Sp1/Sp3 binding is associated with cell-specific expression of the glucose-dependent insulinotropic polypeptide receptor gene.
The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between -1 and -100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5'-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between -85 and -40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions -77, -60, and -50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at -50 and -60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at -70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between -100 and -40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter.
Boylan
Michael O
MO
Section of Gastroenterology, Boston Medical Center, 650 Albany St., Boston, MA 02118, USA.
Jepeal
Lisa I
LI
Wolfe
M Michael
MM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D005749:Gastric Inhibitory Polypeptide / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D005749:Gastric Inhibitory Polypeptide