Weak interaction induces an ON/OFF switch, whereas strong interaction causes gradual change: folding transition of a long duplex DNA chain by poly-L-lysine

A large-scale conformational change in genomic DNA is an essential feature of gene activation in living cells. Considerable effort has been applied to explain the mechanism in terms of key-lock interaction between sequence-specific regulatory proteins and DNA, in addition to the modification of DNA and histones such as methylation and acetylation. However, it is still unclear whether these mechanisms can explain the ON/OFF switching of a large number of genes that accompanies differentiation, carcinogenesis, etc. In this study, using single-molecule observation of DNA molecules by fluorescence microscopy with the addition of poly-L-lysine with different numbers of monomer units (n = 3, 5, 9, and 92), we found that an ON/OFF discrete transition in the higher-order structure of long duplex DNA is induced by short poly-L-lysine, whereas a continuous gradual change is induced by long poly-L-lysine. On the other hand, polycations with a lower positive charge have less potential to induce DNA compaction. Such a drastic difference in the conformational transition of a giant DNA between short and large oligomers is discussed in relation to the mechanisms of gene regulation in a living cell.     

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。     

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.     

真核生物的V形基因调控模型和基因调控关系的预测

我们研究发现,有些果蝇基因的外显子和插入序列的碱基组成没有明显区别.这种基因编码的蛋白质C-末端氨基酸序列与其转录的mRNA尾随片段高度亲和.这种母基因与其编码的子蛋白质高度亲和的特性,我们称为母子相亲机制.用这些基因的尾随片段进行同源搜索,然后再分析同源序列所在位置的RNA结构,结果发现:1)位于基因启动子位置的,其结构为茎环结构,说明该同源序列是基因启动子的重要组成部分;2)位于插入序列中的,出现了一种象钥匙一样的结构,它的一端为回文形成的茎环结构,为钥匙的柄,另一端为单链结构,为钥匙的舌.这两种序列可能参与了基因的调控.由此,我们建立了一个的V形的基因调控模型.这个模型包括3个主体:被控基因、编程基因、启动基因.编程基因提供蛋白质与被调控基因的启动子序列结合,决定被调控基因是否可以表达的.而启动基因转录成的RNA通过剪切等加工,产生一种结构象钥匙的microRNA(miRNA),它可以启动被调基因.根据母子相亲机制和V形调控模型,以及回文规则,我们设计出了一种尾随片段搜索筛选法,预测真核生物基因的互控关系.     

Oscillatory protein expression dynamics endows stem cells with robust differentiation potential

The lack of understanding of stem cell differentiation and proliferation is a fundamental problem in developmental biology. Although gene regulatory networks (GRNs) for stem cell differentiation have been partially identified, the nature of differentiation dynamics and their regulation leading to robust development remain unclear. Herein, using a dynamical system modeling cell approach, we performed simulations of the developmental process using all possible GRNs with a few genes, and screened GRNs that could generate cell type diversity through cell-cell interactions. We found that model stem cells that both proliferated and differentiated always exhibited oscillatory expression dynamics, and the differentiation frequency of such stem cells was regulated, resulting in a robust number distribution. Moreover, we uncovered the common regulatory motifs for stem cell differentiation, in which a combination of regulatory motifs that generated oscillatory expression dynamics and stabilized distinct cellular states played an essential role. These findings may explain the recently observed heterogeneity and dynamic equilibrium in cellular states of stem cells, and can be used to predict regulatory networks responsible for differentiation in stem cell systems.     

Mutagenic specificity in DNA base sequence by irradiation of health lamp light (UV-B) in Escherichia coli.

A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene, was irradiated with health lamp (HL) light containing UV-B. Plasmid mutations were scored by transforming an indicator strain of Escherichia coli carrying a suppressive blue amber mutation in the beta-galactosidase gene. Plasmid survival was also measured by transforming activity of the indicator strain. The majority of mutations induced by HL light were GC-AT transitions (69%) and the rest were transversions (31%). Some hot-spots in the mutations were observed by sequencing the suppressor gene. Mutagenic specificity in DNA base sequences induced by HL in E. coli agrees well with previous reports about 254-nm or 313-nm light effects on mammalian cells. This agreement may depend on the substitution of the inserted base instead of a G residue at the opposite site of a damaged C residue from conformational change of DNA structure in both bacterial and mammalian cells.     

Functional neuronal cells generated by human parthenogenetic stem cells

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.     

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.     

Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells

Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation.