Dynamic Partnership between TFIIH, PGC-1α and SIRT1 Is Impaired in Trichothiodystrophy

<div><p>The expression of protein-coding genes requires the selective role of many transcription factors, whose coordinated actions remain poorly understood. To further grasp the molecular mechanisms that govern transcription, we focused our attention on the general transcription factor TFIIH, which gives rise, once mutated, to Trichothiodystrophy (TTD), a rare autosomal premature-ageing disease causing inter alia, metabolic dysfunctions. Since this syndrome could be connected to transcriptional defects, we investigated the ability of a TTD mouse model to cope with food deprivation, knowing that energy homeostasis during fasting involves an accurate regulation of the gluconeogenic genes in the liver. Abnormal amounts of gluconeogenic enzymes were thus observed in TTD hepatic parenchyma, which was related to the dysregulation of the corresponding genes. Strikingly, such gene expression defects resulted from the inability of PGC1-α to fulfill its role of coactivator. Indeed, extensive molecular analyses unveiled that wild-type TFIIH cooperated in an ATP-dependent manner with PGC1-α as well as with the deacetylase SIRT1, thereby contributing to the PGC1-α deacetylation by SIRT1. Such dynamic partnership was, however, impaired when TFIIH was mutated, having as a consequence the disruption of PGC1-α recruitment to the promoter of target genes. Therefore, besides a better understanding of the etiology of TFIIH-related disease, our results shed light on the synergistic relationship that exist between different types of transcription factors, which is necessary to properly regulate the expression of protein coding genes.</p></div

Fasting response of WT and TTD mice.

<p>(<b>panel A</b>) Daily food intake of WT (black box, n = 6) and TTD (open box, n = 6) mice during 15 days. Measurement of the body (<b>panel B</b>), liver (<b>panel C</b>) and epididymal white adipose tissue (WAT, <b>panel D</b>) weight of WT (black boxes) and TTD (open boxes) mice fed <i>ad libitum</i> or fasted for 24 h or 48 h. Values for liver weight are percentages relative to the <i>ad libitum</i> weight. Serological levels of triglycerides (<b>panel E</b>), free fatty acids (<b>panel F</b>), β-hydroxybutyrate (<b>panel H</b>), lactate (<b>panel I</b>), glucagon (<b>panel J</b>), insulin (<b>panel K</b>) and blood glucose (<b>panel L</b>) in WT (black boxes) and TTD (open boxes) fed normally or fasted for 24 h or 48 h. Error bars represent standard deviations. (<b>panel M</b>) Pyruvate tolerance tests. WT (solid curves, n = 4) and TTD (dashed curves, n = 4) mice were fasted for 16 h and injected with sodium pyruvate (2 g/Kg of body weight). The data are means ± SEM. (<b>panel G</b>) Hematoxylin & Eosin (H&E) staining of liver sections from WT and TTD mice fed normally (sections 1–2) and Periodic Acid Schiff staining of liver sections from WT and TTD mice fed normally (sections 3–4) and fasted for 24 h (sections 5–6) or 48 h (sections 7–8). PV =  Portal Vein; CV =  Central vein. Magnification is indicated at the bottom left of each section. The statistical symbols reflect significant differences between genotypes (*, p<0.05; **, p<0.01; ***, p<0.001 Student's t-test).</p

Model of the dynamic partnership between TFIIH, PGC-1α and SIRT1.

<p>SIRT1 and PGC-1α physically interact with various subunits of the TFIIH complex: SIRT1 interacts with XPB, p62, cdk7 and MAT1, while PGC-1α interacts with XPB, p34 and MAT1. SIRT1 binds to TFIIH alone, but its interaction is reinforced by the presence of PGC-1α. The simultaneous interaction between TFIIH, PGC-1α and SIRT1 suggests that TFIIH might contribute to the PGC-1α deacetylation by SIRT1. Such assumption is supported by the fact that i) the integrity of TFIIH is crucial for the optimal binding of PGC-1α and SIRT1 and ii) the PGC-1α deacetylation is disrupted by XPD mutation (such as XPD/R722W) that affects the integrity of TFIIH. In parallel, the CDK7 kinase of TFIIH targets SIRT1, but the function of such phosphorylation(s) remains elusive. Finally, the binding of ATP to the XPB subunit of TFIIH influences the release of PGC-1α, which in turn affects the binding of SIRT1.</p

Defective recruitments of transcription factors on the promoter of gluconeogenic genes in TTD hepatocytes.

