RESUMEN
Tubulin is subject to a wide variety of posttranslational modifications, which, as part of the tubulin code, are involved in the regulation of microtubule functions. Glycylation has so far predominantly been found in motile cilia and flagella, and absence of this modification leads to ciliary disassembly. Here, we demonstrate that the correct functioning of connecting cilia of photoreceptors, which are non-motile sensory cilia, is also dependent on glycylation. In contrast to many other tissues, only one glycylase, TTLL3, is expressed in retina. Ttll3-/- mice lack glycylation in photoreceptors, which results in shortening of connecting cilia and slow retinal degeneration. Moreover, absence of glycylation results in increased levels of tubulin glutamylation in photoreceptors, and inversely, the hyperglutamylation observed in the Purkinje cell degeneration (pcd) mouse abolishes glycylation. This suggests that both posttranslational modifications compete for modification sites, and that unbalancing the glutamylation-glycylation equilibrium on axonemes of connecting cilia, regardless of the enzymatic mechanism, invariably leads to retinal degeneration.
Asunto(s)
Ácido Glutámico/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Tubulina (Proteína)/metabolismo , Animales , Apoptosis , Cilios/metabolismo , Glicosilación , Ratones Endogámicos C57BL , Neuroglía/metabolismo , Neuroglía/patología , Péptido Sintasas/metabolismo , Fenotipo , Células de Purkinje/metabolismo , Células de Purkinje/patología , Retina/metabolismo , Retina/patología , Rodopsina/metabolismo , Factores de TiempoRESUMEN
Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell-derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition-like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.
Asunto(s)
Células Madre Pluripotentes Inducidas , Degeneración Retiniana , Retinitis Pigmentosa , Humanos , Retina , Retinitis Pigmentosa/genética , Degeneración Retiniana/genética , Epitelio Pigmentado de la Retina , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Hippo signalling regulates eye growth during embryogenesis through its effectors YAP and TAZ. Taking advantage of a Yap heterozygous mouse line, we here sought to examine its function in adult neural retina, where YAP expression is restricted to Müller glia. We first discovered an unexpected temporal dynamic of gene compensation. At postnatal stages, Taz upregulation occurs, leading to a gain of function-like phenotype characterised by EGFR signalling potentiation and delayed cell-cycle exit of retinal progenitors. In contrast, Yap+/- adult retinas no longer exhibit TAZ-dependent dosage compensation. In this context, Yap haploinsufficiency in aged individuals results in Müller glia dysfunction, late-onset cone degeneration, and reduced cone-mediated visual response. Alteration of glial homeostasis and altered patterns of cone opsins were also observed in Müller cell-specific conditional Yap-knockout aged mice. Together, this study highlights a novel YAP function in Müller cells for the maintenance of retinal tissue homeostasis and the preservation of cone integrity. It also suggests that YAP haploinsufficiency should be considered and explored as a cause of cone dystrophies in human.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Distrofia del Cono/patología , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Haploinsuficiencia/genética , Animales , Animales Recién Nacidos , Proteínas Portadoras/metabolismo , Ciclo Celular , Proliferación Celular , Distrofia del Cono/genética , Receptores ErbB/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Homeostasis , Ratones Noqueados , Modelos Biológicos , Opsinas/metabolismo , Fenotipo , Retina/patología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Células Madre/metabolismo , Transactivadores/metabolismo , Proteínas Señalizadoras YAPRESUMEN
PURPOSE: High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. METHODS: Atm gene expression was analyzed by RT-PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special focus on retinal cells. RESULTS: Using RT-PCR, we detected a band of the expected size, with its sequence matching the amplified Atm cDNA sequence. Atm mRNA was detected in most cell bodies of the adult mouse eye by in situ hybridization of ocular tissue sections with specific digoxigenin-labeled PCR-amplified cDNA probes. Western blotting with different specific antibodies revealed bands corresponding to the expected sizes of ATM and its active forms (ATMp). These bands were not observed in the analysis of protein homogenates from Atm-deficient mouse tissues. ATM immunoreactivity was detected in the nucleus of all adult mice retinal cells and in most non-neuronal ocular cell types. The active phosphorylated form of ATM was also present in the retina as well as in non-neuronal cells of the adult mouse eye. However, its subcellular localization differed as a function of the cell type examined. A major finding of this study was that ATMp immunostaining in photoreceptor cells was exclusively in the cytoplasm, whereas ATM immunostaining was only in the nucleus of these cells. Furthermore, the specific and distinct ATM and ATMp immunolabeling patterns in photoreceptor cells were identical to those observed in the adult mouse cerebellar granule cells. CONCLUSIONS: We report the expression profile of Atm gene and protein in the adult mouse eye. In particular, we observed a difference between the localization patterns of the active and inactive forms of ATM in photoreceptor cells. These localization patterns suggest that ATM and its phosphorylated activated form may be involved in both the protection of cells from oxidative damage and the maintenance of ocular cell structure and function. The protection mechanisms mediated by the two forms of ATM appear to be particularly important in maintaining photoreceptor integrity.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Ojo/metabolismo , Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Retina/metabolismo , Proteínas Supresoras de Tumor/genética , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cerebelo/citología , Cerebelo/metabolismo , Cuerpo Ciliar/citología , Cuerpo Ciliar/metabolismo , Córnea/citología , Córnea/metabolismo , Citoplasma/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Ojo/citología , Histonas/metabolismo , Inmunohistoquímica , Hibridación in Situ , Cristalino/citología , Cristalino/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
PURPOSE: The retina is highly exposed to oxidative stress due to the high level of oxygen consumption in this tissue and its exposure to light. The main DNA base lesion generated by oxygen free radicals is 8-oxoguanine (8-oxoG). However, its presence in retinal cells and the mechanisms underlying its repair remain undetermined. METHODS: 8-oxoguanine DNA glycosylase (Ogg1) gene expression and messenger localization in adult mouse ocular tissues was analyzed by RT-PCR and in situ hybridization. Using immunohistochemistry, we determined the localization of Ogg1 protein and three base excision repair (BER) enzymes: apurinic/apyrimidic endonuclease (APE1), DNA polymerase beta, and X-ray repair cross-complementation group 1 (XRCC1). Ogg1 and AP-lyase activities in the neuroretina were obtained using double-stranded oligonucleotides harboring either an 8-oxoG residue or a tetrahydrofuran. RESULTS: We report here that 8-oxoG is abundant in the retina. Ogg1, the enzyme responsible for the recognition and excision of the oxidized base, is present in its active form and found mainly in ganglion cells and photoreceptor inner segments. We show that APE1 and DNA polymerase beta, two BER proteins involved in 8-oxoG repair, are also present in these cells. The cellular distribution of these proteins was similar to that of Ogg1. XRRC1 is present in both inner nuclear and ganglion cells layers; however, this protein is absent from photoreceptor inner segments. CONCLUSIONS: This is the first study to demonstrate the presence of a functional 8-oxoG BER pathway in retinal neurons. The study of three BER proteins involved in 8-oxoG elimination demonstrates that XRCC1 localization differs from those of Ogg1, APE1, and DNA polymerase beta. This result suggests that the elimination of 8-oxoG is coordinated through two pathways, which differ slightly according to the cellular localization of the abnormally oxidized guanine.
Asunto(s)
ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Expresión Génica , Retina/metabolismo , Análisis de Varianza , Animales , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ojo/metabolismo , Perfilación de la Expresión Génica/métodos , Inmunohistoquímica , Hibridación in Situ , Ratones , Reacción en Cadena de la Polimerasa , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Purpose: During retinal degeneration, Müller glia cells respond to photoreceptor loss by undergoing reactive gliosis, with both detrimental and beneficial effects. Increasing our knowledge of the complex molecular response of Müller cells to retinal degeneration is thus essential for the development of new therapeutic strategies. The purpose of this work was to identify new factors involved in Müller cell response to photoreceptor cell death. Methods: Whole transcriptome sequencing was performed from wild-type and degenerating rd10 mouse retinas at P30. The changes in mRNA abundance for several differentially expressed genes were assessed by quantitative RT-PCR (RT-qPCR). Protein expression level and retinal cellular localization were determined by western blot and immunohistochemistry, respectively. Results: Pathway-level analysis from whole transcriptomic data revealed the Hippo/YAP pathway as one of the main signaling pathways altered in response to photoreceptor degeneration in rd10 retinas. We found that downstream effectors of this pathway, YAP and TEAD1, are specifically expressed in Müller cells and that their expression, at both the mRNA and protein levels, is increased in rd10 reactive Müller glia after the onset of photoreceptor degeneration. The expression of Ctgf and Cyr61, two target genes of the transcriptional YAP/TEAD complex, is also upregulated following photoreceptor loss. Conclusions: This work reveals for the first time that YAP and TEAD1, key downstream effectors of the Hippo pathway, are specifically expressed in Müller cells. We also uncovered a deregulation of the expression and activity of Hippo/YAP pathway components in reactive Müller cells under pathologic conditions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Unión al ADN/genética , Células Ependimogliales/metabolismo , Regulación de la Expresión Génica , Fosfoproteínas/genética , Células Fotorreceptoras/metabolismo , ARN Mensajero/genética , Degeneración Retiniana/genética , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Western Blotting , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/biosíntesis , Modelos Animales de Enfermedad , Células Ependimogliales/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares , Fosfoproteínas/biosíntesis , Células Fotorreceptoras/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Transducción de Señal , Factores de Transcripción de Dominio TEA , Factores de Transcripción/biosíntesis , Proteínas Señalizadoras YAPRESUMEN
A DNA ligase IV (LIG4)-null human pre-B cell line and human cell lines with hypomorphic mutations in LIG4 are significantly impaired in the frequency and fidelity of end joining using an in vivo plasmid assay. Analysis of the null line demonstrates the existence of an error-prone DNA ligase IV-independent rejoining mechanism in mammalian cells. Analysis of lines with hypomorphic mutations demonstrates that residual DNA ligase IV activity, which is sufficient to promote efficient end joining, nevertheless can result in decreased fidelity of rejoining. Thus, DNA ligase IV is an important factor influencing the fidelity of end joining in vivo. The LIG4-defective cell lines also showed impaired end joining in an in vitro assay using cell-free extracts. Elevated degradation of the terminal nucleotide was observed in a LIG4-defective line, and addition of the DNA ligase IV-XRCC4 complex restored end protection. End protection by DNA ligase IV was not dependent upon ligation. Finally, using purified proteins, we demonstrate that DNA ligase IV-XRCC4 is able to protect DNA ends from degradation by T7 exonuclease. Thus, the ability of DNA ligase IV-XRCC4 to protect DNA ends may contribute to the ability of DNA ligase IV to promote accurate rejoining in vivo.
Asunto(s)
ADN Ligasas/metabolismo , Reparación del ADN , Emparejamiento Base/genética , Línea Celular , Células Cultivadas , ADN Ligasa (ATP) , ADN Ligasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Mutación , Plásmidos/genética , Plásmidos/metabolismoRESUMEN
The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.
Asunto(s)
División Celular , Momento de Replicación del ADN , Inestabilidad Genómica , Retina/citología , Células Madre/fisiología , Transactivadores/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Xenopus , Proteínas Señalizadoras YAPRESUMEN
PURPOSE: Sirtuin1 (Sirt1) is an NAD(+)-dependent deacetylase involved in development, cell survival, stress resistance, energy metabolism, and aging. It is expressed in the mammalian central nervous system (CNS) and is activated during processes associated with neuroprotection. The retinal degeneration 10 (rd10) mouse model of retinitis pigmentosa (RP) was used to investigate the possible role of Sirt1 in this type of retinal degeneration. METHODS: Eyes from control and rd10 mice were used. Sirt1 mRNA was detected by in situ hybridization, and its abundance was estimated by semiquantitative RT-PCR. The presence of Sirt1 protein was investigated by immunohistofluorescence and Western blot analysis. The apoptosis of photoreceptor cells was analyzed by terminal dUTP transferase nick-end labeling (TUNEL). Immunolabeling for Sirt1, apoptosis-inducing factor (Aif), and caspase-12 (Casp-12) was performed on retinal tissue sections. RESULTS: Sirt1 mRNA and immunoreactivity were observed in normal adult mouse eyes. In the control retina, Sirt1 was immunolocalized mostly to the nucleus. In rd10 mice with retinal degeneration, changes in Sirt1 immunolabeling were observed only in the retinal outer nuclear layer (ONL). The pathologic pattern of Sirt1 immunoreactivity correlated with the start of retinal degeneration in rd10 mice. CONCLUSIONS: The results suggest a link between Sirt1 production and retinal degeneration in rd10 mice. The anti-apoptotic, neuroprotective role of Sirt1 in the mouse retina is based on the involvement of Sirt1 in double DNA strand-break repair mechanisms and in maintaining energy homeostasis in photoreceptor cells. The results suggest that the neuroprotective properties of Sirt1 may gradually weaken in rd10 mouse photoreceptor cells.
Asunto(s)
Retina/metabolismo , Retinitis Pigmentosa/metabolismo , Sirtuinas/fisiología , Animales , Apoptosis , Factor Inductor de la Apoptosis/metabolismo , Western Blotting , Caspasa 12/metabolismo , Sondas de ADN , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica/fisiología , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Confocal , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , ARN Mensajero/metabolismo , Retina/patología , Retinitis Pigmentosa/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1RESUMEN
The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.
