RESUMEN
Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.
Asunto(s)
Histonas , Proteínas Nucleares , Humanos , Masculino , Andrógenos/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Elementos de Facilitación GenéticosRESUMEN
Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine ß-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous ß-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3'eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3'eRNA leads to a reduction in ß-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult ß-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.
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Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , ARN/metabolismo , Transcripción Genética , Globinas beta/genética , Adulto , Animales , Línea Celular Tumoral , Endorribonucleasas/genética , Feto , Humanos , Hígado/embriología , Hígado/metabolismo , Región de Control de Posición , Ratones Transgénicos , ARN/fisiología , ARN Polimerasa II/metabolismo , Globinas beta/biosíntesisRESUMEN
Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. PCa that relapses after hormonal therapies, referred to as castration resistant PCa (CRPC), often presents with metastases (mCRPC) that are the major cause of mortality. The few available therapies for mCRPC patients include taxanes docetaxel (DTX) and cabazitaxel (CBZ). However, development of resistance limits their clinical use. Mechanistically, resistance arises through upregulation of multidrug resistance (MDR) proteins such as MDR1/ABCB1, making ABCB1 an attractive therapeutic target. Yet, ABCB1 inhibitors failed to be clinically useful due to low specificity and toxicity issues. To study taxanes resistance, we produced CBZ resistant C4-2B cells (RC4-2B) and documented resistance to both CBZ and DTX in cell culture and in 3D prostaspheres settings. RNAseq identified increased expression of ABCB1 in RC4-2B, that was confirmed by immunoblotting and immunofluorescent analysis. ABCB1-specific inhibitor elacridar reversed CBZ and DTX resistance in RC4-2B cells, confirming ABCB1-mediated resistance mechanism. In a cell-based screen using a curated library of cytotoxic drugs, we found that DNA damaging compounds Camptothecin (CPT) and Cytarabine (Ara-C) overcame resistance as seen by similar cytotoxicity in parental C4-2B and resistant RC4-2B. Further, these compounds were cytotoxic to multiple PC cells resistant to taxanes with high ABCB1 expression and, therefore, can be used to conquer the acquired resistance to taxanes in PCa. Finally, inhibition of cyclin-dependent kinases 4/6 (CDK4/6) with small molecule inhibitors (CDK4/6i) potentiated cytotoxic effect of CPT or Ara-C in both parental and resistant cells. Overall, our findings indicate that DNA damaging agents CPT and Ara-C alone or in combination with CDK4/6i can be suggested as a new treatment regimen in CRPC patients, including those that are resistant to taxanes.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Docetaxel , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias de la Próstata Resistentes a la Castración , Taxoides , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Línea Celular Tumoral , Docetaxel/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Taxoides/farmacología , Taxoides/uso terapéutico , Antineoplásicos/farmacologíaRESUMEN
Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. AR KO reduced levels of H3.3 at enhancers, indicating feedback mechanism. In addition, HIRA KO deregulates glucocorticoid-driven transcription, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity. Key points: *H3.3 at enhancers promotes acetylation of H3K27Ac and retention of AR/BRD4 complex for transcription regulation*Knockout of H3.3 chaperone HIRA suppresses PC cells growth and deregulates androgen-induced transcription*H3.3/HIRA pathway regulates both AR and GR, suggesting a common HIRA/H3.3 mechanism of nuclear receptors function.
RESUMEN
E2FBP1/hDRIL1, a DNA-binding A/T-rich interaction domain (ARID) family transcription factor, is expressed ubiquitously in human tissues and plays an essential role in maintaining the proliferation potential of passage-limited human fibroblasts by dissociating promyelocytic leukemia nuclear bodies (PML-NBs). This effect on PML-NBs is similar to that of viral immediate-early gene products, such as infected cellular protein 0 (ICP0) from human herpes simplex virus 1 (HSV-1), which also disrupts PML-NBs to override the intrinsic cellular defense. Here we report that E2FBP1 inhibits accumulation of ICP0 RNA and, at the same time, is degraded via ICP0's herpes ubiquitin ligase 2 (HUL-2) activity upon HSV-1 infection. These reciprocal regulatory roles of ICP0 and E2FBP1 are linked in an ARID-dependent fashion. Our results suggest that E2FBP1 functions as an intrinsic cellular defense factor in spite of its PML-NB dissociation function.
