Hypothesis of initiation of DNA methylation de novo and allelic exclusion by small RNAs

We have established that 5′-CG-3′ dinucleotide and 5′-CNG-3′ trinucleotide are found in published sequences of small interfering RNA and microRNA more often than they should be in random DNA sequences. This circumstance indicates the important biological role played by 5′-CG-3′ dinucleotides and 5′-CNG-3′ trinucleotides in small RNA sequences. We suggest that small RNAs containing these di- and trinucleotides participate in the creation of chromatin marks of epigenetic information through a highly specific search for repressible DNA sequences and through the initiation of the methylation de novo of 5′-CG-3′ and 5′-CNG-3′ sites in DNA fragments appearing to be bound complementary to small RNAs. Several genes can be inactivated simultaneously if they contain the motif recognized by small RNA. Allelic exclusion appears, in our opinion, as a result of initiation by small RNAs of DNA methylation de novo of all but one of the alleles that exist in the cell. The predecessor of this small RNA is transcribed from the antiparallel allele chain. Alleles whose antiparallel chains are less actively read by RNA polymerase, which, as we suggest, in the process of transcribing, releases DNA from small RNA bound to it, are inactivated. However, the quantity of small RNA transcribed from only one allele is insufficient to overcome the level above which the repression process of this allele is initiated de novo.     

Changes in DNA methylation and imprinting disorders in E9.5 mouse fetuses and placentas derived from vitrified eight‐cell embryos

Vitrification is increasingly used in assisted reproductive technology (ART) laboratories worldwide, and potential vitrification‐induced risks require further exploration. The effect of vitrification on changes in DNA methylation and imprinting disorders was investigated in E9.5 mouse fetuses and placentas. Fetus and placental tissues were collected from the natural mating (nautural conception [NC]) group, in vitro culture (IVC) group and vitrified embryo transfer (VET) group. The fetal crown‐rump length at E9.5 in both the IVC (0.210 ± 0.059 mm) and VET (0.205 ± 0.048 mm) groups was significantly reduced compared with the NC group (0.288 ± 0.083 mm). The global methylation levels of fetuses were decreased in the IVC group compared with the NC group and it was increased after vitrification compared with IVC (p < 0.05), similar to what was observed in the NC group (p > 0.05). The changes could be attributed to the disorders of DNA methyltransferases and ten‐eleven translocations. In the IVC and VET fetuses, a majority of maternally expressed genes were upregulated, which repressed fetal growth. Furthermore, vitrification led to a change in the methylation level of KvDMR1, which resulted in the disturbance of gene imprinting. According to our results, vitrification could contribute to increased methylation compared with IVC and contributes to a gene imprinting disorder rather than recovery. Despite the routine use of embryo vitrification in clinical settings, the effect that this procedure may have on genomic imprinting deserves much greater attention.     

DNA methylation regulates tissue-specific expression of Shank3

Tissue-specific gene expression can be controlled by epigenetic modifications such as DNA methylation. SHANK3, together with its homologues SHANK1 and SHANK2, has a central functional and structural role in excitatory synapses and is involved in the human chromosome 22q13 deletion syndrome. In this report, we show by DNA methylation analysis in lymphocytes, brain cortex, cerebellum and heart that the three SHANK genes possess several methylated CpG boxes, but only SHANK3 CpG islands are highly methylated in tissues where protein expression is low or absent and unmethylated where expression is present. SHANK3 protein expression is significantly reduced in hippocampal neurons after treatment with methionine, while HeLa cells become able to express SHANK3 after treatment with 5-Aza-2'-deoxycytidine. Altogether, these data suggest the existence of a specific epigenetic control mechanism regulating SHANK3, but not SHANK1 and SHANK2, expression.     

线粒体DNA与DNA甲基化的关系

线粒体DNA（mitochondrial DNA,mtDNA）遗传信息量虽小,却控制着线粒体一些最基本的性质,对细胞及其功能有着重要影响。mtDNA的损伤与衰老、肿瘤等疾病的发生有关。DNA甲基化是调节基因表达的重要方式之一。mtDNA基因的表达受核DNA（nuclear DNA,nDNA）的调控,mtDNA和nDNA协同作用参与机体代谢调节和发病。本文就近年来mtDNA与DNA甲基化的关系作一综述。     

A systems biology approach to prediction of oncogenes and molecular perturbation targets in B‐cell lymphomas

The computational identification of oncogenic lesions is still a key open problem in cancer biology. Although several methods have been proposed, they fail to model how such events are mediated by the network of molecular interactions in the cell. In this paper, we introduce a systems biology approach, based on the analysis of molecular interactions that become dysregulated in specific tumor phenotypes. Such a strategy provides important insights into tumorigenesis, effectively extending and complementing existing methods. Furthermore, we show that the same approach is highly effective in identifying the targets of molecular perturbations in a human cellular context, a task virtually unaddressed by existing computational methods. To identify interactions that are dysregulated in three distinct non‐Hodgkin's lymphomas and in samples perturbed with CD40 ligand, we use the B‐cell interactome (BCI), a genome‐wide compendium of human B‐cell molecular interactions, in combination with a large set of microarray expression profiles. The method consistently ranked the known gene in the top 20 (0.3%), outperforming conventional approaches in 3 of 4 cases.     

