MethyScan：一种甲基化特异性PCR引物设计及评估工具

DNA甲基化是重要的表观遗传现象,对基因表达发挥重要调控功能.大量研究表明,基因DNA甲基化是重要的临床诊断生物标志物.在临床上,实施快速、准确的DNA甲基化状态检测是诊断应用的前提和关键.甲基化特异性PCR(methylation specific PCR,MSP)通过将两种引物与甲基化、非甲基化模板各自特异性结合和扩增,实现基因甲基化状态的区分,是切实可行、简单便捷的临床诊断实验技术.但是,不同于常规PCR,MSP主要存在如何强化引物-甲基化/非甲基化模板特异性结合、降低引物序列Tm值差异、去除假阳性扩增及提高敏感性等四大难点.尽管大多数MSP引物设计软件对上述难题都提出了各自解决办法,但在引物设计影响因素考虑、设计与评估并行处理及特异性扩增预测等方面仍然存在较大缺陷.为此,本研究通过对MethPrimer、MSPPrimer、MethBlast、BiSearch等现有MSP引物设计软件原理的深入探究,以及对Bowtie、SAMtools和BEDTools等工具的有效综合整合,基于图形库Matplotlib和第三方Python功能库BioPython与Primer3-py实现了具有系列优点的甲基化特异性PCR引物设计与评估可视化工具MethyScan.它具有引物设计、基因组索引、引物评估等三大完整功能模块,不仅可快速进行MSP引物设计,实现巢式(Nested)引物适配,还可基于4种基因组碱基转换模板分析引物结合信息,图形化展示非特异性扩增与目的片段差异,从而综合评估引物特异性-非特异性扩增.同时,对食管癌、结直肠癌等多种恶性肿瘤中6个潜在生物标志物TFPI-2、NDRG4、CDKN2A、CD44、CASP8和SDHD的甲基化引物设计对比结果表明,MethyScan不仅可获得更多CpG位点的检测引物,而且所获得MSP引物位置与其他软件结果相同或相近,且引物间Tm值差值更小.总之,作为首个图形化展示特异性-非特异性扩增差异MSP引物设计工具,MethyScan可有效提高甲基化引物设计准确性,为临床DNA甲基化检测项目开展、检测试验实施及诊断试剂盒研发提供有力支撑.MethyScan工具下载地址:https://github.com/bioinfo-ibms-pumc/MethyScan.     

一种快速高效的全基因组甲基化CpG岛富集方法的建立

高甲基化的CpG岛所致基因表观遗传学转录失活已经成为肿瘤表观基因组学研究的重要内容。现在已有很多检测CpG岛甲基化的方法,但由于各自的局限,还没有建立一种能快速在全基因组水平上进行甲基化CpG岛的富集方法。本研究利用甲基化结合蛋白MBD2b具有特异性结合甲基化DNA的特性, 建立了一种基于DNA免疫共沉淀技术的全基因组甲基化CpG岛的富集方法。在大肠杆菌中表达重组的GST-MBD2b蛋白,通过Glutathione Sepharose 4B对重组蛋白进行纯化,制备成亲和层析柱,利用在不同的盐离子强度下甲基化DNA和非甲基化DNA的结合能力不同,对甲基化DNA进行富集。用甲基化酶SssI处理过的DNA片段与非甲基化DNA片段进行富集效率的检测,发现0.5M KCl的浓度是甲基化DNA片段和非甲基化DNA片段得以分开的临界条件。样品的富集效率用Real Time PCR进行检测。结果表明,这种方法能够实现对全基因组甲基化DNA的有效富集且最高的富集倍数可达到100多倍。富集到的甲基化DNA可以进行后续的定量PCR, DNA测序和全基因组芯片的分析等工作,为大规模分析全基因组CpG 岛甲基化的改变奠定了基础。     

