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1.
Mol Cell ; 74(2): 223-224, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31002802

RESUMO

In this issue of Molecular Cell, Bugai et al. (2019) unveil that a key step of the pro-survival cellular response to a genotoxic attack is the activation of P-TEFb by RBM7. This crucial step triggers RNA polymerase II release from promoter-proximal pausing and expression of DNA damage response genes.


Assuntos
Fator B de Elongação Transcricional Positiva , RNA Polimerase II , Dano ao DNA , Regiões Promotoras Genéticas
2.
Mol Cell ; 65(3): 504-514.e4, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28157507

RESUMO

Transcription starts with the assembly of pre-initiation complexes on promoters followed by their opening. Current models suggest that class II gene transcription requires ATP and the TFIIH XPB subunit to open a promoter. Here, we observe that XPB depletion surprisingly leaves transcription virtually intact. In contrast, inhibition of XPB ATPase activity affects transcription, revealing that mRNA expression paradoxically accommodates the absence of XPB while being sensitive to the inhibition of its ATPase activity. The XPB-depleted TFIIH complex is recruited to active promoters and contributes to transcription. We finally demonstrate that the XPB ATPase activity is only used to relieve a transcription initiation block imposed by XPB itself. In the absence of this block, transcription initiation can take place without XPB ATPase activity. These results suggest that a helicase is dispensable for mRNA transcription, thereby unifying the mechanism of promoter DNA opening for the three eukaryotic RNA polymerases.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Transcrição Gênica , Adenosina Trifosfatases/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Humanos , Mutação , Regiões Promotoras Genéticas , Análise de Sequência de RNA/métodos , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo
3.
Mol Cell ; 68(6): 1054-1066.e6, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29225035

RESUMO

Cockayne syndrome (CS) is caused by mutations in CSA and CSB. The CSA and CSB proteins have been linked to both promoting transcription-coupled repair and restoring transcription following DNA damage. We show that UV stress arrests transcription of approximately 70% of genes in CSA- or CSB-deficient cells due to the constitutive presence of ATF3 at CRE/ATF sites. We found that CSB, CSA/DDB1/CUL4A, and MDM2 were essential for ATF3 ubiquitination and degradation by the proteasome. ATF3 removal was concomitant with the recruitment of RNA polymerase II and the restart of transcription. Preventing ATF3 ubiquitination by mutating target lysines prevented recovery of transcription and increased cell death following UV treatment. Our data suggest that the coordinate action of CSA and CSB, as part of the ubiquitin/proteasome machinery, regulates the recruitment timing of DNA-binding factors and provide explanations about the mechanism of transcription arrest following genotoxic stress.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Síndrome de Cockayne/patologia , Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator 3 Ativador da Transcrição/genética , Células Cultivadas , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Ubiquitina/metabolismo
4.
EMBO Rep ; 22(9): e51683, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34296805

RESUMO

Melanoma cell phenotype switching between differentiated melanocytic and undifferentiated mesenchymal-like states drives metastasis and drug resistance. CDK7 is the serine/threonine kinase of the basal transcription factor TFIIH. We show that dedifferentiation of melanocytic-type melanoma cells into mesenchymal-like cells and acquisition of tolerance to targeted therapies is achieved through chronic inhibition of CDK7. In addition to emergence of a mesenchymal-type signature, we identify a GATA6-dependent gene expression program comprising genes such as AMIGO2 or ABCG2 involved in melanoma survival or targeted drug tolerance, respectively. Mechanistically, we show that CDK7 drives expression of the melanocyte lineage transcription factor MITF that in turn binds to an intronic region of GATA6 to repress its expression in melanocytic-type cells. We show that GATA6 expression is activated in MITF-low melanoma cells of patient-derived xenografts. Taken together, our data show how the poorly characterized repressive function of MITF in melanoma participates in a molecular cascade regulating activation of a transcriptional program involved in survival and drug resistance in melanoma.


Assuntos
Melanoma , Fator de Transcrição Associado à Microftalmia , Linhagem Celular Tumoral , Tolerância a Medicamentos , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo
5.
Mol Cell ; 59(4): 513-4, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26295956

RESUMO

In this issue of Molecular Cell, Nilson et al. (2015) took advantage of THZ1, a recently described covalent inhibitor of the TFIIH kinase CDK7, to further characterize the role of this enzyme in the early stages of transcription and postprocessing events. They unveiled an unexpected function of CDK7 in RNA polymerase II pausing and mRNA capping.


