1. SIRT1 Deacetylates and Positively Regulates the Nuclear Receptor LXR
Xiaoling Li, Songwen Zhang, Gil Blander, Jeanette G. Tse, Monty Krieger, Leonard Guarente 
Molecular Cell 
Volume 28, Issue 1, Pages (October 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 SIRT1 Interacts with and Deacetylates LXRs
(A) SIRT1 interacts with LXRα and LXRβ. Human HEK293T cells expressing the indicated proteins were lysed and immunoprecipitated (IP) with anti-HA monoclonal antibodies as described in the Experimental Procedures. The total lysates and immunoprecipitation samples were probed with anti-SIRT1 polyclonal antibodies. Note that HA-LXR pulls down not only overexpressed murine SIRT1 protein (mT1) but also the endogenous human SIRT1 protein (hT1).
(B) LXR is acetylated in vivo and activation by 22(R)-HC, and 9-cisRA reduces acetylation. The levels of immunoprecipitated HA-LXR were detected with Coomassie brilliant blue staining and [3H]-acetyl-labeled HA-LXR on the same gel was detected by [3H] autoradiography. The same samples were also immunoblotted with anti-SIRT1 antibodies.
(C) SIRT1 deacetylates HA-LXR. HA-LXR was immunoprecipitated, and [3H]-labeled HA-LXR, HA-LXR, and SIRT1 were detected as described in (B) except that HA-LXRα was visualized by blotting with HA antibodies.
(D) Knockdown of SIRT1 by RNA interference increases the acetylation of LXRα. HEK293T cells were stably infected either with vector or with pSuper-SIRT1 RNAi construct as indicated. Cells were then transfected with HA-LXRα and labeled with sodium [3H]acetate. HA-LXRα was immunoprecipitated, and [3H]-labeled HA-LXRα, HA-LXRα, and SIRT1 were detected as described in (B).
(E) Loss of function of SIRT1 in hepatocytes increases the acetylation of LXRα. Primary hepatocytes isolated from SIRT1+/+ and SIRT1−/− mice transfected with vector (V), wild-type LXRα (WT), or the K432 mutant (KR) were incubated with 25 μM MG132, 22(R)-HC and 9-cis-RA for 6 hr. HA-LXRα was immunopurified with anti-HA antibodies and eluted with SDS buffer. The purified protein was then diluted in NP-40 buffer and immunoprecipitated with anti-acetyl-lysine antibodies. The purified HA-LXRα and precipitated acetyl- HA-LXRα were then detected by blotting with anti-HA-HRP antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 SIRT1 Localizes on the LXRE of LXR Targets
(A) SIRT1 localizes to the LXRE of ABCA1 promoter and the association increases upon LXR/RXR ligand treatment in primary MEFs. Primary MEFs from SIRT1+/+ and SIRT1−/− animals were treated with or without 10 μM 22(R)-HC and 1 μM 9-cisRA for 24 hr. Cells were then subjected to ChIP assays with anti-SIRT1 or anti-Gal4DBD antibodies as described.
(B) SIRT1 is associated with LXRE of LXR targets in H2.35 hepatoma cells. H2.35 cells were treated with or without 22(R)-HC and 9-cisRA for 4 hr. Cells were then subjected to ChIP assays with anti-SIRT1 or anti-Gal4DBD antibodies, and PCR was performed using primers as indicated.
(C) Human SIRT1 is associated with the LXRE of human ABCA1 promoter. HEK293T cells transfected with control vector or FLAG-tagged human SIRT1 (FLAG-hT1) were fixed and harvested for ChIP assays. THP-1 monocytes were fixed and harvested for ChIP with either anti-SIRT1 antibodies or anti-Gal4DBD antibodies.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
Lysine 432 of LXR in the Loop Region Near the AF2 Domain Is Essential for Its Activation by SIRT1
(A) LXRα lysine acetylation site. HA-LXRα is acetylated on a single lysine K432 (corresponding to K434 in human protein, see Figure S1), a loop region connecting helix H11 and the AF2 domain (Protein Data Bank, 1UHL).
