UHRF1 plays a role in maintaining DNA methylation in mammalian cells

Epigenetic inheritance in mammals relies in part on robust propagation of DNA methylation patterns throughout development. We show that the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), also known as NP95 in mouse and ICBP90 in human, is required for maintaining DNA methylation. UHRF1 colocalizes with the maintenance DNA methyltransferase protein DNMT1 throughout S phase. UHRF1 appears to tether DNMT1 to chromatin through its direct interaction with DNMT1. Furthermore UHRF1 contains a methyl DNA binding domain, the SRA (SET and RING associated) domain, that shows strong preferential binding to hemimethylated CG sites, the physiological substrate for DNMT1. These data suggest that UHRF1 may help recruit DNMT1 to hemimethylated DNA to facilitate faithful maintenance of DNA methylation.     

Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation

Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.     

硒缺乏对鸡肌肉组织DNA甲基化水平的影响

选取健康1日龄雏鸡200只,随机分为2组,低硒组(饲料硒含量为0.033 mg·kg-1)和对照组(饲料硒含量为0.15 mg·kg-1),分别于0、15、25、35、45、55日龄取胸肌、翅肌、腿肌组织,高效液相色谱法检测DNA总甲基化水平,实时荧光定量PCR方法检测DNA甲基转移酶1(DNMT1)、DNA甲基转移酶3A(DNMT3A)、DNA甲基转移酶3B(DNMT3B)mRNA表达水平.结果表明,DNA总甲基化水平整体呈降低趋势,低硒组与对照组相比DNMT1、DNMT3A mRNA表达量先增加后减少,DNMT3B各组均低于正常组并且随日龄增加组内呈降低趋势.硒缺乏可导致鸡肌肉组织DNA甲基化水平降低.     

Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2

The sequence and the structure of DNA methyltransferase-2 (Dnmt2) bear close affinities to authentic DNA cytosine methyltransferases. A combined genetic and biochemical approach revealed that human DNMT2 did not methylate DNA but instead methylated a small RNA; mass spectrometry showed that this RNA is aspartic acid transfer RNA (tRNA(Asp)) and that DNMT2 specifically methylated cytosine 38 in the anticodon loop. The function of DNMT2 is highly conserved, and human DNMT2 protein restored methylation in vitro to tRNA(Asp) from Dnmt2-deficient strains of mouse, Arabidopsis thaliana, and Drosophila melanogaster in a manner that was dependent on preexisting patterns of modified nucleosides. Indirect sequence recognition is also a feature of eukaryotic DNA methyltransferases, which may have arisen from a Dnmt2-like RNA methyltransferase.     

Aconitase couples metabolic regulation to mitochondrial DNA maintenance

Mitochondrial DNA (mtDNA) is essential for cells to maintain respiratory competency and is inherited as a protein-DNA complex called the nucleoid. We have identified 22 mtDNA-associated proteins in yeast, among which is mitochondrial aconitase (Aco1p). We show that this Krebs-cycle enzyme is essential for mtDNA maintenance independent of its catalytic activity. Regulation of ACO1 expression by the HAP and retrograde metabolic signaling pathways directly affects mtDNA maintenance. When constitutively expressed, Aco1p can replace the mtDNA packaging function of the high-mobility-group protein Abf2p. Thus, Aco1p may integrate metabolic signals and mtDNA maintenance.     