Assuntos
Antineoplásicos/química , Quinases Ciclina-Dependentes/química , Fenilenodiaminas/química , Pirimidinas/química , Iniciação da Transcrição Genética , Humanos
6.
Nucleic Acids Res ; 48(4): 1652-1668, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31930303

RESUMO

The excision of mutagenic DNA adducts by the nucleotide excision repair (NER) pathway is essential for genome stability, which is key to avoiding genetic diseases, premature aging, cancer and neurologic disorders. Due to the need to process an extraordinarily high damage density embedded in the nucleosome landscape of chromatin, NER activity provides a unique functional caliper to understand how histone modifiers modulate DNA damage responses. At least three distinct lysine methyltransferases (KMTs) targeting histones have been shown to facilitate the detection of ultraviolet (UV) light-induced DNA lesions in the difficult to access DNA wrapped around histones in nucleosomes. By methylating core histones, these KMTs generate docking sites for DNA damage recognition factors before the chromatin structure is ultimately relaxed and the offending lesions are effectively excised. In view of their function in priming nucleosomes for DNA repair, mutations of genes coding for these KMTs are expected to cause the accumulation of DNA damage promoting cancer and other chronic diseases. Research on the question of how KMTs modulate DNA repair might pave the way to the development of pharmacologic agents for novel therapeutic strategies.


Assuntos
Cromatina/genética , Dano ao DNA/genética , Histona Metiltransferases/genética , Histonas/genética , Cromatina/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Instabilidade Genômica/genética , Instabilidade Genômica/efeitos da radiação , Histona Metiltransferases/química , Metilação/efeitos da radiação , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta
7.
Mol Cell ; 48(5): 785-98, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23102699

RESUMO

Poly-(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers synthesized by poly-(ADP-ribose) polymerases. Here, transcriptome profiling and differentiation assay revealed a requirement of PARG for retinoic acid receptor (RAR)-mediated transcription. Mechanistically, PARG accumulates early at promoters of RAR-responsive genes upon retinoic acid treatment to promote the formation of an appropriate chromatin environment suitable for transcription. Silencing of PARG or knockout of its enzymatic activity maintains the H3K9me2 mark at the promoter of the RAR-dependent genes, leading to the absence of preinitiation complex formation. In the absence of PARG, we found that the H3K9 demethylase KDM4D/JMJD2D became PARsylated. Mutation of two glutamic acids located in the Jumonji N domain of KDM4D inhibited PARsylation. PARG becomes dispensable for ligand-dependent transcription when either a PARP inhibitor or a non-PARsylable KDM4D/JMJD2D mutant is used. Our results define PARG as a coactivator regulating chromatin remodeling during RA-dependent gene expression.


Assuntos
Glicosídeo Hidrolases/metabolismo , Receptores do Ácido Retinoico/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Ácido Glutâmico , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/genética , Células HeLa , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Metilação , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Interferência de RNA , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor alfa de Ácido Retinoico , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Tretinoína/farmacologia
8.
EMBO J ; 31(17): 3550-63, 2012 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-22863773

RESUMO

Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylation (γH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients.


Assuntos
Reparo do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Animais , Linhagem Celular , Dano ao DNA , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Raios Ultravioleta
9.
Nat Chem Biol ; 15(2): 97-98, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30598542
10.
Mol Cell ; 31(1): 9-20, 2008 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-18614043

RESUMO

The transcription/DNA repair factor TFIIH is organized into a core that associates with the CDK-activating kinase (CAK) complex. Using chromatin immunoprecipitation, we have followed the composition of TFIIH over time after UV irradiation of repair-proficient or -deficient human cells. We show that TFIIH changes subunit composition in response to DNA damage. The CAK is released from the core during nucleotide excision repair (NER). Using reconstituted in vitro NER assay, we show that XPA catalyzes the detachment of the CAK from the core, together with the arrival of the other NER-specific factors. The release of the CAK from the core TFIIH promotes the incision/excision of the damaged oligonucleotide and thereby the repair of the DNA. Following repair, the CAK reappears with the core TFIIH on the chromatin, together with the resumption of transcription. Our findings demonstrate that the composition of TFIIH is dynamic to adapt its engagement in distinct cellular processes.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Reparo do DNA , Fator de Transcrição TFIIH/metabolismo , Trifosfato de Adenosina/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Humanos , Modelos Biológicos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/efeitos da radiação , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Quinase Ativadora de Quinase Dependente de Ciclina
11.
Cell Mol Life Sci ; 72(11): 2177-86, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25681868

RESUMO

To safeguard the genome from the accumulation of deleterious effects arising from DNA lesions, cells developed several DNA repair mechanisms that remove specific types of damage from the genome. Among them, Nucleotide Excision Repair (NER) is unique in its ability to remove a very broad spectrum of lesions, the most important of which include UV-induced damage, bulky chemical adducts and some forms of oxidative damage. Two sub-pathways exist in NER; Transcription-Coupled Repair (TC-NER) removes lesion localized exclusively in transcribed genes while Global Genome Repair (GG-NER) removes lesions elsewhere. In TC- or GG-NER, more than 30 proteins detect, open, incise and resynthesize DNA. Intriguingly, half of them are involved in the detection of DNA damage, implying that this is a crucial repair step requiring a high level of regulation. We review here the complex damage recognition step of GG-NER with a focus on post-translational modifications that help the comings and goings of several protein complexes on the same short damaged DNA locus.


Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , DNA/genética , Genoma/genética , Proteínas de Ligação a DNA/genética , Humanos , Processamento de Proteína Pós-Traducional/genética , Fator de Transcrição TFIIH/genética , Transcrição Gênica , Xeroderma Pigmentoso/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética
12.
Proc Natl Acad Sci U S A ; 110(3): E212-20, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23267107

RESUMO

Specific mutations in the XPD subunit of transcription factor IIH result in combined xeroderma pigmentosum (XP)/Cockayne syndrome (CS), a severe DNA repair disorder characterized at the cellular level by a transcriptional arrest following UV irradiation. This transcriptional arrest has always been thought to be the result of faulty transcription-coupled repair. In the present study, we showed that, following UV irradiation, XP-D/CS cells displayed a gross transcriptional dysregulation compared with "pure" XP-D cells or WT cells. Furthermore, global RNA-sequencing analysis showed that XP-D/CS cells repressed the majority of genes after UV, whereas pure XP-D cells did not. By using housekeeping genes as a model, we demonstrated that XP-D/CS cells were unable to reassemble these gene promoters and thus to restart transcription after UV irradiation. Furthermore, we found that the repression of these promoters in XP-D/CS cells was not a simple consequence of deficient repair but rather an active heterochromatinization process mediated by the histone deacetylase Sirt1. Indeed, RNA-sequencing analysis showed that inhibition of and/or silencing of Sirt1 changed the chromatin environment at these promoters and restored the transcription of a large portion of the repressed genes in XP-D/CS cells after UV irradiation. Our work demonstrates that a significant part of the transcriptional arrest displayed by XP-D/CS cells arises as a result of an active repression process and not simply as a result of a DNA repair deficiency. This dysregulation of Sirt1 function that results in transcriptional repression may be the cause of various severe clinical features in patients with XP-D/CS that cannot be explained by a DNA repair defect.


Assuntos
Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , RNA/biossíntese , Sirtuína 1/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Células Cultivadas , Síndrome de Cockayne/complicações , Reparo do DNA , Heterocromatina/genética , Heterocromatina/metabolismo , Heterocromatina/efeitos da radiação , Humanos , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Xeroderma Pigmentoso/complicações , Proteína Grupo D do Xeroderma Pigmentoso/química
13.
PLoS Genet ; 9(7): e1003611, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23861670

RESUMO

UV-induced DNA damage causes repression of RNA synthesis. Following the removal of DNA lesions, transcription recovery operates through a process that is not understood yet. Here we show that knocking-out of the histone methyltransferase DOT1L in mouse embryonic fibroblasts (MEF(DOT1L)) leads to a UV hypersensitivity coupled to a deficient recovery of transcription initiation after UV irradiation. However, DOT1L is not implicated in the removal of the UV-induced DNA damage by the nucleotide excision repair pathway. Using FRAP and ChIP experiments we established that DOT1L promotes the formation of the pre-initiation complex on the promoters of UV-repressed genes and the appearance of transcriptionally active chromatin marks. Treatment with Trichostatin A, relaxing chromatin, recovers both transcription initiation and UV-survival. Our data suggest that DOT1L secures an open chromatin structure in order to reactivate RNA Pol II transcription initiation after a genotoxic attack.


Assuntos
Cromatina/genética , Dano ao DNA/genética , Metiltransferases/genética , Animais , Cromatina/efeitos da radiação , Reparo do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase , Ácidos Hidroxâmicos/farmacologia , Hipersensibilidade , Camundongos , Camundongos Knockout , RNA Polimerase II/metabolismo , Ativação Transcricional , Raios Ultravioleta
14.
Proc Natl Acad Sci U S A ; 110(25): E2261-70, 2013 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-23733932

RESUMO

Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation, immediate early genes such as activating transcription factor 3 (ATF3) are overexpressed. Although the ATF3 target genes, including dihydrofolate reductase (DHFR), were unable to recover RNA synthesis in CSB-deficient cells, transcription was restored rapidly in normal cells. There the synthesis of DHFR mRNA restarts on the arrival of RNA polymerase II and CSB and the subsequent release of ATF3 from its cAMP response element/ATF target site. In CSB-deficient cells ATF3 remains bound to the promoter, thereby preventing the arrival of polymerase II and the restart of transcription. Silencing of ATF3, as well as stable introduction of wild-type CSB, restores RNA synthesis in UV-irradiated CSB cells, suggesting that, in addition to its role in DNA repair, CSB activity likely is involved in the reversal of inhibitory properties on a gene-promoter region. We present strong experimental data supporting our view that the transcriptional defects observed in UV-irradiated CSB cells are largely the result of a permanent transcriptional repression of a certain set of genes in addition to some defect in DNA repair.


Assuntos
Fator 3 Ativador da Transcrição/genética , Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Reparo do DNA/genética , Estresse Fisiológico/genética , Fator 3 Ativador da Transcrição/metabolismo , Linhagem Celular Transformada , Síndrome de Cockayne/metabolismo , DNA Helicases/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Expressão Gênica/fisiologia , Expressão Gênica/efeitos da radiação , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , Cultura Primária de Células , RNA Interferente Pequeno/genética , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Transcrição Gênica/fisiologia , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta/efeitos adversos
15.
Proc Natl Acad Sci U S A ; 110(44): 17927-32, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24127601

RESUMO

DNA lesions that block transcription may cause cell death even when repaired, if transcription does not restart to reestablish cellular metabolism. However, transcription resumption after individual DNA-lesion repair remains poorly described in mechanistic terms and its players are largely unknown. The general transcription factor II H (TFIIH) is a major actor of both nucleotide excision repair subpathways of which transcription-coupled repair highlights the interplay between DNA repair and transcription. Using an unbiased proteomic approach, we have identified the protein eleven-nineteen lysine-rich leukemia (ELL) as a TFIIH partner. Here we show that ELL is recruited to UV-damaged chromatin in a Cdk7- dependent manner (a component of the cyclin-dependent activating kinase subcomplex of TFIIH). We demonstrate that depletion of ELL strongly hinders RNA polymerase II (RNA Pol II) transcription resumption after lesion removal and DNA gap filling. Lack of ELL was also observed to increase RNA Pol II retention to the chromatin during this process. Identifying ELL as an essential player for RNA Pol II restart during cellular DNA damage response opens the way to obtaining a mechanistic description of transcription resumption after DNA repair.


Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/metabolismo , Ativação Transcricional/fisiologia , Fatores de Elongação da Transcrição/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Clonagem Molecular , Primers do DNA/genética , Recuperação de Fluorescência Após Fotodegradação , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
16.
EMBO J ; 28(19): 2971-80, 2009 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-19713942

RESUMO

XPB and XPD subunits of TFIIH are central genome caretakers involved in nucleotide excision repair (NER), although their respective role within this DNA repair pathway remains difficult to delineate. To obtain insight into the function of XPB and XPD, we studied cell lines expressing XPB or XPD ATPase-deficient complexes. We show the involvement of XPB, but not XPD, in the accumulation of TFIIH to sites of DNA damage. Recruitment of TFIIH occurs independently of the helicase activity of XPB, but requires two recently identified motifs, a R-E-D residue loop and a Thumb-like domain. Furthermore, we show that these motifs are specifically involved in the DNA-induced stimulation of the ATPase activity of XPB. Together, our data demonstrate that the recruitment of TFIIH to sites of damage is an active process, under the control of the ATPase motifs of XPB and suggest that this subunit functions as an ATP-driven hook to stabilize the binding of the TFIIH to damaged DNA.


Assuntos
Dano ao DNA , Fator de Transcrição TFIIH/metabolismo , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , DNA Helicases/química , DNA Helicases/metabolismo , Humanos , Modelos Moleculares , Alinhamento de Sequência , Fator de Transcrição TFIIH/química
17.
Nat Genet ; 36(7): 714-9, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15220921

RESUMO

DNA repair-deficient trichothiodystrophy (TTD) results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH. In a third form of DNA repair-deficient TTD, called group A, none of the nine subunits encoding TFIIH carried mutations; instead, the steady-state level of the entire complex was severely reduced. A new, tenth TFIIH subunit (TFB5) was recently identified in yeast. Here, we describe the identification of the human TFB5 ortholog and its association with human TFIIH. Microinjection of cDNA encoding TFB5 (GTF2H5, also called TTDA) corrected the DNA-repair defect of TTD-A cells, and we identified three functional inactivating mutations in this gene in three unrelated families with TTD-A. The GTF2H5 gene product has a role in regulating the level of TFIIH. The identification of a new evolutionarily conserved subunit of TFIIH implicated in TTD-A provides insight into TFIIH function in transcription, DNA repair and human disease.


