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SIRT1 deacetylates RORγt and enhances Th17 cell generation.

Lim HW, Kang SG, Ryu JK, Schilling B, Fei M, Lee IS, Kehasse A, Shirakawa K, Yokoyama M, Schnölzer M, Kasler HG, Kwon HS, Gibson BW, Sato H, Akassoglou K, Xiao C, Littman DR, Ott M, Verdin E - J. Exp. Med. (2015)

Bottom Line: The balance of effector and regulatory T cell function, dependent on multiple signals and epigenetic regulators, is critical to immune self-tolerance.SIRT1 increases RORγt transcriptional activity, enhancing Th17 cell generation and function.These findings reveal an unexpected proinflammatory role of SIRT1 and, importantly, support the possible therapeutic use of SIRT1 inhibitors against autoimmunity.

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Affiliation: Gladstone Institute of Virology and Immunology, Gladstone Institute of Neurological Disease, School of Medecine, Department of Neurology, Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158 Gladstone Institute of Virology and Immunology, Gladstone Institute of Neurological Disease, School of Medecine, Department of Neurology, Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158.

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SIRT1 interacts with RORγt. (A) Flag-tagged WT or H363Y mutant SIRT1 was immunoprecipitated from transfected 293T cells and probed as indicated. (B) RORγt was immunoprecipitated from thymocytes and Th17 cells, and probed with antibody against SIRT1. (C) Immunoprecipitation using lysates of 293T cells co-transfected with constructs encoding SIRT1 and various deletion mutants of RORγt. Relative binding was calculated by normalizing the ratio of immunoprecipitated RORγt/SIRT1 to the ratio of input RORγt/SIRT1. (D) Acetylation of Flag-tagged RORγt immunoprecipitated from 293T cells transfected with various acetyltransferases and Flag-RORγt. (E and F) Acetylation of Flag-RORγt co-transfected with p300 and WT or H363Y mutant SIRT1, in the absence (E) or in the presence (F) of nicotinamide and Ex-527. Equal amounts of Flag-RORγt were loaded (D–F). Representative data are shown from four (A), three (B and E), and two (D) independent experiments, and combined data are shown from three (C and F) independent experiments with error bars representing ±SEM.
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fig2: SIRT1 interacts with RORγt. (A) Flag-tagged WT or H363Y mutant SIRT1 was immunoprecipitated from transfected 293T cells and probed as indicated. (B) RORγt was immunoprecipitated from thymocytes and Th17 cells, and probed with antibody against SIRT1. (C) Immunoprecipitation using lysates of 293T cells co-transfected with constructs encoding SIRT1 and various deletion mutants of RORγt. Relative binding was calculated by normalizing the ratio of immunoprecipitated RORγt/SIRT1 to the ratio of input RORγt/SIRT1. (D) Acetylation of Flag-tagged RORγt immunoprecipitated from 293T cells transfected with various acetyltransferases and Flag-RORγt. (E and F) Acetylation of Flag-RORγt co-transfected with p300 and WT or H363Y mutant SIRT1, in the absence (E) or in the presence (F) of nicotinamide and Ex-527. Equal amounts of Flag-RORγt were loaded (D–F). Representative data are shown from four (A), three (B and E), and two (D) independent experiments, and combined data are shown from three (C and F) independent experiments with error bars representing ±SEM.

Mentions: Because RORγt is the major lineage-specific transcription factor for Th17 differentiation and RORγt protein induction was not affected by SIRT1, we hypothesized that SIRT1 might regulate RORγt activity. In support of this model, we found that RORγt interacts with both WT SIRT1 and SIRT1-H363Y, a catalytically inactive mutant, in transiently transfected 293T cells (Fig. 2 A). Similarly, endogenous RORγt could be co-immunoprecipitated with endogenous SIRT1 in both thymocytes and Th17 cells (Fig. 2 B). Immunoprecipitation experiments using deletion mutants of RORγt showed that SIRT1 binds to the C-terminal part of RORγt (aa 99–495). The ligand-binding domain (aa 304–495) of RORγt was sufficient for SIRT1 binding, whereas the DNA binding domain of RORγt (aa 1–99) did not bind (Fig. 2 C).


