RESUMEN
Unless mitigated, external and physiological stresses are detrimental for cells, especially in mitosis, resulting in chromosomal missegregation, aneuploidy, or apoptosis. Heat shock proteins (Hsps) maintain protein homeostasis and promote cell survival. Hsps are transcriptionally regulated by heat shock factors (HSFs). Of these, HSF1 is the master regulator and HSF2 modulates Hsp expression by interacting with HSF1. Due to global inhibition of transcription in mitosis, including HSF1-mediated expression of Hsps, mitotic cells are highly vulnerable to stress. Here, we show that cells can counteract transcriptional silencing and protect themselves against proteotoxicity in mitosis. We found that the condensed chromatin of HSF2-deficient cells is accessible for HSF1 and RNA polymerase II, allowing stress-inducible Hsp expression. Consequently, HSF2-deficient cells exposed to acute stress display diminished mitotic errors and have a survival advantage. We also show that HSF2 expression declines during mitosis in several but not all human cell lines, which corresponds to the Hsp70 induction and protection against stress-induced mitotic abnormalities and apoptosis.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Mitosis/genética , ARN Polimerasa II/genética , Factores de Transcripción/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular , Cromatina/genética , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/biosíntesis , Humanos , Células MCF-7 , Ratones , Índice Mitótico , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño , Factores de Transcripción/biosíntesis , Transcripción GenéticaRESUMEN
Heat shock factors (HSFs) are the master regulators of transcription under protein-damaging conditions, acting in an environment where the overall transcription is silenced. We determined the genomewide transcriptional program that is rapidly provoked by HSF1 and HSF2 under acute stress in human cells. Our results revealed the molecular mechanisms that maintain cellular homeostasis, including HSF1-driven induction of polyubiquitin genes, as well as HSF1- and HSF2-mediated expression patterns of cochaperones, transcriptional regulators, and signaling molecules. We characterized the genomewide transcriptional response to stress also in mitotic cells where the chromatin is tightly compacted. We found a radically limited binding and transactivating capacity of HSF1, leaving mitotic cells highly susceptible to proteotoxicity. In contrast, HSF2 occupied hundreds of loci in the mitotic cells and localized to the condensed chromatin also in meiosis. These results highlight the importance of the cell cycle phase in transcriptional responses and identify the specific mechanisms for HSF1 and HSF2 in transcriptional orchestration. Moreover, we propose that HSF2 is an epigenetic regulator directing transcription throughout cell cycle progression.
Asunto(s)
Ciclo Celular/genética , Cromatina/genética , Respuesta al Choque Térmico/genética , Mitosis/genética , Transcripción Genética , Sitios de Unión/genética , Western Blotting , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Células K562 , Masculino , Chaperonas Moleculares/genética , Poliubiquitina/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma.
Asunto(s)
Neoplasias Encefálicas/terapia , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Resistencia a Antineoplásicos/genética , Meduloblastoma/terapia , Proteínas Tirosina Quinasas/genética , Interferencia de ARN , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Cisplatino/uso terapéutico , Terapia Combinada , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Tiazolidinedionas/farmacología , Quinasa Tipo Polo 1RESUMEN
The ubiquitin E3 ligase anaphase-promoting complex/cyclosome (APC/C) drives degradation of cell cycle regulators in cycling cells by associating with the coactivators Cdc20 and Cdh1. Although a plethora of APC/C substrates have been identified, only a few transcriptional regulators are described as direct targets of APC/C-dependent ubiquitination. Here we show that APC/C, through substrate recognition by both Cdc20 and Cdh1, mediates ubiquitination and degradation of heat shock factor 2 (HSF2), a transcription factor that binds to the Hsp70 promoter. The interaction between HSF2 and the APC/C subunit Cdc27 and coactivator Cdc20 is enhanced by moderate heat stress, and the degradation of HSF2 is induced during the acute phase of the heat shock response, leading to clearance of HSF2 from the Hsp70 promoter. Remarkably, Cdc20 and the proteasome 20S core α2 subunit are recruited to the Hsp70 promoter in a heat shock-inducible manner. Moreover, the heat shock-induced expression of Hsp70 is increased when Cdc20 is silenced by a specific small interfering RNA (siRNA). Our results provide the first evidence for participation of APC/C in the acute response to protein-damaging stress.
Asunto(s)
Cadherinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/fisiología , Estrés Fisiológico , Factores de Transcripción/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Antígenos CD , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Cadherinas/genética , Proteínas Cdc20 , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Células HEK293 , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
miR-18 belongs to the Oncomir-1 or miR-17~92 cluster that is intimately associated with the occurrence and progression of different types of cancer. However, the physiological roles of the Oncomir-1 cluster and its individual miRNAs are largely unknown. Here, we describe a novel function for miR-18 in mouse. We show that miR-18 directly targets heat shock factor 2 (HSF2), a transcription factor that influences a wide range of developmental processes including embryogenesis and gametogenesis. Furthermore, we show that miR-18 is highly abundant in testis, displaying distinct cell-type-specific expression during the epithelial cycle that constitutes spermatogenesis. Expression of HSF2 and of miR-18 exhibit an inverse correlation during spermatogenesis, indicating that, in germ cells, HSF2 is downregulated by miR-18. To investigate the in vivo function of miR-18 we developed a novel method, T-GIST, and demonstrate that inhibition of miR-18 in intact seminiferous tubules leads to increased HSF2 protein levels and altered expression of HSF2 target genes. Our results reveal that miR-18 regulates HSF2 activity in spermatogenesis and link miR-18 to HSF2-mediated physiological processes such as male germ cell maturation.