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。     

利用甲基化特异性引物高通量检测DNA甲基化

建立一种基于甲基化特异性引物和SAGE技术的高通量DNA甲基化定量检测新方法（MSP-SAGE）,首先利用亚硫酸氢钠对基因组DNA进行处理,使未甲基化的C转变为U,而甲基化的CpG不变.将处理和未处理的DNA双链变性后用随机引物PNNNNCG对存在含有CG的单链进行延伸,而无甲基化CG的单链处则不能延伸;将差异延伸的单链序列和频次信息经过系列分子操作后,引入PCR扩增模板;对中间带有未知序列的PCR扩增产物进行串连克隆测序.将来自于未处理组和处理组的某一CpG位点的序列出现的次数定义为［Tags］A和［Tags］B,将标准系列的实际甲基化水平和［Tags］B/［Tags］A之间建立线性回归方程.根据每一CpG位点的［Tags］B/［Tags］A比值可反推该位点的甲基化水平.MSP-SAGE具有良好的线性,基于标准系列的［Tags］B/［Tags］A与其实际甲基化水平的标准曲线方程为y＝1.455x（R²＝0.984,P<0.01）.MSP-SAGE的回收率在95%到110%之间,精确度位于4.2％和10.5%,检测限在3％左右,单次检测通量可达24个CpG位点.MSP-SAGE是一种很有应用前途的高通量DNA甲基化定量检测方法.     

宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态的研究

为了检测宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态, 以找到无创伤性诊断宫颈癌的新指标, 选取151例宫颈癌患者的血浆和30例患者的宫颈癌组织为研究对象,用MSP的方法检测FHIT基因5′端CpG岛甲基化状态, 并对MSP产物进行克隆和测序。结果在宫颈癌患者血浆和组织中, FHIT基因5′端CpG岛甲基化率为30.46%和53.33%, 血浆和组织的总体符合率为80%。而对照中均未检测到甲基化状态。随着患者临床分期和组织学分级的增加, FHIT基因甲基化的检出率也在逐渐的增加。表明宫颈癌患者的血浆和肿瘤组织中FHIT基因5′端CpG岛甲基化的发生是高频事件, 使用FHIT基因作为标记可以对宫颈癌患者进行无创伤诊断和预后的评估。     

宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态的研究

为了检测宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态, 以找到无创伤性诊断宫颈癌的新指标, 选取151例宫颈癌患者的血浆和30例患者的宫颈癌组织为研究对象,用MSP的方法检测FHIT基因5′端CpG岛甲基化状态, 并对MSP产物进行克隆和测序。结果在宫颈癌患者血浆和组织中, FHIT基因5′端CpG岛甲基化率为30.46%和53.33%, 血浆和组织的总体符合率为80%。而对照中均未检测到甲基化状态。随着患者临床分期和组织学分级的增加, FHIT基因甲基化的检出率也在逐渐的增加。表明宫颈癌患者的血浆和肿瘤组织中FHIT基因5′端CpG岛甲基化的发生是高频事件, 使用FHIT基因作为标记可以对宫颈癌患者进行无创伤诊断和预后的评估。     

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.     

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。     

牛体细胞中Gadd45a的表达与DNA甲基化修饰

目的 检测成年牛心脏、肾脏、肝脏及睾丸组织中Gadd45a基因表达情况及DNA甲基化状态,并说明二者之间的关系.结果 牛Gadd45a基因在心脏中没有表达,而在其他3种组织中有表达且略有差异.选定该基因调控区中的7个CpG位点,利用甲基化特异性PCR检测CpG位点的DNA甲基化状态,结果心脏中的甲基化程度明显高于其他3种组织.结论 DNA甲基化对牛Gadd45a基因的组织特异性表达有一定影响.     

DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制探究

目的：探究DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制。方法：应用甲基化特异性PCR（MSP）检测人乳腺癌细胞MCF-7的DLC-1基因甲基化状态,不同浓度的5-氮杂-2＇-脱氧胞嘧啶（5-Aza-CdR）处理人乳腺癌细胞MCF-7,RT-PCR及Real-time PCR定量检测用药前后细胞中DLC-1基因mRNA表达水平变化。结果：DLC-1基因启动子区CpG岛呈甲基化状态,经过5-Aza-CdR处理后,DLC-1基因启动子区呈去甲基化状态,并且其mRNA恢复表达。结论：抑癌基因DLC-1 CpG岛甲基化是导致该基因低表达的原因之一,5-Aza-CdR能逆转DLC-1基因甲基化状态。     

The mop1 (mediator of paramutation1) mutant progressively reactivates one of the two genes encoded by the MuDR transposon in maize

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mu1 elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop1 for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mu1 and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.     

Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.     

