Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity

Abstract

Longevity regulatory genes include the Forkhead transcription factor FOXO and the NAD-dependent histone deacetylase silent information regulator 2 (Sir2). Genetic studies demonstrate that Sir2 acts to extend lifespan in Caenorhabditis elegans upstream of DAF-16, a member of the FOXO family, in the insulin-like signaling pathway. However, the molecular mechanisms underlying the requirement of DAF-16 activity in Sir2-mediated longevity remain unknown. Here we show that reversible acetylation of Foxo1 (also known as FKHR), the mouse DAF-16 ortholog, modulates its transactivation function. cAMP-response element-binding protein (CREB)-binding protein binds and acetylates Foxo1 at the K242, K245, and K262 residues, the modification of which is involved in the attenuation of Foxo1 as a transcription factor. Conversely, Sir2 binds and deacetylates Foxo1 at residues acetylated by cAMP-response element-binding protein-binding protein. Sir2 is recruited to insulin response sequence-containing promoter and increases the expression of manganese superoxide dismutase and p27(kip1) in a deacetylase-activity-dependent manner. Our findings establish Foxo1 as a direct and functional target for Sir2 in mammalian systems.

Fig. 1.

CBP binds and acetylates Foxo1. (A) Interaction of endogenous CBP and Foxo1. Cell extracts from serum-starved HepG2 cells were immunoprecipitated (IP) with anti-Foxo1 or anti-CBP antibodies and analyzed by Western blotting (WB). The input lanes represent 5% of the total volume of whole-cell extracts used for the binding assay. (B) In vitro interaction of CBP and Foxo1. Cell extracts from HEK293T cells transfected with HA-CBP WT were incubated with GST or various GST-Foxo1 deletion mutants. (C) Foxo1 is acetylated by CBP. Cell extracts from HEK293T cells transfected with indicated plasmids were immunoprecipitated with anti-FLAG antibody and probed with anti-acetylated lysine or anti-FLAG antibodies. The expression of CBP-HA in the cell extract was shown by Western blotting. (D–G) Identification of Foxo1 acetylation sites. Schematics of Foxo1 deletion mutants are shown in D. The GST-Foxo1 proteins, indicated by arrowheads, were subjected to in vitro acetylation assays with immunoprecipitated CBP. Reaction products were analyzed by Coomassie brilliant blue staining and autoradiography (¹⁴C). The asterisk shows the autoacetylated CBP. (H) The anti-acetylated Foxo1 (K242/K245) antibody specifically recognizes the acetylated K242 and K245 residues in Foxo1. The GST-FHD WT or -FHD 2KA (KK242, 245AA) proteins were incubated with GST-CBP (HAT) protein in the presence or absence of acetyl-CoA (0.1 mM) followed by immunoblotting with anti-acetylated-Foxo1 (K242/K245) antibody and staining with Ponceau-S solution.

Fig. 2.

CBP regulates Foxo1-mediated transcription through its acetyltransferase activity on the chromosomal reporter. (A) Effect of CBP acetyltransferase activity on Foxo1-mediated transcription. FK-1 cells were transfected with Foxo1 together with either CBP WT or CBP ΔHAT, and the luciferase activity was measured. (B) KR mutations alter Foxo1 transactivation function. FK-1 cells were transfected with the indicated Foxo1 mutants, and the luciferase activity was measured. (Lower) Equal amounts of FLAG-Foxo1 proteins were detected by Western blotting. (C) Coactivation of Foxo1 WT and 3KR by CBP. FK-1 cells were transfected with Foxo1 WT (black boxes) or 3KR (gray boxes) together with CBP, and the luciferase activity was presented as fold induction above the activity obtained by Foxo1 WT or 3KR alone.

Fig. 3.

Sir2 binds and deacetylates Foxo1. (A) Interaction of endogenous Sir2 and Foxo1. HepG2 cells were coimmunoprecipitated (IP) with anti-Foxo1 antibody. The input lanes represent 5% of the total volume of whole-cell extracts used for the binding assay. WB, Western blotting. (B) Association between Sir2 and Foxo1 in vitro. Cell extracts from HEK293T cells transfected with HA-Sir2 were incubated with GST or various GST-Foxo1 deletion mutants. (C and D) Sir2 deacetylates Foxo1 in vitro. Acetylated GST-Foxo1 (FHD) (C) or (C1) (D) proteins were incubated with immunoprecipitated Sir2. NAD (50 μM), NIA (5 mM), and/or trichostatin A (TSA, 1 μM) were added as indicated, and the reactions were analyzed by Western blotting (Upper) and Ponceau-S stain (Lower). (E) Sir2 deacetylates Foxo1 in vivo. Cell extracts from HEK293T cells were transfected with FLAG-Foxo1, HA-CBP, HA-Sir2 WT, or HA-Sir2 H355A as indicated and analyzed by immunoprecipitation and Western blotting.

Fig. 4.

Sir2 coactivates Foxo1-mediated transcription. (A) Sir2 and CBP facilitates Foxo1 transcription in chromatin context. FK-1 cells were transfected with either empty vector or Foxo1 WT together with or without Sir2 and/or CBP as indicated, and the luciferase activity was measured. (B) Sir2 potentiates GAL4-Foxo1-mediated transcription via its deacetylase activity. GM-1 cells were cotransfected with GAL4-Foxo1 together with or without Sir2 WT or H355A as indicated. (C) Recruitments of Foxo1, CBP, and Sir2 onto the MnSOD and p27^kip1 promoters. Chromatin immunoprecipitation assays were performed with the indicated antibodies in HEK293 cells. Immunoprecipitated (IP) DNA was analyzed by PCR using specific primer sets. (D) NIA reduces Foxo1-mediated gene expression. HEK293 cells were treated with NIA (0, 5, and 10 mM) for 24 h in serum-starved conditions. (E) Overexpression of Sir2 affects Foxo1-mediated gene expression. HEK293 cells stably transfected with Sir2 WT or Sir2 H355A were cultured with or without 10% FBS or insulin (100 nM) for 18 h.