RESUMO
Somatic inactivation of PTEN occurs in different human tumors including glioblastoma, endometrial carcinoma and prostate carcinoma. Germline mutations in PTEN result in a range of phenotypic abnormalities that occur with variable penetrance, including neurological features such as macrocephaly, seizures, ataxia and Lhermitte-Duclos disease (also described as dysplastic gangliocytoma of the cerebellum). Homozygous deletion of Pten causes embryonic lethality in mice. To investigate function in the brain, we used Cre-loxP technology to selectively inactivate Pten in specific mouse neuronal populations. Loss of Pten resulted in progressive macrocephaly and seizures. Neurons lacking Pten expressed high levels of phosphorylated Akt and showed a progressive increase in soma size without evidence of abnormal proliferation. Cerebellar abnormalities closely resembled the histopathology of human Lhermitte-Duclos disease. These results indicate that Pten regulates neuronal size in vivo in a cell-autonomous manner and provide new insights into the etiology of Lhermitte-Duclos disease.
Assuntos
Tamanho Celular/genética , Doenças Cerebelares/genética , Genes Supressores de Tumor , Neurônios/patologia , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Doenças Cerebelares/patologia , Inibidor de Quinase Dependente de Ciclina p27 , Modelos Animais de Doenças , Deleção de Genes , Proteína Glial Fibrilar Ácida/genética , Imuno-Histoquímica , Integrases/genética , Camundongos , Camundongos Transgênicos , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Convulsões/genética , Proteínas Supressoras de Tumor/genética , Proteínas Virais/genéticaRESUMO
EWS is an RNA-binding protein involved in human tumor-specific chromosomal translocations. In approximately 85% of Ewing's sarcomas, such translocations give rise to the chimeric gene EWS/FLI. In the resulting fusion protein, the RNA binding domains from the C terminus of EWS are replaced by the DNA-binding domain of the ETS protein FLI-1. EWS/FLI can function as a transcription factor with the same DNA binding specificity as FLI-1. EWS and EWS/FLI can associate with the RNA polymerase II holoenzyme as well as with SF1, an essential splicing factor. Here we report that U1C, one of three human U1 small nuclear ribonucleoprotein-specific proteins, interacts in vitro and in vivo with both EWS and EWS/FLI. U1C interacts with other splicing factors and is important in the early stages of spliceosome formation. Importantly, co-expression of U1C represses EWS/FLI-mediated transactivation, demonstrating that this interaction can have functional ramifications. Our findings demonstrate that U1C, a well characterized splicing protein, can also function in transcriptional regulation. Furthermore, they suggest that EWS and EWS/FLI may function both in transcriptional and post-transcriptional processes.
Assuntos
Proteínas de Fusão Oncogênica/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular , Biblioteca Gênica , Glutationa Transferase/genética , Células HL-60 , Células HeLa , Humanos , Luciferases/genética , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1 , RNA Polimerase II/metabolismo , Proteína EWS de Ligação a RNA , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae , Sarcoma de Ewing/genética , Fatores de Transcrição/genética , Translocação GenéticaRESUMO
The chimeric gene EWS/FLI is present in at least 85% of Ewing's sarcomas as a result of chromosomal translocations. The resulting fusion protein contains the N terminus of the RNA-binding protein EWS and the ETS DNA-binding domain of the transcription factor FLI-1. Although EWS/FLI binds DNA and activates transcription, both EWS and EWS/FLI also interact with SF1 and U1C, essential components of the splicing machinery. Therefore, we tested the ability of EWS and EWS/FLI to alter 5'-splice site selection using an E1A gene in vivo splicing assay. We found that EWS/FLI, but not EWS, interfered with heterogeneous nuclear ribonucleoprotein A1-dependent splice site selection of E1A. Mutational analysis of EWS/FLI revealed that the ability to affect pre-mRNA splicing coincided with transforming activity. Therefore, EWS/FLI has the ability to influence splicing as well as transcription.