Glucose-stimulated expression of Txnip is mediated by carbohydrate response element-binding protein, p300, and histone H4 acetylation in pancreatic beta cells

Abstract

Recently, we identified Txnip (thioredoxin-interacting protein) as a mediator of glucotoxic beta cell death and discovered that lack of Txnip protects against streptozotocin- and obesity-induced diabetes by preventing beta cell apoptosis and preserving endogenous beta cell mass. Txnip has therefore become an attractive target for diabetes therapy, but although we have found that txnip transcription is highly induced by glucose through a unique carbohydrate response element, the factors controlling this effect have remained unknown. Using transient transfection experiments, we now show that overexpression of the carbohydrate response element-binding protein (ChREBP) transactivates the txnip promoter, whereas ChREBP knockdown by small interfering RNA completely blunts glucose-induced txnip transcription. Moreover, chromatin immunoprecipitation demonstrated that glucose leads to a dose- and time-dependent recruitment of ChREBP to the txnip promoter in vivo in INS-1 beta cells as well as human islets. Furthermore, we found that the co-activator and histone acetyltransferase p300 co-immunoprecipitates with ChREBP and also binds to the txnip promoter in response to glucose. Interestingly, this is associated with specific acetylation of histone H4 and recruitment of RNA polymerase II as measured by chromatin immunoprecipitation. Thus, with this study we have identified ChREBP as the transcription factor that mediates glucose-induced txnip expression in human islets and INS-1 beta cells and have characterized the chromatin modification associated with glucose-induced txnip transcription. In addition, the results reveal for the first time that ChREBP interacts with p300. This may explain how ChREBP induces H4 acetylation and sheds new light on glucose-mediated regulation of chromatin structure and transcription.

FIGURE 1.

ChREBP effects on txnip promoter activity. A, conserved ChREBP binding site (gray) in the txnip promoter of mouse, rat, and human. The core sequence of the four highest conserved consecutive positions is shown in capital letters (MatInspector), and E-boxes are shown in boldface type. B, mutation of the core sequence and first E-box. Shown are the effects of ChREBP (C) and NeuroD (D) overexpression on txnip promoter activity, as measured by luciferase activity. Bars, means ± S.E. of three independent experiments performed in duplicates.

FIGURE 2.

In vivo binding of ChREBP to txnip promoter in INS-1 beta cells by ChIP. INS-1 beta cells were incubated at 5 or 25 mm glucose for 6 h, and ChIP assays were performed using ChREBP (A), Pol II (B), and β-galactosidase (as IgG control) (C) antibodies. The ChoRE regions of rat txnip and LPK as well as the GAPDH coding region were amplified by quantitative real time RT-PCR, and the percentage of bound promoter was calculated. Bars, means ± S.E. of at least three independent ChIP assays.

FIGURE 3.

Dose response and time course of glucose-induced recruitment of ChREBP to txnip promoter. A and B, dose response. INS-1 cells were treated with increasing glucose concentrations (0, 5, 11, and 25 mm) for 6 h prior to analysis by ChIP. C–E, time course. INS-1 cells were incubated at 25 mm glucose for 0, 1.5, 6, and 24 h prior to ChIP. Bars, means ± S.E. of at least three independent ChIP assays.

FIGURE 4.

Effects of ChREBP knockdown on glucose-induced txnip promoter activity. INS-1 cells were transfected with siChREBP or scrambled oligonucleotide, and successful ChREBP knockdown was confirmed at the mRNA (A) and at the protein level (B) by quantitative RT-PCR and immunoblotting, respectively. INS-1 cells were co-transfected with the txnip promoter-driven luciferase reporter plasmid (C) (or the SV40-driven pGL3 control plasmid (D)) and with scrambled oligonucleotide or siChREBP and incubated at low (5 mm) or high (25 mm) glucose for 24 h. Bars, -fold changes in luciferase activity; means ± S.E. of three independent experiments performed in triplicates.

FIGURE 5.

TSA effects on txnip mRNA expression and histone H3/H4 acetylation. A, INS-1 cells were incubated at 5 mm glucose (gray), 5 mm glucose with 60 nm HDAC inhibitor TSA (black), 25 mm glucose (white), or 25 mm glucose with 60 nm of TSA (shaded) for 6 h, and RNA was isolated and analyzed for txnip expression by quantitative RT-PCR. Bars, mean -fold changes ± S.E. of three independent experiments. INS-1 cells were again incubated at 5 mm glucose in the presence or absence of TSA (60 nm) for 6 h, and ChIP assays were performed using antibodies directed against acetylated histone H3 (Ac-H3) (B) and H4 (Ac-H4) (C). Bars, means ± S.E. of at least three independent ChIP experiments.

FIGURE 6.

Glucose-induced histone modification of the txnip gene. INS-1 cells were treated with 25 mm glucose for different times, as indicated in the figure. ChIP assays were performed using antibodies directed against acetylated histone H4 (Ac-H4) (A), H3 (Ac-H3) (B), and lysine 4-trimethylated H3 (H3-K4me3) (C). Bars, means ± S.E. of at least three independent ChIP experiments.

FIGURE 7.

ChREBP and p300 interaction and recruitment to the txnip promoter in human islets. Human islets were incubated at low or high glucose for 24 h, and ChIP assays were performed using antibodies directed against ChREBP (A) and p300 (B). Bars, means ± S.E. of at least three independent ChIP assays using islets of different donors, whereby each donor served as its own control. Co-immunoprecipitation experiments were performed using nuclear extracts of human islets incubated for 24 h at high glucose and immunoprecipitating with ChREBP (C) or p300 (D) and immunoblotting for p300, ChREBP, and Mlx. One representative of at least three independent experiments is shown.

FIGURE 8.

Schematic of glucose-induced transactivating complex formation on the txnip promoter. Glucose induces the binding of ChREBP/Mlx heterodimers to the ChoRE, which in turn recruits p300 to the promoter via direct (or indirect) physical interaction between ChREBP and p300. The recruited p300 stimulates acetylation of histone H4 through its HAT activity and promotes Pol II promoter occupancy, resulting in increased txnip transcription.