(B) Constructs of control (FXR), wild-type, and mutant LXRα ligand-binding domains (LBD) fused to the Gal4 DNA-binding domain (DBD). FXR, wild-type LXRα, LXRα K432R, and LXRα K432Q LBD were fused to Gal4 DBD together with a HA epitope. The expression of these fusion proteins was adjusted so that all of these receptor chimeras were at comparable levels (Figure 3B, right).
(C) Both K432R and K432Q mutations of LXRα reduce ligand-dependent transcriptional activity and fail to respond to SIRT1. (Left panel) HEK293T cells transfected either with vector (pSuper), with pSuper-DMAP1 RNAi, or with pSuper-SIRT1 RNAi construct were cotransfected with luciferase reporters that contains either 5 (G5) or no (G0) Gal4 binding sites upstream from a HSV-tk promoter and constructs encoding indicated Gal4 DBD fusion proteins. 24 hr later, cells were incubated with or without 22(R)-HC for an additional 16 hr and analyzed for luciferase activity as described in the Experimental Procedures. (Right panel) CaCo-2 cells were transfected similarly. Twenty-four hours later, cells were incubated with either DMSO, 22(R)-HC, or 22(R)-HC plus 5 mM nicotinamide for an additional 16 hr and analyzed for luciferase activity as described in the Experimental Procedures. F-luc, firefly luciferase gene; R-luc, Renilla luciferase gene; TK pro, thymidine kinase promoter.
(D) Activities of unfused wild-type (LXRα), K432R (KR), or K432Q (KQ) HA-LXRα on the ABCA1 promoter. HEK293T cells were transfected with wild-type (WT) or LXRE mutant (−LXRE) murine ABCA1 promoter-driven luciferase reporter vectors together with constructs expressing indicated proteins. Twenty-four hours later, cells were incubated with or without 22(R)-HC and 9-cis-RA for an additional 16 hr and analyzed for luciferase activity as described in the Experimental Procedures.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 SIRT1 Regulates LXR Stability
(A) SIRT1 knockout animals have increased hepatic LXRα protein, determined by western blotting using antibodies specific for LXRα.
(B) Inhibition of SIRT1 activity by nicotinamide increases the protein levels of LXRα by interfering with proteasome-mediated degradation. HEK293T cells expressing HA-LXRα were treated with control medium or medium with 0.1 μM TSA, 10 mM nicotinamide, 25 μM MG132, or 10 mM nicotinamide plus 25 μM MG132 for 6 hr as indicated. The levels of HA-LXRα and SIRT1 were analyzed by western blotting using antibodies to HA and SIRT1.
(C) SIRT1 promotes the ubiquitination of LXRα. (Left panel) Overexpression of SIRT1 promotes the ubiquitination of LXRα in HEK293T cells. HEK293T cells were cotransfected with constructs expressing the indicated proteins. Forty hours later, cells were incubated with 25 μM MG132, 22(R)-HC and 9-cis-RA for 1 hr in the absence or presence of 10 mM nicotinamide. HA-LXRα was then immunoprecipitated using antibodies to HA follow by western blotting using antibodies to ubiquitin. HA-LXRα and SIRT1 were detected by western blotting of total cell lysates. (Right panel) Ubiquitination of LXRα is dependent on the deacetylation at K432 by SIRT1. Primary hepatocytes isolated from SIRT1+/+ and SIRT1−/− mice transfected with constructs expressing indicated proteins were incubated with 25 μM MG132, 22(R)-HC, and 9-cis-RA for 6 hr. HA-LXRα was then immunoprecipitated and analyzed as described in the left panel. V, vector; WT, wild-type HA-LXRα; KR, K432R mutant of HA-LXRα; KQ, K432Q mutant of HA-LXRα.
(D) Mutations of K432 block ligand-induced degradation of LXRα. (Left panel) HEK293T cells transfected with constructs expressing the indicated proteins were incubated with or without 22(R)-HC and 9-cis-RA for 16 hr; the levels of HA-LXRα and mutants were analyzed by immunoblotting with antibodies to HA. (Right panel) Primary hepatocytes isolated from SIRT1+/+ and SIRT1−/− mice transfected with indicated constructs were incubated with 10 μg/ml of cycloheximide (CHX), 22(R)-HC, and 9-cis-RA for 30 min; the levels of HA-LXRα and mutants were analyzed by immunoblotting with antibodies to HA.
(E) Inhibition of SIRT1 or proteasome-mediated LXR degradation inhibits the transcription activity of LXR on the ABCA1 promoter. HEK293T cells transfected with wild-type (WT) or LXRE mutant (−LXRE) mouse ABCA1 promoter-driven luciferase reporter vectors were treated with 10 mM nicotinamide (Nico), 25 μM MG132, or 10 mM nicotinamide + 25 μM MG132 in the absence (filled bar) or presence (open bar) of 10 μM 22(R)-HC and 1 μM 9-cisRA for 6 hr. Luciferase activities were measured and normalized as described.
(F) A model for the role of SIRT1 in the promoter resetting and activation of LXR targets. In this model, SIRT1 is required for the deacetylation of LXR after one round of transcription. This deacetylation allows the used LXR molecule to be ubiquitinated and degraded by proteasome. The degradation of the used LXR then allows recruitment of new LXR for the second round of transcription.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Loss of SIRT1 Reduces the Expression of LXR Targets
(A) Inhibition of SIRT1 activity reduces ABCA1 promoter activity. HEK293T cells transfected with wild-type (WT) or LXRE mutant (−LXRE) murine ABCA1 promoter-driven luciferase reporter vectors were treated with 0.1 μM TSA (TSA), 10 mM nicotinamide (Nico), or 10 mM nicotinamide μM TSA in the absence (filled bar) or presence (open bar) of 10 μM 22(R)-HC and 1 μM 9-cisRA for 6 hr. Luciferase activities were determined as described in the Experimental Procedures.
(B) SIRT1 deficiency reduces ABCA1 promoter activity in MEFs. SIRT1+/+ and SIRT1−/− MEFs were generated and infected with pBabe-mT1 (+) or a pBabe control vector (−) as described in the Experimental Procedures. The insert shows immunoblotting analysis of the expression levels of SIRT1 in these four cell lines; tubulin was used as a loading control. MEFs were transfected with wild-type (WT) or LXRE mutant (−LXRE) murine ABCA1 promoter-driven luciferase reporter vectors in the absence (filled bar) or presence (open bar) of 10 μM 22(R)-HC and 1 μM 9-cisRA. Luciferase activities were measured after 24 hr.
(C) SIRT1 deficiency in macrophages reduces the induction of LXR targets by LXR/RXR ligands. Primary macrophages were cultured in RPMI1640 medium with or without 10 μM 22(R)-HC + 1 μM 9-cisRA for 16 hr. Cells were then harvested and analyzed for the expression of LXR targets by quantitative real-time PCR (left) or by immunoblotting with antibodies against ABCA1 proteins (right).
(D) SIRT1−/− mice have reduced levels of an array of LXR targets in liver. Total RNA from livers of wild-type control (filled bars) and SIRT1−/− (open bars) animals was analyzed for the expression of LXR targets and nontargets by real-time PCR as described in the Experimental Procedures (n = 3, ∗p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Loss of Function of SIRT1 Results in Altered Cholesterol and Triglyceride Metabolism
(A–D) SIRT1 deficiency reduces plasma HDL cholesterol in mice. Plasma samples from wild-type (filled), SIRT1+/− (gray), and SIRT1−/− (open) mice were analyzed for (A) total cholesterol, (B) HDL cholesterol, and (C) LDL cholesterol. Eighteen wild-type, 18 SIRT1+/−, and 18 littermate SIRT1−/− male mice in each group were analyzed, and HDL cholesterol was determined to be lower in SIRT1+/− and SIRT1−/− mice compared to wild-type by Student's t test (∗p < 0.01; ∗∗p < 0.001). (D) FPLC plasma lipoprotein cholesterol profiles in wild-type (filled diamond) and SIRT1−/− (open diamond) mice. A representative profile is shown from three independent experiments (n = 4; total of 12 SIRT1−/− and 12 littermate SIRT1+/+).
