Both XPD alleles contribute to the phenotype of compound heterozygote xeroderma pigmentosum patients

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in the rare recessive genetic disorder xeroderma pigmentosum (XP). Many XP patients are compound heterozygotes with a “causative” XPD point mutation R683W and different second mutant alleles, considered “null alleles.” However, there is marked clinical heterogeneity (including presence or absence of skin cancers or neurological degeneration) in these XPD/R683W patients, thus suggesting a contribution of the second allele. Here, we report XP patients carrying XPD/R683W and a second XPD allele either XPD/Q452X, /I455del, or /199insPP. We performed a systematic study of the effect of these XPD mutations on several enzymatic functions of TFIIH and found that each mutation exhibited unique biochemical properties. Although all the mutations inhibited the nucleotide excision repair (NER) by disturbing the XPD helicase function, each of them disrupted specific molecular steps during transcription: XPD/Q452X hindered the transactivation process, XPD/I455del disturbed RNA polymerase II phosphorylation, and XPD/199insPP inhibited kinase activity of the cdk7 subunit of TFIIH. The broad range and severity of clinical features in XP patients arise from a broad set of deficiencies in NER and transcription that result from the combination of mutations found on both XPD alleles

Dysregulation of LXR responsive genes contribute to ichthyosis in trichothiodystrophy

International audienceBackground: Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterised by brittle hairs and various systemic symptoms, including photosensitivity and ichthyosis. While photosensitivity could result from DNA repair defects, other TTD clinical features might be due to deficiencies in certain molecular processes.Objectives: The aim of this study was to understand the pathophysiological mechanism of ichthyosis in TTD, focused on the transcriptional dysregulation.Methods: TTD mouse skin tissue and keratinocytes were pathologically and physiologically examined to identify the alteration of lipid homeostasis in TTD with ichtyosis. Gene expression of certain lipid transporter was assessed in fibroblasts derived from TTD patients and TTD mouse keratinocytes.Results: Histopathology and electron microscopy revealed abnormal lipid composition in TTD mice skin. In addition to abnormal cholesterol dynamics, TTD mouse keratinocytes exhibit impaired expression of Liver X receptor (LXR) responsive genes, including Abca12, a key regulator of Harlequin ichthyosis, and Abcg1 that is involved in the cholesterol transport process in the epidermis. Strikingly, dysregulation of LXR responsive genes has been only observed in cells isolated from TTD patients who developed ichthyosis.Conclusions: Our results suggest that the altered expression of the LXR-responsive genes contribute to the pathophysiology of ichthyosis in TTD. These findings provide a new drug discovery target for TTD

TFIIE orchestrates the recruitment of the TFIIH kinase module at promoter before release during transcription

The general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. Here the authors provide evidence that the TFIIEα and TFIIEβ subunits anchor the TFIIH kinase module within the preinitiation complex before their release during transcription

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

Dysregulation of the Peroxisome Proliferator-Activated Receptor Target Genes by XPD Mutations

Mutations in the XPD subunit of TFIIH give rise to human genetic disorders initially defined as DNA repair syndromes. Nevertheless, xeroderma pigmentosum (XP) group D (XP-D) patients develop clinical features such as hypoplasia of the adipose tissue, implying a putative transcriptional defect. Knowing that peroxisome proliferator-activated receptors (PPARs) are implicated in lipid metabolism, we investigated the expression of PPAR target genes in the adipose tissues and the livers of XPD-deficient mice and found that (i) some genes are abnormally overexpressed in a ligand-independent manner which parallels an increase in the recruitment of RNA polymerase (pol) II but not PPARs on their promoter and (ii) upon treatment with PPAR ligands, other genes are much less induced compared to the wild type, which is due to a lower recruitment of both PPARs and RNA pol II. The defect in transactivation by PPARs is likely attributable to their weaker phosphorylation by the cdk7 kinase of TFIIH. Having identified the phosphorylated residues in PPAR isotypes, we demonstrate how their transactivation defect in XPD-deficient cells can be circumvented by overexpression of either a wild-type XPD or a constitutively phosphorylated PPAR S/E. This work emphasizes that underphosphorylation of PPARs affects their transactivation and consequently the expression of PPAR target genes, thus contributing in part to the XP-D phenotype

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