Article: article from journal or magazin.
Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.
Journal of Biological Chemistry
Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells.
Adenoviridae/genetics, Amino Acid Motifs, Animals, Base Sequence, Blotting, Northern, Cell Line, Cell Line, Tumor, Cell Nucleus/metabolism, Connexins/metabolism, DNA, Complementary/metabolism, Gene Library, Gene Transfer Techniques, Genes, Reporter, Hela Cells, Histone Deacetylases/metabolism, Humans, Hydroxamic Acids/pharmacology, Insulin/metabolism, Islets of Langerhans/metabolism, Luciferases/metabolism, Macrophages/metabolism, Mice, Microscopy, Fluorescence, Molecular Sequence Data, Mutation, Neurons/metabolism, Phenotype, Plasmids/metabolism, Promoter Regions, Genetic, Protein Binding, Rats, Repressor Proteins/metabolism, Repressor Proteins/physiology, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Nucleic Acid, Transcription Factors/metabolism, Transcription Factors/physiology, Transcription, Genetic, Transfection
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