RESUMEN
DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B-EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.
Asunto(s)
Cromatina , Reparación del ADN/genética , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Transcripción Genética/genética , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Silenciador del Gen , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrKRESUMEN
Protein ubiquitination has emerged as a pivotal regulatory reaction that promotes cellular responses to DNA damage. With a goal to delineate the DNA damage signal transduction cascade, we systematically analyzed the human E2 ubiquitin- and ubiquitin-like-conjugating enzymes for their ability to mobilize the DNA damage marker 53BP1 onto ionizing radiation-induced DNA double strand breaks. An RNAi-based screen identified UBE2U as a candidate regulator of chromatin responses at double strand breaks. Further mining of the UBE2U interactome uncovered its cognate E3 RNF17 as a novel factor that, via the radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties (RIDDLE) syndrome protein RNF168, enforces DNA damage responses. Our screen allowed us to uncover new players in the mammalian DNA damage response and highlights the instrumental roles of ubiquitin machineries in promoting cell responses to genotoxic stress.
Asunto(s)
Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Cromatina/genética , Células HeLa , Humanos , Factores de Transcripción/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The Fanconi anemia protein PALB2, also known as FANCN, protects genome integrity by regulating DNA repair and cell cycle checkpoints. Exactly how PALB2 functions may be temporally coupled with detection and signaling of DNA damage is not known. Intriguingly, we found that PALB2 is transformed into a hyperphosphorylated state in response to ionizing radiation (IR). IR treatment specifically triggered PALB2 phosphorylation at Ser-157 and Ser-376 in manners that required the master DNA damage response kinase Ataxia telangiectasia mutated, revealing potential mechanistic links between PALB2 and the Ataxia telangiectasia mutated-dependent DNA damage responses. Consistently, dysregulated PALB2 phosphorylation resulted in sustained activation of DDRs. Full-blown PALB2 phosphorylation also required the breast and ovarian susceptible gene product BRCA1, highlighting important roles of the BRCA1-PALB2 interaction in orchestrating cellular responses to genotoxic stress. In summary, our phosphorylation analysis of tumor suppressor protein PALB2 uncovers new layers of regulatory mechanisms in the maintenance of genome stability and tumor suppression.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína BRCA1/metabolismo , Reparación del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Femenino , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Fosforilación , Radiación Ionizante , Serina/genética , Serina/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/genéticaRESUMEN
Ubiquitylation plays key roles in DNA damage signal transduction. The current model envisions that lysine63-linked ubiquitin chains, via the concerted action of E3 ubiquitin ligases RNF8-RNF168, are built at DNA double-strand breaks (DSBs) to effectively assemble DNA damage-repair factors for proper checkpoint control and DNA repair. We found that RNF168 is a short-lived protein that is stabilized by the deubiquitylating enzyme USP34 in response to DNA damage. In the absence of USP34, RNF168 is rapidly degraded, resulting in attenuated DSB-associated ubiquitylation, defective recruitment of BRCA1 and 53BP1 and compromised cell survival after ionizing radiation. We propose that USP34 promotes a feed-forward loop to enforce ubiquitin signaling at DSBs and highlight critical roles of ubiquitin dynamics in genome stability maintenance.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Proteasas Ubiquitina-Específicas/fisiología , Ubiquitinación , Estabilidad de Enzimas , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/análisisRESUMEN
Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.
Asunto(s)
Lisina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Animales , Células Cultivadas , Daño del ADN , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
MCPH1 is especially important for linking chromatin remodeling to DNA damage response. It contains three BRCT (BRCA1-carboxyl terminal) domains. The N-terminal region directly binds with chromatin remodeling complex SWI-SNF, and the C-terminal BRCT2-BRCT3 domains (tandem BRCT domains) are involved in cellular DNA damage response. The MCPH1 gene associates with evolution of brain size, and its variation can cause primary microcephaly. In this study we solve the crystal structures of MCPH1 natural variant (A761) C-terminal tandem BRCT domains alone as well as in complex with γH2AX tail. Compared with other structures of tandem BRCT domains, the most significant differences lie in phosphopeptide binding pocket. Additionally, fluorescence polarization assays demonstrate that MCPH1 tandem BRCT domains show a binding selectivity on pSer +3 and prefer to bind phosphopeptide with free COOH-terminus. Taken together, our research provides new structural insights into BRCT-phosphopeptide recognition mechanism.
