Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells.

• DOI: 10.1128/mcb.12.10.4578
• Publication Date: 2015-10-06T00:31:35Z
• Authors: K D Shelton; A J Franklin; A Khoor; J Beechem; M A Magnuson
• Keywords: CAAT box; TATA box; Glucokinase; Transcription

beta-cell type-specific expression of the upstream glucokinase promoter was studied by transfection fusion genes and analysis DNA-protein interactions. A construct containing 1,000 bp 5'-flanking DNA efficiently expressed in HIT M2.2.2 cells, a beta-cell-derived line that makes both insulin glucokinase, but not NIH 3T3 heterologous cell line. In series 5' deletion mutations between bases -1000 -100 (relative to base previously designated +1), efficient cells maintained until -280 bp, after which transcription decreased stepwise manner. The sequences -180 -1 contributing transcriptional activity were identified studying 28 block transversion mutants spanned this region 10-bp steps. Two reduced 10-fold or more, while six 3- 10-fold. Three mutationally sensitive regions found bind factor preferentially pancreatic islet beta cells. binding sites, elements (UPEs), shared consensus sequence CAT(T/C)A(C/G). Methylation adenine guanine residues within prevented factor, as did at positions 2, 3, 5. Analysis nuclear extracts from different lines UPE-binding beta-TC-3 AtT-20, 3T3, HeLa cells; possibility greatly amount alpha-TC-6 could be excluded. UV laser cross-linking experiments supported type showed it approximately 50 kDa size. Gel mobility shift competition is same binds similar elements, termed CT boxes, promoter. Thus, role for these (UPEs boxes), them, determining islets suggested.