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An Epigenetic Mechanism of High Gdnf Transcription in Glioma Cells Revealed by Specific Sequence Methylation.
- Source :
-
Molecular neurobiology [Mol Neurobiol] 2016 Sep; Vol. 53 (7), pp. 4352-62. Date of Electronic Publication: 2015 Aug 01. - Publication Year :
- 2016
-
Abstract
- Glioma cells express high levels of GDNF. When investigating its transcriptional regulation mechanism, we observed increased or decreased methylation of different cis-acting elements in the gdnf promoter II. However, it is difficult to determine the contributions of methylation changes of each cis-acting element to the abnormally high transcription of gdnf gene. To elucidate the contributions of methylation changes of specific cis-acting elements to the regulation of gdnf transcription, we combined gene site-directed mutation, molecular cloning, and dual luciferase assay to develop the "specific sequence methylation followed by plasmid recircularization" method to alter methylation levels of specific cis-acting elements in the gdnf promoter in living cells and assess gene transcriptional activity. This method successfully introduced artificial changes in the methylation of different cis-acting elements in the gdnf promoter II. Moreover, compared with unmethylated gdnf promoter II, both silencer II hypermethylation plus enhancer II unmethylation and hypermethylation of the entire promoter II (containing enhancer II and silencer II) significantly enhanced gdnf transcriptional activity (P < 0.05), and no significant difference was noted between these two hypermethylation patterns (P > 0.05). Enhancer II hypermethylation plus silencer II unmethylation did not significantly affect gene transcription (P > 0.05). Furthermore, we found significantly increased DNA methylation in the silencer II of the gdnf gene in high-grade astroglioma cells with abnormally high gdnf gene expression (P < 0.01). The absence of silencer II significantly increased gdnf promoter II activity in U251 cells (P < 0.01). In conclusion, our specific sequence methylation followed by plasmid recircularization method successfully altered the methylation levels of a specific cis-acting element in a gene promoter in living cells. This method allows in-depth investigation of the impact of methylation changes of different cis-acting elements in the same promoter on gene transcriptional activity. Our findings provide preliminary evidence that silencer II hypermethylation in the gdnf promoter II may underlie high gene transcription in high-grade glioma cells.
- Subjects :
- Base Sequence
Cell Line, Tumor
CpG Islands genetics
DNA, Circular genetics
Gene Expression Regulation, Neoplastic
Humans
Mutation genetics
Plasmids metabolism
Promoter Regions, Genetic
Sequence Deletion
Brain Neoplasms genetics
DNA Methylation genetics
Epigenesis, Genetic
Glial Cell Line-Derived Neurotrophic Factor genetics
Glioma genetics
Transcription, Genetic
Subjects
Details
- Language :
- English
- ISSN :
- 1559-1182
- Volume :
- 53
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Molecular neurobiology
- Publication Type :
- Academic Journal
- Accession number :
- 26232065
- Full Text :
- https://doi.org/10.1007/s12035-015-9365-1