Genome‐wide screen of promoter methylation analysis of ES cells and ES derived epidermal‐like cells

Embryonic stem cells (ESCs) are a population of pluripotent cells which can differentiate into different cell types. However, there are few reports with regard to differentiate ESCs into epidermal cells in vitro. In this study, we aimed to investigate differentially methylated promoters involved in process of differentiation from ESCs into epidermal‐like cells (ELCs) induced by human amnion. We successfully induced ESCs into ELCs, which expressed the surface markers of CK19, CK15 and β1‐integrin. With MeDIP‐chip arrays, we identified 3435 gene promoters to be differentially methylated, involving 894 HCP (high CpG‐containing promoter), 974 ICP (intermediate CpG‐containing promoter) and 1567 LCP (low CpG‐containing promoter) among all the 17 500 DNA methylation regions of gene promoters in both ESCs and ELCs. Gene oncology and pathway analysis demonstrated that these genes were involved in all the three categories of GO enrichment analysis, including biological process, molecular function and cellular component. All these data suggested that embryonic stem cells can differentiate into epidermal‐like cells and promoter methylation is of great importance in this process. Copyright © 2015 John Wiley & Sons, Ltd.     

多能性胚胎干细胞与DNA甲基化的关系

胚胎干细胞是一种能够维持自我更新、具有无限扩增能力的多能性干细胞。灵长类多能干细胞（iPSCs）根据其发育能力、细胞形态、基因表达谱以及表观遗传学的差异分为初始态多能干细胞（pPSCs）和原始态多能干细胞（nPSCs）。nPSCs因其容易进行基因工程处理以及体内外再生出功能组织器官等优势而在临床潜在应用上备受关注,因而有效维持ESCs的原始状态对其用于基础及临床研究具有重要意义。nPSCs的线粒体活性和自我更新能力高于pPSCs,且这两种多能性干细胞在DNA甲基化等方面都存在明显差别,DNA甲基化在nPSCs的转化及代谢中起到重要的作用。本文综述了DNA甲基化对ESCs的作用,特别是维持原始态的作用。     

DNA methylation in embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent, self‐renewing cells. These cells can be used in applications such as cell therapy, drug development, disease modeling, and the study of cellular differentiation. Investigating the interplay of epigenetics, genetics, and gene expression in control of pluripotence and differentiation could give important insights on how these cells function. One of the best known epigenetic factors is DNA methylation, which is a major mechanism for regulation of gene expression. This phenomenon is mostly seen in imprinted genes and X‐chromosome inactivation where DNA methylation of promoter regions leads to repression of gene expression. Differential DNA methylation of pluripotence‐associated genes such as Nanog and Oct4/Pou5f1 has been observed between pluripotent and differentiated cells. It is clear that tight regulation of DNA methylation is necessary for normal development. As more associations between aberrant DNA methylation and disease are reported, the demand for high‐throughput approaches for DNA methylation analysis has increased. In this article, we highlight these methods and discuss recent DNA methylation studies on ESCs. J. Cell. Biochem. 109: 1–6, 2010. © 2009 Wiley‐Liss, Inc.     

Targeting DNA 5mCpG sites with chimeric endonucleases

Cytosine modification of the dinucleotide CpG in the DNA regulatory region is an important epigenetic marker during early embryo development, cellular differentiation, and cancer progression. In clinical settings, such as anti-cancer drug treatment, it is desirable to develop research tools to characterize DNA sequences affected by epigenetic perturbations. Here, we describe the construction and characterization of two fusion endonucleases consisting of the ⁵mCpG-binding domain of human MeCP2 (hMeCP2) and the cleavage domains of BmrI and FokI restriction endonucleases (REases). The chimeric (CH) endonucleases cleave M.HpaII (C⁵mCGG)—and M.SssI (⁵mCpG)-modified DNA. Unmodified DNA and M.MspI-modified DNA (⁵mCCGG) are poor substrates for the CH-endonucleases. Sequencing cleavage products of modified λ DNA indicates that cleavage takes place outside the ⁵mCpG recognition sequence, predominantly 4-17 bp upstream of the modified base (/N_4-17⁵mCpG, where / indicates the cleavage site). Such ⁵mCpG-specific endonucleases will be useful to study CpG island modification of the regulatory regions of tumor suppressor genes, and for the construction of cell-specific and tumor-specific modified CpG island databases.     

The value-added genome: building and maintaining genomic cytosine methylation landscapes

Epigenetic marks, such as cytosine methylation and post-translational histone modifications, are important for interpreting and managing eukaryotic genomes. Recent genetic studies in plants have uncovered details on the different interwoven mechanisms that are responsible for specification of genomic cytosine methylation patterns. These mechanisms include targeting cytosine methylation using heterochromatic histone modifications and RNA guides. Genomic cytosine methylation patterns also reflect locus-specific demethylation initiated by specialized DNA glycosylases. While genetics continues to more fully define these mechanisms, genomic studies in Arabidopsis have yielded an unprecedented high-resolution view of how epigenetic marks are layered over a genome.     

