Functional identification of an intronic promoter of the human glucose-dependent insulinotropic polypeptide gene.
Glucose-dependent insulinotropic polypeptide (GIP), a physiological incretin and enterogastrone, plays a vital role in regulating glucose-dependent insulin release from the pancreas and gastric acid secretion from the stomach. By using a transgenic mouse approach, we previously reported that the distal 1.2kb promoter region of the human GIP (hGIP) gene (-2545/-346, relative to the ATG) was able to target the transgene expression in the stomach but not in the small intestine where the majority of GIP-producing cells are located. In the present study, in order to identify the cis-acting element(s) that is/are required for intestinal expression, a 1.6kb (-1580/-) DNA fragment within the first intron of the hGIP gene was isolated and characterized in three GIP-expressing cell lines including HuTu80 (duodenal cells), PANC-1 (pancreatic ductal cells) and Hs746T (stomach cells). By 5' and 3' deletion analysis, a proximal promoter element was confined within the nucleotides -102/-1. This promoter element, functions in an orientation-dependent manner, was able to drive 15.1 and 18.3 fold increases in promoter activities in HuTu80 and PANC-1 cells, respectively. Site-directed mutation analysis indicated that the region -54/-23 was essential for promoter function while the region -22/-1 might possess opposite effects in HuTu80 and PANC-1 cells. In competitive and antibody supershift assays, interactions of the progesterone receptor (PR) and some unknown protein factors from HuTu80 and PANC-1 with the motif(s) at -54/-23 were evident. Consistent with this finding, we demonstrated the transcriptional regulation of the hGIP promoter by progesterone via the PR-B isoform and that progesterone treatment in both HuTu80 and PANC-1 cells resulted in an increase in hGIP transcript level. In addition, a sequence motif (ACATGT) residing -48/-43 was found to be responsible for the binding of potential TFII regulator(s). Taken together, our results suggest that the proximal intronic sequences contain essential cis-acting elements for the cell-specific expression of the hGIP gene.
Hoo
Ruby L C
RL
School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, PR China.
Chu
Jessica Y S
JY
Yuan
Y
Y
Yeung
C M
CM
Chan
Kathy Y Y
KY
Chow
Billy K C
BK
D045744:Cell Line, Tumor; D018387:Codon, Initiator; D004379:Duodenal Neoplasms / Q000378:metabolism; D005749:Gastric Inhibitory Polypeptide / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007438:Introns; D016297:Mutagenesis, Site-Directed; D010190:Pancreatic Neoplasms / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011980:Receptors, Progesterone / Q000378:metabolism; D013274:Stomach Neoplasms / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000378:metabolism
D018387:Codon, Initiator; D011980:Receptors, Progesterone; D019473:Transcription Factors, TFII; C097314:progesterone receptor B; D005749:Gastric Inhibitory Polypeptide