Activation of gastrin transcription in pancreatic insulinoma cells by a CACC promoter element and a 70-kDa sequence-specific DNA-binding protein.
Gastrin gene expression in the pancreatic islets is developmentally regulated and occurs largely during fetal life. Deletional analysis of transiently transfected rat insulinoma cells with gastrin 5'-flanking sequences in luciferase reporter genes demonstrated that the gastrin promoter sequence proximal to -111 base pairs (bp) contains the cis-regulatory elements necessary for maximal transcription. Mutational analysis identified the sequence CCCCACCCCA (-109 to -100 bp) as a positive cis-regulatory element (CACC) located 5' to a previously described negative element (-100 to -90 bp) and E-box positive element at -82 bp. Multimers of the CACC element in a heterologous promoter activated transcription independent of the other cis-regulatory elements. CACC binding proteins were purified from insulinoma cell nuclear extracts by cation exchange and affinity chromatography. Southwestern blot of nuclear extracts identified a 70-kDa CACC-binding protein. Mutational analysis of the CACC element showed a close correlation between DNA binding of this protein and transcriptional activation. Transcriptional activation by multimers of the CACC element in a heterologous promoter was detected in a variety of cell lines but was strongest in those of islet lineage. Likewise, the presence of the 70-kDa CACC-binding protein was found in many cell lines but was most abundant in the insulinoma cells. The CACC-binding protein has not been previously identified among the known pancreatic regulatory factors and may have an important role in the developmental expression of gastrin.
Tillotson
L G
LG
Gastrointestinal Unit, Massachusetts General Hospital, Boston 02114.
Wang
T C
TC
Brand
S J
SJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005755:Gastrins / Q000096:biosynthesis*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007340:Insulinoma / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D005755:Gastrins; D009687:Nuclear Proteins; C085482:Zbtb10 protein, rat