Metabolic regulation of SIRT1 transcription via a HIC1:CtBP corepressor complex

Abstract

The Sir2 histone deacetylases are important for gene regulation, metabolism, and longevity. A unique feature of these enzymes is their utilization of NAD(+) as a cosubstrate, which has led to the suggestion that Sir2 activity reflects the cellular energy state. We show that SIRT1, a mammalian Sir2 homologue, is also controlled at the transcriptional level through a mechanism that is specific for this isoform. Treatment with the glycolytic blocker 2-deoxyglucose (2-DG) decreases association of the redox sensor CtBP with HIC1, an inhibitor of SIRT1 transcription. We propose that the reduction in transcriptional repression mediated by HIC1, due to the decrease of CtBP binding, increases SIRT1 expression. This mechanism allows the specific regulation of SIRT1 in response to nutrient deprivation.

Fig. 1.

2-DG decreases free NADH levels and reduces CtBP binding to HIC1. (A) 2-DG changes the cellular lactate:pyruvate ratio. Primary human fibroblasts were treated with 10 mM 2-DG and the pyruvate and lactate concentrations were determined as described (15). (B) 2-DG decreases nuclear NAD(P)H levels. Two-photon fluorescence measurement of the nuclear NAD(P)H intensity of control and 2-DG-treated fibroblasts is shown. (C) 2-DG blocks the CtBP-HIC1 interaction. CtBP1 and FLAG-tagged HIC1 were transfected into Cos7 cells. Cells were treated with 2-DG before immunoprecipitation with anti-FLAG antibody. CtBP1 associated with HIC1 was assayed by Western blotting. con, control. (D) Interaction of endogenous CtBP and HIC1 is prevented by 2-DG treatment. Human primary fibroblasts were treated with 2-DG and immunoprecipitated with an antibody directed against CtBP. The associated HIC1 was assayed by Western blotting.

Fig. 2.

2-DG decreases CtBP recruitment to the SIRT1 promoter and increases SIRT1 expression. (A) CtBP recruitment to the SIRT1 promoter is blocked by 2-DG. Human primary fibroblasts were treated with 2-DG for 3 h, and ChIP was performed with antibodies directed against CtBP. Recruitment of CtBP to the HIC1-binding sites on the SIRT1 promoter was assayed by PCR. LINE-1 served as nonspecific control. (B) 2-DG increases SIRT1 transcript levels. Primary human fibroblasts were treated with 2-DG for 6 or 9 h, and the SIRT1 transcript was measured by RT-PCR. Data were normalized to 18S RNA and are represented as mean ± SEM from three independent experiments. (C) 2-DG increases SIRT1 protein levels. Primary human fibroblasts were treated with 2-DG for 6 or 9 h, and SIRT1 protein was assayed by Western blotting. Tubulin served as a loading control.

Fig. 3.

HIC1 is required for the transcriptional regulation of SIRT1 by 2-DG. (A) Decreased HIC1 levels in tumor cells are associated with loss of 2-DG-induced SIRT1 up-regulation. Human lung cancer cells (H1299) and primary human fibroblasts (CCD) were treated with 2-DG. Western blot (Left) shows the HIC1 and CtBP levels. SIRT1 RNA levels were measured by RT-PCR (Right). Data were normalized to 18S RNA and are represented as mean ± SEM from three independent experiments. (B) HIC1 rescues the 2-DG-induced SIRT1 up-regulation in tumor cells. H1299 lung cancer cells were transfected with HIC1, either WT or the GLDLSKK-deleted (mt) version, and treated with 2-DG. SIRT1 RNA was measured by RT-PCR and normalized to 18S RNA (Left). Data are represented as mean ± SEM from three independent experiments. Western blot shows the decreased binding of CtBP to the HIC1 GLDLSKK-deletion mutant compared with the WT (Right).

Fig. 4.

CtBP is required for the transcriptional regulation of SIRT1 by 2-DG. (A) Increased levels of SIRT1 transcript in CtBP-null MEFs. SIRT1 transcription in CtBP-null and heterozygous MEFs was measured by RT-PCR. Data were normalized to 18S RNA and are represented as mean ± SEM from three independent experiments. (B) CtBP knockdown in primary human fibroblasts up-regulates SIRT1 transcription. (Left) CtBP1 and -2 in human fibroblasts were knocked down by siRNA. (Right) Cells treated with siRNA (or control) were exposed to 2-DG before measurement of SIRT1 transcript levels. Data were normalized to 18S RNA and are represented as mean ± SEM from three independent experiments. con, control.

Fig. 5.

SIRT1 regulation by hypoxia and 2-DG. (A) Hypoxia decreases SIRT1 transcription. Primary human fibroblasts were treated overnight with hypoxia (1% O₂) alone or in combination with 30 mM pyruvate (pyr) or 10 mM 2-DG. SIRT1 transcripts were measured by RT-PCR. Data were normalized to 18S RNA and are represented as mean ± SEM from three independent experiments. con, control. (B) 2-DG specifically increases SIRT1 transcript levels. Primary human fibroblasts were treated with 2-DG for 6 h, and SIRT1–7 transcripts were measured by RT-PCR. Data were normalized to 18S RNA and are represented as mean ± SEM from three independent experiments.

Fig. 6.

Models for CtBP-regulation of SIRT1 expression. (Upper) The reduction in free nuclear NADH after 2-DG treatment (designated by the smaller oval representing NADH) decreases CtBP association with HIC1, thus increasing SIRT1 transcription. (Lower) Posttranslational modification of CtBP induced by the block in glycolysis prevents association with HIC1.