Homo sapiens
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PROVIDER: PRJNA135405 | ENA |
REPOSITORIES: ENA
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Project description: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.
2011-06-03 | E-GEOD-25981 | biostudies-arrayexpress
Project description: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.
2011-06-03 | GSE25981 | GEO
Project description:Epigenetic gene silencing induced by aberrant DNA methylation is an important mechanism leading to the loss of key cellular pathways in tumorigenesis. Although DNA demethylating agents reverse DNA methylation and have significant cytostatic and cytotoxic effects, it remains elusive whether their effects on cellular phenotypes are solely on reactivation of hypermethylated genes. To address this issue, we constructed LNCaP-derived human prostate cancer cell lines that can pharmacologically induce the expression of fusion gene comprising NFkB transcriptional activation domain (AD) and methyl-CpG binding domain (MBD). Tetracycline induction of NFkB (AD)-MBD protein led to so-called methyl-CpG targeted transcriptional activation (MeTA), as demonstrated by the reactivation of hypermethylated genes such as MT1M, NEFH, and NEFM. The cell proliferation assay indicated that MeTA suppressed the growth of LNCaP cells. Furthermore, both flow cytometry and TUNEL assay clearly demonstrated that MeTA induced apoptosis. In order to search genes responsible for apoptosis, we performed gene expression microarray analysis of MeTA-uninduced and -induced LNCaP cells: Several tumor necrosis factor receptor superfamily (TNFRSF) genes upregulated in accordance with the induction of MeTA. These results suggest that DNA methylation confers cancer cells the ability to avoid apoptosis and MeTA may provide an efficient mean to analyze the change in cancer cell phenotypes by DNA methylation alterations. This is the first report showing that the reactivation of hypermethylated genes by the method other than DNMT inhibition induces growth arrest and apoptosis in cancer cells.
2021-03-02 | GSE74743 | GEO
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-2’-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 “methyl-CpG targeted transcriptional activation (MeTA)” 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-2’-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 “MeTA” 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 “MeTA” 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.
2012-12-11 | GSE26215 | GEO
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.
2012-12-11 | E-GEOD-26215 | biostudies-arrayexpress
Project description:Aberrant DNA hypermethylation, the most well defined epigenetic changes in cancer, is associated with inappropriate gene silencing and this feature is utilized to search for tumor-specific DNA methylation biomarkers. Methyl-CpG binding domain (MBD) proteins (MBPs) can elicit the repressive potential of methylated DNA and play a major role in gene silencing mechanisms. Therefore, if the genes governed by MBPs are specifically reactivated, it should be possible to uncover them. We developed a method termed “methyl-CpG targeted transcriptional activation (MeTA)” that employs a fusion gene comprised of the MBD from MBD2 and the NFkB transcriptional activation domain. Microarray coupled with MeTA (MeTA-array) provides not only the information about methylated genes but also the one about transcriptional repression in a single experiment. We applied MeTA-array to 12 pancreatic cancer cell lines along with HPDE (normal pancreatic ductal epithelial cell line) and identified 31 candidate tumor-specific hypermethylated genes; 26 of them have never been reported previously using the conventional DNA demethylating agents. Seven genes, IRX4, LHX6, NEFH, NEFL, NEFM, NPTX2 and TMEM204 were further examined their methylation statuses by MSP, and we found that 100% (21/21) of IRX4, 62% (13/21) of LHX6, 100% (21/21) of NEFH, 100% (21/21) of NEFL, 100% (21/21) of NEFM, 100% (21/21) of NPTX2 and 95% (20/21) of TMEM204 were methylated in our series of pancreatic cancer cell lines. Furthermore, 68% (15/22) of IRX4, 55% (12/22) of LHX6, 55% (12/22) of NEFH, 59% (23/22) of NEFL, 82% (18/22) of NEFM and 82% (18/22) of NPTX2 were also hypermethylated in primary pancreatic cancer specimens in a tumor-specific manner. Our results suggest that MeTA-array is a highly efficient method to identify methylation-mediated transcriptionally silenced genes in human cancer.
2020-10-11 | GSE141247 | GEO
Project description:Identification of genes upregulated by MeTA-array in human pancreatic cancers
| PRJNA134971 | ENA
Project description:Methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor-suppressor genes. We developed a novel and robust technique that allows the unbiased, genome wide detection of CpG-methylation in limited DNA samples, without applying methylation-sensitive restriction endonucleases or bisulfite-treatment. The approach is based on a recombinant, methyl-CpG binding protein that efficiently binds CpG-methylated DNA depending on its degree of CpG methylation. Its application in methyl-CpG immunoprecipitation (MCIp) facilitates the monitoring of CpG-island methylation on a genome wide level (in combination with CpG-island microarrays). The power of this novel approach was demonstrated by the profiling of three myeloid cell lines leading to the identification of more than a hundred aberrantly methylated CpG islands and many novel, putative tumor-suppressor genes. Keywords: MCIp on Chip
2006-05-10 | GSE4009 | GEO
Project description:DNA demethylating agents are epigenetic drugs for the therapy of myeloid leukemias. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects, however, the relationships between these characteristics have not been established yet due to the lack of method to induce only DNA demethylation. Here we show that a fusion protein comprising the methyl-CpG binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. Gene expression microarray analyses using human embryonic kidney cell line 293T indicate that cells expressing wild-type (wt) TET1 catalytic domain with MBD (MBD-TET1-CDwt) upregulated more genes than ones expressing TET1-CDwt without MBD or catalytically inactive TET1-CD mutant (mut) with MBD (MBD-TET1-CDmut) and their upregulated genes frequently contained CpG islands (CGIs) within ± 1,000 bp of the transcription start site (TSS). Interestingly, 88% of genes upregulated 5-fold or more by MBD-TET1-CDwt were also reactivated after treatment with DNA demethylating agent, 5-azacytidine, suggesting that gene reactivation by both methods is primarily based on DNA demethylation.
2017-02-09 | GSE74744 | GEO
Project description:Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. DNA methylation-mediated silenced genes in pancreatic cancer were searched by methyl-CpG targeted transcriptional activation (MeTA) method and LHX6 (LIM homeobox 6), a transcription factor involved in embryogenesis and head development, was selected as one of candidate genes. LHX6 was downregulated in most pancreatic cancer cell lines (83%: 10/12) mainly through promoter hypermethylation and histone deacetylation. Furthermore, LHX6 was also methylated in primary pancreatic cancers in a tumor-specific manner (57%: 16/28). In order to assess the biological significance of LHX6 in pancreatic tumorigenesis, we first performed colony formation assay and found that LHX6 re-expression inhibited colony formation in pancreatic cancer cell lines. Similarly, inducible expression of LHX6 inhibited cell proliferation and migration in LHX6 low-expressing pancreatic cancer cell lines. On the other hand, knockdown of LHX6 accelerated cell proliferation in LHX6 high-expressing pancreatic cancer cell lines. Our present results suggest that epigenetic inactivation of LHX6 plays an important role in pancreatic tumorigenesis by promoting cell proliferation.
2020-10-11 | GSE135736 | GEO