Asunto(s)
Histonas/química , Proteínas del Tejido Nervioso/química , Fosfoproteínas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Proteínas del Citoesqueleto , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Propiedades de SuperficieRESUMEN
Histone ubiquitylation is emerging as an important protective component in cellular responses to DNA damage. The ubiquitin ligases RNF8 and RNF168 assemble ubiquitin chains onto histone molecules surrounding DNA breaks and facilitate retention of DNA repair proteins. Although RNF8 and RNF168 play important roles in repair of DNA double strand breaks, their requirement for cell protection from replication stress is largely unknown. In this study, we uncovered RNF168-independent roles of RNF8 in repair of replication inhibition-induced DNA damage. We showed that RNF8 depletion, but not RNF168 depletion, hyper-sensitized cells to hydroxyurea and aphidicolin treatment. Consistently, hydroxyurea induced persistent single strand DNA lesions and sustained CHK1 activation in RNF8-depleted cells. In line with strict requirement for RAD51-dependent repair of hydroxyurea-stalled replication forks, RNF8 depletion compromised RAD51 accumulation onto single strand DNA lesions, suggesting that impaired replication fork repair may underlie the enhanced cellular sensitivity to replication arrest observed in RNF8-depleted cells. In total, our study highlights the differential requirement for the ubiquitin ligase RNF8 in facilitating repair of replication stress-associated DNA damage.
Asunto(s)
Daño del ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Replicación del ADN/efectos de los fármacos , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/deficiencia , Fase G2/efectos de los fármacos , Fase G2/genética , Células HeLa , Histonas/metabolismo , Humanos , Hidroxiurea/farmacología , Proteínas Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Recombinasa Rad51/metabolismo , Ubiquitina-Proteína Ligasas , Ubiquitinación/efectos de los fármacosRESUMEN
Homologous recombination mediated by RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-strand breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2: to enhance RAD51's ability to form the D loop. We show that PALB2 binds DNA and physically interacts with RAD51. Notably, although PALB2 alone stimulates D-loop formation, it has a cooperative effect with RAD51AP1, an enhancer of RAD51. This stimulation stems from the ability of PALB2 to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results demonstrate the multifaceted role of PALB2 in chromosome damage repair. Because PALB2 mutations can cause cancer or Fanconi anemia, our findings shed light on the mechanism of tumor suppression in humans.
Asunto(s)
Proteína BRCA2/fisiología , Neoplasias de la Mama/metabolismo , Reparación del ADN/fisiología , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Nucleares/fisiología , Recombinasa Rad51/fisiología , Recombinación Genética/fisiología , Proteínas Supresoras de Tumor/fisiología , Proteínas Reguladoras de la Apoptosis , Proteína BRCA2/química , Proteínas de Unión al ADN/química , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Femenino , Humanos , Complejos Multiproteicos , Proteínas de Neoplasias/química , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas de Unión al ARN , Recombinasa Rad51/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/fisiología , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genéticaRESUMEN
The eukaryotic RNA splicing machinery is dedicated to the daunting task of excising intronic sequences on the many nascent RNA transcripts in a cell, and in doing so facilitates proper translation of its transcriptome. Notably, emerging evidence suggests that RNA splicing may also play direct roles in maintaining genome stability. Here we report the identification of the RNA/DNA-binding protein SON as a component of spliceosome that plays pleiotropic roles during mitotic progression. We found that SON is essential for cell proliferation, and that its inactivation triggers a MAD2-dependent mitotic delay. Moreover, SON deficiency is accompanied by defective chromosome congression, compromised chromosome segregation and cytokinesis, which in turn contributes to cellular aneuploidy and cell death. In summary, our study uncovers a specific link between SON and mitosis, and highlights the potential of RNA processing as additional regulatory mechanisms that govern cell proliferation and division.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Mitosis , Empalmosomas/metabolismo , Supervivencia Celular , Citocinesis , Silenciador del Gen , Células HeLa , Humanos , Antígenos de Histocompatibilidad Menor , Unión Proteica , Huso Acromático/metabolismoRESUMEN
Dynamic changes of chromatin structure facilitate diverse biological events, including DNA replication, repair, recombination, and gene transcription. Recent evidence revealed that DNA damage elicits alterations to the chromatin to facilitate proper checkpoint activation and DNA repair. Here we report the identification of the PWWP domain-containing protein EXPAND1/MUM1 as an architectural component of the chromatin, which in response to DNA damage serves as an accessory factor to promote cell survival. Depletion of EXPAND1/MUM1 or inactivation of its PWWP domain resulted in chromatin compaction. Upon DNA damage, EXPAND1/MUM1 rapidly concentrates at the vicinity of DNA damage sites via its direct interaction with 53BP1. Ablation of this interaction impaired damage-induced chromatin decondensation, which is accompanied by sustained DNA damage and hypersensitivity to genotoxic stress. Collectively, our study uncovers a chromatin-bound factor that serves an accessory role in coupling damage signaling with chromatin changes in response to DNA damage.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Secuencia de Aminoácidos , Animales , Supervivencia Celular , Células Cultivadas , Cromatina/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Datos de Secuencia Molecular , ARN Interferente Pequeño/genética , Elementos de Respuesta , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
The breast and ovarian cancer type 1 susceptibility protein (BRCA1) has pivotal roles in the maintenance of genome stability. Studies support that BRCA1 exerts its tumour suppression function primarily through its involvement in cell cycle checkpoint control and DNA damage repair. In addition, recent proteomic and genetic studies have revealed the presence of distinct BRCA1 complexes in vivo, each of which governs a specific cellular response to DNA damage. Thus, BRCA1 is emerging as the master regulator of the genome through its ability to execute and coordinate various aspects of the DNA damage response.
Asunto(s)
Proteína BRCA1/metabolismo , Daño del ADN , Inestabilidad Genómica , Animales , Proteína BRCA1/genética , Ciclo Celular , Reparación del ADN , HumanosRESUMEN
PALB2 is an integral component of the BRCA complex important for recombinational DNA repair. However, exactly how this activity is regulated in vivo remains unexplored. Here we provide evidence to show that MRG15 is a novel PALB2-associated protein that ensures regulated recombination events. We found that the direct interaction between MRG15 and PALB2 is mediated by an evolutionarily conserved region on PALB2. Intriguingly, although damage-induced RAD51 foci formation and mitomycin C sensitivity appeared normal in MRG15-binding defective PALB2 mutants, these cells exhibited a significant increase in gene conversion rates. Consistently, we found that abrogation of the PALB2-MRG15 interaction resulted in elevated sister chromatid exchange frequencies. Our results suggest that loss of the PALB2-MRG15 interaction relieved the cells with the suppression of sister chromatid exchange and therefore led to a hyper-recombination phenotype in the gene conversion assay. Together, our study indicated that although PALB2 is required for proficient homologous recombination, it could also govern the choice of templates used in homologous recombination repair.
Asunto(s)
Proteínas Nucleares/biosíntesis , Recombinación Genética , Factores de Transcripción/fisiología , Proteínas Supresoras de Tumor/biosíntesis , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Relación Dosis-Respuesta a Droga , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Eliminación de Gen , Células HeLa , Humanos , Mitomicina/farmacología , Modelos Genéticos , Unión Proteica , Estructura Terciaria de Proteína , Intercambio de Cromátides Hermanas , Factores de Transcripción/biosíntesisRESUMEN
Maintenance of genomic stability ensures faithful transmission of genetic information and helps suppress neoplastic transformation and tumorigenesis. Although recent progress has advanced our understanding of DNA damage checkpoint regulations, little is known as to how DNA repair, especially the RAD51-dependent homologous recombination repair pathway, is executed in vivo. Here, we reveal novel properties of the BRCA2-associated protein PALB2 in the assembly of the recombinational DNA repair machinery at DNA damage sites. Although the chromatin association of PALB2 is a prerequisite for subsequent BRCA2 and RAD51 loading, the focal accumulation of the PALB2 x BRCA2 x RAD51 complex at DSBs occurs independently of known DNA damage checkpoint and repair proteins. We provide evidence to support that PALB2 exists as homo-oligomers and that PALB2 oligomerization is essential for its focal accumulation at DNA breaks in vivo. We propose that both PALB2 chromatin association and its oligomerization serve to secure the BRCA2 x RAD51 repair machinery at the sites of DNA damage. These attributes of PALB2 are likely instrumental for proficient homologous recombination DNA repair in the cell.