(E) SIRT1−/− mice moderately accumulate cholesterol in liver and testis. Total cholesterol from wild-type control (filled bar) and littermate SIRT1−/− (open bar) testes and livers were extracted and analyzed as described in the Experimental Procedures (n = 8, ∗p < 0.01).
(F) SIRT1 deficiency reduces plasma triglyceride levels (n = 12, ∗p < 0.01).
(G–I) Plasma lipids and lipoproteins in SIRT1+/− mice backcrossed four times into C57BL/6 background. SIRT1+/− mice in mixed 129/sv-CD1 background were backcrossed with C57BL/6 mice for four generations, using satellite DNA markers to select progeny with highest (95%) C57BL/6 contribution. SIRT1+/+ and SIRT1+/− mice from this background were then analyzed for plasma lipids ([G], triglycerides; [H], total cholesterol) and lipoproteins ([I], FPLC lipoprotein cholesterol profiles) at 6–8 weeks of age (n = 15 for SIRT1+/+ and SIRT1+/−; ∗p < 0.01).
(J–L) Loss of function reduces apoA-I-mediated cholesterol efflux in primary cells. Loss of SIRT1 reduces apoA-I-mediated cholesterol efflux in primary macrophages (J), hepatocytes (K), and MEFs (L) (n = 3, ∗p < 0.001). SIRT1+/+ (filled) and SIRT1−/− (open) MEFs were infected with retroviral vector pBabe, pBabe-hSIRT1 (hT1), or pBabe-hSIRT1HY(hT1HY).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
SIRT1 Deficiency Compromises the Normal Responses to LXR Agonist In Vivo
(A) Relative levels of LXR targets in livers in gender-matched littermates with or without oral administration of TO901317, a LXR agonist. Total RNA from livers of wild-type animals without (black filled bar) or with (black striped bars) 50 mg/kg TO for 18 hr and SIRT1−/− mice without (open bars) or with (gray striped bars) TO were analyzed by real-time PCR (n = 3–4, #p < 0.05; ∗p < 0.01).
(B) Protein levels of LXR targets in control (+/+ or +/−) and SIRT1−/− mice treated with vehicle or TO (Top) Wild-type (+/+) and SIRT1−/− (−/−) mice from 129/CD1 mix background were fed without or with 50 mg/kg TO (TO) for 18 hr. Total protein lysates were obtained from livers of the indicated mice and analyzed for the levels of ABCA1 protein by immunoblotting. (Bottom) Pregnant females in the 95% C57BL/6 background were fed with vehicle or 30 mg/kg TO daily from E17.5 to E19.5; livers of control (+/+ or +/−) and SIRT1−/− (−/−) mice of newborns at P0.5 were then extracted, and total protein lysates were analyzed for the levels of ABCA1 and SREBP-1 by immunoblotting. As previously observed, ABCA1 proteins comprise multiple bands by immunoblotting (Denis et al., 2004).
(C) Macroscopic appearance of livers from SIRT1+/+ and SIRT1−/− mice fed without or with 10 mg/kg TO for 8 days. Scale bar, 2 cm. Increasing triglyceride accumulation (“fatty liver”) is manifest by livers turning from brown to gray to white.
(D) Triglycerides from plasma and liver of wild-type mice without (black filled bars) or with TO (black striped bars) and SIRT1−/− animals without (open bars) or with TO (gray striped bars) were analyzed as described in the Experimental Procedures (n = 8, ∗∗p < 0.001). Plasma results in (D), (E), and (F) represent the same mice before and after 7–8 days of administration of 10 mg/kg TO Results for livers in (D) (left) compare littermates without or with TO901317, as in (A) and (B) above.
(E) SIRT1−/− mice show blunted induction of plasma cholesterol by TO feeding (n = 8, ##p < 0.02).
(F) Representative FPLC plasma lipoprotein profiles for wild-type (+/+) and SIRT1−/− mice before (filled diamond) and after (open square) TO feeding (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions