Cloning and DNA-binding properties of the rat pancreatic beta-cell-specific factor Nkx6.1.
The homeodomain (HD) protein Nkx6.1 is the most beta-cell-specific transcription factor known in the pancreas and its function is critical for the formation of the insulin-producing beta-cells. However, the target genes, DNA-binding site, and transcriptional properties of Nkx6.1 are unknown. Using in vitro binding site selection we have identified the DNA sequence of the Nkx6.1 binding site to be TTAATTG/A. A reporter plasmid containing four copies of this sequence is activated by an Nkx6.1HD/VP16 fusion construct. Full-length Nkx6.1 fails to activate this reporter plasmid in spite of robust interaction with the binding site in vitro. Stable expression of Nkx6.1 in the glucagon-producing alpha-cell-like MSL-G-AN cells induces expression of the endogenous insulin gene in a subset of the cell population. The expression of other known beta-cell-specific factors such as Pax4, Pax6, Pdx1, GLUT2 and GLP1-R is unchanged by the introduction of Nkx6.1.
Jørgensen
M C
MC
Department of Developmental Biology, Hagedorn Research Institute, Niels Steensensvej 6, DK-2820, Gentofte, Denmark.
Vestergård Petersen
H
H
Ericson
J
J
Madsen
O D
OD
Serup
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D005935:Glucagonoma / Q000473:pathology; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000473:pathology; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C491327:Nkx6-1 protein, rat; D011993:Recombinant Fusion Proteins; D004247:DNA