Isolation and characterization of two human transcription factor IIH (TFIIH)-related complexes: ERCC2/CAK and TFIIH.

Transcription factor IIH (TFIIH) is a multisubunit protein complex essential for both the initiation of RNA polymerase class II (pol II)-catalyzed transcription and nucleotide excision repair of DNA. Recent studies have shown that TFIIH copurifies with the cyclin-dependent kinase (cdk)-activating kinase complex (CAK) that includes cdk7, cyclin H, and p36/MAT1. Here we report the isolation of two TFIIH-related complexes: TFIIH* and ERCC2/CAK. TFIIH* consists of a subset of the TFIIH complex proteins including ERCC3 (XPB), p62, p44, p41, and p34 but is devoid of detectable levels of ERCC2 (XPD) and CAK. ERCC2/CAK was isolated as a complex that exhibits CAK activity that cosediments with the three CAK components (cdk7, cyclin H, and p36/MAT1) as well as the ERCC2 (XPD) protein. TFIIH* can support pol II-catalyzed transcription in vitro with lower efficiency compared with TFIIH. This TFIIH*-dependent transcription reaction was stimulated by ERCC2/CAK. The ERCC2/CAK and TFIIH* complexes are each active in DNA repair as shown by their ability to complement extracts prepared from ERCC2 (XPD)- and ERCC3 (XPB)-deficient cells, respectively, in supporting the excision of DNA containing a cholesterol lesion. These data suggest that TFIIH* and ERCC2/CAK interact to form the TFIIH holoenzyme capable of efficiently assembling the pol II transcription initiation complex and directly participating in excision repair reactions.

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

Defective transcription/repair factor IN recruitment to specific UV lesions in trichothiodystrophy syndrome

Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFHH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 64PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some mutations affect the recruitment of TFHH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFHH complexes carrying an NH2-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFUH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFHH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions.

Études structurales d’interactions protéine/protéine impliquées dans l’érythropoïèse

Le développement hématopoïétique est régulé par l’action combinée de facteurs de transcription lignée spécifiques et de la machinerie transcriptionnelle de base, permettant ainsi l’expression de gènes en temps et lieu appropriés. Les travaux présentés dans cette thèse portent sur l’étude structurale et fonctionnelle d’interactions décisives pour la régulation de l’expression de gènes et impliquant des domaines de transactivation (TAD). En effet, les interactions faisant intervenir les TAD d’activateurs permettent de réguler l’activation de la transcription de façon spécifique. La première étude présentée dans cette thèse relate l'identification et la caractérisation d'une nouvelle interaction entre deux facteurs de transcription : le facteur hématopoïétique GATA-1 et la protéine suppresseur de tumeur p53. En combinant des études in vitro par titrage calorimétrique en condition isotherme (ITC) et par spectroscopie RMN et des études in vivo, nous avons identifié et caractérisé cette nouvelle interaction. Il s'avère que le TAD de p53 et le domaine de liaison à l’ADN de GATA-1 sont les domaines minimaux requis pour la formation de ce complexe. L'inhibition de la voie p53 par GATA-1 s’est avérée être la conséquence majeure de cette interaction, permettant ainsi le maintien en vie des précurseurs érythrocytaires via l’inhibition de l’apoptose. Un deuxième type d’interaction a fait l’objet d’études : l’interaction entre divers TAD et la machinerie transcriptionnelle de base, plus spécifiquement avec le Facteur général de Transcription IIH (TFIIH). La structure des complexes constitués par la sous-unité Tfb1/p62 du facteur TFIIH en interaction avec le TAD viral de VP16 d’une part, et avec le TAD humain du facteur érythrocytaire « Erythroid Krüppel-like factor» (EKLF) d’autre part, ont été résolues par spectroscopie RMN. La structure du complexe Tfb1/VP16 a révélée que le mode de liaison de VP16 à Tfb1 est similaire au mode de liaison du TAD de p53 avec le même partenaire. En effet, les TAD de VP16 et de p53 forment tous deux une hélice α de 9 résidus en interaction avec Tfb1. En dépit de partager avec p53 et VP16 le même site de liaison sur Tfb1/p62, la structure RMN du complexe EKLF/Tfb1 démontre que le mode d’interaction de ce TAD se distingue du mode de liaison canonique des activeurs transcriptionnels. Etonnamment, EKLF adopte un mécanisme de liaison semblable au mécanisme de liaison du facteur général de transcription TFIIEα avec p62, leurs conformations demeurent étendues en interaction avec Tfb1/p62. En se basant sur nos données structurales, nous avons identifié un résidu dans le TAD d'EKLF décisif pour la formation du complexe EKLF/p62 : le Trp73. La mutation de cet acide aminé perturbe son interaction avec Tfb1PH/p62PH et réduit significativement l'activité transcriptionnelle d'EKLF dans les érythrocytes.

Caractérisation structurale et fonctionnelle des interactions impliquant TFIIH et les domaines de transactivation viraux

Le facteur de transcription IIH (TFIIH) joue un rôle crucial dans la transcription et dans la réparation de l’ADN. La sous-unité Tfb1/p62 (levure et humain) de TFIIH interagit avec de nombreux facteurs de transcription (p53, NFκB, TFIIEα) et de réparation (Rad2/XPG and Rad4/XPC) (1). La majorité des interactions avec Tfb1/p62 requiert le domaine d’homologie à la Pleckstrin (PH) localisé dans la région N-terminal de la protéine (2, 3). Ce domaine PH forme des complexes avec des domaines de transactivation acide provenant de protéines cibles impliquées dans la transcription et la réparation de l’ADN. De récentes études ont montré que Tfb1/p62 est une cible pour les protéines virales telles que la protéine VP16 du virus de l’herpès simplex (HSV) de type 1, la protéine E1 du virus du papillome humain (VPH) et la protéine EBNA-2 du virus Epstein-Barr (EBV) (4, 5). Ces protéines virales interagissent avec la sous-unité Tfb1/p62 par un domaine de transactivation acide suggérant une interaction similaire à ce qui est observé chez les facteurs de transcription humains comme p53. Ce mémoire présente une caractérisation structurelle et fonctionnelle du complexe formé par la protéine virale EBNA2 et la protéine humaine Tfb1/p62. L’analyse est faite en utilisant le titrage calorimétrique isotherme (ITC), la résonance magnétique nucléaire (RMN) et une expérience de transactivation chez la levure. Cette étude amène une plus grande compréhension des protéines impliquées dans les maladies comme le lymphome de Burkitt et le lymphome de Hodgkin qui sont souvent associées à l’infection à l’EBV (revue dans (6)) et caractérise une cible potentielle pour un antiviral.

Functional XPB/RAD25 redundancy in Arabidopsis genome: characterization of AtXPB2 and expression analysis

The xeroderma pigmentosum complementation group B (XPB) protein is involved in both DNA repair and transcription in human cells. It is a component of the transcription factor IIH (TFIIH) and is responsible for DNA helicase activity during nucleotide (nt) excision repair (NER). Its high evolutionary conservation has allowed identification of homologous proteins in different organisms, including plants. In contrast to other organisms, Arabidopsis thaliana harbors a duplication of the XPB orthologue (AtXPB1 and AtXPB2), and the proteins encoded by the duplicated genes are very similar (95% amino acid identity). Complementation assays in yeast rad25 mutant strains suggest the involvement of AtXPB2 in DNA repair, as already shown for AtXPB1, indicating that these proteins may be functionally redundant in the removal of DNA lesions in A. thaliana. Although both genes are expressed in a constitutive manner during the plant life cycle, Northern blot analyses suggest that light modulates the expression level of both XPB copies, and transcript levels increase during early stages of development. Considering the high similarity between AtXPB1 and AtXPB2 and that both of predicted proteins may act in DNA repair, it is possible that this duplication may confer more flexibility and resistance to DNA damaging agents in thale cress. (C) 2004 Elsevier B.V. All rights reserved.

On the traces of XPD: cell cycle matters - untangling the genotype-phenotype relationship of XPD mutations

Abstract Mutations in the human gene coding for XPD lead to segmental progeria - the premature appearance of some of the phenotypes normally associated with aging - which may or may not be accompanied by increased cancer incidence. XPD is required for at least three different critical cellular functions: in addition to participating in the process of nucleotide excision repair (NER), which removes bulky DNA lesions, XPD also regulates transcription as part of the general transcription factor IIH (TFIIH) and controls cell cycle progression through its interaction with CAK, a pivotal activator of cyclin dependent kinases (CDKs). The study of inherited XPD disorders offers the opportunity to gain insights into the coordination of important cellular events and may shed light on the mechanisms that regulate the delicate equilibrium between cell proliferation and functional senescence, which is notably altered during physiological aging and in cancer. The phenotypic manifestations in the different XPD disorders are the sum of disturbances in the vital processes carried out by TFIIH and CAK. In addition, further TFIIH- and CAK-independent cellular activities of XPD may also play a role. This, added to the complex feedback networks that are in place to guarantee the coordination between cell cycle, DNA repair and transcription, complicates the interpretation of clinical observations. While results obtained from patient cell isolates as well as from murine models have been elementary in revealing such complexity, the Drosophila embryo has proven useful to analyze the role of XPD as a cell cycle regulator independently from its other cellular functions. Together with data from the biochemical and structural analysis of XPD and of the TFIIH complex these results combine into a new picture of the XPD activities that provides ground for a better understanding of the patophysiology of XPD diseases and for future development of diagnostic and therapeutic tools.

A Drosophila XPD model links cell cycle coordination with neuro-development and suggests links to cancer

XPD functions in transcription, DNA repair and in cell cycle control. Mutations in human XPD (also known as ERCC2) mainly cause three clinical phenotypes: xeroderma pigmentosum (XP), Cockayne syndrome (XP/CS) and trichothiodystrophy (TTD), and only XP patients have a high predisposition to developing cancer. Hence, we developed a fly model to obtain novel insights into the defects caused by individual hypomorphic alleles identified in human XP-D patients. This model revealed that the mutations that displayed the greatest in vivo UV sensitivity in Drosophila did not correlate with those that led to tumor formation in humans. Immunoprecipitations followed by targeted quantitative MS/MS analysis showed how different xpd mutations affected the formation or stability of different transcription factor IIH (TFIIH) subcomplexes. The XP mutants most clearly linked to high cancer risk, Xpd R683W and R601L, showed a reduced interaction with the core TFIIH and also an abnormal interaction with the Cdk-activating kinase (CAK) complex. Interestingly, these two XP alleles additionally displayed high levels of chromatin loss and free centrosomes during the rapid nuclear division phase of the Drosophila embryo. Finally, the xpd mutations showing defects in the coordination of cell cycle timing during the Drosophila embryonic divisions correlated with those human mutations that cause the neurodevelopmental abnormalities and developmental growth defects observed in XP/CS and TTD patients.

Xpd/Ercc2 regulates CAK activity and mitotic progression

General transcription factor IIH (TFIIH) consists of nine sub- units: cyclin-dependent kinase 7 (Cdk7), cyclin H and MAT1 (forming the Cdk-activating-kinase or CAK complex), the two helicases Xpb/Hay and Xpd, and p34, p44, p52 and p62 (refs 1–3). As the kinase subunit of TFIIH, Cdk7 participates in basal transcription by phosphorylating the carboxy-terminal domain of the largest subunit of RNA polymerase II1,4,5. As part of CAK, Cdk7 also phosphorylates other Cdks, an essential step for their activation6–9. Here we show that the Drosophila TFIIH com- ponent Xpd negatively regulates the cell cycle function of Cdk7, the CAK activity. Excess Xpd titrates CAK activity, resulting in decreased Cdk T-loop phosphorylation, mitotic defects and lethality, whereas a decrease in Xpd results in increased CAK activity and cell proliferation. Moreover, Xpd is downregulated at the beginning of mitosis when Cdk1, a cell cycle target of Cdk7, is most active. Downregulation of Xpd thus seems to contribute to the upregulation of mitotic CAK activity and to regulate mitotic progression positively. Simultaneously, the downregulation of Xpd might be a major mechanism of mitotic silencing of basal transcription.

Assembly, subunit composition, and footprint of human DNA repair excision nuclease

The assembly and composition of human excision nuclease were investigated by electrophoretic mobility shift assay and DNase I footprinting. Individual repair factors or any combination of up to four repair factors failed to form DNA–protein complexes of high specificity and stability. A stable complex of high specificity can be detected only when XPA/RPA, transcription factor IIH, XPC⋅HHR23B, and XPG and ATP are present in the reaction mixture. The XPF⋅ERCC1 heterodimer changes the electrophoretic mobility of the DNA–protein complex formed with the other five repair factors, but it does not confer additional specificity. By using proteins with peptide tags or antibodies to the repair factors in electrophoretic mobility shift assays, it was found that XPA, replication protein A, transcription factor IIH, XPG, and XPF⋅excision repair cross-complementing 1 but not XPC⋅HHR23B were present in the penultimate and ultimate dual incision complexes. Thus, it appears that XPC⋅HHR23B is a molecular matchmaker that participates in the assembly of the excision nuclease but is not present in the ultimate dual incision complex. The excision nuclease makes an assymmetric DNase I footprint of ≈30 bp around the damage and increases the DNase I sensitivity of the DNA on both sides of the footprint.

Human and Yeast Cdk-activating Kinases (CAKs) Display Distinct Substrate Specificities

Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40MO15/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40MO15 have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40MO15 preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40MO15 only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21CIP1, p27KIP1, p57KIP2, p16INK4a, and p18INK4c, could block phosphorylation by p40MO15 but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40MO15 activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.

Human cyclin-dependent kinase-activating kinase exists in three distinct complexes.

Transcription factor IIH (TFIIH) is a multisubunit complex required for transcription and for DNA nucleotide excision repair. TFIIH possesses three enzymatic activities: (i) an ATP-dependent DNA helicase, (ii) a DNA-dependent ATPase, and (iii) a kinase with specificity for the carboxyl-terminal domain of RNA polymerase II. The kinase activity was recently identified as the cdk (cyclin-dependent kinase) activating kinase, CAK, composed of cdk7, cyclin H, and MAT-1. Here we report the isolation and characterization of three distinct CAK-containing complexes from HeLa nuclear extracts: CAK, a novel CAK-ERCC2 complex, and TFIIH. CAK-ERCC2 can efficiently associate with core-TFIIH to reconstitute holo-TFIIH transcription activity. We present evidence proposing a critical role for ERCC2 in mediating the association of CAK with core TFIIH subunits.

Functional XPB/RAD25 redundancy in Arabidopsis genome: characterization of AtXPB2 and expression analysis

The xeroderma pigmentosum complementation group B (XPB) protein is involved in both DNA repair and transcription in human cells. It is a component of the transcription factor IIH (TFIIH) and is responsible for DNA helicase activity during nucleotide (nt) excision repair (NER). Its high evolutionary conservation has allowed identification of homologous proteins in different organisms, including plants. In contrast to other organisms, Arabidopsis thaliana harbors a duplication of the XPB orthologue (AtXPB1 and AtXPB2), and the proteins encoded by the duplicated genes are very similar (95% amino acid identity). Complementation assays in yeast rad25 mutant strains suggest the involvement of AtXPB2 in DNA repair, as already shown for AtXPB1, indicating that these proteins may be functionally redundant in the removal of DNA lesions in A. thaliana. Although both genes are expressed in a constitutive manner during the plant life cycle, Northern blot analyses suggest that light modulates the expression level of both XPB copies, and transcript levels increase during early stages of development. Considering the high similarity between AtXPB1 and AtXPB2 and that both of predicted proteins may act in DNA repair, it is possible that this duplication may confer more flexibility and resistance to DNA damaging agents in thale cress. (C) 2004 Elsevier B.V. All rights reserved.

Growth Factor Stimulation Improves The Structure And Properties Of Scaffold-free Engineered Auricular Cartilage Constructs.

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.