RESUMEN
The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif in vitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.
Asunto(s)
Centrómero , Proteína EWS de Unión a ARN , Humanos , Autoantígenos/metabolismo , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteínas Cromosómicas no Histona/metabolismo , ARN , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de EwingRESUMEN
Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle. The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteína A Centromérica/metabolismo , Proteínas Luminiscentes/metabolismo , Espectrometría de Masas , Proteínas Recombinantes de Fusión/metabolismo , Ubiquitinación , Supervivencia Celular , Centrómero , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Péptidos/metabolismoRESUMEN
CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity, but how CENP-A is deposited at the centromere remains obscure. We previously reported that CENP-A K124 ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere. However, a recent report stated that CENP-A K124R mutants show no defects in centromere localization and cell viability. In the present study, we found that EYFP tagging induces additional ubiquitylation of EYFP-CENP-A K124R, which allows the mutant protein to bind to HJURP. Using a previously developed conditional CENP-A knockout system and our CENP-A K124R knockin mutant created by the CRISPR-Cas9 system, we show that the Flag-tagged or untagged CENP-A K124R mutant is lethal. This lethality is rescued by monoubiquitin fusion, indicating that CENP-A ubiquitylation is essential for viability.
Asunto(s)
Proteína A Centromérica/metabolismo , Ubiquitinación , Supervivencia Celular , Femenino , Humanos , Proteínas Mutantes/metabolismo , Péptidos/metabolismo , Unión ProteicaRESUMEN
The centromere plays an essential role in accurate chromosome segregation, and the chromosomal location of the centromere is determined by the presence of a histone H3 variant, centromere protein A (CENP-A), in centromeric nucleosomes. However, the precise mechanisms of deposition, maintenance, and inheritance of CENP-A at centromeres are unclear. We have reported that CENP-A deposition requires ubiquitylation of CENP-A lysine 124 mediated by the E3 ligase activity of Cullin 4A (CUL4A)-RING-box protein 1 (RBX1)-COP9 signalsome complex subunit 8 (COPS8). We have proposed a model of inheritance for CENP-A ubiquitylation, through dimerization between rounds of cell divisions, that maintains the position of centromeres.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Centrómero/genética , Epigénesis Genética , Células HeLa , Humanos , Modelos Biológicos , Unión Proteica , UbiquitinaciónRESUMEN
The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A-containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. However, the mechanism that controls the E3 ligase activity of the CUL4A-RBX1-COPS8 complex remains obscure. We have discovered that the SGT1-HSP90 complex is required for recognition of CENP-A by COPS8. Thus, the SGT1-HSP90 complex contributes to the E3 ligase activity of the CUL4A complex that is necessary for CENP-A ubiquitylation and CENP-A deposition at the centromere.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Complejo del Señalosoma COP9/metabolismo , Proteínas Cullin/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Modelos Biológicos , Transporte de Proteínas , Proteínas/metabolismo , UbiquitinaciónRESUMEN
CENP-A (Centromere protein A) is a histone H3 variant that epigenetically determines the centromere position, but the mechanism of its centromere inheritance is obscure. We propose that CENP-A ubiquitylation, which is inherited through dimerization between rounds of cell division, is a candidate for the epigenetic mark of centromere identity.
RESUMEN
The presence of chromatin containing the histone H3 variant CENP-A dictates the location of the centromere in a DNA sequence-independent manner. But the mechanism by which centromere inheritance occurs is largely unknown. We previously reported that CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. Here, we show that pre-existing ubiquitylated CENP-A is necessary for recruitment of newly synthesized CENP-A to the centromere and that CENP-A ubiquitylation is inherited between cell divisions. In vivo and in vitro analyses using dimerization mutants and dimerization domain fusion mutants revealed that the inheritance of CENP-A ubiquitylation requires CENP-A dimerization. Therefore, we propose models in which CENP-A ubiquitylation is inherited and, through dimerization, determines centromere location. Consistent with this model is our finding that overexpression of a monoubiquitin-fused CENP-A mutant induces neocentromeres at noncentromeric regions of chromosomes.
Asunto(s)
Autoantígenos/metabolismo , División Celular , Proteínas Cromosómicas no Histona/metabolismo , Multimerización de Proteína , Ubiquitinación , Autoantígenos/genética , Centrómero/metabolismo , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Células HeLa , Humanos , Unión ProteicaRESUMEN
"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.(1-4) Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.
Asunto(s)
Centrómero , Técnica del Anticuerpo Fluorescente/métodos , Cinetocoros , Western Blotting , Técnicas de Cultivo de Célula , Humanos , Microscopía Fluorescente/métodos , Fijación del Tejido/métodos , TransfecciónRESUMEN
CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere. Addition of monoubiquitin is sufficient to restore CENP-A K124R to centromeres and the interaction with HJURP, indicating that "signaling" ubiquitylation is required for CENP-A loading at centromeres. The CUL4A-RBX1 complex is required for loading newly synthesized CENP-A and maintaining preassembled CENP-A at centromeres. Thus, CENP-A K124R ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere.
