Abstract
The ionotropic glutamate receptor 6 (GluR6 or GRIK2) gene is transcribed by two cell-type-specific promoters in neuronal and non-neuronal cells, which results in five different transcript variants. The purpose of this study was to explore cell-type-specific silencing of these promoters by epigenetic mechanisms. The neuronal and non-neuronal promoter sequences were cloned upstream of the luciferase gene in the pGL3 luciferase reporter vector. Promoter susceptibility to methylation was confirmed by 5-azacytidine and trichostatin treatment, and the status of CpG dinucleotides was determined by bisulfite sequencing of the promoter was determined by bisulfite sequences. GluR6A transcript variant was expressed in the brain, and GluR6B was most abundant in tumor cell lines. The neuronal promoter was methylated in non-neuronal cell lines. The treatment with 5-azacytidine and trichostatin upregulated transcription of the GluR6 gene, and methylation of the GluR6 promoter sequence in the luciferase reporter system led to downregulation of the luciferase gene transcription. Bisulfite sequencing revealed methylation of 3 and 41 CpG sites in non-neuronal and neuronal promoters, respectively. The differential activation/silencing of GluR6 promoters suggests that the transcript variants of GluR6 are involved in tissue-specific biological processes and their aberrant regulation in tumor cells may contribute to distinct properties of tumor cells.
Highlights
The ionotropic glutamate receptors are multimeric ligand-gated channels that regulate the flux of sodium and calcium ions across the neuronal cell membranes
By using the chromosome transfer technique, we have shown that the smallest region of chromosome 6 responsible for senescence harbors the GluR6 gene
We investigated the expression of GluR6 in 18 cell lines representing different tumors and compared them to the abundance of GluR6 transcript in chemically immortalized fibroblasts (SUSM1) as well as normal fibroblasts (FS2)
Summary
The ionotropic glutamate receptors are multimeric ligand-gated channels that regulate the flux of sodium and calcium ions across the neuronal cell membranes. These receptors are classified based on their binding preference for N-methyl-D-aspartate (NMDA), α-amino-3hydroxy-5-methyl-4-isoazolepropionic acid (AMPA), and 2-carboxy-3-carboxymethyl-4isopropenylpyrrolidine (kainate) [1]. The loss of heterozygosity for markers D6S1543, D6S449, D6S283, and D6S434 in breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, lymphomas, and leukemia are suggestive of the presence of a tumor critical gene in this region of chromosome 6 [8–12]. The biological significance of chromosome 6q is evident by its ability to induce senescence of normal and tumor cells [14–16]. The role of GluR6 in apoptosis is indirectly supported by other observations in the literature [5]
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