Asunto(s)
Cromatina/metabolismo , Reparación del ADN/fisiología , Inestabilidad Genómica/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis , Proteína BRCA2/genética , Línea Celular , Roturas del ADN de Doble Cadena , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Estructura Terciaria de Proteína , ARN Interferente Pequeño , Recombinasa Rad51/genética , Proteínas Supresoras de Tumor/químicaRESUMEN
Mutations in breast cancer susceptibility gene 1 and 2 (BRCA1 and BRCA2) predispose individuals to breast and ovarian cancer development. We previously reported an in vivo interaction between BRCA1 and BRCA2. However, the biological significance of their association is thus far undefined. Here, we report that PALB2, the partner and localizer of BRCA2, binds directly to BRCA1, and serves as the molecular scaffold in the formation of the BRCA1-PALB2-BRCA2 complex. The association between BRCA1 and PALB2 is primarily mediated via apolar bonding between their respective coiled-coil domains. More importantly, BRCA1 mutations identified in cancer patients disrupted the specific interaction between BRCA1 and PALB2. Consistent with the converging functions of the BRCA proteins in DNA repair, cells harboring mutations with abrogated BRCA1-PALB2 interaction resulted in defective homologous recombination (HR) repair. We propose that, via its direct interaction with PALB2, BRCA1 fine-tunes recombinational repair partly through its modulatory role in the PALB2-dependent loading of BRCA2-RAD51 repair machinery at DNA breaks. Our findings uncover PALB2 as the molecular adaptor between the BRCA proteins, and suggest that impaired HR repair is one of the fundamental causes for genomic instability and tumorigenesis observed in patients carrying BRCA1, BRCA2, or PALB2 mutations.
Asunto(s)
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Reparación del ADN/genética , ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Proteína BRCA1/genética , Proteína BRCA2/genética , Línea Celular , ADN/genética , Roturas del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Humanos , Proteínas Nucleares/genética , Unión Proteica , Proteínas Supresoras de Tumor/genéticaRESUMEN
Proper activation of checkpoint during mitotic stress is an important mechanism to prevent genomic instability. Chfr (Check point protein with FHA (Forkhead-associated domain) and RING domains) is a ubiquitin-protein isopeptide ligase (E3) that is important for the control of an early mitotic checkpoint, which delays entry into metaphase in response to mitotic stress. Because several lines of evidence indicate that Chfr is a potential tumor suppressor, it is critically important for us to identify Chfr substrates and understand how Chfr may regulate these substrates, control mitotic transitions, and thus, act as a tumor suppressor in vivo. Here, we report the discovery of a new Chfr-associated protein Kif22, a chromokinesin that binds to both DNA and microtubules. We demonstrated that Kif22 is a novel substrate of Chfr. We showed that Chfr-mediated Kif22 down-regulation is critical for the maintenance of chromosome stability. Collectively, our results reveal a new substrate of Chfr that plays a role in the maintenance of genome integrity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Cinesinas/metabolismo , Proteínas de Neoplasias/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Riñón/citología , Riñón/metabolismo , Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Unión a Poli-ADP-Ribosa , ARN Interferente Pequeño/farmacología , Huso Acromático/metabolismo , Transfección , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Chromatin endowed by histone modifications governs chromatin structure, which in turn represents a means to regulate cellular processes, including transcription and heterochromatin formation. Recent evidence revealed a plethora of enzymes that catalyze specific histone modifications for epigenetic maintenance, and dysregulation of which contributes to tumorigenesis and developmental defects. The histone methyltransferase SET8 (also known as Pr-Set7) was previously reported to monomethylate Lys(20) of histone H4. However, the temporal and spatial control of SET8 activity remains elusive. Here, we provide evidence to support that SET8 monomethylates Lys(20) of histone H4 during S phase by tethering to proliferating cell nuclear antigen via a putative proliferating cell nuclear antigen-interacting protein box. In addition, we show that SET8 function is required for S phase progression. Finally, deletion of SET8 in mice causes embryonic lethality, suggesting that SET8 plays an important role in mammalian embryogenesis.
