Artikel
Identification and characterization of c-Jun-N-terminal phosphorylation as an epigenetic regulator of highly methylated gene promoter regions in glioblastoma multiforme
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Veröffentlicht: | 13. Mai 2014 |
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Gliederung
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Objective: High-grade gliomas (HGG) are the most common brain tumors with an average survival of 14 months. A large number of highly methylated gene loci (G-CIMP) are associated with the proneural subgroup and better clinical outcome. Our aim was to identify and characterize putative transcription factors (TF) which are involved in the establishment of methylator phenotype in GBM.
Method: Brain tumor tissue of eight patients was classified as mesenchymal (n=4) and proneural (n=4) subgroup according to expression array analysis. Genome-wide methylation status was detected by Infinium Human Methylation 450k and analyzed with HOMER motif enrichment. Expression of the best ranked transcription factors was analyzed in tissue and primary cell lines. Cell behavior was evaluated using a scratch assay in one proneural and one mesenchymal cell line with activation (Anisomycin) or inhibition (SP600125) of the JNK pathway. Methylation status of treated cells was detected by Infinium Human Methylation 450k and analysed with RnBeads. Chromatin-bound phosphorylated c-Jun was immunoprecipitated to evaluate the enrichment of highly ranked gene promoters (b-value differences). In addition, immunoprecipitation (IP) was performed to detect P-c-Jun protein interaction with the DNA Methyltransferase family members.
Results: The AP-1 binding side was identified as a motif which was highly enriched in methylated gene promoters. By influencing the JNK pathway and consequently the phosphorylation status of c-Jun, we detected changes in cell behavior and expression of mesenchymal signature genes. An increase of P-c-Jun resulted in a less aggressive cell behavior with lower expression of genes connected to the mesenchymal subtype and increased expression of DNMT family members (DNMT1) whereas reduction in phosphorylation had the opposite effect. The methylation array detected an increase in methylation events after increasing P-c-Jun and a reduction in JNK-inhibited cells. Interestingly, a protein interaction between DNMT family members (DNMT1) and P-c-Jun was observed.
Conclusions: The phosporylation status of c-Jun influences the genome-wide methylation status. These data suggest that the JNK pathway plays an important role in development of GBM methylated phenotype and less aggressive behavior. This pathway might therefore be a potential target for personalized GBM treatment.