5-Lipoxygenase and epigenetic DNA methylation in aging cultures of cerebellar granule cells
| Autor: | Svetlana Dzitoyeva, Louisa W. Ng, Marta Imbesi, Hari Manev |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
DNA (Cytosine-5-)-Methyltransferase 1
Methyltransferase Cellular differentiation Molecular Sequence Data Biology DNA methyltransferase Article DNA Methyltransferase 3A Epigenesis Genetic Mice Cerebellum Animals DNA (Cytosine-5-)-Methyltransferases RNA Messenger Epigenetics Promoter Regions Genetic Cells Cultured Cellular Senescence Arachidonate 5-Lipoxygenase Base Sequence General Neuroscience Cell Differentiation Methylation DNA Methylation Molecular biology Mice Inbred C57BL Animals Newborn embryonic structures DNA methylation DNMT1 5' Untranslated Regions Cell aging |
| Zdroj: | Neuroscience. 164:1531-1537 |
| ISSN: | 0306-4522 |
| Popis: | 5-Lipoxygenase (5-LOX), an enzyme involved in the metabolism of arachidonic acid, participates in the modulation of the proliferation and differentiation of neural stem cells and cerebellar granule cell (CGC) precursors. Since epigenetic mechanisms including DNA methylation regulate 5-LOX expression and have been suggested as possible modulators of stem cell differentiation and aging, using primary cultures of mouse CGC (1, 5, 10, 14, 30 days in vitro; DIV), we studied DNA methylation patterns of the 5-LOX promoter and 5-LOX mRNA levels. We also measured the mRNA and protein content of the DNA methyltransferases DNMT1 and DNMT3a. 5-LOX, DNMT1, and DNMT3a mRNA levels were measured by real-time PCR. We observed that 5-LOX expression and the expression of maintenance DNMT1 is maximal at 1 DIV (proliferating neuronal precursors), whereas the expression of the de novo DNA methyltransferase DNMT3a mRNA increased in aging cultures. We analyzed the methylation status of the 5-LOX promoter using the methylation-sensitive restriction endonucleases AciI, BstUI, HpaII, and HinP1I, which digest unmethylated CpGs while leaving methylated CpGs intact. The 5-LOX DNA methylation increased with the age of the cells. Taken together, our data show that as cultured CGC mature and age in-vitro, a decrease in 5-LOX mRNA content is accompanied by an increase in the methylation of the gene DNA. In addition, an increase in DNMT3a but not DNMT1 expression accompanies an increase of 5-LOX methylation during in-vitro maturation. |
| Databáze: | OpenAIRE |
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