SIRT1 is a Direct Coactivator of Thyroid Hormone Receptor β1 with Gene-Specific Actions

<div><p>Sirtuin 1 (SIRT1) NAD⁺-dependent deacetylase regulates energy metabolism by modulating expression of genes involved in gluconeogenesis and other liver fasting responses. While many effects of SIRT1 on gene expression are mediated by deacetylation and activation of peroxisome proliferator activated receptor coactivator α (PGC-1α), SIRT1 also binds directly to DNA bound transcription factors, including nuclear receptors (NRs), to modulate their activity. Since thyroid hormone receptor β1 (TRβ1) regulates several SIRT1 target genes in liver and interacts with PGC-1α, we hypothesized that SIRT1 may influence TRβ1. Here, we confirm that SIRT1 cooperates with PGC-1α to enhance response to triiodothyronine, T₃. We also find, however, that SIRT1 stimulates TRβ1 activity in a manner that is independent of PGC-1α but requires SIRT1 deacetylase activity. SIRT1 interacts with TRβ1 <i>in vitro</i>, promotes TRβ1 deacetylation in the presence of T₃ and enhances ubiquitin-dependent TRβ1 turnover; a common response of NRs to activating ligands. More surprisingly, SIRT1 knockdown only strongly inhibits T₃ response of a subset of TRβ1 target genes, including glucose 6 phosphatase (G-6-Pc), and this is associated with blockade of TRβ1 binding to the G-6-Pc promoter. Drugs that target the SIRT1 pathway, resveratrol and nicotinamide, modulate T₃ response at dual TRβ1/SIRT1 target genes. We propose that SIRT1 is a gene-specific TRβ1 co-regulator and TRβ1/SIRT1 interactions could play important roles in regulation of liver metabolic response. Our results open possibilities for modulation of subsets of TR target genes with drugs that influence the SIRT1 pathway.</p></div

SIRT1 deacetylates TRβ1.

<p>Immunoprecipitation analysis of HepG2-TRβ1 cells infected with null adenovirus control or adSIRT1 and treated +/− T₃. TRβ1 was immunoprecipitated with anti-Flag antibodies and precipitates were blotted with anti-acetyl-lysine, TRβ1 or SIRT1 antibodies. IgG control precipitation is shown at right. Acetylated TRβ1 levels relative to total TRβ1 were quantified by Phosphor Imager (right panel).</p

SIRT1 knockdown inhibits some TRβ1 target genes.

<p>(A–D) qPCR analysis of HepG2-TRβ1 cells extracts treated +/− T₃ and SIRT1 siRNA. G-6-Pc (A), PCK1 (B), FGF21 (C) and <u>Hairless</u> HR (D). The data are representative of at least three independent experiments. All values represent the mean ± SD of duplicate samples. **, <i>P</i> < 0.01; *, <i>P</i> < 0.05.</p

SIRT1 is recruited to TREs of TRβ1 target genes.

<p>ChIP assays performed in HepG2-TRβ cells treated +/− SIRT1 siRNA and T₃. Antibodies used for immunoprecipitation were Flag, SIRT1 or IgG control. 10 % (v/v) of the supernatant was represented as ‘input’ chromatin prior to immunoprecipitation by antibodies.</p

SIRT1 regulates TRβ1 target gene promoter activity.

<p>(<b>A</b>) Schematic representation of TRβ1 response regions (TREs) of TRβ1 target genes with sequences and positions of DR-4 sites for G-6-Pc gene and PCK1 gene. <b>(B–C)</b> Luciferase assays performed on extracts of 293T cells that were cotransfected with indicated reporters along with TRβ1 and SIRT1 expression vectors and treated +/− T₃. <b>(D, E)</b> Luciferase assays performed on extracts of HepG2-TRβ1 cells transfected with indicated reporters and SIRT1 siRNA and treated +/− T₃. The levels of luciferase activity were normalized to the lacZ expression. Data are representative of at least three independent experiments with similar results. All values represent mean ± SD of duplicate samples. **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001.</p

SIRT1 interacts with TRβ1.

<p><b>(A, B)</b> Co-immunoprecipitation assays from 293T cells transfected with expression vectors for Flag tagged SIRT1 and Myc tagged TRβ1 and treated +/− 10 nM T₃ for 12 hr. Antibody used for immunoprecipitation is indicated at the top of the panel and antibody used for western analysis is indicated at the right hand side. Panels below represent western blots of input proteins or GAPDH loading control and quantitative scans of amounts of each protein detected in western analysis of input protein panels. (<b>C</b>) Co-immunoprecipitation assays from HepG2 cells which stably express Flag tagged TRβ1. TRβ1 was immunoprecipitated with anti-flag and western analysis of precipitants was performed with antibodies indicated at the right of each panel. Lower panels represent western blots of input proteins or loading control. (<b>D</b>) GST pull-down assays to demonstrate SIRT1 directly interacts with TRβ1 <i>in vitro</i>. The image represents a western blot of an SDS-PAGE gel used to separate input and retained SIRT1 after binding reaction with GST- or GST- full length TRβ1 fusions linked to a solid support and probed with SIRT1 antibody. Input represents 10% of the total volume of SIRT1 used in the binding assay.</p

A subset of TRβ1 target genes that are inhibited by SIRT1 knockdown.

<p>Heat map representing results of array analysis performed on HepG2-TRβ1 cells treated +/− T₃ and SIRT1 siRNA and displaying probe sets in which T₃ response is inhibited by SIRT1 siRNA. Scale is shown at top. The first lane (1) represents T₃ responses obtained in the presence of control siRNA, second lane (2) represents T₃ responses obtained in the presence of SIRT1 siRNA. The third lane (3) represents comparison of mRNA expression levels in the presence of control siRNA or SIRT1 siRNA. Note that, in most instances in which SIRT1 inhibits these T₃ responses, this effect is not accompanied by SIRT1-dependent changes in basal gene expression.</p

A Small Molecule SIRT1 activator and inhibitor regulate expression of TRβ1 target genes.

<p><b>(A∼C)</b> qPCR analysis performed upon HepG2-TRβ cells treated with 100 µM resveratrol +/−T₃. Genes are indicated at top. <b>(D∼F)</b> As for 10A–C, except that cells were treated with 10 mM nicotimamide instead of resveratrol. Data are representative of at least two independent experiments. All values represent the mean ± SD of duplicate samples. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01.</p

SIRT1 induces proteasome-dependent TRβ1 degradation and ubiquitination.

<p>(<b>A</b>) Western analysis of 293T cells transfected with myc-TRβ1 and SIRT1 expression vectors and treated with T₃ for 24 hours and 20 µM MG-132 for 6 hours. Note the MG132-dependent increase in TRβ1 levels observed with SIRT1 and T₃. The lower panel represents GAPDH loading control. (<b>B</b>) SIRT1 leads to ubiquitination of TRβ1 protein. The panel represents western analysis of extracts of 293T cells transfected with myc-TRβ1 and SIRT1 expression vectors and treated with T₃ for 24 hours and 20 µM MG-132 for 6 hour, immunoprecipitated with anti-myc antibody and blotted with anti-ubiquitin antibody.</p