Development of MeTA-inducible system in LNCaP cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE74743 | GEO | 2021/03/02
REPOSITORIES: GEO
Similar Datasets
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: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: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:Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour suppressor genes The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the meditation of gene silencing by interaction with histone deacetylases and histone methyltransferases. However the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD may proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in promoting DNA methylation. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer, highlighting a potential active role of MBD2 in promoting cancer specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 reveals that MBD2 associates with DNA methyltransferase (DNMT) enzymes 1 and 3A. Together our results demonstrate that MBD2 plays a critical role in â??rewritingâ?? the cancer methylome at specific regulatory regions. LNCaP prostate cancer cell line clones with reduced MBD2 expression were establised by using shRNA to MBD2 and scrambled control clones were established with scrambled control shRNA. To interrogate methylation changes induced by MBD2 knock-down we profiled three stably transfected scrambled control clones and three MBD2 knockdown clones on Illumina HumanMethylation450K arrays. Differential methylation analysis was carried out to identified CpG sites hypo-/hyper-methylated as a result of MBD2 knockdown.
2016-08-24 | E-GEOD-77201 | biostudies-arrayexpress
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:Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour suppressor genes The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the meditation of gene silencing by interaction with histone deacetylases and histone methyltransferases. However the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD may proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in promoting DNA methylation. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer, highlighting a potential active role of MBD2 in promoting cancer specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 reveals that MBD2 associates with DNA methyltransferase (DNMT) enzymes 1 and 3A. Together our results demonstrate that MBD2 plays a critical role in ârewritingâ the cancer methylome at specific regulatory regions. LNCaP prostate cancer cell line clones with reduced MBD2 expression were establised by using shRNA to MBD2 and scrambled control clones were established with scrambled control shRNA. To interrogate expression changes induced by MBD2 knock-down we profiled three stably transfected scrambled control clones and three MBD2 knockdown clones on Affymetrix HuGene 1.0ST expression arrays. Differential expression analysis was carried out to identified genes up-/down-regulated by MBD2 knockdown.
2016-08-24 | E-GEOD-77187 | 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:Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour suppressor genes The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the meditation of gene silencing by interaction with histone deacetylases and histone methyltransferases. However the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD may proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in promoting DNA methylation. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer, highlighting a potential active role of MBD2 in promoting cancer specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 reveals that MBD2 associates with DNA methyltransferase (DNMT) enzymes 1 and 3A. Together our results demonstrate that MBD2 plays a critical role in “rewriting” the cancer methylome at specific regulatory regions.
2016-08-24 | GSE77201 | GEO
Project description:Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour suppressor genes The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the meditation of gene silencing by interaction with histone deacetylases and histone methyltransferases. However the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD may proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in promoting DNA methylation. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer, highlighting a potential active role of MBD2 in promoting cancer specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 reveals that MBD2 associates with DNA methyltransferase (DNMT) enzymes 1 and 3A. Together our results demonstrate that MBD2 plays a critical role in “rewriting” the cancer methylome at specific regulatory regions.
2016-08-24 | GSE77187 | GEO
Project description:Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to the loss of key cellular pathways in tumorigenesis. We previously reported that PYCARD (PYD and CARD domain containing), an apoptosis inducing gene, was frequently hypermethylated in prostate cancer in a tumor-specific manner (46/51: 90%), by methylation specific PCR (MSP). In order to examine the relationship between methylation status and level of the PYCARD protein expression in prostate cancer, we further performed immunohistochemical analyses of the 51 MSP-analyzed prostate cancer specimens and observed reduced or absent protein expression in tumors with aberrant methylation status of PYCARD. In contrast, PYCARD was unmethylated and expressed in normal prostatic tissues including epithelial, basal and inflammatory cells. Furthermore, we tried to identify downstream genes of PYCARD by microarray analyses using PYCARD inducible LNCaP prostate cancer cells. Interestingly, PYCARD induction upregulated PYDC1, another PYCARD homolog that associates with PYCARD and accelerates inflammation and apoptosis. Our present results suggest that aberrant methylation of PYCARD is an extremely frequent event and contributes to escape from apoptosis by reducing PYCARD expression in prostate cancer.
2020-12-19 | GSE142283 | GEO