DNA甲基化在肿瘤形成中的作用（综述）

DNA甲基化改变是肿瘤细胞中常见的现象,DNA甲基化与肿瘤的发生有密切关系。从以下几方面对此做一综述。（1）简介哺乳动物细胞的DNA甲基化;（2）DNA甲基化与肿瘤基因突变;（3）肿瘤DNA甲基化的基因外作用,其中包括：原癌基因的低甲基化和抑癌基因的高甲基化。     

Influence of DNA methylation on transgene expression

DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene b-glu- curonidase (GUS) gene ( uidA ) was introduced into tobaccos via Agrobacterium-mediated transformation method, and the foreign uidA gene became inactive in some transgenic tobaccos. No mRNA of uidA was detected in these plants by Northern blotting analysis, and DNA methylation of promoter region was found. The results indicated that gene silencing might be caused by DNA methylation of promoter.     

Aberrant DNA methylation in cloned ovine embryos

By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine （5MeC）, the present study detected the DNA methylation patterns of cloned ovine embryos. The embryos derived from in vitro fertilization were also examined for reference purpose. The results showed that： （1） during the preimplantation development, cloned embryos displayed a similar demethylation profile to the fertilized embryos; that is, the methylation level decreased to the lowest at 8-cell stage, and then increased again at morulae stage. However, methylation level was obviously higher in cloned embryos than in stage-matched fertilized embryos, especially at 8-cell stage and afterwards; （2） at blastocyst stage, the methylation pattern in cloned embryos was different from that in fertilized embryos. In cloned blastocyst, inner cell mass （ICM） exhibited a comparable level to trophectoderm cells （TE）, while in in-vitro fertilized blastocyst the methylation level of ICM was lower than that of TE, which is not consistent with that reported by other authors. These results indicate that DNA methylation is abnormally reprogrammed in cloned embryos, implying that aberrant DNA methylation reprogramming may be one of the factors causing cloned embryos developmental failure.     

CpG-rich islands and the function of DNA methylation

It is likely that most vertebrate genes are associated with 'HTF islands'--DNA sequences in which CpG is abundant and non-methylated. Highly tissue-specific genes, though, usually lack islands. The contrast between islands and the remainder of the genome may identify sequences that are to be constantly available in the nucleus. DNA methylation appears to be involved in this function, rather than with activation of tissue specific genes.     

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.     

Aberrant DNA methylation patterns in cultured mouse embryos

Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofluorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.     

Genome-scale DNA methylation maps of pluripotent and differentiated cells

DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.     

The Polycomb group protein EZH2 directly controls DNA methylation

The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. The essential epigenetic systems involved in heritable repression of gene activity are the Polycomb group (PcG) proteins and the DNA methylation systems. Here we show that the corresponding silencing pathways are mechanistically linked. We find that the PcG protein EZH2 (Enhancer of Zeste homolog 2) interacts-within the context of the Polycomb repressive complexes 2 and 3 (PRC2/3)-with DNA methyltransferases (DNMTs) and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, we show by bisulphite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems.     

DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation.

The promoter for a weakly expressed constitutive gene, the lactose repressor gene (lacI), has been sequenced, along with an 'up' promoter mutation Iq. The 10-fold enhancement in I expression found in Iq is the result of a single base change at position -35. To facilitate the sequencing, the lacI gene was cloned in a small plasmid.     

水稻基因组DNA总甲基化水平测定方法

对高效液相色谱法测定DNA总甲基化水平的关键因素进行研究,即基因组DNA的提取及纯化和高效液相色谱条件的选择。结果表明:CTAB法Ⅰ提取和纯化效果优于CTAB法Ⅱ;较优高效液相色谱条件为:采用Diamonsil C18(2)(250 mm×4.6 mm,5μm)的色谱柱,以甲醇-10 mmol/L磷酸二氢钾(10-90,v/v)为流动相构成,流动相pH为4.7,流速为0.5 mL/min,柱温为30℃,紫外检测器波长为285 nm时,是分离胞嘧啶和5-甲基胞嘧啶的较优条件。以试验优化的DNA提取方法和HPLC色谱条件,基因组DNA水解液的胞嘧啶(C)和5-甲基胞嘧啶(5 mC)可得到较好的分离效果。