title: The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase expression.
abstract: OBJECTIVE
  Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing beta-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency.
  
  
  METHODS
  Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of beta-cells from endocrine progenitors, and the expression of mature beta-cell markers during development in Rfx3(-/-) and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 beta-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes.
  
  
  RESULTS
  Reduced development of insulin-positive cells in Rfx3(-/-) mice was not due to deficiencies in endocrine progenitors or beta-lineage specification, but reflected the accumulation of insulin-positive beta-cell precursors and defective beta-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated beta-cells developed in pancreas-specific Rfx3-deficient embryos. Defective beta-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference-mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene.
  
  
  CONCLUSIONS
  Our results show that Rfx3 is required for the differentiation and function of mature beta-cells and regulates the beta-cell promoter of the glucokinase gene.
authors.affiliation: Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Centre Médical Universitaire, Geneva, Switzerland.
authors.forename: Aouatef
authors.initials: A
authors.lastname: Ait-Lounis
authors.forename: Claire
authors.initials: C
authors.lastname: Bonal
authors.forename: Queralt
authors.initials: Q
authors.lastname: Seguín-Estévez
authors.forename: Christoph D
authors.initials: CD
authors.lastname: Schmid
authors.forename: Philipp
authors.initials: P
authors.lastname: Bucher
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Herrera
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Durand
authors.forename: Paolo
authors.initials: P
authors.lastname: Meda
authors.forename: Walter
authors.initials: W
authors.lastname: Reith
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D000071701:Regulatory Factor X Transcription Factors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D007328:Insulin; C000605540:RFX3 protein, human; D000071701:Regulatory Factor X Transcription Factors; C000605541:Rfx3 protein, mouse; D014157:Transcription Factors; D005941:Glucokinase