表观遗传因子在不同基因组区域的分布模式研究

应用人类CD4阳性T细胞表观遗传因子高通量定位、DNA甲基化修饰和基因表达谱数据,对基因启动子、外显子和内含子区域间的表观遗传因子分布模式进行了分析;同时,研究了不同基因组区域基因表达状态与表观遗传因子分布模式的内在关系,并应用功能富集分析阐释了表观遗传因子协同调控模式与基因功能的相关性.结果表明:DNA甲基化等表观遗传因子的分布模式在启动子、外显子和内含子区域间存在显著差异,并且表观遗传因子分布模式与基因表达状态密切相关.DNA甲基化与其他表观遗传因子间存在组合调控,拥有特定调控模式的相关基因与人类CD4阳性T细胞所行使的免疫系统过程、免疫应答、白细胞活性和淋巴细胞活性等生物学功能显著相关.     

人类乳腺癌异常甲基化基因分析

运用Illumina HumanMethylation27BeadChip数据,利用sam函数中Delta值与FDR值的关系,选取最佳Delta值,识别人类乳腺肿瘤组织和正常乳腺组织中显著差异甲基化基因.分析这些差异甲基化基因在人类各条染色体的分布,进一步通过这些差异甲基化基因对肿瘤样本和正常样本进行双向非监督聚类分析.接着,利用limma包计算乳腺肿瘤组织样本和正常乳腺组织样本中差异表达基因,对既发生差异甲基化又同时发生差异表达的基因,进行GO生物分子功能富集分析.结果发现低甲基化的基因皆与蛋白质及核苷酸结合功能相关,高甲基化基因则主要富集到与运输载体活性相关的分子功能上.这些研究结果,有助于我们从基因功能角度进一步理解乳腺癌发生的原因,对乳腺癌的预后和临床诊断起到帮助作用.     

甲基化数量性状位点研究及其在精神分裂症中的作用

人类基因组DNA序列上存在着被称为“甲基化数量性状位点”的位点驱动着DNA甲基化的水平,其在基因组上的分布情况、功能等问题仍在很大程度上是未知的.越来越多的证据表明,甲基化数量性状位点的研究或许能为精神分裂症的发病机制提供新的见解.本研究收集了来自499名中国汉族人群首发精神分裂症患者,以及年龄性别相匹配的500名中国汉族健康对照组的甲基化数量性状位点数据,从不同的角度证明了甲基化数量性状位点与基因表达调控之间存在着紧密的关系,并展示了甲基化数量性状位点在探究精神分裂症遗传因素中的巨大潜力.最后,结合大规模精神分裂症全基因组关联研究数据提出了精神分裂症风险位点可能通过调控AS3MT基因上的甲基化水平,从而影响精神分裂症患病风险的潜在机制.     

DNA甲基化数据的可视化分析

DNA甲基化作为一种重要的表观遗传修饰,在生长发育、基因调控、染色质结构、分子印记以及许多疾病中起着至关重要的作用.随着各种测序技术的不断发展产生了大量的DNA甲基化数据,对其数据进行分析是目前DNA甲基化研究的一个热点和难点.目前针对于DNA甲基化数据的研究主要体现在基因组的局部区域上,而针对全基因组的分析则无法直观表现.为了更加直观的分析同一位点不同修饰信号的DNA甲基化数据间的差别,本文采用了一种专门针对表观遗传研究的数据库—WashU Epigenome Browser,针对人类表观遗传学药物数据库(HEDD)中的疾病数据,可视化的分析不同修饰信号间的差异并用数据波峰图来解释说明.     

DNA甲基化异常与人类肿瘤

DNA甲基化是表遗传学上研究最深入的一种机制,是一种酶介导的化学修饰过程,在DNA的某些碱基上增加一个甲基.在人类的肿瘤中都可以发现不同程度的DNA异常甲基化现象.介绍DNA甲基化在基因表达中的作用及其抑制基因转录、表达的机理,尤其发生在抑癌基因CpG岛和其他相关基因的甲基化异常与肿瘤发生、演进的关系,甲基化的检测方法以及去甲基化在肿瘤治疗方面的应用前景.     

抑癌基因与原癌基因在正常细胞与癌细胞中的表达水平与甲基化比较

选取抑癌基因和癌基因转录起始位点和转录终止位点上下2000bp,研究它们在癌细胞和正常细胞中甲基化分布差别,结果表明抑癌基因在癌细胞比正常细胞甲基化分布高,并且抑癌基因甲基化分布集中于转录起始位点前后1000bp,而原癌基因的甲基化分布要比抑癌基因的分布广.然后又对每个抑癌基因和原癌基因转录起始位点和转录终止位点的甲基化水平进行了研究,找出了在所选的癌细胞比正常细胞甲基化高的抑癌基因RUNX3,WT1,发现它们都是转录因子且生物学功能相似.     

肺癌组织中AKAP12启动子区域甲基化状态研究

目的:探讨肺癌组织中A激酶锚定蛋白12(AKAP12)基因启动子区域异常甲基化情况及其临床意义。方法:用甲基化特异性PCR(MSP)检测肺癌组织中AKAP12基因启动子区域Cp G岛甲基化状态,分析其与肺癌患者病理参数的关系;采用亚硫酸盐测序法对包含17个Cp G岛的选定片段发生甲基化的频率进行分析。结果:通过甲基化特异性PCR(MSP)检测50例患者肺癌标本,32份标本(64.0%)被检测出AKAP12基因启动子区域存在异常甲基化。AKAP12基因甲基化水平在不同年龄组以及性别组中的差异无统计学意义(P0.05),而与肺癌的病理分期和分化程度的差异有显著统计学意义(P0.05)。不同标本在17个Cp G位点中发生甲基化的情况并不相同,其中7、9、10、11和13五个位点出现甲基化的比例较其余位点明显增高(P0.05)。结论:AKAP12启动子甲基化改变与肺癌恶性程度及肿瘤进展相关,7、9、10、11和13五个位点出现异常甲基化频率较高可能提示其对AKAP12正常表达具有一定的调控作用。     

基于PWM扫描算法的启动子区域统计分析

为了寻找启动子区域上转录因子结合位点的分布规律,进而研究这种规律与真核基因表达调控机制之间的关系,该文从已有数据出发,运用位置权重矩阵(PWM)扫描算法对启动子区域上4种与肝脏特异表达相关的转录因子结合位点分布情况进行了初步研究,并提出了一种新的序列评分方法。通过该方法提取的统计特征,肝脏特异基因的鉴别准确率可以达93.33%。实验结果表明:肝脏特异基因的启动子区域上结合位点的分布情况与其他基因相比存在显著差异。新的序列评分方法可以更好地反映这种差异性,实现肝脏特异基因的精确鉴别。     

SFRP1基因启动子区甲基化与宫颈癌及癌前病变的相关性研究

应用MassARRAY DNA甲基化定量分析方法检测58例宫颈癌组织、57例CIN2/3组织、36例CIN1组织及28例正常对照组织中SFRP1基因启动子区各CpG位点甲基化,探讨SFRP1基因启动子区甲基化与宫颈癌演进及与HPV16感染的相关性。结果显示:2110个CpG单位中甲基化率低于30%的CpG单位1612个,占总数的76.4%;甲基化率大于80%的CpG单位为4.4%,多分布于CIN2/3和宫颈癌组织之中。其中SFRP1基因启动子区CpG12.13和CpG18位点的甲基化率宫颈癌组高于对照、CIN1和CIN2/3组,差异具有统计学意义(P0.05)。CpG12.13和CpG18位点甲基化率与HPV16感染无相关性(P0.05)。提示SFRP1基因在CpG12.13和CpG18位点上高甲基化可能与宫颈癌的演进相关。     

子宫颈癌抑癌基因P16第一外显子CpG岛异常甲基化

利用限制性核酸内切酶-PCR方法分析33例肿瘤组织和15例正常组织抑癌基因p16第一外显子SacⅡ和SmaⅠ酶切位点甲基化,应用SPSS软件进行统计分析。结果显示,18例子宫颈癌在抑癌基因p16第一外显子SacⅡ位点甲基化,8例子宫颈癌在SmaⅠ位点甲基化。正常组织仅有两例在抑癌基因p16第一外显子SacⅡ位点甲基化,正常组织在SmaⅠ位点没有甲基化。另外,抑癌基因p16第一外显子SacⅡ和SmaⅠ酶切位点甲基化易发生在肿瘤的早期。抑癌基因p16第一外显子SacⅡ和SmaⅠ酶切位点甲基化与子宫颈癌相关。     

Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation

Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin. DNA methyltransferase 3a (Dnmt3a) and its regulatory factor, DNA methyltransferase 3-like protein (Dnmt3L), are both required for the de novo DNA methylation of imprinted genes in mammalian germ cells. Dnmt3L interacts specifically with unmethylated lysine 4 of histone H3 through its amino-terminal PHD (plant homeodomain)-like domain. Here we show, with the use of crystallography, that the carboxy-terminal domain of human Dnmt3L interacts with the catalytic domain of Dnmt3a, demonstrating that Dnmt3L has dual functions of binding the unmethylated histone tail and activating DNA methyltransferase. The complexed C-terminal domains of Dnmt3a and Dnmt3L showed further dimerization through Dnmt3a-Dnmt3a interaction, forming a tetrameric complex with two active sites. Substitution of key non-catalytic residues at the Dnmt3a-Dnmt3L interface or the Dnmt3a-Dnmt3a interface eliminated enzymatic activity. Molecular modelling of a DNA-Dnmt3a dimer indicated that the two active sites are separated by about one DNA helical turn. The C-terminal domain of Dnmt3a oligomerizes on DNA to form a nucleoprotein filament. A periodicity in the activity of Dnmt3a on long DNA revealed a correlation of methylated CpG sites at distances of eight to ten base pairs, indicating that oligomerization leads Dnmt3a to methylate DNA in a periodic pattern. A similar periodicity is observed for the frequency of CpG sites in the differentially methylated regions of 12 maternally imprinted mouse genes. These results suggest a basis for the recognition and methylation of differentially methylated regions in imprinted genes, involving the detection of both nucleosome modification and CpG spacing.     

Maternal inhibition of hepatitis B surface antigen gene expression in transgenic mice correlates with de novo methylation

Differential modifications of the genome during gametogenesis result in a functional difference between the paternal and maternal genomes at the moment of fertilization. A possible cause of this imprinting is the methylation of DNA. The insertion of foreign DNA into transgenic mice allows the tagging of regions that are differentially methylated during gametogenesis. We describe here a transgenic mouse strain in which the expression of the hepatitis B surface antigen gene is irreversibly repressed following its passage through the female germ line. This inhibition is accompanied by the methylation of all the HpaII and HhaI sites within the foreign gene, which we have shown to be integrated into a site on chromosome 13. The irreversibility reported here contrasts with what is found with other transgenic mice sequences which are reversibly methylated after passage through the male or female germ line, though in both cases methylation appears to be important in the imprinting process.     

Detection of DNA methylation changes during seed germination in rapeseed （Brassica napus）

DNA methylation is a common yet important modi- fication of DNA in eukaryotic organisms. DNA methy- lation, especially methylation of cytosine (m5C), have both epigenetic and mutagenic effects on various cellu- lar activities such as differential gene exp…     

Spontaneous epigenetic variation in the Arabidopsis thaliana methylome

Heritable epigenetic polymorphisms, such as differential cytosine methylation, can underlie phenotypic variation. Moreover, wild strains of the plant Arabidopsis thaliana differ in many epialleles, and these can influence the expression of nearby genes. However, to understand their role in evolution, it is imperative to ascertain the emergence rate and stability of epialleles, including those that are not due to structural variation. We have compared genome-wide DNA methylation among 10 A. thaliana lines, derived 30 generations ago from a common ancestor. Epimutations at individual positions were easily detected, and close to 30,000 cytosines in each strain were differentially methylated. In contrast, larger regions of contiguous methylation were much more stable, and the frequency of changes was in the same low range as that of DNA mutations. Like individual positions, the same regions were often affected by differential methylation in independent lines, with evidence for recurrent cycles of forward and reverse mutations. Transposable elements and short interfering RNAs have been causally linked to DNA methylation. In agreement, differentially methylated sites were farther from transposable elements and showed less association with short interfering RNA expression than invariant positions. The biased distribution and frequent reversion of epimutations have important implications for the potential contribution of sequence-independent epialleles to plant evolution.     

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.     

CpG methylation is maintained in human cancer cells lacking DNMT1

Hypermethylation is associated with the silencing of tumour susceptibility genes in several forms of cancer; however, the mechanisms responsible for this aberrant methylation are poorly understood. The prototypic DNA methyltransferase, DNMT1, has been widely assumed to be responsible for most of the methylation of the human genome, including the abnormal methylation found in cancers. To test this hypothesis, we disrupted the DNMT1 gene through homologous recombination in human colorectal carcinoma cells. Here we show that cells lacking DNMT1 exhibited markedly decreased cellular DNA methyltransferase activity, but there was only a 20% decrease in overall genomic methylation. Although juxtacentromeric satellites became significantly demethylated, most of the loci that we analysed, including the tumour suppressor gene p16INK4a, remained fully methylated and silenced. These results indicate that DNMT1 has an unsuspected degree of regional specificity in human cells and that methylating activities other than DNMT1 can maintain the methylation of most of the genome.     

A unique regulatory phase of DNA methylation in the early mammalian embryo

DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)--including many CpG island promoters--that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.