DNA甲基化模式及其在癌症中的作用

随着对癌症研究的不断深入,表观遗传调控在癌症发生发展中的作用也越来越受到人们的关注。DNA基化作为一种重要的表观遗传修饰机制,在基因表达调控中起着十分重要的作用。该文对DNA基化模式及其在癌症中的作用作了综述,并对DNA甲基化作为癌症早期诊断的生物标记以及癌症表观治疗的新策略作了总结和展望。     

不同水陆环境中喜旱莲子草甲基化调控因子表达水平和差异表达基因启动子区甲基化状态分析

DNA甲基化是一种重要的表观遗传调控方式,参与对植物生长发育的调控,并在植物逆境胁迫响应中发挥作用。DNA甲基化的建立和维持是胞嘧啶甲基转移酶、染色质重塑酶、组蛋白修饰因子和去甲基化因子等协同作用的结果,环境胁迫能诱导植物体内DNA甲基化状态改变,进而改变基因表达水平和式样,影响植物的适应性。喜旱莲子草是一种恶性入侵植物,种内遗传多样性很低,主要依靠极强的表型可塑性侵占不同水陆生境。对克隆繁殖的喜旱莲子草个体进行不同时间的淹水处理,并用定量PCR方法检测16个DNA甲基化调控基因在不同处理条件下的表达水平和变化趋势,发现其中13个基因在不同处理时间点的表达水平有明显变化,且在淹水植株中多数基因在处理前期被强烈诱导上调表达。运用亚硫酸氢钠测序技术对两个在不同水陆条件下明显差异表达基因(Contig942和Contig23336)的上游启动子区甲基化动态进行分析,发现启动子区多个胞嘧啶位点的甲基化修饰状态在不同水陆条件下及淹水处理的不同时期呈现快速且可逆的动态变化,可能影响这些基因在不同环境条件下的表达水平。本研究结果能帮助了解喜旱莲子草表型可塑性变异和适应性发生的分子机理。     

牛不同供体核克隆囊胚Xist基因DNA甲基化程度的比较研究

利用亚硫酸氢盐测序法分析Holstein奶牛胎儿成纤维细胞(FFB)和输卵管上皮细胞(FOV)来源的克隆囊胚Xist基因DNA甲基化状况,以体外受精囊胚(IVF)和供体细胞作对照.克隆囊胚Xist基因处于较低程度的DNA甲基化状态,其中,FFB来源的克隆囊胚Xist基因DNA甲基化程度为43%,而FOV来源的克隆囊胚仅为17%.在体外受精囊胚中,Xist基因DNA甲基化处于中等状态,为49%.然而,在体细胞中,Xist基因的甲基化程度较高,FFB为66%,FOV为63%.这些结果说明,Xist基因DNA甲基化是可以被重编程的,所检测的CpG岛可能调节Xist基因的表达.结合已发表的实验数据,在同一个体中,FFB来源的克隆囊胚发育率比FOV的低,但其克隆牛胎儿的妊娠率和产犊率比FOV的高,这暗示不同供体核克隆囊胚的重编程是有差异的,并可能影响到胚胎及个体的发育.     

The Dynamics of DNA Methylation Covariation Patterns in Carcinogenesis

Recently it has been observed that cancer tissue is characterised by an increased variability in DNA methylation patterns. However, how the correlative patterns in genome-wide DNA methylation change during the carcinogenic progress has not yet been explored. Here we study genome-wide inter-CpG correlations in DNA methylation, in addition to single site variability, during cervical carcinogenesis. We demonstrate how the study of changes in DNA methylation covariation patterns across normal, intra-epithelial neoplasia and invasive cancer allows the identification of CpG sites that indicate the risk of neoplastic transformation in stages prior to neoplasia. Importantly, we show that the covariation in DNA methylation at these risk CpG loci is maximal immediately prior to the onset of cancer, supporting the view that high epigenetic diversity in normal cells increases the risk of cancer. Consistent with this, we observe that invasive cancers exhibit increased covariation in DNA methylation at the risk CpG sites relative to normal tissue, but lower levels relative to pre-cancerous lesions. We further show that the identified risk CpG sites undergo preferential DNA methylation changes in relation to human papilloma virus infection and age. Results are validated in independent data including prospectively collected samples prior to neoplastic transformation. Our data are consistent with a phase transition model of carcinogenesis, in which epigenetic diversity is maximal prior to the onset of cancer. The model and algorithm proposed here may allow, in future, network biomarkers predicting the risk of neoplastic transformation to be identified.     

