Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.
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.
Martin
David
D
Department of Internal Medicine, University Hospital, CHUV-1011 Lausanne, Switzerland.
Tawadros
Thomas
T
Meylan
Laure
L
Abderrahmani
Amar
A
Condorelli
Daniele F
DF
Waeber
Gérard
G
Haefliger
Jacques-Antoine
JA
D000256:Adenoviridae / Q000235:genetics; D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D017630:Connexins / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006367:HeLa Cells; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D000097004:Gap Junction delta-2 Protein; D000099229:RE1-Silencing Transcription Factor
D017630:Connexins; D018076:DNA, Complementary; D006655:Histone Deacetylases; D006877:Hydroxamic Acids; D007328:Insulin; D008156:Luciferases; D012097:Repressor Proteins; D014157:Transcription Factors; D000097004:Gap Junction delta-2 Protein; D000099229:RE1-Silencing Transcription Factor; C012589:trichostatin A