Identification of the human insulin negative regulatory element as a negative glucocorticoid response element

被引:32
作者
Goodman, PA
MedinaMartinez, O
FernandezMejia, C
机构
[1] UNIV NACL AUTONOMA MEXICO, INP, INST INVEST BIOMED, UNIDAD GENET NUTR, MEXICO CITY 04530, DF, MEXICO
[2] CEDARS SINAI MED CTR, RES INST, LOS ANGELES, CA 90048 USA
[3] CEDARS SINAI MED CTR, DEPT MED, LOS ANGELES, CA 90048 USA
关键词
glucocorticoid receptor; negative regulation; insulin;
D O I
10.1016/0303-7207(96)03830-0
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Insulin gene transcription in adults is restricted to pancreatic beta cells. Studies with both transgenic mice and islet cell lines have demonstrated that beta cell specific expression is conferred by the 5' flanking region of the insulin gene. Transfection analysis has shown that cell specific expression involved an interaction between both positive and negative promoter cis elements. An upstream region (between - 258 and - 279) of the human insulin promoter served as a site of negative regulation. Transfection analysis in the pancreatic cell line HIT T-15 M 2.2.2 revealed that a DNA fragment containing this region causes a 45% reduction in promoter activity when linked to the native insulin promoter and a 72% reduction when linked to a heterologous tk promoter. Electrophoretic mobility shift analysis of this negative regulatory region (NRE) reveals a complex pattern of binding, wherein two major and several minor complexes are observed. Competition experiments demonstrated that formation of the fastest mobility complex is completely inhibited with excess cold glucocorticoid responsive element (GRE) consensus oligonucleotide. Purified glucocorticoid receptor binding domain (T7X556) demonstrated binding to the NRE oligonucleotide. Functional studies showed that dexamethasone treatment of HIT T-15 M 2.2.2 cells containing an NRE-tk CAT plasmid decreased CAT gene expression by 48%. Analysis of the NRE revealed 73% homology with the negative GRE consensus sequence. These data show that the human insulin NRE is a negative glucocorticoid response element.
引用
收藏
页码:139 / 146
页数:8
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