巢式甲基化特异性PCR检测不同类型组织标本中WIF-1基因启动子异常甲基化

目的:采用一种高灵敏度的DNA甲基化分析方法,即巢式甲基化特异性PCR法(nested-MSP,nMSP),检测外科手术切除新鲜组织、石蜡包埋组织及纤维支气管镜活检组织中WIF-1基因启动子的异常甲基化状态。方法:将基因组DNA变性成为单链,用亚硫酸氢盐修饰单链DNA,所有未甲基化的胞嘧啶被转变为尿嘧啶,而甲基化的胞嘧啶则不变。设计针对甲基化和非甲基化等位基因的特异引物,进行巢式PCR扩增,最后经凝胶电泳检测目的片段。结果:在3种类型的原发性非小细胞肺癌标本中都检测出了WIF-1基因启动子的异常甲基化。结论:巢式甲基化特异性PCR是一种灵敏度高、特异性强的甲基化检测方法,可广泛应用于不同类型标本基因启动子甲基化的分析。     

Influence of DNMT Genotype on Global and Site Specific DNA Methylation Patterns in Neonates and Pregnant Women

This study examines the relationship between common genetic variation within DNA methyltransferase genes and inter-individual variation in DNA methylation. Eleven polymorphisms spanning DNMT1 and DNMT3B were genotyped. Global and gene specific (IGF2, IGFBP3, ZNT5) DNA methylation was quantified by LUMA and bisulfite Pyrosequencing assays, respectively, in neonatal cord blood and in maternal peripheral blood. Associations between maternal genotype and maternal methylation (n ^≈ 333), neonatal genotype and neonatal methylation (n ^≈ 454), and maternal genotype and neonatal methylation (n ^≈ 137) were assessed. The findings of this study provide some support to the hypothesis that genetic variation in DNA methylating enzymes influence DNA methylation at global and gene-specific levels; however observations were not robust to correction for multiple testing. More comprehensive analysis of the influence of genetic variation on global and site specific DNA methylation is warranted.     

Comprehensive Genomic Characterization of RNA-Binding Proteins across Human Cancers

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核小体定位模式及其与DNA甲基化位点分布的关系

核小体定位是指DNA双螺旋相对于组蛋白八联体的位置.核小体定位通过限制蛋白结合位点参与基因转录调控.本文利用实验检测的人类CD4+ T细胞核小体定位数据,研究了核小体定位在转录因子结合位点(TFBS)和转录起始位点(TSS)附近的分布模式,并分析了在TFBS和TSS周围,核小体定位与DNA甲基化之间的关系.结果表明,在休眠和激活的人类CD4+ T细胞中,部分TFBS和TSS周围的核小体定位在动态改变,即在定位和缺失两种状态之间切换.在TFBS周围,核小体定位和DNA甲基化存在一种互补模式,核小体定位与DNA低甲基化相联系;而在TSS周围,两者呈现同步模式,DNA高甲基化伴随高核小体水平.而且,在TFBS和TSS周围,DNA甲基化位点的分布呈周期模式.CD4+ T细胞被激活时,较少的转录因子启动了较多的基因.     

A Comparison of Genome-Wide DNA Methylation Patterns between Different Vascular Tissues from Patients with Coronary Heart Disease