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

Evidence for an epigenetic role in inbreeding depression

Inbreeding depression (i.e. negative fitness effects of inbreeding) is central in evolutionary biology, affecting numerous aspects of population dynamics and demography, such as the evolution of mating systems, dispersal behaviour and the genetics of quantitative traits. Inbreeding depression is commonly observed in animals and plants. Here, we demonstrate that, in addition to genetic processes, epigenetic processes may play an important role in causing inbreeding effects. We compared epigenetic markers of outbred and inbred offspring of the perennial plant Scabiosa columbaria and found that inbreeding increases DNA methylation. Moreover, we found that inbreeding depression disappears when epigenetic variation is modified by treatment with a demethylation agent, linking inbreeding depression firmly to epigenetic variation. Our results suggest an as yet unknown mechanism for inbreeding effects and demonstrate the importance of evaluating the role of epigenetic processes in inbreeding depression.     

Stem cell therapy: an exercise in patience and prudence

In recent times, the epigenetic study of pluripotency based on cellular reprogramming techniques led to the creation of induced pluripotent stem cells. It has come to represent the forefront of a new wave of alternative therapeutic approaches in the field of stem cell therapy. Progress in drug development has saved countless lives, but there are numerous intractable diseases where curative treatment cannot be achieved through pharmacological intervention alone. Consequently, there has been an unfortunate rise in incidences of organ failures, degenerative disorders and cancers, hence novel therapeutic interventions are required. Stem cells have unique self-renewal and multilineage differentiation capabilities that could be harnessed for therapeutic purposes. Although a number of mature differentiated cells have been characterized in vitro, few have been demonstrated to function in a physiologically relevant context. Despite fervent levels of enthusiasm in the field, the reality is that other than the employment of haematopoietic stem cells, many other therapies have yet to be thoroughly proven for their therapeutic benefit and safety in application. This review shall focus on a discussion regarding the current status of stem cell therapy, the issues surrounding it and its future prospects with a general background on the regulatory networks underlying pluripotency.     

Human AP endonuclease possesses a significant activity as major 3'-5' exonuclease in human leukemia cells

Apurinic/apyrimidinic (AP) endonuclease (Ape1) is the major cellular enzyme responsible for repairing AP-sites in DNA. It can cleave the DNA phosphodiester backbone immediately 5(') to an AP-site. Ape1 also shows 3(')-phosphodiesterase activity, a 3(')-phosphatase activity, and an RNaseH activity. However, regarding its exonuclease activity, it remains controversial whether human Ape1 may possess a 3(')-5(') exonuclease activity. During the course of study to search for the major nuclease activity to double-stranded DNA in human leukemia cells, we purified a 37 kDa Mg(2+)-dependent exonuclease from cytosolic fraction of human leukemia U937 cells. Surprisingly, this exonuclease is Ape1. We demonstrated for the first time that Ape1 possesses a significant activity as major 3(')-5(') exonuclease in human leukemia cells. In addition, we also observed that translocation of cytoplasmic Ape1 into nucleus occurs during DNA damage.     

The expanding field of poly(ADP-ribosyl)ation reactions. 'Protein Modifications: Beyond the Usual Suspects' Review Series

Poly(ADP-ribosyl)ation is a post-translational modification of proteins that is mediated by poly(ADP-ribose) polymerases (PARPs). Although the existence and nature of the nucleic acid-like molecule poly(ADP-ribose) (PAR) has been known for 40 years, understanding its biological functions--originally thought to be only the regulation of chromatin superstructure when DNA is broken--is still the subject of intense research. Here, we review the mechanisms controlling the biosynthesis of this complex macromolecule and some of its main biological functions, with an emphasis on the most recent advances and hypotheses that have developed in this rapidly growing field.     

综述: 表观遗传学研究的模式生物——粗糙脉孢菌

丝状真菌粗糙脉孢菌是一种作为遗传学研究的经典模式生物．通过对粗糙脉孢菌5S rRNA基因的组成和在染色体上分布的研究,揭示了丝状真菌中存在的一种基因组防御机制——重复序列诱导的DNA点突变(RIP)．通过对发生突变的5S rRNA假基因的研究还发现,粗糙脉孢菌中存在一种重要的表观遗传修饰——DNA甲基化,随后的深入研究使粗糙脉孢菌成为解析DNA甲基化机制的最重要模式生物之一．粗糙脉孢菌基因转化操作引起的营养生长阶段同源基因的沉默(quelling)是由RNAi途径调控的,同时该途径也是调控减数分裂过程中非配对DNA诱发的基因沉默(meiotic silencing)的关键．由于粗糙脉孢菌基因组简单,且存在与高等真核生物相同的DNA甲基化和多种组蛋白的修饰,使其成为今后深入研究组蛋白修饰与染色质重塑等表观遗传现象参与基因表达调控和基因组稳定性维持的重要模式生物之一．