Relative contribution of PDX-1, MafA and E47/beta2 to the regulation of the human insulin promoter.
The insulin promoter binds a number of tissue-specific and ubiquitous transcription factors. Of these, the homoeodomain protein PDX-1 (pancreatic duodenal homeobox factor-1), the basic leucine zipper protein MafA and the basic helix-loop-helix heterodimer E47/BETA2 (beta-cell E box transactivator 2; referred to here as beta2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta2 to activate the rat insulin 1 promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/beta2 in regulating the human insulin promoter, and whether these factors could interact synergistically in the context of the human promoter. Mutagenesis of the PDX-1, MafA and E47/beta2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 beta-cells. In the islet glucagonoma cell line alphaTC1.6, overexpression of PDX-1 and MafA separately increased promoter activity approx. 2.5-3-fold, and in combination approx. 6-fold, indicating that their overall effect was additive. Overexpression of E47 and beta2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and beta2 each increased activity by less than 2-fold. There was no indication of any synergistic effects on the human insulin promoter. On the other hand, the rat insulin 1 promoter and a mutated version of the human insulin promoter, in which the relevant regulatory elements were separated by the same distances as in the rat insulin 1 promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin 1 gene in alphaTC1.6 cells, whereas MafA activated the insulin 2 gene. In combination, PDX-1 and MafA activated both insulin genes. Chromatin immunoprecipitation assays confirmed that PDX-1 increased the association of acetylated histones H3 and H4 with the insulin 1 gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.
Docherty
Hilary M
HM
School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK.
Hay
Colin W
CW
Ferguson
Laura A
LA
Barrow
John
J
Durward
Elaine
E
Docherty
Kevin
K
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D024002:HMGB Proteins / Q000502:physiology*; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics; D051473:Maf Transcription Factors, Large; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000502:physiology*; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000502:physiology*
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D024002:HMGB Proteins; D018398:Homeodomain Proteins; D007328:Insulin; D051473:Maf Transcription Factors, Large; D051820:TCF Transcription Factors; D015534:Trans-Activators; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; C485159:MAFA protein, human; C093970:NEUROD1 protein, human; C544912:TCF7L1 protein, human; C553060:Tcf7l1 protein, rat; C084010:pancreatic and duodenal homeobox 1 protein