The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase expression.
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.
Ait-Lounis
Aouatef
A
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Centre Médical Universitaire, Geneva, Switzerland.
Bonal
Claire
C
Seguín-Estévez
Queralt
Q
Schmid
Christoph D
CD
Bucher
Philipp
P
Herrera
Pedro L
PL
Durand
Bénédicte
B
Meda
Paolo
P
Reith
Walter
W
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*
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