DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells

Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.     

Immortal DNA or epigenetic signature ?

During mitosis each daughter cell inherits a full copy of the maternal genomic material. DNA replication, however, is an imprecise process, thus errors can arise resulting in potentially deleterious mutations over extended rounds of cell division and these may lead to cancinogenesis. Over thirty years ago, J. Cairns proposed that a cell could avoid the accumulation of mutations arising from DNA replication if all template DNA strands are inherited in one daughter cell during cell division, thus giving rise to the notion of < immortal > DNA strands. In this model the stem cells would retain the template DNA (older) strands. Proving or disproving this notion experimentally has been challenging. Further, it has recently become apparent that epigenetic regulation of gene expression plays a critical role in governing cell states, self-renewal and differentiation. In light of these data, can the phenomenon on template DNA strand segregation also reflect this epigenetic signature? In this review we explore these notions, discuss the evidence in support of this theory, the implications, and some of the mechanisms which could explain this phenomenon.     

Spatiotemporal expression of Prdm genes during Xenopus development

Epigenetic regulation is known to be important in embryonic development, cell differentiation and regulation of cancer cells. Molecular mechanisms of epigenetic modification have DNA methylation and histone tail modification such as acetylation, phosphorylation and ubiquitination. Until now, many kinds of enzymes that modify histone tail with various functional groups have been reported and regulate the epigenetic state of genes. Among them, Prdm genes were identified as histone methyltransferase. Prdm genes are characterized by an N-terminal PR/SET domain and C-terminal some zinc finger domains and therefore they are considered to have both DNA-binding ability and methylation activity. Among vertebrate, fifteen members are estimated to belong to Prdm genes family. Even though Prdm genes are thought to play important roles for cell fate determination and cell differentiation, there is an incomplete understanding of their expression and functions in early development. Here, we report that Prdm genes exhibit dynamic expression pattern in Xenopus embryogenesis. By whole mount in situ hybridization analysis, we show that Prdm genes are expressed in spatially localized manners in embryo and all of Prdm genes are expressed in neural cells in developing central nervous systems. Our study suggests that Prdm genes may be new candidates to function in neural cell differentiation.     

斑马鱼中囊胚过渡调控机制

斑马鱼中囊胚过渡(MBT)始于受精卵的第10次卵裂,此时亦伴有细胞周期延长,分裂同步性丧失,合子型基因开始转录活化,胚胎细胞开始具备运动迁移能力等现象。斑马鱼MBT。的发生依赖于胚胎细胞的核质比,胚胎细胞周期中的G1时相则只有在合子型基因组开始被转录活化后才能出现。细胞周期检验点的激活可能也是受转录调控的,但中期检验点对DNA复制抑制状态的响应不仅在MBT前后、甚至在MBT前的不同阶段也可能有具体作用途径的差异。活化的P38蛋白在胚胎中的不对称分布是维持卵裂阶段细胞分裂同步性的关键因素。尽管大规模的合子型基因的表达发生在MBT开始后,也有少数与胚层分化有关的合子型基因是在MBT。前表达的,还有一些既有母型表达也有合子型表达的基因在MBT前后分别参与不同的信号途径来调控胚胎的发育与分化。     

GADD45A protein plays an essential role in active DNA demethylation during terminal osteogenic differentiation of adipose-derived mesenchymal stem cells

Methylation and demethylation of DNA are the complementary processes of epigenetic regulation. These two types of regulation influence a diverse array of cellular activities, including the maintenance of pluripotency and self-renewal in embryonic stem cells. It was generally believed that DNA demethylation occurs passively over several cycles of DNA replication and that active DNA demethylation is rare. Recently, evidence for active DNA demethylation has been obtained in several cancer, neuronal, and embryonic stem cell lines. Studies in embryonic stem cell models, however, suggested that active DNA demethylation might be restricted to the early development of progenitor cells. Whether active demethylation is involved in terminal differentiation of adult stem cells is poorly understood. We provide evidence that active DNA demethylation does occur during terminal specification of stem cells in an adipose-derived mesenchymal stem cell-derived osteogenic differentiation model. The medium CpG regions in promoters of the Dlx5, Runx2, Bglap, and Osterix osteogenic lineage-specific genes were demethylated during the increase in gene expression associated with osteogenic differentiation. The growth arrest and DNA damage-inducible protein GADD45A was up-regulated in these processes. Knockdown of GADD45A led to hypermethylation of Dlx5, Runx2, Bglap, and Osterix promoters, followed by suppression of the expression of these genes and interruption of osteogenic differentiation. These results reveal that GADD45A plays an essential role in gene-specific active DNA demethylation during adult stem cell differentiation. They enhance the current knowledge of osteogenic specification and may also lead to a better understanding of the common mechanisms underlying epigenetic regulation in adult stem cell differentiation.     

