical issues, limiting the stem cells to treat a wider range of diseases. It has been demonstrated that somatic cells could directly convert to embryonic stem-like cells with some defined transcriptional factors.This is called induced pluripotent stem cells(iPSCs), which.makes the production of patient-specific cells possible. But so far, the molecular mechanism of reprogramming remains elusive, it still needs further research. Elucidating the molecular mechanism behind this process will alarmingly promote the study of reprogramming theory, regenerative medicine, disease-specific model and developmental biology. It has great significance in detrimental diseases that threatening the human life.In recent years, researchers found a widespread histone variant H2A.Z which is necessary in the development of higher eukaryotes and performs an important role in gene transcription, chromosome segregation, DNA pairing and promote cell cycle, it also could regulate the gene expression by incorporation and deletion in different biological processes.We do the following research by using the mice as experimental animals:(1) obtain mouse embryonic fibroblasts (MEF) and transmit three retroviral vectors Oct4, Sox2, and Klf4into MEF genome for producing induced pluripotent stem cells (iPSCs), then its pluripotency were identified.(2) microarray analysis the expression of MEF, viral infection7days later-7D, MEF, iPSC and mouse embryonic stem cells (mESCs), to dig the deeper molecular mechanisms of reprogramming.(3) ChIP-Seq locate the incorporation sites of the histone variant H2A.Z at different stages in the genome and combine gene expression microarray to analyze the relationship between the H2A.Z regulation of gene expression and reprogramming.We can draw the following conclusions throughout comprehensive results of this study: (1) Establishment and characterization of induced pluripotent stem cells modelMEF would aggregate at4-5days after the virus infection, and gradually strengthen cohesion as the induction time extension.13-14days later, mESC-like colonies formed and AP staining was positive. The monoclonal had a normal karyotype, stem cell-specific marker protein cell immunofluorescence staining was positive, stem cell marker genes such as Nanog and Res-1had a similar expression levels with mESC. After injected into nude mice, teratoma would appear after one month, its diameter was about1.5cm and three germ layers could be observed after HE staning.(2) Molecular mechanism in the process of reprogrammingWe found that iPSC had similar gene expression profiles with mESC while it had big difference with MEF. The difference between iPSC and MEF mainly occured at the induction late stage(7D to iPSC), Virus infection could cause immune stress, DNA damage, apoptosis, and hinder cell proliferation, immortalization, thereby inhibiting the process of cell reprogramming. But the expression of pluripotency genes could cause epigenetic modification change and chromatin remodeling, thus rebuilding the regulatory network. P53pathway played an important role during the reprogramming process by regulating cell cycle and genomic stability.(3) Histone variant H2A.Z and reprogrammingBased on the H2A.Z genome-wide location analysis, we found that H2A.Z had high enrichment with the form of two peaks near the transcription start site (TSS). The degree of enrichment first increased and then decreased during the induction process, so iPSC had the lowest level. One interesting thing was H2A.Z tended to enrich in the TSS region which had high gene expression. By comparing the gene expression level at different induction stage, we found both the MEF-7D and7D-iPSC, H2A.Z had shedding in the TSS region of the gene expression changing significantly. Moreover, we observed H2A.Z gain enrichment near the TSS region of the gene expression small changes in7D-iPSC.