CpG island methylation in human lymphocytes is highly correlated with DNA sequence, repeats, and predicted DNA structure

CpG island methylation plays an important role in epigenetic gene control during mammalian development and is frequently altered in disease situations such as cancer. The majority of CpG islands is normally unmethylated, but a sizeable fraction is prone to become methylated in various cell types and pathological situations. The goal of this study is to show that a computational epigenetics approach can discriminate between CpG islands that are prone to methylation from those that remain unmethylated. We develop a bioinformatics scoring and prediction method on the basis of a set of 1,184 DNA attributes, which refer to sequence, repeats, predicted structure, CpG islands, genes, predicted binding sites, conservation, and single nucleotide polymorphisms. These attributes are scored on 132 CpG islands across the entire human Chromosome 21, whose methylation status was previously established for normal human lymphocytes. Our results show that three groups of DNA attributes, namely certain sequence patterns, specific DNA repeats, and a particular DNA structure, are each highly correlated with CpG island methylation (correlation coefficients of 0.64, 0.66, and 0.49, respectively). We predicted, and subsequently experimentally examined 12 CpG islands from human Chromosome 21 with unknown methylation patterns and found more than 90% of our predictions to be correct. In addition, we applied our prediction method to analyzing Human Epigenome Project methylation data on human Chromosome 6 and again observed high prediction accuracy. In summary, our results suggest that DNA composition of CpG islands (sequence, repeats, and structure) plays a significant role in predisposing CpG islands for DNA methylation. This finding may have a strong impact on our understanding of changes in CpG island methylation in development and disease.     

Histone methylation marks play important roles in predicting the methylation status of CpG islands

The methylation status of CpG islands is highly correlated with gene expression. Current methods for computational prediction of DNA methylation only utilize DNA sequence features. In this study, besides 35 DNA sequence features, we added four histone methylation marks to predict the methylation status of CpG islands, and improved the accuracy to 89.94%. Also we applied our model to predict the methylation pattern of all the CpG islands in the human genome, and the results are consistent with the previous reports. Our results imply the important roles of histone methylation marks in affecting the methylation status of CpG islands. H3K4me enriched in the methylation-resistant CpG islands could disrupt the contacts between nucleosomes, unravel chromatin and make DNA sequences accessible. And the established open environment may be a prerequisite for or a consequence of the function implementation of zinc finger proteins that could protect CpG islands from DNA methylation.     

Reconstructing the ancestral germ line methylation state of young repeats

One of the key objectives of comparative genomics is the characterization of the forces that shape genomes over the course of evolution. In the last decades, evidence has been accumulated that for vertebrate genomes also epigenetic modifications have to be considered in this context. Especially, the elevated mutation frequency of 5-methylcytosine (5mC) is assumed to facilitate the depletion of CpG dinucleotides in species that exhibit global DNA methylation. For instance, the underrepresentation of CpG dinucleotides in many mammalian genomes is attributed to this effect, which is only neutralized in so-called CpG islands (CGIs) that are preferentially unmethylated and thus partially protected from rapid CpG decay. For primate-specific CpG-rich transposable elements from the ALU family, it is unclear whether their elevated CpG frequency is caused by their small age or by the absence of DNA methylation. In consequence, these elements are often misclassified in CGI annotations. We present a method for the estimation of germ line methylation from pairwise ancestral-descendant alignments. The approach is validated in a simulation study and tested on DNA repeats from the AluSx family. We conclude that a predicted unmethylated state in the germ line is highly correlated with epigenetic activity of the respective genomic region. Thus, CpG-rich repeats can be facilitated as in silico probes for the epigenetic potential of their genomic neighborhood.     

Estimating the total mouse DNA methylation according to the B1 repetitive elements

Measuring the degree of methylation of the B1 element in mouse may represent the global DNA methylation status because about 30,000 copies of the B1 element are randomly dispersed in the total mouse genome. Six CpG dinucleotides are located within each 163 bp size of B1 element, and each CpG dinucleotide was partially methylated. We quantitated the DNA methylation of the B1 repetitive elements by performing PCR for the methylation specific PCR (MSP) and also by the pyrosequencing. Each CpG dinucleotide was methylated at an average of 9% in the mouse genome by the pyrosequencing and MSP. Especially, we checked whether CpG methylation of the B1 element could respond to a treatment of the DNA methylation inhibitor, 5-azacytidine (5-AzaC). Consequently, the calibration graph resulting from measuring the relative CpG methylation percentage of the B1 element is linearly decreased with the increasing amount of 5-AzaC (up to 50 ng/ml concentration) in the NIH3T3 cells with a standard deviation of only 1.73% between three independent assays. Our methods can be applied to the routine analysis of the global DNA methylation changes in mouse in vivo and in vitro in pharmaceuticals and basic epigenetic research with efforts being less labor-intensive.