Methylation changes in the apolipoprotein AI gene during embryonic development of the mouse.

We report here a detailed study of developmental changes in the methylation status of specific sites in a single-copy tissue-specific gene, from the germ cell through the early embryo to adult tissues. Two sites at the 5' end of the mouse apolipoprotein AI gene were unmethylated in the ovulated unfertilized oocytes and methylated in the sperm. In contrast, a third site, located upstream of the gene, was methylated and a CpG island within the gene was unmethylated in both oocyte and sperm. The methylated sites, regardless of maternal or paternal origin, underwent demethylation in the early embryo (8-16 cells) and stayed unmethylated through the late blastocyst stage. During gastrulation, non-CpG island sites underwent methylation, followed by gradual demethylation at specific sites in tissues parallel to expression of the gene (liver and intestine). The formation of the mature tissue-specific methylation pattern of the apolipoprotein AI gene, therefore, involves the following three major events: (i) erasure of the germ-cell methylation pattern (at the 8- to 16-cell stage), (ii) formation of a new methylation pattern by de novo methylation of non-CpG island sites (during gastrulation), and (iii) tissue-specific demethylation associated with the onset of expression of the gene.     

Epigenomic analysis of Alu repeats in human ependymomas

Global loss of DNA methylation has been known for decades as an epigenomic aberration associated with carcinogenesis and cancer progression. Loss of DNA methylation affects predominantly repetitive elements, which encompass >50% of the CpG dinucleotides present in the human genome. Because of the lack of an effective approach, no studies have been conducted to reveal such genome-wide methylation changes at a single-base resolution. To precisely determine the CpG sites with methylation loss during progression of pediatric intracranial ependymomas, we exploited a high-throughput bisulfite sequencing approach that simultaneously generates methylation profiles for thousands of Alu elements and their flanking sequences. Comparison of the methylation profiles of normal and tumor tissues revealed that the methylation status of the majority of CpG sites adjacent to or within Alu repeats remain unaltered, while a small set of CpG sites gain or lose methylation in ependymomas. Compared to the CpG sites with stable methylation level between normal control and ependymomas, the differentially methylated CpG sites are enriched in the sequences with low CpG density in the flanking regions of Alu repeats, rather than within the Alu sequences themselves. In addition, the CpG sites that are hypermethylated in ependymomas are proximal to CpG islands, whereas those that are hypomethylated are overrepresented in intergenic regions. Lastly, aberrant methylation of several genomic loci was confirmed to be associated with the aggressive primary tumors and the relapsed ependymomas.     

Hepatitis B viral DNA is methylated in liver tissues

The mechanisms that regulate hepatitis B virus (HBV) replication within the liver are poorly understood. Given that methylation of CpG islands regulates gene expression in human tissues, we sought to identify CpG islands in HBV-DNA and to determine if they are methylated in human tissues. In silico analysis demonstrated three CpG islands in HBV genotype A sequences, two of which were of particular interest because of their proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2). Human sera with intact virions that were largely unmethylated were used to transfect HepG2 cells and HBV-DNA became partially methylated at both islands 1 and 2 by day 6 following exposure of HepG2 to virus. Examination of three additional human sera and 10 liver tissues showed no methylation in sera but tissues showed methylation of island 1 in six of 10 cases and of island 2 in five of 10 cases. The cell line Hep3B, with integrated HBV, showed complete methylation of island 1 but no methylation of island 2. In conclusion, HBV-DNA can be methylated in human tissues and methylation may play an important role in regulation of HBV gene expression.     

Hedgehog通路中Ptchl基因在人胃癌中高甲基化状态分析

目的 研究Hedgehog通路中Ptchl基因表达及其甲基化状态在胃癌发生中的变化.方法 分别抽提10例胃癌组织及其癌旁＞3 cm组织和胃癌细胞株AGS的总RNA和基因组DNA.实时定量逆转录多聚酶链反应(QRT-PCR)检测Ptch基因的mRNA表达.应用软件分析Ptchl基因5'调控序列的CpG岛状态,亚硫酸氢盐测序PCR(BSP)分析甲基化水平.结果 对Ptchl mRNAla转录体转录起始位点(计为0点)上游-3950 bp和下游+2050 bp进行CpG岛分析,发现存在两个CpG岛,第1个为-1139 bp～+860 bp,第2个为+875 bp～+1692 bp.以第1个CpG岛的-870 bp～+229 bp区段内的19个CpG位点的BSP测序结果显示,胃癌细胞株AGS全部发生甲基化,胃癌组织中甲基化程度为16%～100%,平均64%±32%,癌旁组织甲基化程度为0%～42%,平均13%±14%,两组问差异有统计学意义(P＜0.05).Spearman秩相关分析发现,Ptchl基因甲基化同其表达呈负相关(r=-0.558,P=0.011).结论 Ptchl基因高甲基化参与胃癌的发生,可能为胃癌新的肿瘤标志物.     

Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer

AIM： TO screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza2-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line. METHODS： Methylated DNA sequences in genome were enriched with methylated CpG islands amplification （MCA） to undergo representational difference analysis （RDA）, with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR （MSP） and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer. Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent. RESULTS： Nineteen differentially methylated sequences were obtained and located at 5＇ end, exons, introns and 3＇ end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, as the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signalsexcept with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent. CONCLUSION： Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical methods. Significant     

De novo methylation of the MyoD1 CpG island during the establishment of immortal cell lines.

CpG dinucleotides are unevenly distributed in the vertebrate genome. Bulk DNA is depleted of CpGs and most of the cytosines in the dinucleotide in this fraction are methylated. On the other hand, CpG islands, which are often associated with genes, are unmethylated at testable sites in all normal tissues with the exception of genes on the inactive X chromosome. We used Hpa II/Msp I analysis and ligation-mediated polymerase chain reaction to examine the methylation of the MyoD1 CpG island in adult mouse tissues, early cultures of mouse embryo cells, and immortal fibroblastic cell lines. The island was almost devoid of methylation at CCGG sites in adult mouse tissues and in low-passage mouse embryo fibroblasts. In marked contrast, the island was methylated in 10T 1/2 cells and in six other immortal cell lines showing that methylation of this CpG island had occurred during escape from senescence. The island became even more methylated in chemically transformed derivatives of 10T 1/2 cells. Thus, CpG islands not methylated in normal tissues may become modified to an abnormally high degree during immortalization and transformation.