Unknown,Transcriptomics,Genomics,Proteomics

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Identification of genes upregulated by methyl-CpG targeted transcriptional activation (MeTA) in 293T

ABSTRACT: The identification of genes transcriptionally silenced by DNA hypermethylation is important in understanding the molecular basis of epigenetically regulated biological processes such as X chromosome inactivation, genomic imprinting, and cancer development. Our previously developed methyl-CpG targeted transcriptional activation (MeTA) method reactivates epigenetically silenced genes by using a methyl-CpG binding domain from MBD2 with a transcriptional activation domain. We applied either MeTA or a conventional DNA demethylating agent, 5-aza-cytidine (Aza-CR), to a human embryonic kidney cell line 293T and analyzed gene expression profiles by microarray; 138 and 202 genes that are upregulated 5-fold or more were identified by MeTA and Aza-CR, respectively. The top ten upregulated genes detected by MeTA were further analyzed. We found associations between expressional restorations by MeTA, methylation status, and NFkB(AD)-MBD fusion protein bindings in CpG islands (CGIs) around the transcription start site of the genes. Importantly, MeTA can upregulate genes meeting the stringent criteria of CGIs defined by Takai and Jones at the promoter region at higher frequency; 109 of 138 (79.0%) genes in MeTA vs. 121 of 202 (59.9%) genes in Aza-CR. Interestingly, only 27 genes were upregulated by both methods; MeTA may identify methylated genes that show low levels of induction by the DNA demethylating agents; demethylating agents may also induce factors that help re-expression of genes that harbor less stringent or no CGIs. These results suggest that microarray coupled with MeTA (MeTA-array) is an efficient alternative way to identify transcriptionally silenced genes by DNA hypermethylation. 293T cells were transfected with pcDNA6/myc-His vector or pcDNA6-3xFLAG-NFkB (AD)-MBD and were harvested 48 h after transfection. In contrast, 293T cells were treated with 5-aza-cytidine (Aza-CR, 25 uM) or the same volume of PBS for 96 h and medium replaced every 24 h.

ORGANISM(S): Homo sapiens

SUBMITTER: Shinichi Fukushige

PROVIDER: E-GEOD-25981 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Microarray coupled with methyl-CpG targeted transcriptional activation (MeTA-array) identifies hypermethylated genes containing the stringent criteria of CpG islands at high frequency.

Sato Yuko Y Horii Akira A Fukushige Shinichi S

Epigenetics 20110601 6

The identification of genes transcriptionally silenced by DNA hypermethylation is important in understanding the molecular basis of epigenetically regulated biological processes such as X chromosome inactivation, genomic imprinting and cancer development. Our previously developed methyl-CpG targeted transcriptional activation (MeTA) method reactivates epigenetically silenced genes by using a methyl-CpG binding domain from MBD2 with a transcriptional activation domain. We applied either MeTA or a ...[more]

PMID: 21586901

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Project description:Identification and characterization of epigenetically silenced genes is very important for cancer research. Particularly, information of hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses, and genes frequently methylated in a tumor-specific manner can be used as tumor markers. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2M-bM-^@M-^Y-deoxycytidine were widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting such methylation-mediated silenced genes in cancer and successfully developed a new method termed M-bM-^@M-^\methyl-CpG targeted transcriptional activation (MeTA)M-bM-^@M-^] by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD) that specifically recognizes and binds to methylated DNAs. Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the present study, we applied this method to three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control). All of these cell lines have already been analyzed their expression profiles by 5-aza-2M-bM-^@M-^Y-deoxycytidine. We first analyzed the expression of five genes by RT-PCR with Southern hybridization, NEFH, NPTX2, SFRP1, TIMP3, and UCHL1; these genes are known to be methylated in at least any one of these cancer cell lines. Upregulation by M-bM-^@M-^\MeTAM-bM-^@M-^] was confirmed in all of these genes. Then we searched for upregulated-genes, by two-folds or more, in all the three cancer cell lines after MeTA; nineteen such upregulated genes were identified. Among these, sixteen genes except NEFH, HOXA9, and CLDN5 have not been reported previously using the conventional DNA methylation inhibitors. Methylation status of two genes, SLC32A1 and CSMD2, were further analyzed by methylation-specific PCR and found that SLC32A1 and CSMD2 were methylated in 100% (21/21) and 83% (15/18) pancreatic cancer cell lines analyzed, respectively. Our results suggest that M-bM-^@M-^\MeTAM-bM-^@M-^] is a highly efficient method to isolate methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer. Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control) were transfected with pcDNA6/myc-His vector or pcDNA6-3xFLAG-NFkB (AD)-MBD and were harvested 48 h after transfection.

Dataset Information

Identification of genes upregulated by methyl-CpG targeted transcriptional activation (MeTA) in 293T

Publications

Microarray coupled with methyl-CpG targeted transcriptional activation (MeTA-array) identifies hypermethylated genes containing the stringent criteria of CpG islands at high frequency.

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