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Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 1/patogenicidad , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Factores de Virulencia/metabolismo , Línea Celular , Humanos , Proteínas Represoras/genéticaRESUMEN
Androgen ablation therapy is the standard of care for newly diagnosed prostate cancer (PC) patients. PC that relapsed after hormonal therapy, referred to as castration-resistant PC (CRPC), often presents with metastasis (mCRPC) and is the major cause of disease lethality. The few available therapies for mCRPC include the Taxanes Docetaxel (DTX) and Cabazitaxel (CBZ). Alas, clinical success of Taxanes in mCRPC is limited by high intrinsic and acquired resistance. Therefore, it remains essential to develop rationally designed treatments for managing therapy-resistant mCRPC disease. The major effect of Taxanes on microtubule hyper-polymerization is a prolonged mitotic block due to activation of the Spindle Assembly Checkpoint (SAC). Taxane-sensitive cells eventually inactivate SAC and exit mitosis by mitotic catastrophe, resulting in genome instability and blockade of proliferation. Resistant cells remain in mitotic block, and, upon drug decay, resume mitosis and proliferation, underlying one resistance mechanism. In our study we explored the possibility of forced mitotic exit to elevate Taxane efficacy. Inactivation of the SAC component, mitotic checkpoint kinase Mps1/TTK with a small molecule inhibitor (Msp1i), potentiated efficacy of Taxanes treatment in both 2D cell culture and 3D prostasphere settings. Mechanistically, Mps1 inhibition forced mitotic catastrophe in cells blocked in mitosis by Taxanes. Androgen receptor (AR), the main driver of PC, is often mutated or truncated in mCRPC. Remarkably, Mps1i significantly potentiated CBZ cytotoxicity regardless of AR status, in both AR-WT and in AR-truncated CRPC cells. Overall, our data demonstrate that forced mitotic exit by Mps1 inhibition potentiates Taxanes efficacy. Given that several Mps1i's are currently in different stages of clinical trials, our results point to Mps1 as a new therapeutic target to potentiate efficacy of Taxanes in mCRPC patients.
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Proteínas de Ciclo Celular/metabolismo , Neoplasias de la Próstata Resistentes a la Castración , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores Androgénicos , Andrógenos/farmacología , Hidrocarburos Aromáticos con Puentes , Docetaxel/farmacología , Docetaxel/uso terapéutico , Resistencia a Antineoplásicos/genética , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Taxoides/farmacología , Taxoides/uso terapéuticoRESUMEN
BACKGROUND: Functional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies. RESULTS: Using a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites. To confirm our data, we next performed in situ hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to in silico predicted several extra-chromosomes were positive for TR by in situ analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions. CONCLUSIONS: Chromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified in silico and confirmed in situ 3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.
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ADN Satélite/genética , ADN Satélite/metabolismo , Genoma , Animales , Biología Computacional , Sondas de ADN/química , Hibridación Fluorescente in Situ , Cariotipificación , RatonesRESUMEN
Genes under control of super-enhancers are expressed at extremely high levels and are frequently associated with nuclear speckles. Recent data suggest that the high concentration of unphosphorylated RNA polymerase II (Pol II) and Mediator recruited to super-enhancers create phase-separated condensates. Transcription initiates within or at the surface of these phase-separated droplets and the phosphorylation of Pol II, associated with transcription initiation and elongation, dissociates Pol II from these domains leading to engagement with nuclear speckles, which are enriched with RNA processing factors. The transitioning of Pol II from transcription initiation domains to RNA processing domains effectively co-ordinates transcription and processing of highly expressed RNAs which are then rapidly exported into the cytoplasm.
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ARN Polimerasa II , Transcripción Genética , Fosforilación , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismoRESUMEN
Proteins that combine PML NBs (ND10) can be divided into two groups: "transient" (that accumulate at PML NBs upon over-expression, interferon-induced up-regulation, block of proteosomal degradation, environmental stress or viral infection) and "constitutive" that co-localize with PML in the majority of cultured cells. One of the few "constitutive" components of PML NBs is the death domain-associated protein Daxx. While PML NBs are the most obvious depositories of Daxx, there are multiple alternative localization of this protein in the nucleus and cytoplasm, suggesting differential functionality of Daxx at different cellular compartments and stages of the cell cycle. The purpose of this review is to analyze Daxx spatiotemporal behavior within and outside of PML NBs and to discuss functions attributed to these localizations. We suggest that Daxx can participate in numerous cellular functions as a mediator of protein interactions, thus acting as a fine tuning instrument in highly orchestrated cellular processes; we also envision PML NBs accumulation of Daxx as an "out of action" storage depot.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Núcleo Celular/fisiología , Neutrófilos/fisiología , Proteínas Nucleares/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Nucléolo Celular/fisiología , Cromatina/genética , Cromatina/fisiología , Proteínas Co-Represoras , Citoplasma/fisiología , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Ratones , Chaperonas Moleculares , Proteínas Nucleares/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
INTRODUCTION: Overcoming resistance to antimitotic drugs, such as paclitaxel (PTX), would represent a major advance in breast cancer treatment. PTX induces mitotic block and sensitive cells exit mitosis dying by mitotic catastrophe. Resistant cells remain in block and continue proliferation after drug decay, denoting one of the PTX resistance mechanisms. Mild hyperthermia (HT) triggers mitotic exit of PTX-pretreated cells, overcoming PTX resistance and suggesting HT-forced mitotic exit as a promising strategy to potentiate PTX. METHODS AND RESULTS: Superparamagnetic iron oxide nanoparticles (SPIONs) were used to deliver mild HT at 42°C in PTX-pretreated breast adenocarcinoma MCF-7 cells sensitive and resistant to PTX. To evaluate mechanism of cell death, cells were classified based on nuclear morphology into interphase, mitotic, micronucleated, and apoptotic. The combined PTXâSPION treatment resulted in an increase in the percentage of micronucleated cells, an indication of forced mitotic exit. Importantly, in PTX-resistant cells, the combination therapy using SPION HT helps to overcome resistance by reducing the number of cells relative to the control. CONCLUSION: SPION HT potentiates PTX by significantly reducing cell survival, suggesting potential of combined treatment for future clinical translation.
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Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/terapia , Resistencia a Antineoplásicos , Hipertermia Inducida , Nanopartículas de Magnetita/química , Paclitaxel/farmacología , Neoplasias de la Mama/patología , Supervivencia Celular/efectos de los fármacos , Terapia Combinada , Femenino , Humanos , Células Tumorales CultivadasRESUMEN
BACKGROUND: The main chromatin unit, the nucleosome, can be modulated by the incorporation of histone variants that, in combination with posttranslational histones modifications, determine epigenetics properties of chromatin. Understanding the mechanism that creates a histone variants landscape at different genomic elements is expected to elevate our comprehension of chromatin assembly and function. The Daxx chaperone deposits transcription-associated histone H3.3 at centromeres, but mechanism of centromere-specific Daxx targeting remains unclear. RESULTS: In this study, we identified an unexpected function of the constitutive centromeric protein CENP-B that serves as a "beacon" for H3.3 incorporation. CENP-B depletion reduces Daxx association and H3.3 incorporation at centromeres. Daxx/CENP-B interaction and Daxx centromeric association are SUMO dependent and requires SIMs of Daxx. Depletion of SUMO-2, but not SUMO-1, decreases Daxx/CENP-B interaction and reduces centromeric accumulation of Daxx and H3.3, demonstrating distinct functions of SUMO paralogs in H3.3 chaperoning. Finally, disruption of CENP-B/Daxx-dependent H3.3 pathway deregulates heterochromatin marks H3K9me3, ATRX and HP1α at centromeres and elevates chromosome instability. CONCLUSION: The demonstrated roles of CENP-B and SUMO-2 in H3.3 loading reveal a novel mechanism controlling chromatin maintenance and genome stability. Given that CENP-B is the only centromere protein that binds centromere-specific DNA elements, our study provides a new link between centromere DNA and unique epigenetic landscape of centromere chromatin.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína B del Centrómero/fisiología , Cromatina/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Centrómero/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Co-Represoras , HumanosRESUMEN
Androgen receptor (AR) mediates initiation and progression of prostate cancer (PCa); AR-driven transcription is activated by binding of androgens to the ligand-binding domain (LBD) of AR. Androgen ablation therapy offers only a temporary relief of locally advanced and metastatic PCa, and the disease eventually recurs as a lethal castration-resistant PCa (CRPC) as there is no effective treatment for CRPC patients. Thus, it is critical to identify novel targeted and combinatorial regimens for clinical management of CRPC. Reduction of the repressive epigenetic modification H3K27me2/3 correlates with PCa aggressiveness, while corresponding demethylases JMJD3/UTX are overexpressed in PCa. We found that JMJD3/UTX inhibitor GSK-J4 reduced more efficiently proliferation of AR-ΔLBD cells (CRPC model) compared with isogenic AR-WT cells. Inhibition of JMJD3/UTX protects demethylation of H3K27Me2/3, thus reducing levels of H3k27Me1. We observed that the reduction dynamics of H3K27Me1 was faster and achieved at lower inhibitor concentrations in AR-ΔLBD cells, suggesting that inhibition of JMJD3/UTX diminished proliferation of these cells by hindering AR-driven transcription. In addition, we observed synergy between GSK-J4 and Cabazitaxel, a taxane derivative that is approved for CRPC treatment. Collectively, our results point at the H3K27 demethylation pathway as a new potential therapeutic target in CRPC patients.
