Abstract
O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl DNA adducts such as those induced by alkylating agents. Loss of MGMT expression through transcriptional silencing by hypermethylation of its CpG island (CGI) is found in diverse human cancers including glioblastomas. Glioblastomas that have MGMT methylation respond to temozolomide, an alkylating agent, resulting in improved survival. Consequently, assessment of MGMT methylation has become a therapy response and prognostic indicator. However, it is not clear whether the region of the MGMT CGI commonly analysed is the critical region involved in transcriptional control. We measured methylation levels at each CpG site for the entire MGMT CGI using bisulfite modification and pyrosequencing, and compared them with MGMT mRNA expression in glioblastoma cell lines, xenografts and normal brain tissues (41 samples). Two critical regions were identified (DMR1 and DMR2). DMR2 encompasses the commonly analysed region and was always methylated when DMR1 was methylated. A luciferase reporter assay showed that substitutions of several specific CpG sites within DMR2 significantly attenuated the promoter activity of the MGMT CGI. Our results indicate that several CpG sites within DMR2 play a critical role in the transcriptional control of MGMT, making DMR2 the optimal target for methylation testing. However, given the highly variable patterns of MGMT methylation associated with transcriptional silencing observed in this region among the tumours in this study, methylation levels need to be measured at a number of individual CpGs within DMR2 to confidently predict transcriptional silencing and thus sensitivity to alkylating agents.
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Abbreviations
- CGI:
-
CpG island
- DMR:
-
Differentially methylated region
- FDR:
-
Benjamini and Hochberg false discover rate
- MGMT:
-
O6-Methylguanine-DNA methyltransferase
- MSP:
-
Methylation-specific PCR
- PCR:
-
Polymerase chain reaction
- qPCR:
-
Real-time quantitative PCR
- TF:
-
Transcription factor
- TMZ:
-
Temozolomide
- TSS:
-
Transcription start site
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Acknowledgments
This work was supported by funds from Samantha Dickson Brain Tumour Trust and Cambridge Fund for the Prevention of Disease (CAMPOD).
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401_2011_803_MOESM1_ESM.ppt
Supplementary Fig. 1. Two-way clustering and heat map of MGMT CGI methylation as determined by pyrosequencing (red, high methylation; green, low methylation). CpG sites clustered together are indicated on the right of the heat map. (PPT 231 kb)
401_2011_803_MOESM2_ESM.ppt
Supplementary Fig. 2. Superimposed plots of the multiple test-corrected p-values using different thresholds for expression to group samples (0.1%, blue; 1%, black; 30%, red) at each CpG site against their distance from TSS (=0, arrow) to indicate that the patterns of p-values are similar. Grid lines represent p=0.05, 0.01 and 0.001. The CpG numbers are indicated at selected sites. The positions of DMRs and MSP primers are shown with grey bars. (PPT 204 kb)
401_2011_803_MOESM3_ESM.ppt
Supplementary Fig. 3. Nucleosome occupancy prediction within MGMT CGI. The posterior probability to start a nucleosome (P start, orange line, right axis) and the nucleosome occupancy prediction (P occupied, black line, left axis) within the MGMT CpG island (see Materials and Methods for the source) are plotted. A High P start value indicates the position nucleosome binding is likely to start. A High P occupied value indicates the position nucleosome is likely to be bound. Positions of CpGs are indicated by grey dots (the dots for CpG83, 86 and 89 are larger) and the numbers of selected CpGs are shown above the dots. Binding sites for various transcription factors predicted by TFSEARCH (http://molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html) are represented by green lines. Among them, the putative STAT3 binding site is indicated. AP2 and SP1 binding sites predicted by Harris et al. [13] and Costello et al. [4] are shown respectively as red and yellow lines. Frequently methylated regions such as CpG1-20, DMR1 and DMR2 are indicated by dark blue lines (see also Fig. 2) whereas pale blue lines correspond to the minimal promoter and enhancer. TSS, transcription start site. (PPT 147 kb)
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Malley, D.S., Hamoudi, R.A., Kocialkowski, S. et al. A distinct region of the MGMT CpG island critical for transcriptional regulation is preferentially methylated in glioblastoma cells and xenografts. Acta Neuropathol 121, 651–661 (2011). https://doi.org/10.1007/s00401-011-0803-5
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DOI: https://doi.org/10.1007/s00401-011-0803-5