Asunto(s)
Autoantígenos/metabolismo , Proteínas Portadoras/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cullin/metabolismo , Proteínas/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Autoantígenos/genética , Western Blotting , Complejo del Señalosoma COP9 , Proteínas Portadoras/genética , Células Cultivadas , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Proteínas Cullin/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Histonas/metabolismo , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Lisina/química , Lisina/genética , Lisina/metabolismo , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Unión Proteica , Proteínas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , UbiquitinaciónRESUMEN
The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Genoma Humano , Inestabilidad Genómica , Cinetocoros/metabolismo , Mitosis/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Aurora Quinasa B , Aurora Quinasas , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Activación Enzimática , Citometría de Flujo , Silenciador del Gen/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/farmacología , Serina/metabolismo , Huso Acromático/genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
Wnt signalling is known to promote G1/S progression through the stimulation of gene expression, but whether this signalling regulates mitotic progression is not clear. Here, the function of dishevelled 2 (Dvl2), which transmits the Wnt signal, in mitosis was examined. Dvl2 localized to the spindles and spindle poles during mitosis. When cells were treated with nocodazole, Dvl2 was observed at the kinetochores (KTs). Dvl2 bound to and was phosphorylated at Thr206 by a mitotic kinase, Polo-like kinase 1 (Plk1), and this phosphorylation was required for spindle orientation and stable microtubule (MT)-KT attachment. Dvl2 was also found to be involved in the activation of a spindle assembly checkpoint (SAC) kinase, Mps1, and the recruitment of other SAC components, Bub1 and BubR1, to the KTs. However, the phosphorylation of Dvl2 by Plk1 was dispensable for SAC. Furthermore, Wnt receptors were involved in spindle orientation, but not in MT-KT attachment or SAC. These results suggested that Dvl2 is involved in mitotic progression by regulating the dynamics of MT plus-ends and the SAC in Plk1-dependent and -independent manners.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitosis/fisiología , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Dishevelled , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Humanos , Cinetocoros/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Nocodazol/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Huso Acromático/metabolismo , Moduladores de Tubulina/metabolismo , Proteínas Wnt/genética , Quinasa Tipo Polo 1RESUMEN
The spindle checkpoint, which monitors kinetochore-microtubule attachment, is required for high fidelity of chromosome transmission. A failure in this mechanism causes aneuploidy, thereby promoting progression to tumorigenesis. However, the cell death mechanism that prevents the aneuploidy caused by failure of the spindle checkpoint is yet unknown. We have recently identified a novel type of mitotic cell death, which we term caspase-independent mitotic death (CIMD). In BUB1-deficient (but not MAD2-deficient) cells, CIMD is induced by conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel or 17-AAG [17-allylaminogeldanamycin]). CIMD depends on p73, a homolog of p53, but not on p53. It also depends on the apoptosis-inducing factor (AIF) and endonuclease G (Endo G), which are effectors of caspase-independent cell death. When BUB1 is completely depleted, aneuploidy occurs instead of CIMD. We propose that CIMD can be the cell death mechanism that protects cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation. Our study also shows that previous evaluations of the spindle checkpoint activity in mutant or cancer cells by monitoring mitotic index could be misleading.
Asunto(s)
Muerte Celular/fisiología , Segregación Cromosómica/fisiología , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/fisiología , Animales , Factor Inductor de la Apoptosis/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Humanos , Ratones , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The spindle checkpoint that monitors kinetochore-microtubule attachment has been implicated in tumorigenesis; however, the relation between the spindle checkpoint and cell death remains obscure. In BUB1-deficient (but not MAD2-deficient) cells, conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG) induced DNA fragmentation during early mitosis. This mitotic cell death was independent of caspase activation; therefore, we named it caspase-independent mitotic death (CIMD). CIMD depends on p73, a homologue of p53, but not on p53. CIMD also depends on apoptosis-inducing factor and endonuclease G, which are effectors of caspase-independent cell death. Treatment with nocodazole, paclitaxel, or 17-AAG induced CIMD in cell lines derived from colon tumors with chromosome instability, but not in cells from colon tumors with microsatellite instability. This result was due to low BUB1 expression in the former cell lines. When BUB1 is completely depleted, aneuploidy rather than CIMD occurs. These results suggest that cells prone to substantial chromosome missegregation might be eliminated via CIMD.
Asunto(s)
Caspasas/metabolismo , Mitosis/fisiología , Proteínas Quinasas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Factor Inductor de la Apoptosis/metabolismo , Benzoquinonas/farmacología , Células CACO-2 , Caspasas/ultraestructura , Línea Celular Tumoral , Cromosomas/genética , Cromosomas/metabolismo , Frío , Fragmentación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Activación Enzimática , Inestabilidad Genómica , Células HCT116 , Células HeLa , Humanos , Lactamas Macrocíclicas/farmacología , Modelos Biológicos , Nocodazol/farmacología , Proteínas Nucleares/metabolismo , Paclitaxel/farmacología , Proteínas Quinasas/genética , Proteínas Quinasas/ultraestructura , Proteínas Serina-Treonina Quinasas , ARN Interferente Pequeño/farmacología , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
We identified a novel splice variant of human SGT1 (SUGT1), a suppressor of the G2 allele of SKP1, by analysis of 8 human EST clones whose open reading frame encoded 365 amino acids. We termed this variant SGT1B (SUGT1B) and the original SGT1A (SUGT1A). The putative SGT1B and SGT1A proteins are 91% identical, and both contain a tetratricopeptide repeat (TPR) domain, two variable regions, a CS domain, and a SGS domain. The NCBI human genome database showed that SGT1B and SGT1A are located on chromosome band 13q14.13. SGT1B contains an additional 33 amino acids encoded by a region between exons 5 and 6 of SGT1A and lacks Ser110 of SGT1A. Immunoblotting using antibodies to N-terminal (amino acids 1-157) and C-terminal (amino acids 182-333) regions of SGT1A detected 2 bands whose sizes corresponded to those predicted for SGT1A and SGT1B. Overexpression experiments confirmed this finding. Additional immunoblot analysis demonstrated that both are highly expressed in human brain, liver, lung, and testis.