RESUMEN
BACKGROUND: The death domain-associated protein (DAXX) collaborates with accessory proteins to deposit the histone variant H3.3 into mouse telomeric and pericentromeric repeat DNA. Pericentromeric repeats are the main genetic contributor to spatially discrete, compact, constitutive heterochromatic structures called chromocentres. Chromocentres are enriched in the H3K9me3 histone modification and serve as integral, functionally important components of nuclear organization. To date, the role of DAXX as an H3.3-specific histone chaperone has been investigated primarily using biochemical approaches which provide genome-wide views on cell populations and information on changes in local chromatin structures. However, the global chromatin and subnuclear reorganization events that coincide with these changes remain to be investigated. RESULTS: Using electron spectroscopic imagine (ESI), a specialized form of energy-filtered transmission electron microscopy that allows us to visualize chromatin domains in situ with high contrast and spatial resolution, we show that in the absence of DAXX, H3K9me3-enriched domains are structurally altered and become uncoupled from major satellite DNA. In addition, the structural integrity of nucleoli and the organization of ribosomal DNA (rDNA) are disrupted. Moreover, the absence of DAXX leads to chromatin that is more sensitive, on a global level, to micrococcal nuclease digestion. CONCLUSIONS: We identify a novel role of DAXX as a major regulator of subnuclear organization through the maintenance of the global heterochromatin structural landscape. As well, we show, for the first time, that the loss of a histone chaperone can have severe consequences for global nuclear organization.
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USP7 (Ubiquitin Specific processing Protease-7) is a deubiquitinase which, over the past decade emerged as a critical regulator of cellular processes. Deregulation of USP7 activity has been linked to cancer, making USP7 inhibition an appealing anti-cancer strategy. The identification of novel USP7 substrates and additional USP7-dependent cellular activities will broaden our knowledge towards potential clinical application of USP7 inhibitors. Results presented in this study uncover a novel and pivotal function of USP7 in the maintenance of genomic stability. Upon USP7 depletion we observed prolonged mitosis and mitotic abnormalities including micronuclei accumulation, lagging chromosomes and karyotype instability. Inhibition of USP7 with small molecule inhibitors stabilizes cyclin B and causes mitotic abnormalities. Our results suggest that these USP7-dependent effects are mediated by decreased levels of spindle assembly checkpoint (SAC) component Bub3, which we characterized as an interacting partner and substrate of USP7. In silico analysis across the NCI-60 panels of cell lines supports our results where lower levels of USP7 strongly correlate with genomic instability. In conclusion, we identified a novel role of USP7 as regulator of the SAC component Bub3 and genomic stability.
