RESUMEN
In eukaryotes, transcription of protein-coding genes requires the assembly at core promoters of a large preinitiation machinery containing RNA polymerase II (RNAPII) and general transcription factors (GTFs). Transcription is potentiated by regulatory elements called enhancers, which are recognized by specific DNA-binding transcription factors that recruit cofactors and convey, following chromatin remodeling, the activating cues to the preinitiation complex. This review summarizes nearly five decades of work on transcription initiation by describing the sequential recruitment of diverse molecular players including the GTFs, the Mediator complex, and DNA repair factors that support RNAPII to enable RNA synthesis. The elucidation of the transcription initiation mechanism has greatly benefited from the study of altered transcription components associated with human diseases that could be considered transcription syndromes.
Asunto(s)
ARN Polimerasa II/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIIH/genética , Iniciación de la Transcripción Genética/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Complejo Mediador/genética , Complejo Mediador/metabolismo , Mutación , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , SíndromeRESUMEN
Transcription factor IIH (TFIIH) is a multiprotein complex involved in both transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies.
Asunto(s)
Reparación del ADN/efectos de los fármacos , Factor de Transcripción TFIIH/genética , Transcripción Genética/efectos de los fármacos , Síndromes de Tricotiodistrofia/genética , Xerodermia Pigmentosa/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , ADN/genética , ADN/metabolismo , Daño del ADN , Humanos , Modelos Moleculares , Terapia Molecular Dirigida , Mutación , Fenilendiaminas/uso terapéutico , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pirimidinas/uso terapéutico , Espironolactona/uso terapéutico , Factor de Transcripción TFIIH/antagonistas & inhibidores , Factor de Transcripción TFIIH/metabolismo , Síndromes de Tricotiodistrofia/tratamiento farmacológico , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patología , Xerodermia Pigmentosa/tratamiento farmacológico , Xerodermia Pigmentosa/metabolismo , Xerodermia Pigmentosa/patologíaRESUMEN
The transcription initiation factor TFIIH is a remarkable protein complex that has a fundamental role in the transcription of protein-coding genes as well as during the DNA nucleotide excision repair pathway. The detailed understanding of how TFIIH functions to coordinate these two processes is also providing an explanation for the phenotypes observed in patients who bear mutations in some of the TFIIH subunits. In this way, studies of TFIIH have revealed tight molecular connections between transcription and DNA repair and have helped to define the concept of 'transcription diseases'.
Asunto(s)
Reparación del ADN/genética , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética , Animales , Enfermedad , Humanos , Modelos GenéticosRESUMEN
The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I2 synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Neoplasias/patología , Síndromes de Tricotiodistrofia/enzimología , Animales , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Epoprostenol , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de la radiación , Ratones , Piel/patología , Transcripción Genética , Síndromes de Tricotiodistrofia/genética , Rayos Ultravioleta , Xerodermia Pigmentosa/genéticaRESUMEN
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.
Asunto(s)
Melanoma , Factor de Transcripción Asociado a Microftalmía , Línea Celular Tumoral , Tolerancia a Medicamentos , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismoRESUMEN
Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystem disorder trichothiodystrophy (TTD), which share only cutaneous photosensitivity. Gene-expression profiles of primary dermal fibroblasts revealed overexpression of matrix metalloproteinase 1 (MMP-1), the gene encoding the metalloproteinase that degrades the interstitial collagens of the extracellular matrix (ECM), in TTD patients mutated in XPD compared with their healthy parents. The defect is observed in TTD and not in XP and is specific for fibroblasts, which are the main producers of dermal ECM. MMP-1 transcriptional up-regulation in TTD is caused by an erroneous signaling mediated by retinoic acid receptors on the MMP-1 promoter and leads to hypersecretion of active MMP-1 enzyme and degradation of collagen type I in the ECM of cell/tissue systems and TTD patient skin. In agreement with the well-known role of ECM in eliciting signaling events controlling cell behavior and tissue homeostasis, ECM alterations in TTD were shown to impact on the migration and wound-healing properties of patient dermal fibroblasts. The presence of a specific inhibitor of MMP activity was sufficient to restore normal cell migration, thus providing a potential approach for therapeutic strategies. This study highlights the relevance of ECM anomalies in TTD pathogenesis and in the phenotypic differences between TTD and XP.