Asunto(s)
Replicación del ADN , Regulación del Desarrollo de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Metilación de ADN , Perfilación de la Expresión Génica , Células HeLa , N-Metiltransferasa de Histona-Lisina/química , Humanos , Ratones , Modelos Biológicos , Fase S , TransfecciónRESUMEN
UNLABELLED: Metastasis is a major cause of cancer morbidity and mortality in individuals with hepatocellular carcinoma (HCC), yet little is known about the underlying molecular basis. Using genetic information derived from chromosome-based comparative genomic hybridization, we have reported previously on regional chromosome 7q21-q22 gains in close association with HCC progression. In this study, we undertook cDNA microarray-based comparative genomic hybridization, to examine the 7q21-q22 region for the involved gene(s) in HCC. High-resolution mapping analysis highlighted 7 candidates, namely PFTAIRE protein kinase 1 (PFTK1), ODAG, CDK6, CAS1, PEX1, SLC25A, and PEG10, within the region. Quantitative reverse transcription (RT)-PCR evaluation further indicated upregulation of a single candidate gene, PFTK1, that correlated significantly with both advanced metastatic HCCs (P = 0.032) and tumor microvascular invasion (P = 0.012). Given that little is known about the function(s) of PFTK1, which is a novel cell division cycle (Cdc)2-related gene, we examined its potential role in the motile phenotype of HCC cells by both ectopic expression and knockdown investigations. RNA-interference knockdown of PFTK1 in invasive Hep3B cells resulted in a significant reduction in cell invasion, chemotactic migration, and cell motility (P < 0.001). Conversely, ectopic expression of PFTK1 in noninvasive HKCI-C3 cells induced substantial cellular invasion and migration (P < or = 0.007). In neither cell line was there any effect on cell viability. Immunofluorescence showed marked filamentous actin polymerizations in PFTK1-expressing cells. CONCLUSION: In this study, we have thus provided preliminary evidence that overexpression of PFTK1 may confer a motile phenotype in malignant hepatocytes that accounts for the association of upregulation of this gene in metastatic HCC.
Asunto(s)
Proteína Quinasa CDC2/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Adulto , Anciano , Anciano de 80 o más Años , Proteína Quinasa CDC2/análisis , Quinasas Ciclina-Dependientes , Femenino , Dosificación de Gen , Humanos , Masculino , Persona de Mediana Edad , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Mensajero/análisisRESUMEN
Widespread DNA copy number alterations are well recognized in hepatocellular carcinoma (HCC), although the affected genes expression remained largely undefined. In this study, we performed genome-wide analysis on HCC to examine the relationship between gene copy number and corresponding transcriptional changes. To ensure analysis on a homogenous population of tumor cells, integrative analysis of array-based CGH and expression profilings was performed on 20 HCC cell lines using a 19,200-element cDNA microarray platform. Further validation studies were carried out on a large series of primary HCC tumors and paired adjacent non-malignant liver to ascertain finding. Correlative analyses highlighted 31 candidate genes that manifested both copy gains and gene up-regulations (R2>0.5; p<0.05). Of interest was over-expressed paternally expressed 10 (PEG10) resided within the chromosome region 7q21 that has been implicated in the progression of HCC. Quantitative PCR and qRT-PCR studies verified concurrent genomic gains and over-expression of PEG10 in HCC cell lines and primary tumors (34/40 cases; 85%). In addition, qRT-PCR demonstrated a significant progressive trend of increasing PEG10 expressions from the putative pre-malignant adjacent livers to early resectable HCC tumors, and to late inoperable HCCs (p=0.007). In summary, the present study demonstrated the usefulness of integrated genomic and expression profilings in identifying candidate genes within regions of genomic alteration. Our results also suggested that PEG10 may be a potential biomarker in the progressive development of HCC, and that genomic gain represents one of the major mechanisms in the induction of PEG10 over-expressions.