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Identification of the acetylated lysine residues of Dnmt3L protein in murine embryonic stem cells

ABSTRACT: 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.

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ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Kyunggon Kim

LAB HEAD: Kyunggon Kim

PROVIDER: PXD032953 | Pride | 2025-05-26

REPOSITORIES: Pride

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Project description:A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur prior to any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Illumina Expresson BeadChip arrays (MouseRef-8v2.0) were used to assess gene expression changes in neural stem cells (n=5; derived from mouse embryonic stem cells) and their differentiated neuronal progeny (n=3; FACS-purified based on Tau-RedStar expression).

Project description:A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur prior to any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Neural stem cells derived from mouse embryonic stem cells were differentiated into neurons and FACS purified based on RedStar fluorescence driven by the Tau promoter. Chromatin was prepared from NSCs and neurons (n=1), sonicated to roughly 300bp and immunoprecipitated with antibodies against H3K4me3, H3K27me3, total Histone H3 and total IgG, alongside a 5% input sample. K4/K27 and corresponding input samples were analysed by ChIPSeq

			Action	DRS
	20180705_K8R_E01.mgf	Mgf
	20180705_K8R_E01.raw	Raw
	20180705_K8R_E02.mgf	Mgf
	20180705_K8R_E02.raw	Raw
	20180705_LOT_E01.mgf	Mgf

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Identification of the acetylated lysine residues of Dnmt3L protein in murine embryonic stem cells

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