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Proteínas de Ciclo Celular/genética , Neoplasias/genética , Ubiquitina Tiolesterasa/genética , Proteínas de Ciclo Celular/metabolismo , Expresión Génica , Inestabilidad Genómica , Células HCT116 , Células HEK293 , Humanos , Neoplasias/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Transfección , Ubiquitina Tiolesterasa/metabolismo , Peptidasa Específica de Ubiquitina 7RESUMEN
Microtubule-poisoning drugs, such as Paclitaxel (or Taxol, PTX), are powerful and commonly used anti-neoplastic agents for the treatment of several malignancies. PTX triggers cell death, mainly through a mitotic arrest following the activation of the spindle assembly checkpoint (SAC). Cells treated with PTX slowly slip from this mitotic block and die by mitotic catastrophe. However, cancer cells can acquire or are intrinsically resistant to this drug, posing one of the main obstacles for PTX clinical effectiveness. In order to override PTX resistance and increase its efficacy, we investigated both the enhancement of mitotic slippage and the block of mitotic exit. To test these opposing strategies, we used physiological hyperthermia (HT) to force exit from PTX-induced mitotic block and the anaphase-promoting complex/cyclosome (APC/C) inhibitor, proTAME, to block mitotic exit. We observed that application of HT on PTX-treated cells forced mitotic slippage, as shown by the rapid decline of cyclin B levels and by microscopy analysis. Similarly, HT induced mitotic exit in cells blocked in mitosis by other antimitotic drugs, such as Nocodazole and the Aurora A inhibitor MLN8054, indicating a common effect of HT on mitotic cells. On the other hand, proTAME prevented mitotic exit of PTX and MLN8054 arrested cells, prolonged mitosis, and induced apoptosis. In addition, we showed that proTAME prevented HT-mediated mitotic exit, indicating that stress-induced APC/C activation is necessary for HT-induced mitotic slippage. Finally, HT significantly increased PTX cytotoxicity, regardless of cancer cells' sensitivity to PTX, and this activity was superior to the combination of PTX with pro-TAME. Our data suggested that forced mitotic exit of cells arrested in mitosis by anti-mitotic drugs, such as PTX, can be a more successful anticancer strategy than blocking mitotic exit by inactivation of the APC/C.
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Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Antineoplásicos/farmacología , Hipertermia Inducida/métodos , Mitosis/fisiología , Neoplasias/tratamiento farmacológico , Paclitaxel/farmacología , Western Blotting , Ciclina B/metabolismo , Humanos , Mitosis/efectos de los fármacosRESUMEN
Nuclear structures ND10/PML NBs are linked to multiple processes, including the maintenance of intranuclear homeostasis by sequestering proteins into "nuclear depot." This function presumes release of proteins from PML NBs and their redistribution to the alternative, supposedly "active" locations, in response to the external stress application. To further investigate this nuclear depot function, we focused on the intranuclear distribution of protein Daxx that in normal conditions is mainly accumulated at PML NBs, and has a minor association with centromeres and pericentromeres (CEN/periCEN). Here we report that application of physiological Heat Shock (HS) changes this balance forcing very robust and reversible accumulation of Daxx on CEN/periCEN heterochromatin. Heterochromatin architecture is essential for the proper orchestration of nuclear processes, while transcription from this part of genome is required for its maintenance. To understand functional consequences of Daxx deposition at CEN/periCEN, we tested for Daxx-dependency of heterochromatin transcription. Depletion of Daxx reduces accumulation of CEN RNA in normal conditions and periCEN RNA after HS application. Searching for the mechanism of Daxx-dependent regulation of heterochromatin transcription, we found that depletion of Daxx decreases incorporation of transcription-associated histone H3 variant, H3.3, into both CEN and periCEN. Surprisingly, HS-induced deposition of Daxx does not further elevate incorporation of H3.3 into CEN/periCEN that remained steady during stress and recovery. Instead, depletion of Daxx leads to HS-induced changes in the balance of epigenetic modifications at heterochromatin, most dramatically elevating levels of active H3K4Me2 modification at periCEN. We propose dualistic function of Daxx-containing complexes at CEN/periCEN: (1) regulation of H3.3 loading in normal conditions and (2) protection of epigenetic status upon stress-induced accumulation, thus collectively guarding epigenetic identity of CEN/periCEN heterochromatin.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Centrómero/metabolismo , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Proteínas Co-Represoras , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , ARN/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Estrés Fisiológico , TemperaturaRESUMEN
DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (K(D) â¼60 µM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Sitios de Unión , Proteínas Co-Represoras , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Chaperonas Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Eliminación de SecuenciaRESUMEN
The intracellular translocation of Daxx to the cytoplasm is a phenomenon often attributed to cells undergoing stress, opposite to predominant nuclear localization of this protein under normal homeostatic conditions. Moreover, a number of reports have suggested that export to the cytosol upon several stress conditions, including oxidative stress, glucose deprivation and beta-amyloid peptide treatment, is indispensable for the proper execution of Daxx-induced apoptosis. On the contrary, other studies have described translocation of Daxx from cytoplasm to nucleus upon stress application. Here, we examined cellular distribution of Daxx by sub-cellular fractionation and immunofluorescent localization of endogenous protein, using SH-SY5Y neuroblastoma cell line previously reported to exhibit cytoplasmic translocation of Daxx after oxidative stress and beta-amyloid exposure. In control conditions, Daxx is an exclusively nuclear protein in SH-SY5Y cells. Short treatment by either H(2)O(2) or beta-amyloid did not show any significant change in nuclear localization of Daxx. Prolonged exposure of cells to stress compounds did not alter the intracellular deposition of Daxx that remains exclusively in the nucleus. A cohort of other cell lines, including human prostate cancer cell line DU-145, previously reported to exhibit stress-induced cytosol translocation was examined for Daxx distribution and none were confirmed to show re-localization of Daxx to the cytoplasm after either short or long stress. Time-lapse visualization of Daxx-GFP upon H(2)O(2) treatment or glucose deprivation did not show cytoplasmic translocation either. Thus, while several Daxx-dependent apoptotic mechanisms have been described, the cytosolic association and function of this protein is questionable in light of these findings.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Estrés Fisiológico , Línea Celular Tumoral , Proteínas Co-Represoras , Citoplasma/metabolismo , Humanos , Chaperonas Moleculares , Estrés Oxidativo , Transporte de ProteínasRESUMEN
Resistance to the anti-neoplastic drug paclitaxel is frequent in breast cancer patients. Most studies of paclitaxel resistance have focused on pathways that elicit cellular response, while little is known about players involved in the acquirement of taxane resistance. By screening a cohort of breast cancer cell lines, we observed a correlation between level of protein Daxx and response to paclitaxel. Cells lines expressing increased level of Daxx displayed a robust paclitaxel response with nearly all cells undergoing micronucleation, while cell lines with low amount of Daxx showed a decrease in micronucleation, and accumulation in mitosis. At used paclitaxel concentrations, apoptotic levels were negligible in all cell lines tested. Human cell lines expressing anti-Daxx siRNA as well as Daxx-/- mouse fibroblasts showed similar cellular response to paclitaxel. Importantly, absence or depletion of Daxx resulted in cell survival after paclitaxel treatment, as measured by colony formation assay. We conclude that Daxx may be an important predictive factor in cellular response to paclitaxel, which emphasizes a critical but unknown function of this protein in mitotic progression, which, when disabled, leads to survival advantages upon paclitaxel treatment.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/fisiología , Mitosis/efectos de los fármacos , Proteínas Nucleares/metabolismo , Paclitaxel/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Línea Celular Tumoral , Proteínas Co-Represoras , Femenino , Humanos , Ratones , Pruebas de Micronúcleos , Chaperonas Moleculares , Proteínas Nucleares/genética , Paclitaxel/metabolismo , Interferencia de ARN , Ensayo de Tumor de Célula MadreRESUMEN
The cellular protein Daxx was identified as an interactor with avian sarcoma virus (ASV) integrase (IN) in a yeast two-hybrid screen. After infection, Daxx-IN interactions were detected by coimmunoprecipitation. An association between Daxx and viral DNA, likely mediated by IN, was also detected by chromatin immunoprecipitation. Daxx was not required for early events in ASV replication, including integration, as Daxx-null cells were transduced as efficiently as Daxx-expressing cells. However, viral reporter gene expression from ASV-based vectors was substantially higher in the Daxx-null cells than in Daxx-complemented cells. Consistent with this observation, histone deacetylases (HDACs) were found to associate with viral DNA in Daxx-complemented cells but not in Daxx-null cells. Furthermore, Daxx protein was induced in an interferon-like manner upon ASV infection. We conclude that Daxx interacts with an IN-viral DNA complex early after infection and may mediate the repression of viral gene expression via the recruitment of HDACs. Our findings provide a novel example of cellular immunity against viral replication in which viral transcription is repressed via the recruitment of antiviral proteins to the viral DNA.