Asunto(s)
Matriz Extracelular/patología , Metaloproteinasa 1 de la Matriz/metabolismo , Factor de Transcripción TFIIH/fisiología , Síndromes de Tricotiodistrofia/enzimología , Humanos , Metaloproteinasa 1 de la Matriz/genética , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/metabolismo , Síndromes de Tricotiodistrofia/patología , Cicatrización de HeridasRESUMEN
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.
Asunto(s)
Sirtuina 1/genética , Factor de Transcripción TFIIH/genética , Factores de Transcripción/genética , Transcripción Genética , Síndromes de Tricotiodistrofia/genética , Animales , Reparación del ADN/genética , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Ratones , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Sirtuina 1/biosíntesis , Factor de Transcripción TFIIH/biosíntesis , Factores de Transcripción/biosíntesis , Síndromes de Tricotiodistrofia/patologíaRESUMEN
Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.
Asunto(s)
Colágeno Tipo VI/genética , Regulación hacia Abajo , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética , Síndromes de Tricotiodistrofia/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Colágeno Tipo VI/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Factor de Transcripción TFIIH/genética , Síndromes de Tricotiodistrofia/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismoRESUMEN
In response to hormonal stimuli, a cascade of hierarchical post-translational modifications of nuclear receptors are required for the correct expression of target genes. Here, we show that the transcription factor TFIIH, via its cdk7 kinase, phosphorylates the androgen receptor (AR) at position AR/S515. Strikingly, this phosphorylation is a key step for an accurate transactivation that includes the cyclic recruitment of the transcription machinery, the MDM2 E3 ligase, the subsequent ubiquitination of AR at the promoter of target genes and its degradation by the proteasome machinery. Impaired phosphorylation disrupts the transactivation, as observed in cells either overexpressing the non-phosphorylated AR/S515A, isolated from xeroderma pigmentosum patient (bearing a mutation in XPD subunit of TFIIH), or in which cdk7 kinase was silenced. Indeed, besides affecting the cyclic recruitment of the transcription machinery, the AR phosphorylation defect favourizes to the recruitment of the E3 ligase CHIP instead of MDM2, at the PSA promoter, that will further attract the proteasome machinery. These observations illustrate how the TFIIH phosphorylation might participate to the transactivation by regulating the nuclear receptors turnover.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores Androgénicos/metabolismo , Factor de Transcripción TFIIH/metabolismo , Activación Transcripcional/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Inmunoprecipitación de Cromatina , Células HeLa , Humanos , Fosforilación , Ubiquitinación , Xerodermia Pigmentosa/genéticaRESUMEN
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.
Asunto(s)
Daño del ADN , Reparación del ADN , Cromatina/genética , Transcripción Genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Melanoma , Humanos , Línea Celular Tumoral , Melanoma/patología , Secuencias Reguladoras de Ácidos Nucleicos , Inestabilidad Genómica , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Elementos de Facilitación Genéticos , Proteínas/metabolismoRESUMEN
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.
Asunto(s)
Reparación del ADN , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismo , ADN Helicasas/metabolismo , Humanos , Quinasas Relacionadas con NIMA/genética , Fosforilación , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo D/genéticaRESUMEN
The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented.
RESUMEN
Small-Cell Lung Cancer (SCLC) is an aggressive neuroendocrine malignancy with a poor prognosis. Here, we focus on the neuroendocrine SCLC subtypes, SCLC-A and SCLC-N, whose transcription addiction was driven by ASCL1 and NEUROD1 transcription factors which target E-box motifs to activate up to 40% of total genes, the promoters of which are maintained in a steadily open chromatin environment according to ATAC and H3K27Ac signatures. This leverage is used by the marine agent lurbinectedin, which preferentially targets the CpG islands located downstream of the transcription start site, thus arresting elongating RNAPII and promoting its degradation. This abrogates the expression of ASCL1 and NEUROD1 and of their dependent genes, such as BCL2, INSM1, MYC, and AURKA, which are responsible for relevant SCLC tumorigenic properties such as inhibition of apoptosis and cell survival, as well as for a part of its neuroendocrine features. In summary, we show how the transcription addiction of these cells becomes their Achilles's heel, and how this is effectively exploited by lurbinectedin as a novel SCLC therapeutic endeavor.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Carbolinas , Compuestos Heterocíclicos de 4 o más Anillos , Neoplasias Pulmonares , Proteínas Represoras , Carcinoma Pulmonar de Células Pequeñas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carbolinas/farmacología , Línea Celular Tumoral , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Represoras/metabolismo , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismoRESUMEN
Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH yield the rare genetic disorder trichothiodystrophy (TTD). Although this syndrome was initially associated with a DNA repair defect, individuals with TTD develop neurological features, such as microcephaly and hypomyelination that could be connected to transcriptional defects. Here we show that an XPD mutation in TTD mice results in a spatial and selective deregulation of thyroid hormone target genes in the brain. Molecular analyses performed on the mice brain tissue demonstrate that TFIIH is required for the stabilization of thyroid hormone receptors (TR) to their DNA-responsive elements. The limiting amounts of TFIIH found in individuals with TTD thus contribute to the deregulation of TR-responsive genes. The discovery of an unexpected stabilizing function for TFIIH deepens our understanding of the pathogenesis and neurological manifestations observed in TTD individuals.
