Project description:The intestinal epithelium is replaced weekly by non-quiescent stem cells with kinetics that rely on a rapid loss of stemness and choice for secretory or absorptive lineage differentiation. To determine how the cellular transcriptome and proteome changes during these transitions, we developed a new cell sorting method to purify stem cells, secretory and absorptive progenitor cells, and mature, differentiated cells. Transcriptome analyses revealed that as stem cells transition to the progenitor stage, alternative mRNA splicing and polyadenylation dominate changes in the transcriptome. In contrast, as progenitors differentiate into mature cell types, alterations in gene expression and mRNA levels drive the changes. RNA processing targets mRNAs encoding regulators of cell cycle, RNA regulators, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, carrier-assisted mass spectrometry of sorted cell populations detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.
Project description:During in vitro differentiation, pluripotent stem cells undergo extensive remodeling of their gene expression profiles. While studied extensively at the transcriptome level, much less is known about protein dynamics, which might differ significantly from their mRNA counterparts. Here, we present deep proteome-wide measurements of protein levels during the differentiation of embryonic stem cells.
Project description:The core purpose of this project is to identify the novel epigenetic factors which play a crucial role on regulating stem cells developed from embryo development to ageing through the longitudinal analysis of their transcriptome, protein interactome, and post- translational modification network. Thus, this study would not only advance our understanding of epigenetic nature of stem cells through whole life, but also provide the novel strategy to develop the next generation stem cell therapeutics. According to our previous reports, we found that DNA methylation, particularly mediated by Dnmt3L could play a common epigenetic signature for modulating the stemness potency of both embryonic and adult stem cells. To investigate the post-translational modifications of Dnmt3L protein, we performed mass spectrometry analysis of Flag-tagged Dnmt3L proteins in murine embryonic stem cells (ESCs) and then identified several acetylated lysine residues. To examine their functional roles, we manufactured the lentivirus containing wild type Dnmt3L or the acetylated lysine mutants and they established the ESC cell lines stably harboring these constructs. To focus on epigenetic mechanism for stem cell biology, we will narrow down the key acetylated lysine sites of Dnmt3L, which can characteristically regulate the epigenetic status or transcription gene ontology. To get mechanistic insights, the transcriptome and DNA methylome features of the established ESC cell lines will be analyzed. The functional significance was examined by a series of experiments for in vitro and in vivo self-renewal and differentiation assays. Finally, we would extend these results to develop the efficient ex vivo expansion protocol for adult stem cells, particularly focusing on human mesenchymal stem cells. We expect that this proposal will not only advance our understating the epigenetic landscape of stem cell population but also contribute to establish the clinically best-suitable stem cell population. In addition, this study can be appliable to get a novel conceptional advances in other biological research such as cancer biology and ageing research.
Project description:Genome-wide bisulfite sequencing and corresponding transcriptome are analyzed together to seek for methylation target during neural differentiation H1 human embryonic stem cells are able to differentiated into neural stem cells with induction of compound C in 72h. We extracted genomic DNA from 0, 24, 48h samples during induction and measured their DNA methylation level by BS-seq.
Project description:We utilized quantitative analyses of the proteome, transcriptome, and ubiquitinome to study how ubiquitination and NEDD4 control neural crest cell survival and stem-cell-like properties. We report 276 novel NEDD4 targets in neural crest cells and show that loss of NEDD4 leads to a striking global reduction in specific ubiquitin lysine linkages.