Epigenetic mechanisms of gene regulation in context of cardiovascular diseases are of considerable interest. So far, our current knowledge of the DNA methylation profiles for atherosclerosis affected and healthy human vascular tissues is still limited. Using the Illumina Infinium Human Methylation27 BeadChip, we performed a genome-wide analysis of DNA methylation in right coronary artery in the area of advanced atherosclerotic plaques, atherosclerotic-resistant internal mammary arteries, and great saphenous veins obtained from same patients with coronary heart disease. The resulting DNA methylation patterns were markedly different between all the vascular tissues. The genes hypomethylated in athero-prone arteries to compare with atherosclerotic-resistant arteries were predominately involved in regulation of inflammation and immune processes, as well as development. The great saphenous veins exhibited an increase of the DNA methylation age in comparison to the internal mammary arteries. Gene ontology analysis for genes harboring hypermethylated CpG-sites in veins revealed the enrichment for biological processes associated with the development. Four CpG-sites located within the MIR10B gene sequence and about 1 kb upstream of the HOXD4 gene were also confirmed as hypomethylated in the independent dataset of the right coronary arteries in the area of advanced atherosclerotic plaques in comparison with the other vascular tissues. The DNA methylation differences observed in vascular tissues of patients with coronary heart disease can provide new insights into the mechanisms underlying the development of pathology and explanation for the difference in graft patency after coronary artery bypass grafting surgery.     

Comparison of the Prognostic Utility of the Diverse Molecular Data among lncRNA,DNA Methylation,microRNA, and mRNA across Five Human Cancers

IntroductionAdvances in high-throughput technologies have generated diverse informative molecular markers for cancer outcome prediction. Long non-coding RNA (lncRNA) and DNA methylation as new classes of promising markers are emerging as key molecules in human cancers; however, the prognostic utility of such diverse molecular data remains to be explored.ResultsUsing the IDFO approach, we obtained good predictive performance of the molecular datasets (bootstrap accuracy: 0.71–0.97) in five cancer types. Impressively, lncRNA was identified as the best prognostic predictor in the validated cohorts of four cancer types, followed by DNA methylation, mRNA, and then microRNA. We found the incorporating of multi-type molecular data showed similar predictive power to single-type molecular data, but with the exception of the lncRNA + DNA methylation combinations in two cancers. Survival analysis of proportional hazard models confirmed a high robustness for lncRNA and DNA methylation as prognosis factors independent of traditional clinical variables.ConclusionOur study provides insight into systematically understanding the prognostic performance of diverse molecular data in both single and aggregate patterns, which may have specific reference to subsequent related studies.     

The Pattern of Change in the Abundances of Specific Bacterioplankton Groups Is Consistent across Different Nutrient-Enriched Habitats in Crete

A common source of disturbance for coastal aquatic habitats is nutrient enrichment through anthropogenic activities. Although the water column bacterioplankton communities in these environments have been characterized in some cases, changes in α-diversity and/or the abundances of specific taxonomic groups across enriched habitats remain unclear. Here, we investigated the bacterial community changes at three different nutrient-enriched and adjacent undisturbed habitats along the north coast of Crete, Greece: a fish farm, a closed bay within a town with low water renewal rates, and a city port where the level of nutrient enrichment and the trophic status of the habitat were different. Even though changes in α-diversity were different at each site, we observed across the sites a common change pattern accounting for most of the community variation for five of the most abundant bacterial groups: a decrease in the abundance of the Pelagibacteraceae and SAR86 and an increase in the abundance of the Alteromonadaceae, Rhodobacteraceae, and Cryomorphaceae in the impacted sites. The abundances of the groups that increased and decreased in the impacted sites were significantly correlated (positively and negatively, respectively) with the total heterotrophic bacterial counts and the concentrations of dissolved organic carbon and/or dissolved nitrogen and chlorophyll α, indicating that the common change pattern was associated with nutrient enrichment. Our results provide an in situ indication concerning the association of specific bacterioplankton groups with nutrient enrichment. These groups could potentially be used as indicators for nutrient enrichment if the pattern is confirmed over a broader spatial and temporal scale by future studies.     

Using DNA Methylation Patterns to Infer Tumor Ancestry

Background

Exactly how human tumors grow is uncertain because serial observations are impractical. One approach to reconstruct the histories of individual human cancers is to analyze the current genomic variation between its cells. The greater the variations, on average, the greater the time since the last clonal evolution cycle (“a molecular clock hypothesis”). Here we analyze passenger DNA methylation patterns from opposite sides of 12 primary human colorectal cancers (CRCs) to evaluate whether the variation (pairwise distances between epialleles) is consistent with a single clonal expansion after transformation.

