Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma

Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (-575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco(2) (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC(3)/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2'-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco(2), and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.     

Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers

The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in approximately 25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from -373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.     

DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines

CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.     

翘嘴鲌胰岛素样生长因子igf3基因的克隆及表达分析

为研究翘嘴鲌(Culter alburnus Basilewsky)胰岛素样生长因子(Insulin-like growth factor 3, igf3)基因在性别调控过程中的作用,基于分子克隆手段RT-PCR和RACE技术相结合方法扩增到翘嘴鲌igf3基因完整cDNA序列,利用qRT-PCR技术检测其在不同组织中的表达水平,最后通过甲基化检测方法比较分析了翘嘴鲌性腺组织igf3基因组水平的CpG岛修饰水平。实验结果显示igf3 cDNA序列全长901 bp,包含92 bp的5′端非编码区和203 bp的3′端非编码区,开放阅读框606 bp,编码201个氨基酸。序列分析显示,类似于其他IGF家族成员igf1和igf2, igf3同样存在保守的特征结构域,主要划分为前体信号肽、B、C、A、D和E区。igf3基因在翘嘴鲌不同组织中的表达水平差异明显(P<0.05),在卵巢中表达量最高,其次是精巢组织,而在脑、心脏、脾脏、肝脏、肌肉和肾脏中表达丰度极低。甲基化检测结果显示在卵巢中CG位点几乎不发生甲基化,然而精巢中的甲基化程度非常高,这与其表达水平正好呈负相关。研究结果表明igf3...     

Quantitation of inhibition of DNA methylation of the retinoic acid receptor beta gene by 5-Aza-2'-deoxycytidine in tumor cells using a single-nucleotide primer extension assay

The expression of several cancer-related genes has been reported to be silenced by DNA methylation of their promoter region. 5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent and specific inhibitor of DNA methylation, can reactivate the in vitro expression of these genes. In future clinical trials in tumor therapy with 5-AZA-CdR a method to quantitate its inhibition of methylation of specific tumor suppressor genes would provide important data for the analysis of the therapeutic efficacy of this analogue. We have modified the methylation-sensitive single-nucleotide primer extension assay reported by Gonzalgo and Jones (Nucleic Acids Res. 25, 2529-2531, 1997). Genomic DNA was treated with bisulfite and a fragment of the promoter region of the human retinoic acid receptor beta (RARbeta) gene, a tumor suppressor gene, was amplified using seminested PCR. Using two different primers we quantitated the inhibition of methylation produced by 5-AZA-CdR at two specific CpG sites in the RARbeta promoter in a human colon and a breast carcinoma cell line. The results obtained with the modified assay show a precise and reproducible quantitation of inhibition of DNA methylation produced by 5-AZA-CdR in tumor cells.     

Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.     

HaCaT细胞中FUT4表达与其启动子区甲基化的相关性分析

岩藻糖基转移酶Ⅳ(Fucosyltransferase Ⅳ,FUT4)在正常细胞中表达量很低,但其低表达的调控机制以及是否受其启动子甲基化调控并不十分清楚。文章采用Western blot、免疫荧光和Real-time PCR的方法检测正常人永生化表皮细胞系HaCaT细胞FUT4的表达,观察DNA甲基转移酶抑制剂5-aza-dC处理对FUT4表达的影响。应用甲基化特异性PCR方法分析HaCaT细胞中FUT4启动子甲基化状态。结果表明,HaCaT细胞中FUT4的表达水平明显低于人表皮鳞癌细胞A431和SCC12。5 μmol/L的5-aza-dC处理72 h的HaCaT细胞,其FUT4 mRNA水平明显升高,并且与未经5-aza-dC处理的对照组相比,U引物扩增检测到的产物量增加,M 引物扩增检测到的产物量明显减少。这些结果表明,HaCaT细胞中FUT4的低表达可能与其启动子区CpG岛甲基化有关。     

Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene,Tact1/Actl7b,represses its expression in somatic cells

Methylation of CpG islands spanning promoter regions is associated with control of gene expression. However, it is considered that methylation of exonic CpG islands without promoter is not related to gene expression, because such exonic CpG islands are usually distant from the promoter. Whether methylation of exonic CpG islands near the promoter, as in the case of a CpG-rich intronless gene, causes repression of the promoter remains unknown. To gain insight into this issue, we investigated the distribution and methylation status of CpG dinucleotides in the mouse Tact1/Actl7b gene, which is intronless and expressed exclusively in testicular germ cells. The region upstream to the gene was poor in CpG, with CpG dinucleotides absent from the core promoter. However, a CpG island was found inside the open reading frame (ORF). Analysis of the methylation status of the Tact1/Actl7b gene including the 5′-flanking area demonstrated that all CpG sites were methylated in somatic cells, whereas these sites were unmethylated in the Tact1/Actl7b-positive testis. Trans fection experiments with in vitro-methylated constructs indicated that methylation of the ORF but not 5′ upstream repressed Tact1/Actl7b promoter activity in somatic cells. Similar effects of ORF methylation on the promoter activity were observed in testicular germ cells. These are the first results indicating that methylation of the CpG island in the ORF represses its promoter in somatic cells and demethylation is necessary for gene expression in spermatogenic cells.     

Effect of CpG Island Methylation on MicroRNA Expression in the k-562 Cell Line

To test the hypothesis that methylation of a CpG island is associated with regulation of microRNA expression, we investigated CpG islands in the upstream sequences of microRNA precursors (pre-miRNAs) through bioinformatic analysis and determined whether the CpG islands were methylated by methylation-specific PCR in the k-562 cell line. We used 5-azacytidine for DNA demethylation, and changes in microRNA expression were detected by microarray assay, RT-PCR, and real-time PCR after 5-azacytidine induction. We showed that the CpG islands in the upstream regions of 18 pre-miRNAs were methylated, including miR-663, miR-369, miR-615, and miR-410, and promoter activity was detected in the upstream region of pre-miR-663. We found that a decrease in methylation of a CpG island could up-regulate the expression of miR-663, suggesting that miR-663 could be regulated by DNA methylation. Expression levels of miR-369, miR-615, and miR-410 were not regulated by DNA methylation in this cell line.     

Promoter hypomethylation contributes to the expression of MUC3A in cancer cells

MUC3A is a membrane-bound glycoprotein that is aberrantly expressed in carcinomas and is a risk factor for a poor prognosis. However, the exact mechanism of MUC3A expression has yet to be clarified. Here, we provide the first evidence that MUC3A gene expression is controlled by the CpG methylation status of the proximal promoter region. We show that the DNA methylation pattern is intimately correlated with MUC3A expression in breast, lung, pancreas and colon cancer cell lines. The DNA methylation status of 30 CpG sites from −660 to +273 was mapped using MassARRAY analysis. MUC3A-negative cancer cell lines and those with low MUC3A expression (e.g., MCF-7) were highly methylated in the proximal promoter region, corresponding to 9 CpG sites (−345 to −75 bp), whereas MUC3A-positive cell lines (e.g., LS174T) had low methylation levels. Moreover, 5-aza-2′-deoxycytidine and trichostatin A treatment of MUC3A-negative cells or those with low MUC3A expression caused elevation of MUC3A mRNA. Our results suggest that DNA hypomethylation in the 5′-flanking region of the MUC3A gene plays an important role in MUC3A expression in carcinomas of various organs. An understanding of epigenetic changes in MUC3A may contribute to the diagnosis of carcinogenic risk and to prediction of outcome in patients with cancer.     

DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution

Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation.