RESUMO
The HEXIM1 protein, in association with 7SK snRNA, binds and inhibits the kinase activity of P-TEFb (CDK9/cyclin T). P-TEFb activity is crucial for efficient transcription elongation of viral and cellular genes. HEXIM1 was originally isolated as a protein up-regulated by hexamethylene bisacetamide (HMBA), a prototypical inducer of differentiation. To determine the causative role of HEXIM1 during cell differentiation we analyzed the biochemical and functional consequences of HEXIM1 protein levels in several in vitro differentiation systems. We found that HEXIM1 mRNA and protein levels are up-regulated during differentiation of murine erythroleukemia cells upon treatment with HMBA or DMSO. Stimulation of HEXIM1 is not restricted to hematopoietic cells, as induction of phenotypic differentiation of neuroblastoma cells by retinoic acid results in up-regulation of HEXIM1. Moreover, ectopic expression of HEXIM1 causes growth inhibition and promotes neuronal differentiation. These findings highlight a crucial role of HEXIM1 protein during cell differentiation.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia Eritroblástica Aguda/genética , Neuroblastoma/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/genética , Acetamidas/farmacologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Dimetil Sulfóxido/farmacologia , Humanos , Leucemia Eritroblástica Aguda/metabolismo , Camundongos , Neuroblastoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Células U937RESUMO
The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb.HEXIM1.7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb.HEXIM1.7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.
Assuntos
Fator B de Elongação Transcricional Positiva/química , Proteínas de Ligação a RNA/química , RNA/química , Transcrição Gênica , Anisotropia , Centrifugação com Gradiente de Concentração , Reagentes de Ligações Cruzadas/farmacologia , Ciclina T , Ciclinas/química , Dimerização , Glicerol/farmacologia , Células HeLa , Humanos , Imunoprecipitação , Plasmídeos/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Transcrição , Técnicas do Sistema de Duplo-Híbrido , UltracentrifugaçãoRESUMO
BACKGROUND: Mutations in the parkin gene, an E3 protein-ubiquitin ligase, cause autosomal recessive early-onset Parkinson disease (PD). The role of polymorphisms in the parkin gene as risk factors for PD is still unclear, as the results in the literature are contradictory. PATIENTS: We compared the allele and genotype frequencies of the Ser167Asn, Arg366Trp, Val380Leu, and Asp394Asn polymorphisms in 194 patients with PD (92 familial and 102 sporadic) and 125 control subjects. RESULTS: Homozygous Val380 was significantly associated with sporadic PD (P =.008). There was also a trend toward an association of homozygous Asp394 with familial PD (P =.07). CONCLUSIONS: Some parkin polymorphisms appear to be risk factors for sporadic or familial PD. The functional effects of these coding polymorphisms need to be established, and further studies on parkin polymorphisms in PD should be undertaken.