癌症DNA甲基化调控位点的识别

DNA甲基化是一种重要的表观遗传学修饰,在基因的转录调控方面具有重要的作用。异常的DNA甲基化可以导致癌症等复杂疾病发生,癌基因相关的DNA甲基化调控位点的识别对于解析癌症的发生发展机制及识别新的癌症标记具有重要意义。本研究通过整合The Cancer Genome Atlas(TCGA)的泛癌症基因组的高通量甲基化谱和基因表达谱,识别癌基因相关的DNA甲基化调控位点。对于每种癌症分批次计算Cp G位点甲基化与相关基因表达之间的相关性,并筛选调控下游基因的Cp G位点(包括强调控位点、弱调控位点和不调控位点),结果表明仅有一半的Cp G位点对下游基因具有调控作用;对癌症间共享的调控位点的分析发现不同癌症间共享的调控位点不尽相同,表明癌症特异的甲基化调控位点的存在。进一步地,对差异甲基化和差异表达基因的功能富集分析揭示了受甲基化调控的基因确实参与了癌症发生发展相关的功能。本研究的结果是对当前甲基化调控位点集的重要补充,也是识别癌症新型分子标记特征的重要资源。     

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.     

Stimulation of de novo methylation following limited proteolysis of mouse ascites DNA methylase

The ability of mouse Krebs II ascites cell DNA methylase to add methyl groups to native, unmethylated DNA (de novo activity) is stimulated by limited proteolysis. The affinity of the enzyme for DNA is not altered by this treatment but the rate of reaction is increased so that 40% or more of methylatable sites are methylated within 4.5 h. The activation is associated with a decrease in size of the enzyme to 6.2 S.     

DNA methylation variation in cloned mice

Summary: Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue‐specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue‐specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue‐specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned. genesis 30:45–50, 2001. © 2001 Wiley‐Liss, Inc.     

DNA methylation in mammalian development and disease

Epigenetic modification of the cytosine base of DNA by its methylation introduced the possibility that beyond the inherent information contained within the nucleotide sequence there was an additional layer of information added to the underlying genetic code. DNA methylation has been implicated in a wide range of biological functions, including an essential developmental role in the reprogramming of germ cells and early embryos, the repression of endogenous retrotransposons, and a generalized role in gene expression. Special functions of DNA methylation include the marking of one of the parental alleles of many imprinted genes, a group of genes essential for growth and development in mammals with a unique parent-of-origin expression pattern, a role in stabilizing X-chromosome inactivation, and centromere function. In this regard, it is not surprising that errors in establishing or maintaining patterns of methylation are associated with a diverse group of human diseases and syndromes.     

甲基化修饰在细菌表观调控中的功能

为了适应复杂多变的生存环境,微生物通常需要在保证基因组序列不变的前提下不断调整胞内代谢网络。表观调控可以在不改变DNA序列的情况下对基因表达进行调控,因此成为细菌中重要的调控方式。作为一种DNA修饰,DNA甲基化修饰是生物体中最常见的表观调控工具。在本文中我们全面、深入解析了两种孤儿甲基转移酶:DNA腺嘌呤甲基转移酶(DNA adenine methyltransferase,Dam)和细胞周期调控甲基转移酶(Cell cycle-regulated methyltransferase,Ccr M)在原核生物中的表观调控功能。我们主要探讨了DNA甲基化参与的细胞生理过程包括DNA复制起始、DNA错配修复、基因表达调控、致病性和相变异等方面。同时,我们结合三维基因组研究技术基因组结构捕获(Chromosome conformation capture,3C)技术和新型DNA磷硫酰化修饰讨论了该领域的发展前景。     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

肾细胞癌DNA甲基化标记检测的重复性及其与基因表达改变的相关性

DNA甲基化是影响基因表达的重要因素之一。DNA甲基化芯片已广泛应用于寻找癌症的标志物,但是目前还没有研究对这些标志物的重复性进行评价。另外,DNA甲基化对基因表达的影响也存在争议。在本文中,通过分析肾细胞癌的两套甲基化数据,发现它们的差异甲基化基因的方向高度的一致,证明通过甲基化芯片获得的甲基化标记有很高的重复性。进一步分析甲基化基因对应的表达改变,发现肾细胞癌中高甲基化的基因显著影响表达下调,而低甲基化的基因与表达改变无显著关系。最后,通过功能分析,找到了三个同时发生甲基化和表达改变的通路。针对这些通路研究DNA甲基化抑制剂,可能有助于肾细胞癌的靶向治疗。