CpG DNA对鸡传染性喉气管炎病毒DNA疫苗免疫效果的影响

 将分别构建的含有鸡传染性喉气管炎病毒（ＩＬＴＶ）王岗株ｇＢｇＣ和ｇＤ基因的重组真核表达质粒及ＣＰＧＤＮＡ佐剂分组肌肉注射 ＳＰＦ鸡，检测了免疫后的抗体水平，并观测了攻毒后的免疫保护效果。实验结果显示，ＣｐＧＤＮＡ佐剂和ＤＮＡ疫苗联合免疫后的抗体水平比单一使用ＤＮＡ疫苗的要高，而佐剂组的发病率、死亡率均低于非佐剂组，保护率则高于非佐剂组。这表明 ＣｐＧ ＤＮＡ佐剂增强了 ＩＬＴＶ ＤＮＡ疫苗的免疫效果。     

CpG—DNA特征结构与其免疫刺激特性的关系

CpG-DNA是一些具有免疫激活功能的以未甲基化的CpG基序为核心的DNA序列,它包括含CpG基序的人工合成的寡聚脱氧核苷酸(oligodeoxynucleotides,ODN)和自然界中细菌、病毒、无脊椎动物等低等生物的基因组DNA。CpG基序(CpG motifs)是指一类以非甲基化的胞嘧啶和鸟嘌呤核苷酸为核心的寡聚脱氧核糖核苷酸,其碱基排列大多遵循以下规律:5’端为2个嘌呤,3’端为2个嘧啶。研究表明,这种序列可激活多种免疫效应细胞,其特征结构如CpG核心、侧翼序列、骨架长度等都对其免疫刺激特性有重要影响。本文就其特征结构与免疫刺激特性的关系作一介绍。     

cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.     

Epigenetic reprogramming in mammalian development

DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of its function. Genomic methylation patterns in somatic differentiated cells are generally stable and heritable. However, in mammals there are at least two developmental periods-in germ cells and in preimplantation embryos-in which methylation patterns are reprogrammed genome wide, generating cells with a broad developmental potential. Epigenetic reprogramming in germ cells is critical for imprinting; reprogramming in early embryos also affects imprinting. Reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals, and in the erasure of acquired epigenetic information. A role of reprogramming in stem cell differentiation is also envisaged. DNA methylation is one of the best-studied epigenetic modifications of DNA in all unicellular and multicellular organisms. In mammals and other vertebrates, methylation occurs predominantly at the symmetrical dinucleotide CpG (1-4). Symmetrical methylation and the discovery of a DNA methyltransferase that prefers a hemimethylated substrate, Dnmt1 (4), suggested a mechanism by which specific patterns of methylation in the genome could be maintained. Patterns imposed on the genome at defined developmental time points in precursor cells could be maintained by Dnmt1, and would lead to predetermined programs of gene expression during development in descendants of the precursor cells (5, 6). This provided a means to explain how patterns of differentiation could be maintained by populations of cells. In addition, specific demethylation events in differentiated tissues could then lead to further changes in gene expression as needed. Neat and convincing as this model is, it is still largely unsubstantiated. While effects of methylation on expression of specific genes, particularly imprinted ones (7) and some retrotransposons (8), have been demonstrated in vivo, it is still unclear whether or not methylation is involved in the control of gene expression during normal development (9-13). Although enzymes have been identified that can methylate DNA de novo (Dnmt3a and Dnmt3b) (14), it is unknown how specific patterns of methylation are established in the genome. Mechanisms for active demethylation have been suggested, but no enzymes have been identified that carry out this function in vivo (15-17). Genomewide alterations in methylation-brought about, for example, by knockouts of the methylase genes-result in embryo lethality or developmental defects, but the basis for abnormal development still remains to be discovered (7, 14). What is clear, however, is that in mammals there are developmental periods of genomewide reprogramming of methylation patterns in vivo. Typically, a substantial part of the genome is demethylated, and after some time remethylated, in a cell- or tissue-specific pattern. The developmental dynamics of these reprogramming events, as well as some of the enzymatic mechanisms involved and the biological purposes, are beginning to be understood. Here we look at what is known about reprogramming in mammals and discuss how it might relate to developmental potency and imprinting.     

鸭MyoG基因启动子的生物信息学分析及其甲基化频率对基因表达的影响

采用RT–PCR技术扩增和克隆鸭Myo G基因启动子,并对其启动子序列进行生物信息学分析,采用Sequenom Mass Array技术检测Cp G岛在鸭肌肉组织中的甲基化水平,用q RT–PCR检测Myo G基因的表达量。结果表明,扩增得到鸭Myo G基因启动子序列2 730 bp,对启动子序列预测后,发现存在2个Cp G岛,其中Cp G岛(–2 536~–1 997 bp)存在5个转录因子结合位点和多个真核生物结构元件。甲基化检测结果表明:在鸭的个体和组织水平上,启动子甲基化率均未聚类在一起;Cp G位点甲基化频率存在个体差异,22%Cp G位点的甲基化频率与Myo G的m RNA表达量呈负相关(P0.05),78%Cp G位点的甲基化频率呈正相关(P0.05),其中,腿肌甲基化位点Cp G_1、Cp G_26.27.28.29的甲基化频率与Myo G基因表达水平均呈显著相关(P0.05)。Myo G基因在鸭与在哺乳动物中的转录调控机制存在差异。试验中发现多个影响鸭Myo G基因转录的潜在甲基化位点,其中Cp G_1与Cp G_26.27.28.29能通过DNA甲基化修饰影响Myo G基因在鸭腿肌中的转录。本研究结果可为鸭Myo G基因转录调控提供参考依据。     

牛Dgat1基因高GC含量片段的定点突变

将扩增出的1.8 kb目的片段克隆入pUCm-T载体,以构建好的质粒为模板进行突变反应。DpnⅠ酶切反应产物,去除甲基化及半甲基化DNA模板。酶切产物经纯化后转入DH5α感受态细胞,提取质粒测序。结果表明,双核苷酸碱基AA突变为GC,成功得到突变载体。将定点突变试剂盒方法加以改进,完成GC含量高达69.5%模板定点突变,改进后的方法操作简单、经济实用,有效地解决了高GC含量模板难突变难题。     

Genomic sequencing and methylation analysis by ligation mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.     

Enhanced CpG mutability and tumorigenesis in MBD4-deficient mice

The mammalian protein MBD4 contains a methyl-CpG binding domain and can enzymatically remove thymine (T) or uracil (U) from a mismatched CpG site in vitro. These properties suggest that MBD4 might function in vivo to minimize the mutability of 5-methylcytosine by removing its deamination product from DNA. We tested this hypothesis by analyzing Mbd4-/- mice and found that the frequency of of C --> T transitions at CpG sites was increased by a factor of three. On a cancer-susceptible Apc(Min/+) background, Mbd4-/- mice showed accelerated tumor formation with CpG --> TpG mutations in the Apc gene. Thus MBD4 suppresses CpG mutability and tumorigenesis in vivo.     

含CpG序列PRRSV SD2 E基因真核表达载体质粒构建及表达

为了提高猪生殖与呼吸综合征病毒山东株(PRRSV SD2株)基因免疫的效果,将PRRSV SD2 E基因插入哺乳动物表达载体PVAX1中,构建出PVAX1-E质粒。再将已筛选出的CpG-ODN序列通过在其两端的粘性酶切末端定向插入含PVAX 1-E的真核表达载体,构建含CpG基因序列真核重组表达质粒CpG-pVAX 1-E。将重组质粒转染COS-7细胞,经RT-PCR检测E基因mRNA的转录和间接免疫荧光试验(IFA)证实:CpG-pVAX 1-E可表达PRRSV GP5蛋白。试验结果为进一步研究PRRSV SD2 ORF5动物免疫反应奠定基础。     

组织培养紫叶酢浆草DNA甲基化的研究

紫叶酢浆草是重要的观赏植物,是较早建立较为完善组织培养系统之一.本研究提取经组织培养获得的紫叶酢浆草基因组DNA,利用Southern杂交,分析了5S rDNA位点的DNA甲基化情况.结果表明,紫叶酢浆草的5S rDNA位点的CpG高度甲基化,而CNG的甲基化状况则较低.