Defining the Epigenetic Mechanism of Asymmetric Cell Division of Schizosaccharomyces japonicus Yeast

A key question in developmental biology addresses the mechanism of asymmetric cell division. Asymmetry is crucial for generating cellular diversity required for development in multicellular organisms. As one of the potential mechanisms, chromosomally borne epigenetic difference between sister cells that changes mating/cell type has been demonstrated only in the Schizosaccharomyces pombe fission yeast. For technical reasons, it is nearly impossible to determine the existence of such a mechanism operating during embryonic development of multicellular organisms. Our work addresses whether such an epigenetic mechanism causes asymmetric cell division in the recently sequenced fission yeast, S. japonicus (with 36% GC content), which is highly diverged from the well-studied S. pombe species (with 44% GC content). We find that the genomic location and DNA sequences of the mating-type loci of S. japonicus differ vastly from those of the S. pombe species. Remarkably however, similar to S. pombe, the S. japonicus cells switch cell/mating type after undergoing two consecutive cycles of asymmetric cell divisions: only one among four “granddaughter” cells switches. The DNA-strand–specific epigenetic imprint at the mating-type locus1 initiates the recombination event, which is required for cellular differentiation. Therefore the S. pombe and S. japonicus mating systems provide the first two examples in which the intrinsic chirality of double helical structure of DNA forms the primary determinant of asymmetric cell division. Our results show that this unique strand-specific imprinting/segregation epigenetic mechanism for asymmetric cell division is evolutionary conserved. Motivated by these findings, we speculate that DNA-strand–specific epigenetic mechanisms might have evolved to dictate asymmetric cell division in diploid, higher eukaryotes as well.     

Determining to Divide: How Do Cells Decide?

A cell's decision to divide must be regulated with the highest fidelity. Otherwise, abnormalities occurring in the replication of genetic material and cytokinesis would be incompatible with life. It has been known for almost a century that cells comprising a population undergo cellular division at extremely variable rates, even though genetically identical cell clones have been examined. Studies with T lymphocytes at the single cell level have revealed that the rate of cellular division is determined by the accumulation of a critical number of ligand-triggered interleukin-2 (IL2) receptors at the cell surface throughout the G₁ phase of the cell cycle. Thus, the cell “counts” the number of triggered IL2 receptors, and only decides to divide when the critical number has been attained. This information is then transferred to the cellular interior via intracellular sensors comprised of D-type cyclins, which ultimately determine when the cell surpasses the “Restriction Point” in late G₁, and which commits the cell irrevocably to initiate DNA replication. Beyond the R-point, the cell assembles a definite number of macromolecular pre-replication complexes (Pre-RCs) comprised of at least 6 distinct proteins at sites of the origin of replication on DNA. Complete assembly of the Pre-RCs is a prerequisite for their subsequent disassembly, which must occur before the initiation of DNA strand replication, and which occurs asynchronously throughout the S-phase of the cell cycle and only terminates when the entire DNA has been duplicated. Thus, the fidelity of the decision to divide is exquisitely regulated by macromolecular mechanisms initiated at the cell surface and transferred to the cellular interior so that the cell can make the decision in a quantal (all-or-none) fashion. The question before us is how this quantal decision is made at the molecular level. The available data indicate that the assembly and disassembly of a definite number of large multicomponent macromolecular complexes make the quantal decisions. Here, it is postulated that all fundamental cellular decisions, i.e. survival, death, proliferation and differentiation, are regulated in this fashion. It remains to be determined how the cell counts the signals it receives, and what the molecular forces are that dictate the behavior of macromolecular complexes. Alexander Hamilton: “The best security for the fidelity of men, is to make interest coincide with duty.”     

Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus

During cell division complete DNA replication must occur before mitosis is initiated. Using a cell-free extract derived from Xenopus eggs that oscillates between S phase and mitosis, we have investigated how completion of DNA synthesis is coupled to the initiation of mitosis. We find that Xenopus eggs contain a feedback pathway which suppresses mitosis until replication is completed and that activation of this inhibitory system is dependent on the presence of a threshold concentration of unreplicated DNA. We demonstrate that in the presence of unreplicated DNA the active feedback system inhibits initiation of mitosis by blocking the activation of MPF, a regulator of mitosis found in all eukaryotic cells. Our results demonstrate that the feedback system does not inhibit MPF activation by blocking the synthesis or accumulation of cyclin protein, a subunit of MPF, or by blocking association of cyclin with the cdc2 subunit of MPF. We propose that the feedback system blocks mitosis by maintaining MPF in an inactive state by modulating posttranslational modifications critical for MPF activation.