Assuntos
Reparo do DNA , Fatores de Transcrição TFII/fisiologia , Transcrição Gênica , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Microinjeções , Fases de Leitura Aberta , Fator de Transcrição TFIIH , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/genética
18.
Nat Commun ; 14(1): 341, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36670096

RESUMO

The transcriptional response to genotoxic stress involves gene expression arrest, followed by recovery of mRNA synthesis (RRS) after DNA repair. We find that the lack of the EXD2 nuclease impairs RRS and decreases cell survival after UV irradiation, without affecting DNA repair. Overexpression of wild-type, but not nuclease-dead EXD2, restores RRS and cell survival. We observe that UV irradiation triggers the relocation of EXD2 from mitochondria to the nucleus. There, EXD2 is recruited to chromatin where it transiently interacts with RNA Polymerase II (RNAPII) to promote the degradation of nascent mRNAs synthesized at the time of genotoxic attack. Reconstitution of the EXD2-RNAPII partnership on a transcribed DNA template in vitro shows that EXD2 primarily interacts with an elongation-blocked RNAPII and efficiently digests mRNA. Overall, our data highlight a crucial step in the transcriptional response to genotoxic attack in which EXD2 interacts with elongation-stalled RNAPII on chromatin to potentially degrade the associated nascent mRNA, allowing transcription restart after DNA repair.


Assuntos
Dano ao DNA , Reparo do DNA , Cromatina/genética , Transcrição Gênica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética
19.
Cell Rep ; 42(11): 113363, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37924516

RESUMO

Super-enhancers (SEs) are stretches of enhancers ensuring a high level of expression of key genes associated with cell function. The identification of cancer-specific SE-driven genes is a powerful means for the development of innovative therapeutic strategies. Here, we identify a MITF/SOX10/TFIIH-dependent SE promoting the expression of BAHCC1 in a broad panel of melanoma cells. BAHCC1 is highly expressed in metastatic melanoma and is required for tumor engraftment, growth, and dissemination. Integrative genomics analyses reveal that BAHCC1 is a transcriptional regulator controlling expression of E2F/KLF-dependent cell-cycle and DNA-repair genes. BAHCC1 associates with BRG1-containing remodeling complexes at the promoters of these genes. BAHCC1 silencing leads to decreased cell proliferation and delayed DNA repair. Consequently, BAHCC1 deficiency cooperates with PARP inhibition to induce melanoma cell death. Our study identifies BAHCC1 as an SE-driven gene expressed in melanoma and demonstrates how its inhibition can be exploited as a therapeutic target.


Assuntos
Melanoma , Humanos , Linhagem Celular Tumoral , Melanoma/patologia , Sequências Reguladoras de Ácido Nucleico , Instabilidade Genômica , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Elementos Facilitadores Genéticos , Proteínas/metabolismo
20.
Sci Adv ; 8(33): eabp9457, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35977011

RESUMO

The helicase XPD is known as a key subunit of the DNA repair/transcription factor TFIIH. However, here, we report that XPD, independently to other TFIIH subunits, can localize with the motor kinesin Eg5 to mitotic spindles and the midbodies of human cells. The XPD/Eg5 partnership is promoted upon phosphorylation of Eg5/T926 by the kinase CDK1, and conversely, it is reduced once Eg5/S1033 is phosphorylated by NEK6, a mitotic kinase that also targets XPD at T425. The phosphorylation of XPD does not affect its DNA repair and transcription functions, but it is required for Eg5 localization, checkpoint activation, and chromosome segregation in mitosis. In XPD-mutated cells derived from a patient with xeroderma pigmentosum, the phosphomimetic form XPD/T425D or even the nonphosphorylatable form Eg5/S1033A specifically restores mitotic chromosome segregation errors. These results thus highlight the phospho-dependent mitotic function of XPD and reveal how mitotic defects might contribute to XPD-related disorders.


Assuntos
Reparo do DNA , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , DNA Helicases/metabolismo , Humanos , Quinases Relacionadas a NIMA/genética , Fosforilação , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética
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