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas/metabolismo , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Cartilla de ADN , Proteínas de Unión al ADN , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Neoplasias Hepáticas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/genética , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Molecular characterizations of hepatocellular carcinoma have indicated frequent allelic losses on chromosomes 4q, 8p, 16q and 17p, where the minimal deleted regions have been further defined on 4q12-q23, 4q31-q35, 8p21-p22, 16q12.1-q23.1 and 17p13. Despite these regions are now well-recognized in early liver carcinogenesis, few underlying candidate genes have been identified. In an effort to define affected genes within common deleted loci of hepatocellular carcinoma, we conducted transcriptional mapping by high-resolution cDNA microarray analysis. In 20 hepatocellular carcinoma cell lines and 20 primary tumors studied, consistent downregulations of novel transcripts were highlighted throughout the entire genome and within sites of frequent losses. The array-derived candidates including fibrinogen gamma peptide (FGG, at 4q31.3), vitamin D binding protein (at 4q13.3), fibrinogen-like 1 (FGL1, at 8p22), metallothionein 1G (MT1G, at 16q12.2) and alpha-2-plasmin inhibitor (SERPINF2, at 17p13) were confirmed by quantitative reverse transcription-polymerase chain reaction, which also indicated a more profound downregulation of FGL1, MT1G and SERPINF2 relative to reported tumor-suppressor genes, such as DLC1 (8p22), E-cadherin (16q22.1) and TP53 (17p13.1). In primary hepatocellular carcinoma examined, a significant repression of MT1G by more than 100-fold was indicated in 63% of tumors compared to the adjacent nonmalignant liver (P = 0.0001). Significant downregulations of FGG, FGL1 and SERPINF2 were also suggested in 30, 23 and 33% of cases, respectively, compared to their nonmalignant counterparts (P < 0.016). In summary, transcriptional mapping by microarray indicated a number of previously undescribed downregulated genes in hepatocellular carcinoma, and highlighted potential candidates within common deleted regions.
Asunto(s)
Carcinoma Hepatocelular/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Adulto , Anciano , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Humanos , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Genome-wide copy number aberrations are common in hepatocellular carcinoma, although the precise genetic events underlying disease progression remain poorly defined. Previous work from our group has indicated several regional chromosomal gains such as chromosome 7q34-q36 that are associated with advanced metastatic tumors. Although the distal chromosome 7q gains have also been implicated in the progression of other malignancies, information on underlying targeted genes is limited. In this study, we have examined the chromosome 7q34-q36 region for involved gene(s) (or genes of interest). An integrated array-based comparative genomic hybridization and transcriptional mapping analyses has enabled us to identify a single candidate, zyxin on chromosome 7q34-q36. This array-derived finding was supported by quantitative reverse transcription-polymerase chain reaction, which also indicated common upregulations of zyxin in hepatocellular carcinoma tumors compared to their corresponding nonmalignant liver tissue (17/52 cases; 33%). Although there was no correlation between zyxin expression and tumor stagings, there was a significant increase in messenger RNA levels in hepatocellular carcinoma cases that presented with multifocal disease (211.5 +/- 936.9-fold) compared to those with solitary lesions (3.5 +/- 6.3-fold). Moreover, recurrence after resection was common in cases that displayed zyxin overexpressions in the initial resected tumor (P = 0.05). Functional examination of zyxin by small interfering RNA-mediated knockdown in Hep3B cell line indicated a significant inhibition on cell migration through porous membrane (P = 0.002) and invasion through matrigel-coated membrane (P = 0.005). In summary, mapping of chromosome 7q34-q36 has led to the identification of frequent zyxin overexpressions in hepatocellular carcinoma, and a potential role for zyxin in conferring a motile phenotype.