Asunto(s)
Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/metabolismo , Factor de Transcripción TFIIH/fisiología , Síndromes de Tricotiodistrofia/complicaciones , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Línea Celular Transformada , Huella de ADN/métodos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Mutación , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedades del Sistema Nervioso/patología , ARN Interferente Pequeño/farmacología , Factor de Transcripción TFIIH/deficiencia , Transfección , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologíaRESUMEN
Mitochondria are highly dynamic organelles subjected to fission and fusion events. During mitosis, mitochondrial fission ensures equal distribution of mitochondria to daughter cells. If and how this process can actively drive mitotic progression remains largely unknown. Here, we discover a pathway linking mitochondrial fission to mitotic progression in mammalian cells. The mitochondrial fission factor (MFF), the main mitochondrial receptor for the Dynamin-related protein 1 (DRP1), is directly phosphorylated by Protein Kinase D (PKD) specifically during mitosis. PKD-dependent MFF phosphorylation is required and sufficient for mitochondrial fission in mitotic but not in interphasic cells. Phosphorylation of MFF is crucial for chromosome segregation and promotes cell survival by inhibiting adaptation of the mitotic checkpoint. Thus, PKD/MFF-dependent mitochondrial fission is critical for the maintenance of genome integrity during cell division.
Asunto(s)
Proteínas Mitocondriales/genética , Mitosis/fisiología , Proteína Quinasa C/metabolismo , Animales , Humanos , Ratones , Transducción de SeñalRESUMEN
BACKGROUND: 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.
Asunto(s)
Regulación de la Expresión Génica , Ictiosis/genética , Receptores X del Hígado/metabolismo , Piel/patología , Síndromes de Tricotiodistrofia/complicaciones , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Transportadoras de Casetes de Unión a ATP/genética , Animales , Células Cultivadas , Colesterol/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Humanos , Ictiosis/patología , Queratinocitos/metabolismo , Ratones , Cultivo Primario de Células , Piel/citología , Transcripción Genética , Síndromes de Tricotiodistrofia/genéticaRESUMEN
In eukaryotes, the general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. However, the mechanism by which these transcription factors incorporate the preinitiation complex and coordinate their action during RNA polymerase II transcription remains elusive. Here we show that the TFIIEα and TFIIEß subunits anchor the TFIIH kinase module (CAK) within the preinitiation complex. In addition, we show that while RNA polymerase II phosphorylation and DNA opening occur, CAK and TFIIEα are released from the promoter. This dissociation is impeded by either ATP-γS or CDK7 inhibitor THZ1, but still occurs when XPB activity is abrogated. Finally, we show that the Core-TFIIH and TFIIEß are subsequently removed, while elongation factors such as DSIF are recruited. Remarkably, these early transcriptional events are affected by TFIIE and TFIIH mutations associated with the developmental disorder, trichothiodystrophy.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIH/metabolismo , Factores de Transcripción TFII/metabolismo , Transcripción Genética , Síndromes de Tricotiodistrofia/genética , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/genética , Fibroblastos , Humanos , Mutación , Proteínas Nucleares/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Factor de Transcripción TFIIH/genética , Factores de Transcripción TFII/genética , Factores de Elongación Transcripcional/metabolismo , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
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.