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

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

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

Design and Development of a Robotized System Coupled to µCT Imaging for Intratumoral Drug Evaluation in a HCC Mouse Model

<div><p>Hepatocellular carcinoma (HCC) is one of the most common cancer related deaths worldwide. One of the main challenges in cancer treatment is drug delivery to target cancer cells specifically. Preclinical evaluation of intratumoral drugs in orthotopic liver cancer mouse models is difficult, as percutaneous injection hardly can be precisely performed manually. In the present study we have characterized a hepatoma model developing a single tumor nodule by implantation of Hep55.1C cells in the liver of syngeneic C57BL/6J mice. Tumor evolution was followed up by µCT imaging, and at the histological and molecular levels. This orthotopic, poorly differentiated mouse HCC model expressing fibrosis, inflammation and cancer markers was used to assess the efficacy of drugs. We took advantage of the high precision of a previously developed robotized system for automated, image-guided intratumoral needle insertion, to administer every week in the tumor of the Hep55.1C mouse model. A significant tumor growth inhibition was observed using our robotized system, whereas manual intraperitoneal administration had no effect, by comparison to untreated control mice.</p></div

Treatment of Hep55.1C mouse HCC model by Doxorubicin.

<p>Mice bearing orthotopic HCC tumors were treated by recurrent automated robotized intratumoral injection (IT, n = 3) or manual intraperitoneal (IP, n = 3) of Doxorubicin; untreated mice (n = 3) were used as control of tumor growth. (<b>A</b>) Representative 3-weeks follow-up by µCT scan imaging and 3D reconstruction of tumor development. Volumes of the tumors resected at the end of the experiment were measured. (<b>B</b>) Tumor volume was computed at each time point from the 3D reconstructions of the tumors in untreated (left), IT (middle) or IP (right) injected mice (mean +/− SEM, star (*) indicates a difference with untreated control at the same time point with p<0.05).</p

Hep55.1C tumors markers.

<p>Expression of COLA1, ASMA, MMP3, FN1 fibrosis markers, IL6, PDGFB, TGFB inflammation markers and ALB, CCND1, SPP1, AFP cancer markers was assessed by RTqPCR from Hep55.1C tumor and corresponding surrounding normal liver. Relative mRNA level of each gene was normalized to the level of the housekeeping gene 36B4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106675#s2" target="_blank">Results</a> are expressed as the mean +/− SEM for at least 3 animals.</p

Mouse restrainer and robotized needle injector for intratumoral drug administration.

<p>(<b>A</b>) Description of the mouse restrainer bed. (<b>B</b>) View of a mouse restrained under gaseous anesthesia. The animal is tightly fixed on a hemicylindrical shell by a sterile operative field stuck on the abdomen. A sensor placed on the back of the mouse monitors respiration. (<b>C</b>) A dedicated registration cover is screwed on the bed frame during µCT scan imaging and registration by structured light projection.</p