<p>Expression of <i>Pgc-1α</i> (<b>panel A1</b>) <i>Pepck</i> (<b>panel B1</b>) and <i>G6Pase</i> (<b>panel C1</b>) genes in WT (solid curves), TTD (dashed curves) and TTD overexpressing XPDwt (dotted curves) hepatocytes after pyruvate treatment. The results are presented as n-fold induction relative to non-treated cells. Recruitment of RNA pol II, p62, CDK7, PGC-1α and SIRT1 on the proximal promoter of PGC-1α (<b>panels A2 to A6</b>), PEPCK (<b>panels B2 to B6</b>) and G6Pase (<b>panels C2 to C6</b>) in WT (dotted curves) and TTD (dashed curves) hepatocytes. The results of three independent experiments are presented as percentage of DNA immunoprecipitated relative to the input. The shaded areas underline the concomitant recruitments of the transcription factors with the expression profile of the target genes in WT hepatocytes. (<b>panel D</b>) Western blot analyses of TFIIH, illustrated by its p62 (62 kDa) and CDK7 (39 kDa) subunits, PGC-1α (110 kDa) and SIRT1 (110 kDa) with increasing amounts of whole cell extracts isolated from WT (lanes 1–3) and TTD (lanes 4–6) hepatocytes. β-tubulin (β-Tub, 50 kDa) has been used as an internal control. * indicates unspecific band. Measurement of intracellular glucose 6-phosphate (<b>panel E</b>) and glucose output (<b>panel F</b>) levels from WT (black boxes) and TTD (open boxes) hepatocytes after 0 and 12 hours of pyruvate treatment. Values represent the means ± SEM. The statistical symbols reflect significant differences between genotypes (*, p<0.05, Student's t-test).</p

Dysregulation of gluconeogenesis-induced proteins in TTD liver.

<p>PEPCK (<b>panel A</b>) and G6Pase (<b>panel B</b>) immunostainings of liver sections from <i>ad libitum</i> (sections 1–2) and 48 h fasted (sections 3–4) WT and TTD mice. PV =  Portal Vein; CV =  Central vein. Magnification is indicated at the bottom left of each part. Expression of the hepatic fasting-induced <i>Pepck</i> (<b>panel C</b>) and <i>G6pase</i> (<b>panel D</b>) genes in WT (black boxes, n = 4) and TTD (open boxes, n = 4) fed normally or fasted for 24 h or 48 h. Results are expressed as the mean normalized to 18S RNA. (<b>panel E</b>) Western Blot analyses of PGC-1α (110 kDa) levels in the liver of three WT and three TTD fed normally (lanes 1–6) or fasted for 48 h (lanes 7–12). TBP (TATA box Binding Protein, 36 kDa) has been used as an internal control. Diagram represents the mean of the ratios between PGC-1α and TBP for each group. (<b>panel F</b>) Expression of the <i>Pgc-1α</i> gene in WT (black boxes, n = 4) and TTD (open boxes, n = 4) fed normally or fasted for 24 h or 48 h. Results are expressed as the mean normalized to 18S RNA. Error bars represent standard deviations. The statistical symbols reflect significant differences between genotypes (**, p<0.01, Student's t-test).</p

TFIIH influences PGC-1α deacetylation by SIRT1 by interacting with both.

<p>(<b>panel A</b>) After immunoprecipitation of PGC-1α from nuclear extracts of WT (lanes 1–2) and TTD (lanes 3–5) hepatocytes, co-immunoprecipitated proteins were visualized by western blots with antibodies raised against PGC-1α (110 kDa), SIRT1 (110 kDa) and the p62 subunit (62 kDa). TTD nuclear extract was supplemented with recombinant TFIIH (rIIH, lane 5). (<b>panel B</b>) When indicated (+), GST-PGC-1α purified from bacteria (130 kDa) was incubated with lysate of Sf9 cells overexpressing TFIIH (rIIH). After immunoprecipitation with an anti Flag-Tag antibody (that recognized the flagged XPB subunit, 89 kDa), the bound proteins were visualized by western blots using antibodies raised against PGC-1α and XPB. (<b>panel C</b>) Purified SIRT1 (110 kDa) was incubated with lysate of Sf9 cells overexpressing TFIIH (rIIH). Immunoprecipitations were performed as described panel B. The bound proteins were visualized by western blots using antibodies raised against SIRT1 and XPB. (<b>panel D</b>) In vitro pull-down assays were performed with GST alone (-, 26 kDa, lanes 2) or GST-PGC-1α (PGC-1α, 130 kDa, lanes 3) incubated with Sf9 cell extracts overexpressing separately each subunit of TFIIH. The bound proteins were visualized by western blots using antibodies directed against each TFIIH subunit. As a reference, the input lanes (IN, lanes 1) represent 10% of the total volume of extract used for each incubation. (<b>panel E</b>) Purified SIRT1 was incubated with Sf9 cell extracts overexpressing separately each TFIIH subunit. Immunoprecipitations (IP) were done using antibodies directed against the TFIIH subunits. The bound proteins were revealed by western blots. (<b>panel F</b>) Deacetylation profile of PGC-1α in WT (lanes 1–3) and TTD (lanes 4–6) hepatocytes after different times of pyruvate treatment (0, 4 and 6 hours). After immunoprecipitation with specific antibodies (IP Ab-PGC-1α), PGC-1α acetylation has been visualized by western blots with anti-acetyl lysine antibodies. Graph depicts the ratio of acetyl-Lysine (Ac-Lys)/PGC-1α western blots signals. (<b>panel G</b>) PGC-1α was immunoprecipitated with specific antibodies (IP Ab-PGC-1α) from nuclear extracts of WT (lanes 1–3) and TTD (lanes 4–6) hepatocytes after different times of pyruvate treatment (0, 4 and 6 hours). Co-immunoprecipitated proteins were visualized by western blots with anti-PGC-1α and -SIRT1 antibodies. Graph depicts the binding ratio between SIRT1 and PGC-1α.</p