Asunto(s)
Antígenos Nucleares/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Retroviridae/genética , Transgenes , Animales , Antígenos Nucleares/genética , Células CHO , Cricetinae , Cricetulus , Proteínas de Unión al ADN/genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Hibridación Fluorescente in Situ , Autoantígeno Ku , Regiones Promotoras Genéticas , Integración ViralRESUMEN
DNA interstrand cross-links (ICL) can be induced both by natural products (e.g., psoralens with UVA) and by chemical agents, some of which are used in chemotherapy (e.g., Carboplatin and mitomycin C). Here, we report the formation of ICL by UV radiation in brominated DNA, but only for very specific conformations. The quantum yields for strand break and cross-link formation depend on the wavelength with a maximum near 280 nm. It is known that the photosensitization of DNA by bromodeoxyuridine (BrdUrd) results mainly from the electron affinity of bromine, leading to the irreversible formation of 2'-deoxyuridin-5-yl radicals (dUrd*) upon the addition of an electron from an adjacent adenosine. It is well documented that the photolytic loss of the bromine atom is greatly suppressed in single-stranded DNA versus that in double-stranded DNA. To study this behavior, we have used two models of BrdUrd-mediated sensitization: one consists of a DNA duplex containing a bulge, formed by five mismatched bases, including the BrdUrd, and the other consists of completely duplex DNA. UV irradiation induces much higher levels of single-strand breaks (ssb) in the completely duplex DNA at the BrdUrd site compared to the DNA with a bulge. However, in completely duplex DNA, ssb appear only in the brominated strand, whereas in the bulged duplex DNA, ssb occur on both strands. Most importantly, we also observe formation of interstrand cross-links in bulged duplex DNA in the BrdUrd region. Thus, we propose that UV irradiation of cells containing BrdUrd incorporated randomly into duplex DNA will create many ssb, whereas BrdUrd present in DNA bulges or open regions in double-stranded DNA (transcription bubbles, replication forks) will lead to potentially lethal damage in both strands in the form of ICL. These findings may help explain the potent clinical antiviral activity of IdUrd and BrdUrd (e.g., IdUrd is used to treat eye infections caused by the herpes virus) and suggest that ICL formation may be a very specific probe for identifying single-stranded regions in the DNA of living cells. In addition, this model system provides an excellent means of introducing ICL for studies on their repair and biological consequences.
Asunto(s)
Bromodesoxiuridina/química , Daño del ADN , ADN/química , Conformación de Ácido Nucleico/efectos de la radiación , Rayos Ultravioleta , Secuencia de Bases , ADN/genética , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Plásmidos/química , Plásmidos/genéticaRESUMEN
UV light provokes DNA lesions that interfere with replication and transcription. These lesions may compromise cell viability and usually are removed by nucleotide excision repair (NER). In humans, inactivation of NER is associated with three rare autosomal recessive inherited disorders: xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy. The NER earliest step is lesion recognition by a complex formed by XPC and HHR23B proteins. In a subsequent step, XPA protein becomes associated to the repair complex. Here we investigate whether XPA and XPC proteins, involved in global genome repair, may contribute to a signal transduction pathway regulating the response to UVC-induced lesions. We monitored the expression of several UVC-induced genes in cells deficient in either a transduction pathway or mutated on an NER gene. Expression of the KIN17 gene is induced after UVC irradiation independently of p53 and of activating transcription factor 2. However, in human cells derived from XPA or XPC patients the UVC-induced accumulation of KIN17 RNA and protein is abolished. Our results indicate that the presence of functional XPA and XPC proteins is essential for the up-regulation of the KIN17 gene after UVC irradiation. They also show that the integrity of global genome repair is required to trigger KIN17 gene expression and probably other UVC-responsive genes.
Asunto(s)
Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Proteínas Nucleares , Rayos Ultravioleta , Factor de Transcripción Activador 2 , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Proteínas de Unión al ADN/fisiología , Humanos , Melanoma/genética , Mitomicina/farmacología , Proteínas de Unión al ARN , Factores de Transcripción/fisiología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/fisiología , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
Genotoxic agents deform DNA structure thus eliciting a complex genetic response allowing recovery and cell survival. The Kin17 gene is up-regulated during this response. This gene encodes a conserved nuclear protein that shares a DNA-binding domain with the bacterial RecA protein. The KIN17 protein binds DNA and displays enhanced expression levels in proliferating cultured cells, suggesting a role in nuclear metabolism. We investigated this by studying the expression profile of KIN17 protein during mouse spermatogenesis. As expected, the expression level of Kin17 is higher in proliferating than in differentiated cells. KIN17 is selectively extracted from this tissue by detergents and a fraction was tightly associated with the nuclear matrix. Germinal cells ubiquitously express Kin17 and the protein is located mainly in the nucleus except in elongated spermatids where cytoplasmic staining is also observed. Sertoli and germ cells that are no longer mitotically active express KIN17, suggesting a general role in all testicular cell types. In adult testis a significant proportion of KIN17 co-purifies with polyadenylated RNA. KIN17 directly binds RNA, preferentially poly(G) and poly(U) homopolymers. These results together with the identification of KIN17 as a component of the human spliceosome indicate that this protein may participate in RNA processing.