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Background <p><i>MGMT</i> promoter methylation is a well-established biomarker in glioblastoma, its relevance in melanoma brain metastases remains controversial. Recent studies indicate that methylation of <i>MGMT</i> enhancers contributes to <i>MGMT</i> regulation, whereas the role of silencer methylation has not yet been investigated.</p> Methods <p>DNA methylation of 52 CpG sites was analyzed in regions annotated as potential silencers located in the promoter, intronic, and upstream regions of <i>MGMT</i>. These regions were selected based on genomic annotations and their genomic proximity to <i>MGMT</i>, encompassing sites to approximately 1.5&#xa0;Mb upstream, without experimental validation of direct regulatory activity. Analyses were performed in glioblastoma and melanoma brain metastases using in-house developed assays based on bisulfite conversion, PCR amplification of target regions, and pyrosequencing. By integrating previously published promoter and enhancer methylation data, a DNA methylation landscape of <i>MGMT</i> regulatory elements was established. Associations between CpG methylation and MGMT protein expression, overall survival, patient age, and sex were evaluated.</p> Results <p>The degree of methylation strongly depended on the genomic location of the respective regulatory element. Both shared and tumor-type-specific methylation patterns were observed in glioblastoma and melanoma brain metastases. Promoter-associated CpGs and selected intronic silencer-annotated regions showed associations with MGMT protein expression. In glioblastoma, several promoter-associated CpGs were associated with overall survival, whereas sex-specific and age-related effects were absent.</p> Conclusions <p>DNA methylation of <i>MGMT</i> regulatory elements is highly CpG-specific. CpG-resolved analyses reveal a complex epigenetic landscape across the <i>MGMT</i> locus and highlight substantial regional heterogeneity. These findings suggest that extending methylation analyses beyond the classical promoter region provides additional insights into <i>MGMT</i>-associated epigenetic variation and its potentical clinical associations.</p>

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DNA methylation of silencer-annotated regions at the MGMT locus in the context of promoter and enhancer methylation in glioblastoma and melanoma brain metastases

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月1日

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  • Katharina Pühringer,
  • Fabian Firlinger,
  • Benno Fehringer,
  • Serge Weis,
  • Sabine Spiegl-Kreinecker,
  • Margit Cichna-Markl

摘要

Background

MGMT promoter methylation is a well-established biomarker in glioblastoma, its relevance in melanoma brain metastases remains controversial. Recent studies indicate that methylation of MGMT enhancers contributes to MGMT regulation, whereas the role of silencer methylation has not yet been investigated.

Methods

DNA methylation of 52 CpG sites was analyzed in regions annotated as potential silencers located in the promoter, intronic, and upstream regions of MGMT. These regions were selected based on genomic annotations and their genomic proximity to MGMT, encompassing sites to approximately 1.5 Mb upstream, without experimental validation of direct regulatory activity. Analyses were performed in glioblastoma and melanoma brain metastases using in-house developed assays based on bisulfite conversion, PCR amplification of target regions, and pyrosequencing. By integrating previously published promoter and enhancer methylation data, a DNA methylation landscape of MGMT regulatory elements was established. Associations between CpG methylation and MGMT protein expression, overall survival, patient age, and sex were evaluated.

Results

The degree of methylation strongly depended on the genomic location of the respective regulatory element. Both shared and tumor-type-specific methylation patterns were observed in glioblastoma and melanoma brain metastases. Promoter-associated CpGs and selected intronic silencer-annotated regions showed associations with MGMT protein expression. In glioblastoma, several promoter-associated CpGs were associated with overall survival, whereas sex-specific and age-related effects were absent.

Conclusions

DNA methylation of MGMT regulatory elements is highly CpG-specific. CpG-resolved analyses reveal a complex epigenetic landscape across the MGMT locus and highlight substantial regional heterogeneity. These findings suggest that extending methylation analyses beyond the classical promoter region provides additional insights into MGMT-associated epigenetic variation and its potentical clinical associations.