Methodology/Principal Findings

Data from 12 primary CRCs are compared to epigenomic data simulated under a single clonal expansion for a variety of possible growth scenarios. We find that for many different growth rates, a single clonal expansion can explain the population variation in 11 out of 12 CRCs. In eight CRCs, the cells from different glands are all equally distantly related, and cells sampled from the same tumor half appear no more closely related than cells sampled from opposite tumor halves. In these tumors, growth appears consistent with a single “symmetric” clonal expansion. In three CRCs, the variation in epigenetic distances was different between sides, but this asymmetry could be explained by a single clonal expansion with one region of a tumor having undergone more cell division than the other. The variation in one CRC was complex and inconsistent with a simple single clonal expansion.

Conclusions

Rather than a series of clonal expansion after transformation, these results suggest that the epigenetic variation of present-day cancer cells in primary CRCs can almost always be explained by a single clonal expansion.     

人二倍体化合子的DNA 甲基化变化模式

目的:探讨人类三原核合子及二倍体化合子中DNA甲基化模式的变化情况。方法:我们采用显微操作技术去除三原核合子中两个雄原核中的一个,观察恢复了二倍体状态的胚胎的发育情况,并检测了三原核和二倍体化的合子及早期胚胎中DNA甲基化模式的动态变化。结果:二倍体化的合子的囊胚形成率与三原核合子的囊胚形成率无显著性差异;在人三原核合子中两个雄原核发生主动地DNA去甲基化而雌原核在受精后的20h后仍保持甲基化。三原核与二倍体化合子中,DNA甲基化模式没有差别。结论:去除一个雄原核不会影响合子和胚胎的DNA甲基化模式。去除多余雄原核并不能改善胚胎的发育。     

Arginine Methylation of Vasa Protein Is Conserved across Phyla

Recent studies have uncovered an unexpected relationship between factors that are essential for germline development in Drosophila melanogaster: the arginine protein methyltransferase 5 (dPRMT5/Csul/Dart5) and its cofactor Valois, methylate the Piwi family protein Aub, enabling it to bind Tudor. The RNA helicase Vasa is another essential protein in germline development. Here, we report that mouse (mouse Vasa homolog), Xenopus laevis, and D. melanogaster Vasa proteins contain both symmetrical and asymmetrical dimethylarginines. We find that dPRMT5 is required for the production of sDMAs of Vasa in vivo. Furthermore, we find that the mouse Vasa homolog associates with Tudor domain-containing proteins, Tdrd1 and Tdrd6, as well as the Piwi proteins, Mili and Miwi. Arginine methylation is thus emerging as a conserved and pivotal post-translational modification of proteins that is essential for germline development.     

利用分子量阵列平台进行特定序列甲基化分析的方法探讨

目的:探讨利用分子量阵列平台进行特定序列甲基化分析的方法。方法:通过对不同扩增条件和扩增效率及不同条件处理的质谱分析比较了MassArray平台进行甲基化分析的特点。结果:本研究通过与重亚硫酸盐测序结果比较证实,MassArray分子量阵列技术平台能够反映甲基化修饰的真实水平;通过不同条件下PCR扩增效率与甲基化分析的结果,发现扩增效率是制约MassArray分子量阵列技术平台甲基化分析的关键因素,而产物的放置时间和不同的处理没有明显影响甲基化分析。结论:Mas-sArray甲基化分析平台是高效快速检测甲基化修饰的平台,在使用过程中应该根据实际的实验条件,进行合理的质控。     

利用分子量阵列平台进行特定序列甲基化分析的方法探讨

目的：探讨利用分子量阵列平台进行特定序列甲基化分析的方法。方法：通过对不同扩增条件和扩增效率及不同条件处理的质谱分析比较了MassArray平台进行甲基化分析的特点。结果：本研究通过与重亚硫酸盐测序结果比较证实,MassArray分子量阵列技术平台能够反映甲基化修饰的真实水平;通过不同条件下PCR扩增效率与甲基化分析的结果,发现扩增效率是制约MassArray分子量阵列技术平台甲基化分析的关键因素,而产物的放置时间和不同的处理没有明显影响甲基化分析。结论：Mas-sArray甲基化分析平台是高效快速检测甲基化修饰的平台,在使用过程中应该根据实际的实验条件,进行合理的质控。