Bis-Michael Acceptors as Novel Probes to Study the Keap1/Nrf2/ARE Pathway

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator that promotes the transcription of cytoprotective genes in response to oxidative/electrophilic stress. Various Michael-type compounds were designed and synthesized, and their potency to activate the Keap1/Nrf2/ARE pathway was evaluated. Compounds bearing two Michael-type acceptors proved to be the most active. Tether length and rigidity between the acceptors was crucial. This study will help to understand how this feature disrupts the interaction between Keap1 and Nrf2

Synthesis and Characterization of a Phosphate Prodrug of Isoliquiritigenin

Isoliquiritigenin (<b>1</b>) possesses a variety of biological activities in vitro. However, its poor aqueous solubility limits its use for subsequent in vivo experimentation. In order to enable the use of <b>1</b> for in vivo studies without the use of toxic carriers or cosolvents, a phosphate prodrug strategy was implemented relying on the availability of phenol groups in the molecule. In this study, a phosphate group was added to position C-4 of <b>1</b>, leading to the more water-soluble prodrug <b>2</b> and its ammonium salt <b>3</b>, which possesses increased stability compared to <b>2</b>. Herein are reported the synthesis, characterization, solubility, and stability of phosphate prodrug <b>3</b> in biological medium in comparison to <b>1</b>, as well as new results on its anti-inflammatory properties in vivo. As designed, the solubility of prodrug <b>3</b> was superior to that of the parent natural product <b>1</b> (9.6 mg/mL as opposed to 3.9 μg/mL). Prodrug <b>3</b> as an ammonium salt was also found to possess excellent stability as a solid and in aqueous solution, as opposed to its phosphoric acid precursor <b>2</b>

Basal <i>Cox-2</i> mRNA and protein expression in Normal, At Risk and COPD lung fibroblasts.

<p>(A) Basal <i>Cox-2</i> mRNA: There was no significant difference in basal <i>Cox-2</i> mRNA between Normal (fold change: 1 ± 0.04), At Risk (5.9 ± 2.2) and COPD fibroblasts (1.5 ± 0.69). (B) Basal COX-2 protein: Basal COX-2 levels were low in Normal (non-smoker) lung fibroblasts. A detectable increase in COX-2 protein was observed in At Risk lung fibroblasts as well as lung fibroblasts from COPD subjects. Dashed line denotes different gel. (C) Basal COX-2 protein- quantification: There was a significant increase in basal COX-2 protein expression in lung fibroblasts from COPD subjects (fold change was 28.1 ± 6.1, ** p < 0.001 compared to At Risk and *** p < 0.01 compare Normal). Results are expressed as the mean ± SEM (fold-change) of COX-2 protein levels normalized to the Normal lung fibroblasts and each symbol represents fibroblasts from a different individual.</p

Regulation of IL-1β induction of COX-2 protein in human lung fibroblasts by the AhR is at the level of mRNA stability.

<p>(A) HLFs- AhR siRNA: There was significantly more <i>Cox-2</i> mRNA remaining after induction with IL-1β in AhR knock-down cells. (B) A549-AhR^KO: There was a significant decline in the percentage (%) of <i>Cox-2</i> mRNA remaining within one hour after addition of ActD in A549-AhR^WT cells (* p < 0.05; ** p< 0.01) but not in A549-AhR^KO cells (ns). Results are expressed as mean ± SEM of 2 independent experiments.</p