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. 2000 Sep 12;97(19):10412-7.
doi: 10.1073/pnas.190326997.

DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells

N Nasrin  1 , S Ogg, C M Cahill, W Biggs, S Nui, J Dore, D Calvo, Y Shi, G Ruvkun, M C Alexander-Bridges
Affiliations

DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells

N Nasrin et al. Proc Natl Acad Sci U S A. .

Abstract

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. We find that the Caenorhabditis elegans protein DAF-16 binds the IGFBP-1 small middle dotIRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1.IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). We show that DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1.IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, we conclude that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site.

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Figures

Figure 1
Figure 1
Effect of DAF-16 homologs on insulin- and glucocorticoid-responsive gene transcription. HepG2 were cotransfected with a construct encoding the native IGFBP-1 promoter (10 μg/ml) driving luciferase gene expression and the pcDNA expression vector alone (1 μg/ml), or the expression vectors pcDNA⋅DAF-16, pcDNA⋅FKHR, pcDNA⋅FKHRL1, and pcDNA⋅AFX (1 μg/ml). In A, insulin (1 milliunit/ml) was added to serum-starved cells during the last 18 h of incubation. In B, cells were exposed to dexamethasone (0.5 μM) for 18 h. The effect of these agents on endogenous proteins, pcDNA (bars A and B), or the exogenous proteins encoded by pcDNA⋅DAF-16 (bars C and D) and its three mammalian homologs, pcDNA⋅FKHRL1 (bars E and F), pcDNA⋅FKHR (bars G and H), and pcDNA⋅AFX (bars I and J) is shown. Luciferase activity was corrected for β-galactosidase gene expression. The data shown are representative of three experiments.
Figure 2
Figure 2
(A) Effect of DAF-16 and HNF3α on insulin-responsive gene transcription. HepG2 cells were incubated in the presence or absence of insulin (1 milliunit/ml) for 16 h before harvesting. Luciferase activity recovered in the presence of pcDNA (bars A and B), pcDNA⋅DAF-16 (bars C and D), pcDNA⋅DAF-16 (Δ340–511) (bars E and F), and HNF3α (bars G and H) is shown. (B) Effect of E1A on dexamethasone-responsive gene transcription. Cells were transfected with the native IGFBP-1 promoter driving luciferase gene expression (15 μg/ml), and expression vectors including pcDNA alone (bars A–D) or pcDNA⋅DAF-16 (bars E–H); and CMV alone (1 μg/ml) (bars A, B, E, and F); or CMV⋅E1A (bars C, D, G, and H). Cells were inoculated with (bars B, D, F, and H) and without (bars A, C, E, and G) dexamethasone (0.5 μM) for 18 h. Luciferase activity is shown corrected for growth hormone (GH) and normalized to the control value for pcDNA alone. (C) DAF-16 gene expression is inhibited by insulin and by wild-type E1A but not by E1A Δ2–36. HepG2 cells were transiently cotransfected with the native IGFBP-1 promoter-luciferase gene (10 μg/ml), and the pcDNA expression vector alone (1 μg/ml) (bars A–D), or wild-type pcDNA⋅DAF-16 (bars E–H), or pcDNA⋅FKHR (bars I–L) or pcDNA⋅DAF-16 4(S/T-A) (bars M–P). Control and insulin-stimulated activity was assessed in the presence of the expression vector CMV alone (0.2 μg/ml; bars A, B, E, F, I, J, M, and N). The effect of wild-type CMV⋅E1A (bars C, G, K, and O) or a derivative of E1A that fails to interact with CBP, CMV⋅E1A Δ2–36 (bars D, H, L, and P) is shown.
Figure 3
Figure 3
DAF-16 interacts with p300 and SRC. (A) Interaction of [35S]methionine-labeled DAF-16 with the KIX and C/H3 interaction domains of GST⋅CBP. In vitro translated [35S]methionine-labeled DAF-16 or DAF-16 (Δ340–511) was incubated with GST (lane 1); or GST⋅CBP (KIX), which encodes amino acids 450–684 of CBP (lane 2); or GST⋅CBP (C/H3), which encodes amino acids 1890–2441 of CBP (lane 3); or GST⋅SRC, which encodes amino acids 594–780 (lane 4). The bound proteins were washed, eluted, and subjected to SDS/PAGE as described in the text. The autoradiograph of a dried gel is shown. (B) Interaction of GST⋅SRC with [35S]methionine-labeled p300 and DAF-16. In vitro translated [35S]methionine-labeled p300 (lanes 2 and 4) or DAF-16 (lanes 1 and 3) was incubated with bacterially produced GST (lanes 1 and 2) or GST⋅SRC (amino acids 594–780) (lanes 3 and 4) bound to glutathione-Sepharose beads. Eluted proteins are shown. (C) Interaction of [35S]methionine-labeled DAF-16, FKHR, and AFX with GST⋅CBP (KIX) and GST⋅SRC. [35S]Methionine-labeled DAF-16, FKHR, and AFX were incubated with GST (lane 1), GST⋅KIX (lane 2), or GST⋅SRC (lane 3) bound to glutathione-Sepharose beads. Eluted proteins are shown.
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