Abstract
Background: The molecular regulation of increased MGMT expression in human brain tumors, the associated regulatory elements, and linkages of these to its epigenetic silencing are not understood. Because the heightened expression or non-expression of MGMT plays a pivotal role in glioma therapeutics, we applied bioinformatics and experimental tools to identify the regulatory elements in the MGMT and neighboring EBF3 gene loci. Results: Extensive genome database analyses showed that the MGMT genomic space was rich in and harbored many undescribed RNA regulatory sequences and recognition motifs. We extended the MGMT’s exon-1 promoter to 2019 bp to include five overlapping alternate promoters. Consensus sequences in the revised promoter for (a) the transcriptional factors CTCF, NRF1/NRF2, GAF, (b) the genetic switch MYC/MAX/MAD, and (c) two well-defined p53 response elements in MGMT intron-1, were identified. A putative protein-coding or non-coding RNA sequence was located in the extended 3′ UTR of the MGMT transcript. Eleven non-coding RNA loci coding for miRNAs, antisense RNA, and lncRNAs were identified in the MGMT-EBF3 region and six of these showed validated potential for curtailing the expression of both MGMT and EBF3 genes. ChIP analysis verified the binding site in MGMT promoter for CTCF which regulates the genomic methylation and chromatin looping. CTCF depletion by a pool of specific siRNA and shRNAs led to a significant attenuation of MGMT expression in human GBM cell lines. Computational analysis of the ChIP sequence data in ENCODE showed the presence of NRF1 in the MGMT promoter and this occurred only in MGMT-proficient cell lines. Further, an enforced NRF2 expression markedly augmented the MGMT mRNA and protein levels in glioma cells. Conclusions: We provide the first evidence for several new regulatory components in the MGMT gene locus which predict complex transcriptional and posttranscriptional controls with potential for new therapeutic avenues.
Highlights
Human O6-methylguanine DNA methyltransferase (MGMT), called O6-alkylguanine DNA alkyltransferase (AGT) is a simple DNA repair protein involved in the protection of the normal cellular genome from the mutagenic actions of alkylating agents [1,2]
Much of the MGMT transcriptional studies and its negative regulation in gliomas are heavily related to the promoter methylation, the regulatory elements elsewhere in the gene have received scant attention
This study provided the first evidence for alternative promoters, new intronic and exonic regulatory motifs, the involvement of the CTCF and NRF2 transcription factors, and several RNA regulatory determinants within the MGMT and neighboring genomic region
Summary
Human O6-methylguanine DNA methyltransferase (MGMT), called O6-alkylguanine DNA alkyltransferase (AGT) is a simple DNA repair protein involved in the protection of the normal cellular genome from the mutagenic actions of alkylating agents [1,2]. Human cancers including brain tumors express the MGMT protein in abundance [4,5,6,7]; a large fraction of CNS malignancies, are MGMT-deficient due to gene silencing by promoter methylation [8,9]. The MGMT status in brain cancers is of huge importance for treatment because the antimutagenic function of MGMT interferes with the cytotoxic actions of anticancer alkylating agents [7] This is because MGMT effectively repairs the O6-methylguanine and O6-chloroethylguanine lesions induced by methylating agents (temozolomide, TMZ), and chloroethylating agents (BCNU, CCNU), respectively, thereby preventing the generation of mutagenic lesions and interstrand DNA cross-links [7,10]. Conclusions: We provide the first evidence for several new regulatory components in the MGMT gene locus which predict complex transcriptional and posttranscriptional controls with potential for new therapeutic avenues
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