PDX-1 interaction and regulation of the Pancreatic Derived Factor (PANDER, FAM3B) promoter.
Pancreatic Derived Factor (PANDER) is a novel cytokine-like protein dominantly expressed within the endocrine pancreas. Our previous study demonstrated that the PANDER promoter was both tissue-specific and glucose-responsive. Surrounding the PANDER transcriptional start site are several putative A- and E-Box elements that may bind to the various pancreatic transcriptional factors of MafA, BETA2/NeuroD, and Pancreatic Duodenal Homeobox-1 (PDX-1). To characterize the transcriptional regulatory factors involved in PANDER gene expression, we performed co-transfection reporter gene analysis and demonstrated upregulation by all three transcription factors, with the greatest individual increase stemming from PDX-1. Potential binding of PDX-1 to A box (TAAT) regions of the PANDER promoter was demonstrated by chromatin immunoprecipitation (ChIP) and further corroborated by electrophoretic mobility shift assay (EMSA). Binding of PDX-1 to the A box regions was inhibited by mutagenized (TAGT) oligonucleotides. Site-directed mutagenesis of the three PDX-1 A box binding motifs revealed that A box sites 2 and 3 in combination were critical for maximal gene expression and deletion resulted in a 82% reduction in promoter activity. Furthermore, deletion of A box sites 2 and 3 completely diminished the glucose-responsiveness of the PANDER promoter. Our findings demonstrate that PANDER is a potential PDX-1 target gene and the A box sites within the promoter region are critical for basal and glucose-stimulated PANDER expression.
Burkhardt
Brant R
BR
Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318, USA. burkhardtb@email.chop.edu
Cook
Joshua R
JR
Young
Robert A
RA
Wolf
Bryan A
BA
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D050417:Insulin-Secreting Cells / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D014407:Tumor Cells, Cultured
D016207:Cytokines; D018398:Homeodomain Proteins; C504749:PANDER protein, mouse; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D005947:Glucose