Chromatin-bound mitogen-activated protein kinases transmit dynamic signals in transcription complexes in beta-cells

Abstract

MAPK pathways regulate transcription through phosphorylation of transcription factors and other DNA-binding proteins. In pancreatic beta-cells, ERK1/2 are required for transcription of the insulin gene and several other genes in response to glucose. We show that binding of glucose-sensitive transcription activators and repressors to the insulin gene promoter depends on ERK1/2 activity. We also find that glucose and NGF stimulate the binding of ERK1/2 to the insulin gene and other promoters. An ERK1/2 cascade module, including MEK1/2 and Rsk, are found in complexes bound to these promoters. These findings imply that MAPK-containing signaling complexes are positioned on sensitive promoters with their protein substrates to modulate transcription in situ in response to incoming signals.

Fig. 1.

Differential effects of glucose and K⁺ depolarization on promoters in islets and β-cells. (A) Schematic representation of the relative location of primers used to amplify the insulin gene promoter in ChIP assays and the sites of factor binding. (B) ChIP assay on the insulin promoter in Min6 cells showing effects of glucose and K⁺ depolarization on enrichment of transcription factor association detected at 1-, 10-, and 100-fold dilutions of ChIP DNA by PCR. (C) Effects of inhibitors on glucose- and K⁺- induced transcription factor association. (D) Effects of acute glucose (30 min) and K⁺ (5 min) stimulation in cells that were cultured in 5.5 mM (Basal) or 25 mM glucose for three days (long-term). (E) Assay of insulin promoter reporter activity in response to glucose and K⁺ stimulation (4 h) in Min6 cells that were cultured in either basal or long-term high glucose. NS, no stimulation; G and Glc, glucose; U, U0126; FK, FK520; R, rapamycin.

Fig. 2.

Signal-transduced gene regulation and enrichment of ERK1/2 with promoter DNA in islets and β-cells. (A) Quantitative PCR of fos gene expression in Min6 cells exposed to 5.5 or 25 mM glucose in the presence of U0126. (B) Diagram of differential regulation of ERK1/2 by glucose and NGF. (C) ChIP analysis of insulin and fos gene promoters in INS-1 cells in response to glucose, GLP-1, and NGF without or with FK520. (D) ChIP analysis of the insulin promoter in INS-1 cells in response to NGF in the presence of FK520 and U0126 (Upper); pERK1/2 immunoblot (Lower). (E) ChIP assay time course of the fos gene promoter in response to glucose and K⁺ in INS-1 cells without or with FK520. (F) Schematic representation of the relative location of primers used to amplify the fos gene promoter in ChIP assays and the contained elements.

Fig. 3.

Stimulus-dependent association of MAPK signaling components with insulin and fos gene promoter DNA. (A) ChIP analysis of B-raf, MEK1, and ERK2 association with the fos promoter in INS-1 cells in response to glucose, GLP-1, and NGF in the presence of U (U0126), PD (PD098059), and FK (FK520). (B) Immunoprecipitation of Raf1 and B-Raf from glucose-stimulated INS-1 lysates. (C) MEK1/2 and ERK1/2 association with insulin and fos promoters in human islets in response to glucose, GLP-1, and NGF in the presence of U (U0126) and FK (FK506). (D) ERK1/2, RSK2, NFAT, and calcineurin A association with the insulin promoter in human islets in response to glucose and GLP-1 in the presence of U (U0126) and FK (FK506). Rabbit IgG was used as an antibody control in ChIPs.

Fig. 4.

Cooccupancy of ERK1/2 with transcription factors on the insulin gene promoter and requirement for MAPK signaling components. (A) Sequential ChIP assay for cooccupancy of ERK1/2 with transcription factors on the insulin gene promoter in human islets in response to glucose and GLP-1. (B) ChIP analysis of effects of transfected MAPK signaling components on ERK1/2 and MEK1/2 association with insulin gene promoter DNA. Rabbit IgG and goat IgG were used as antibody controls in ChIPs.