SIRT1 deacetylates RORγt and enhances Th17 cell generation.

Lim HW, Kang SG, Ryu JK, Schilling B, Fei M, Lee IS, Kehasse A, Shirakawa K, Yokoyama M, Schnölzer M, Kasler HG, Kwon HS, Gibson BW, Sato H, Akassoglou K, Xiao C, Littman DR, Ott M, Verdin E - J. Exp. Med. (2015)

SIRT1 interacts with RORγt. (A) Flag-tagged WT or H363Y mutant SIRT1 was immunoprecipitated from transfected 293T cells and probed as indicated. (B) RORγt was immunoprecipitated from thymocytes and Th17 cells, and probed with antibody against SIRT1. (C) Immunoprecipitation using lysates of 293T cells co-transfected with constructs encoding SIRT1 and various deletion mutants of RORγt. Relative binding was calculated by normalizing the ratio of immunoprecipitated RORγt/SIRT1 to the ratio of input RORγt/SIRT1. (D) Acetylation of Flag-tagged RORγt immunoprecipitated from 293T cells transfected with various acetyltransferases and Flag-RORγt. (E and F) Acetylation of Flag-RORγt co-transfected with p300 and WT or H363Y mutant SIRT1, in the absence (E) or in the presence (F) of nicotinamide and Ex-527. Equal amounts of Flag-RORγt were loaded (D–F). Representative data are shown from four (A), three (B and E), and two (D) independent experiments, and combined data are shown from three (C and F) independent experiments with error bars representing ±SEM.
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fig2: SIRT1 interacts with RORγt. (A) Flag-tagged WT or H363Y mutant SIRT1 was immunoprecipitated from transfected 293T cells and probed as indicated. (B) RORγt was immunoprecipitated from thymocytes and Th17 cells, and probed with antibody against SIRT1. (C) Immunoprecipitation using lysates of 293T cells co-transfected with constructs encoding SIRT1 and various deletion mutants of RORγt. Relative binding was calculated by normalizing the ratio of immunoprecipitated RORγt/SIRT1 to the ratio of input RORγt/SIRT1. (D) Acetylation of Flag-tagged RORγt immunoprecipitated from 293T cells transfected with various acetyltransferases and Flag-RORγt. (E and F) Acetylation of Flag-RORγt co-transfected with p300 and WT or H363Y mutant SIRT1, in the absence (E) or in the presence (F) of nicotinamide and Ex-527. Equal amounts of Flag-RORγt were loaded (D–F). Representative data are shown from four (A), three (B and E), and two (D) independent experiments, and combined data are shown from three (C and F) independent experiments with error bars representing ±SEM.
Mentions: Because RORγt is the major lineage-specific transcription factor for Th17 differentiation and RORγt protein induction was not affected by SIRT1, we hypothesized that SIRT1 might regulate RORγt activity. In support of this model, we found that RORγt interacts with both WT SIRT1 and SIRT1-H363Y, a catalytically inactive mutant, in transiently transfected 293T cells (Fig. 2 A). Similarly, endogenous RORγt could be co-immunoprecipitated with endogenous SIRT1 in both thymocytes and Th17 cells (Fig. 2 B). Immunoprecipitation experiments using deletion mutants of RORγt showed that SIRT1 binds to the C-terminal part of RORγt (aa 99–495). The ligand-binding domain (aa 304–495) of RORγt was sufficient for SIRT1 binding, whereas the DNA binding domain of RORγt (aa 1–99) did not bind (Fig. 2 C).

Bottom Line: The balance of effector and regulatory T cell function, dependent on multiple signals and epigenetic regulators, is critical to immune self-tolerance.SIRT1 increases RORγt transcriptional activity, enhancing Th17 cell generation and function.These findings reveal an unexpected proinflammatory role of SIRT1 and, importantly, support the possible therapeutic use of SIRT1 inhibitors against autoimmunity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gladstone Institute of Virology and Immunology, Gladstone Institute of Neurological Disease, School of Medecine, Department of Neurology, Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158 Gladstone Institute of Virology and Immunology, Gladstone Institute of Neurological Disease, School of Medecine, Department of Neurology, Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158.

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Related in: MedlinePlus