玉米MuDR转座子DNA甲基化的MSP检测

DNA甲基化作为一种重要的表观遗传修饰,广泛存在于高等动植物中,并在维持基因组稳定性、调节基因表达等方面起着重要作用,因此建立快速有效地DNA甲基化检测技术至关重要.本文以两种不同MuDR活性的玉米转座子材料为研究对象, 探讨了甲基化特异性PCR(MSP)在检测DNA甲基化的有效性.结果表明: MSP技术可快速有效地检测MuDR转座子的末端反向重复(TIRs)序列内的CpG岛DNA甲基化的变化,灵敏度高,特异性强,可作为植物已知基因DNA甲基化检测的一种新方法.同时利用MSP研究发现,玉米MuDR转座子的活性随其TIRs序列内的CpG岛DNA甲基化的变化而改变, DNA甲基化是调控玉米MuDR转座活性的重要分子机制之一.     

基因组差异甲基化片段筛选技术

分离和鉴定细胞之间的差异甲基化片段,不仅有助于了解基因的功能、分离疾病相关基因,而且可以发现与细胞分化或病变相关的甲基化标记。目前筛选差异甲基化DNA片段的方法主要有：甲基化敏感的限制性界标基因组扫描、甲基化敏感的代表性差异分析、甲基化敏感的限制性指纹技术、甲基化CpG岛扩增．代表性差异分析、微阵列技术等。其中微阵列法又先后建立有CpG岛微阵列、寡核苷酸微阵列和表达CpG岛序列标签微阵列。这些方法各有特点和适用范围,应根据具体研究目的和工作条件进行恰当的选择。     

亚硫酸氢钠测序法检测水稻FIE基因CpG岛甲基化状态

建立了适用于水稻基因组特定基因甲基化检测的亚硫酸氢钠测序法,并利用此方法对FIE2A基因CpG岛部分片段的甲基化差异进行了研究。采用CTAB法提取水稻叶片和胚乳细胞的基因组DNA,经亚硫酸氢钠化学修饰后,针对已修饰的FIE基因序列设计特异引物并结合巢式PCR扩增,TA载体克隆、测序,最后对测序结果进行分析。结果表明巢式PCR能够增加特异性产物的产生,FIE基因CpG岛在对称的CG和CNG位点甲基化水平较高,而在非对称CNN位点甲基化水平最低,此外在叶片中的平均甲基化水平较高。由此表明本研究建立的亚硫酸氢钠测序法适用于水稻基因组特定基因甲基化状态的检测。     

结直肠癌中DNA甲基化研究进展

CpG岛是人类基因组中富含CpG二核苷酸的DNA序列,主要位于基因启动子区,大小约为100-1000bp,与约60％编码基因相关。DNA中CpG岛甲基化可导致抑癌基因的表观遗传学转录失活,直接参与肿瘤的发生机制。近年来,甲基化已成为表观遗传学研究的焦点。我们简要综述了DNA甲基化在结直肠癌中的研究进展。     

DNA甲基化与基因表达调控研究进展

表观遗传修饰是指不改变DNA序列的、可遗传的对碱基和组蛋白的化学修饰,主要包括DNA甲基化、组蛋白修饰、染色质重塑以及非编码RNA等.表观遗传修饰是更高层次的基因表达调控手段.DNA甲基化是一种重要的表观遗传修饰,参与基因表达调控、基因印记、转座子沉默、X染色体失活以及癌症发生等重要生物学过程.近年来随着研究方法和技术的进步,全基因组DNA甲基化的研究广泛兴起,多个物种全基因组甲基化图谱被破译,全局水平对DNA甲基化的研究不仅利于在宏观层面上了解DNA甲基化的特性与规律,同时也为深入分析DNA甲基化的生物学功能与调控奠定了基础.结合最新研究进展综述DNA甲基化在基因组中的分布模式、规律以及和基因转录的关系等.     

DNA甲基化微阵列

DNA甲基化微阵列是近年发展起来的高通量分析基因组水平DNA甲基化状态和模式的新型技术,已成为肿瘤表观遗传学组研究的重要工具之一。利用DNA甲基化微阵列研究某种疾病状态下异常甲基化的基因有利于进一步明确该疾病的表观遗传学异常机制,发现与之相关的表观遗传学标志物。现有的DNA甲基化微阵列主要包括CpG岛微阵列和甲基化寡聚核苷探针微阵列,根据已有的文献资料,较为详细地阐述了上述技术的原理、特点和适用范围,对于研究者根据自己的研究目的选择适当的DNA甲基化微阵列技术具有一定的指导价值。     

人类全基因组范围的CpG岛的预测与分析

CpG岛的甲基化是表观遗传中基因表达调控的重要机制。虽然目前已存在几个从DNA序列判别CpG岛的标准,但如何在标准中选择合适的参数仍是研究的焦点。文章通过分析比较两种经典CpG岛判定标准与三种预测方法,提出了改进的CpG岛预测方法——CpGISeeker。应用该预测方法,结合判定标准中的三个基本参数组合出的13组组合参数,在人类全基因组范围内进行了CpG岛预测,并统计分析了CpG岛的重复序列组成以及相对于基因转录起始位点的位置分布情况。分析结果表明CpGISeeker具有更精确判定CpG岛的特性;同时还提示,随着判定标准严格性的增加,CpG岛的重复序列含量降低,与基因转录起始位点的相关性提高。将CpG岛最小尺寸为500bp、GC含量为60%、CpG出现率达到0.65的组合参数作为标准,是目前预测CpG岛的最佳方式。     

CpG甲基化与基因调控

CpG双核苷酸中的胞嘧啶甲基化和去甲基化在哺乳动物的基因表达中有重要的调控作用.哺乳动物基因组中有两类启动子:CpG岛启动子和CpG缺乏启动子.两种蛋白质因子通过与甲基化CpG的相互作用影响基因表达,CpG岛在基因组分析中也有广泛的用途.     

牛组织中MBD1基因表达及其DNA甲基化调节

DNA甲基化是主要的表观遗传调节方式,在转录水平调节基因的表达,甲基化CpG结合蛋白MBD1能够结合甲基化及非甲基化的DNA,通过抑制域抑制基因的转录,在DNA甲基化和转录抑制之间起重要作用,但DNA甲基化对MBD1自身的调节作用还不清楚.本研究首先利用RT-PCR检测成年牛心脏、肾脏、肝脏、睾丸及卵巢5种组织中MBD1基因mRNA的表达;并根据牛MBD1调节区序列,针对其中的12个CpG位点设计引物,利用甲基化PCR测序分析方法,分析该调节区的DNA甲基化状态在牛5种组织中的变化.结果表明,在牛的5种组织中,MBD1基因在心脏和肾脏的表达量低于肝脏、睾丸及卵巢,且差异显著(P＜0.05);DNA甲基化检测显示,心脏和肾脏MBD1调节区的甲基化比率较肝脏、睾丸及卵巢甲基化低,说明调控区DNA甲基化与MBD1基因的组织特异性表达相关.     

应用亚硫酸氢盐修饰后PCR联合TA克隆测序对E-cadherin启动子区甲基化水平的检测

E-cadherin是一种细胞粘附因子,通过增强细胞之间的粘附而起到抑制肿瘤转移的作用.Ecadherin基因启动子区的高甲基化是导致其在众多肿瘤细胞中表达下调甚至缺失的主要原因之一.本实验首先抽提SGC-7901细胞(胃腺癌细胞)、A549细胞(肺腺癌细胞)、MCF-7细胞(乳腺癌细胞)等3个肿瘤细胞株的全基因组DNA,然后对抽提的DNA进行亚硫酸氢盐修饰和纯化回收,根据修饰后的DNA序列设计引物并对其进行PCR扩增.然后将PCR扩增产物与pUC-T TA载体连接并转化入感受态大肠杆菌DH5α中进行培养,对筛选出的含有阳性重组子的菌落进行测序.测序结果显示,3个肿瘤细胞株的E-cadherin基因启动子区的CpG岛都呈现了高度的甲基化,亚硫酸氢盐的修饰效率达到了99.2%.综上研究表明,亚硫酸氢盐修饰后PCR(BSP)联合TA克隆测序可以对肿瘤细胞某基因启动子区CpG岛的甲基化水平进行精确量化,研究所使用的3个肿瘤细胞株均可作为研究肿瘤细胞E-cadherin基因甲基化的细胞模型.     

Densely methylated sequences that are preferentially localized at telomere-proximal regions of human chromosomes

We have constructed a library of densely methylated DNA sequences from human blood DNA by selecting fragments with a high affinity for a methyl-CpG binding domain (MBD) column. PCR analysis of the library confirmed the presence of known densely methylated CpG island sequences. Analysis of random clones, however, showed that the library was dominated by sequences whose G+C content and CpG frequency were intermediate between those of bulk genomic DNA and bona fide CpG islands. When human chromosomes were probed with the library by fluorescent in situ hybridisation (FISH), the predominant sites of labelling were at terminal regions of many chromosomes, approximately corresponding to T-bands. Analysis of the methylation status of random clones indicated that all were heavily methylated at CpGs in blood DNA, but many were under-methylated in sperm DNA. Lack of methylation in germ cells may reduce CpG depletion at some sub-terminal sequences and result in a high density of methyl-CpG when these regions become methylated in somatic cells.     

The Chromosomal Distribution of CpG Islands in the Mouse: Evidence for Genome Scrambling in the Rodent Lineage

We have prepared a library of mouse whole CpG islands using a methyl-CpG binding domain column. The distribution of CpG islands in the mouse genome was determined by FISH, using the library as a probe. Unlike in other vertebrate genomes that have been examined (human and chicken), extreme clustering of CpG islands was not seen in the mouse genome. No individual murine chromosome stood out as being either very gene-rich or very gene-poor. Despite the more even distribution of CpG islands in the mouse at a gross chromosomal level, at finer resolution concentrations of CpG islands are seen to correspond to the R-band early replicating regions of the genome.     

Specific protection of methylated CpGs in mammalian nuclei

We have compared nuclear accessibility of methylated and nonmethylated sequences using restriction enzymes. MspI, which cuts CpG sites in naked DNA regardless of methylation, cut DNA in intact mouse liver or brain nuclei almost exclusively at CpG islands. Bulk chromatin was not significantly cleaved by MspI but was cleaved extensively by enzymes that do not recognize CpG. Quantitative analysis of limit digests showed that MspI and another methyl-CpG insensitive enzyme, Tth, have a strong bias against cutting methylated sites in these nuclei. Southern analysis confirmed this at three genomic loci. Our results suggest that resistance to nucleases is mediated by factors that are bound specifically to methylated CpGs. MeCP, a protein that binds to methylated DNA in vitro, may be one such factor, since nuclease resistance was significantly reduced in an MeCP-deficient cell line.     

Primate CpG islands are maintained by heterogeneous evolutionary regimes involving minimal selection

Mammalian CpG islands are key epigenomic elements that were first characterized experimentally as genomic fractions with low levels of DNA methylation. Currently, CpG islands are defined based on their genomic sequences alone. Here, we develop evolutionary models to show that several distinct evolutionary processes generate and maintain CpG islands. One central evolutionary regime resulting in enriched CpG content is driven by low levels of DNA methylation and consequentially low rates of CpG deamination. Another major force forming CpG islands is biased gene conversion that stabilizes constitutively methylated CpG islands by balancing rapid deamination with CpG fixation. Importantly, evolutionary analysis and population genetics data suggest that selection for high CpG content is not?a significant factor contributing to conservation of CpGs in differentially methylated regions. The heterogeneous, but not selective, origins of CpG islands have direct implications for the understanding of DNA methylation patterns in healthy and diseased cells.     

Methylome profiling of cancer cells by amplification of inter-methylated sites (AIMS)

Alterations of the DNA methylation pattern have been related to generalized chromosomal disruption and inactivation of multiple tumor suppressor genes in neoplasia. To screen for tumor-specific alterations and to make a global assessment of methylation status in cancer cells, we have modified the methylated CpG island amplification method to generate easily readable fingerprints representing the cell’s DNA methylation profile. The method is based on the differential cleavage of isoschizomers with distinct methylation sensitivity. Specific adaptors are ligated to the methylated ends of the digested genomic DNA. The ligated sequences are amplified by PCR using adaptor- specific primers extended at the 3′ end with two to four arbitrarily chosen nucleotidic residues to reduce the complexity of the product. Fingerprints consist of multiple anonymous bands, representing DNA sequences flanked by two methylated sites, which can be isolated and individually characterized. Hybridization of the whole product to metaphase chromosomes revealed that most bands originate from the isochore H3, which identifies the regions of the genome with the highest content of CpG islands and genes. Comparison of the fingerprints obtained from normal colon mucosa, colorectal carcinomas and cell lines revealed tumor-specific alterations that are putative recurrent markers of the disease and include tumor-specific hypo- and hypermethylations.     

Linking the epigenome to the genome: correlation of different features to DNA methylation of CpG islands

DNA methylation of CpG islands plays a crucial role in the regulation of gene expression. More than half of all human promoters contain CpG islands with a tissue-specific methylation pattern in differentiated cells. Still today, the whole process of how DNA methyltransferases determine which region should be methylated is not completely revealed. There are many hypotheses of which genomic features are correlated to the epigenome that have not yet been evaluated. Furthermore, many explorative approaches of measuring DNA methylation are limited to a subset of the genome and thus, cannot be employed, e.g., for genome-wide biomarker prediction methods. In this study, we evaluated the correlation of genetic, epigenetic and hypothesis-driven features to DNA methylation of CpG islands. To this end, various binary classifiers were trained and evaluated by cross-validation on a dataset comprising DNA methylation data for 190 CpG islands in HEPG2, HEK293, fibroblasts and leukocytes. We achieved an accuracy of up to 91% with an MCC of 0.8 using ten-fold cross-validation and ten repetitions. With these models, we extended the existing dataset to the whole genome and thus, predicted the methylation landscape for the given cell types. The method used for these predictions is also validated on another external whole-genome dataset. Our results reveal features correlated to DNA methylation and confirm or disprove various hypotheses of DNA methylation related features. This study confirms correlations between DNA methylation and histone modifications, DNA structure, DNA sequence, genomic attributes and CpG island properties. Furthermore, the method has been validated on a genome-wide dataset from the ENCODE consortium. The developed software, as well as the predicted datasets and a web-service to compare methylation states of CpG islands are available at http://www.cogsys.cs.uni-tuebingen.de/software/dna-methylation/.     

Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.     

A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions

We developed a method for site-selective CpG methylation of the budding yeast genome. The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site. Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site. Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo. This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation.