胚胎干细胞分化过程中的表观遗传调控

作为一类既有自我更新能力,并具有多向分化潜能的细胞,胚胎干细胞具有非常重要的理论研究意义和临床应用前景。近期以胚胎干细胞为模型,研究有关干细胞分化的表观遗传调控已成为新的研究热点。本文就胚胎干细胞分化过程中DNA甲基化、组蛋白修饰、非编码RNA调控以及与胚胎干细胞分化密切相关的表观遗传学动态变化做一概述,对表观遗传学改变与胚胎干细胞分化关系的基础研究进行探讨。     

Genome-wide DNA methylation analysis reveals phytoestrogen modification of promoter methylation patterns during embryonic stem cell differentiation

Background

Environmental challenges during development affect the fetal epigenome, but the period(s) vulnerable to epigenetic dysregulation is(are) not clear. By employing a soy phytoestrogen, genistein, that is known to alter the epigenetic states of the A^vy allele during embryogenesis, we have explored the sensitive period for epigenetic regulation. The post-implantation period, when de novo DNA methylation actively proceeds, is amenable to in vitro analysis using a mouse embryonic stem (ES) cell differentiation system.

Methods and Findings

Mouse ES cells were differentiated in the presence or absence of genistein, and DNA methylation patterns on day 10 were compared by microarray-based promoter methylation analysis coupled with a methylation-sensitive endonuclease (HpaII/McrBC)-dependent enrichment procedure. Moderate changes in methylation levels were observed in a subset of promoters following genistein treatment. Detailed investigation of the Ucp1 and Sytl1 promoters further revealed that genistein does not affect de novo methylation occurring between day 0 and day 4, but interferes with subsequent regulatory processes and leads to a decrease in methylation level for both promoters.

Conclusion

Genistein perturbed the methylation pattern of differentiated ES cells after de novo methylation. Our observations suggest that, for a subset of genes, regulation after de novo DNA methylation in the early embryo may be sensitive to genistein.     

Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling

Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.     

组蛋白修饰对胚胎干细胞分化过程[]的调控机制

选用人类胚胎干细胞系和由人类胚胎干细胞系分化来的神经干细胞系为研究对象,分析组蛋白修饰对胚胎干细胞分化过程的调控作用。得到了两种细胞系差异表达基因转录起始位点侧翼区域内八种组蛋白修饰的分布模式,以及组蛋白修饰功能簇。研究表明在两类细胞系中,八种组蛋白修饰谱分布模式一致,且呈现两种分布类型; H3K27ac,H3K4me3和H3K9ac组成的功能簇是保守的;H3K27me3,H3K36me3和H3K79me1组成的功能簇以及H3K9me3和H3K27me3组成的功能簇在胚胎干细胞向神经干细胞分化的过程中消失。结果揭示了组蛋白修饰对胚胎干细胞系向神经干细胞系分化过程的部分调控机制,为该分化过程分子调控机制的研究提供部分重要的理论基础。     

Fluorescence-activated single cell sorting of human embryonic stem cells

Human embryonic stem cells (hESC) are the subject of intense investigation for use in regenerative medicine, in toxicity testing, and as models for the study of human development. Automated cell sorting will enhance the isolation of homogenous pools of differentiated hESCs both for basic studies and for therapeutic applications. Sorting could also be used to deplete undifferentiated, potentially tumourigenic cells. However, hESCs are sensitive to single cell disaggregation and recover poorly when plated at clonal density. Here we report a method for successful semi-automated single cell sorting of hESCs. This method utilizes an ES-specific promoter-transgene construct and automated FACS-based single cell sorting and plating. Clonal recovery in physiologic oxygen (2%) was increased fourfold over room oxygen (21%; p < 0.01). This automated protocol will help to realize proposed hESC strategies that are hampered by low throughput and poor yields.     

Gene profiling on mixed embryonic stem cell populations reveals a biphasic role for beta-catenin in osteogenic differentiation

The differentiation of embryonic stem cells (ESCs) into osteoblasts is enhanced to 60% when exposed to vitamin D3 (VD3) but leaves a remainder of one half of the cell population unidentified. To increase differentiation outcome, the known osteoinducers retinoic acid (RA) and bone morphogenetic protein-2 (BMP-2) were evaluated. Initial studies using RA and BMP-2 during early osteogenesis in addition to VD3 increased osteogenic yield in the case of RA, but surprisingly decreased osteogenesis when BMP-2 was administered together with VD3 or RA. This paper describes a comprehensive microarray study examining the gene expression profile of differentiating osteoblasts in these mixed ESC populations. In addition to five other families of signaling molecules (insulin growth factors, prostaglandin, follistatin, TGFbeta2, and Wnt molecules), we identified an endogenous expression pattern for BMPs and RA that differed from our previous exogenous administration of these molecules. By mimicking the change in expression of the RA and BMP-2 families with exogenous supplementation at the correct time, it was then possible to increase the number of ESC-derived osteoblasts to 90%. This effect was mediated through alteration in beta-catenin (CatnB) expression levels and nuclear CatnB activity, both of which are modulated by VD3, RA, and BMP-2. Our results suggest that blockage of CatnB activity by VD3 and RA is opposed by induction of CatnB activity through BMP-2 when administered together. Hence, osteoinduction, in vitro, is an intricate process involving both temporal and quantitative changes in gene expression and CatnB activity.     

Cathepsin L proteolytically processes histone H3 during mouse embryonic stem cell differentiation

Chromatin undergoes developmentally-regulated structural and chemical changes as cells differentiate, which subsequently lead to differences in cellular function by altering patterns of gene expression. To gain insight into chromatin alterations that occur during mammalian differentiation, we turned to a mouse embryonic stem cell (ESC) model. Here we show that histone H3 is proteolytically cleaved at its N-terminus during ESC differentiation. We map the sites of H3 cleavage and identify Cathepsin L as a protease responsible for proteolytically processing the N-terminal H3 tail. In addition, our data suggest that H3 cleavage may be regulated by covalent modifications present on the histone tail itself. Our studies underscore the intriguing possibility that histone proteolysis, brought about by Cathepsin L and potentially other family members, plays a role in development and differentiation that was not previously recognized.     

Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells

It has been reported that DNA methyltransferase 1-deficient (Dnmt1-/-) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a-/-,3b-/-] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a-/-,3b-/-] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a-/-,3b-/-] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1-/- ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1.     

Hematopoietic differentiation of embryonic stem cells

This review is focused on methods that are used to derive hematopoietic cells from embryonic stem cells (ESCs). One of the strategies that have been recently used to achieve this goal is an approach of mimicking the hematopoietic niche in vitro by using hematopoiesis-supportive feeder cells, cocktails of soluble hematopoietic growth factors and a variety of matrices. While there is clear evidence that it is possible to derive hematopoietic stem cells (HSCs) and subsequently committed hematopoietic progenitors and mature cells from ESCs, there remains the need to address multiple issues including the efficiency of HSCs derivation in vitro and their proper functionality.     

胚胎干细胞向造血系统的分化

胚胎干细胞是指从囊胚期的内细胞团中分离出来的尚未分化的胚胎细胞,可分化形成各种组织类型。在合适的条件下,胚胎干细胞可发育成造血干细胞及各类成熟血细胞,为造血干细胞移植及血细胞输注开辟了新的来源,同时也为造血发生及造血调控研究提供了有效可靠的模型。本文将综述ES细胞向造血系统分化的诱导条件、调控机制及应用前景。     

Chromatin in embryonic stem cell neuronal differentiation

Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.