Relative contribution of PDX-1, MafA and E47/b2 to the regulation of the human insulin promoter.

Hilary Docherty, Colin Hay, Angela Louise Ferguson, John Barrow, Elaine Durward, Kevin Docherty

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

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 beta 2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta 2 to activate the rat insulin I promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/beta 2 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/beta 2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 beta-cells. In the islet glucagonoma cell line alpha TC1.6, 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 beta 2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and beta 2 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 I 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 I promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin I gene in alpha TC1.6 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 I gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.

Original languageEnglish
Pages (from-to)813-820
Number of pages8
JournalBiochemical Journal
Volume389
Issue number3
DOIs
Publication statusPublished - 1 Aug 2005

Keywords

  • animals
  • basic Helix-Loop-Helix transcription Factors
  • cell line, tumor
  • DNA-binding proteins
  • gene expression regulation
  • HMGB proteins
  • homeodomain proteins
  • humans
  • insulin
  • Maf transcription factors, Large
  • Promoter Regions, Genetic
  • TCF transcription factors
  • trans-activators
  • transcription factors
  • diabetes mellitus
  • gemne transcription
  • islets of Langerhans
  • PDX-1

Cite this

Docherty, H., Hay, C., Ferguson, A. L., Barrow, J., Durward, E., & Docherty, K. (2005). Relative contribution of PDX-1, MafA and E47/b2 to the regulation of the human insulin promoter. Biochemical Journal, 389(3), 813-820. https://doi.org/10.1042/BJ20041891

Relative contribution of PDX-1, MafA and E47/b2 to the regulation of the human insulin promoter. / Docherty, Hilary; Hay, Colin; Ferguson, Angela Louise; Barrow, John; Durward, Elaine; Docherty, Kevin.

In: Biochemical Journal, Vol. 389, No. 3, 01.08.2005, p. 813-820.

Research output: Contribution to journalArticle

Docherty, H, Hay, C, Ferguson, AL, Barrow, J, Durward, E & Docherty, K 2005, 'Relative contribution of PDX-1, MafA and E47/b2 to the regulation of the human insulin promoter.', Biochemical Journal, vol. 389, no. 3, pp. 813-820. https://doi.org/10.1042/BJ20041891
Docherty, Hilary ; Hay, Colin ; Ferguson, Angela Louise ; Barrow, John ; Durward, Elaine ; Docherty, Kevin. / Relative contribution of PDX-1, MafA and E47/b2 to the regulation of the human insulin promoter. In: Biochemical Journal. 2005 ; Vol. 389, No. 3. pp. 813-820.
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abstract = "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 beta 2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta 2 to activate the rat insulin I promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/beta 2 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/beta 2 binding sites reduced promoter activity by 60, 74 and 94{\%} respectively, in INS-1 beta-cells. In the islet glucagonoma cell line alpha TC1.6, 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 beta 2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and beta 2 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 I 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 I promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin I gene in alpha TC1.6 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 I gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.",
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N2 - 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 beta 2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta 2 to activate the rat insulin I promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/beta 2 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/beta 2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 beta-cells. In the islet glucagonoma cell line alpha TC1.6, 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 beta 2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and beta 2 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 I 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 I promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin I gene in alpha TC1.6 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 I gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.

AB - 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 beta 2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta 2 to activate the rat insulin I promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/beta 2 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/beta 2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 beta-cells. In the islet glucagonoma cell line alpha TC1.6, 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 beta 2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and beta 2 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 I 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 I promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin I gene in alpha TC1.6 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 I gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.

KW - animals

KW - basic Helix-Loop-Helix transcription Factors

KW - cell line, tumor

KW - DNA-binding proteins

KW - gene expression regulation

KW - HMGB proteins

KW - homeodomain proteins

KW - humans

KW - insulin

KW - Maf transcription factors, Large

KW - Promoter Regions, Genetic

KW - TCF transcription factors

KW - trans-activators

KW - transcription factors

KW - diabetes mellitus

KW - gemne transcription

KW - islets of Langerhans

KW - PDX-1

U2 - 10.1042/BJ20041891

DO - 10.1042/BJ20041891

M3 - Article

C2 - 15862113

VL - 389

SP - 813

EP - 820

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -