RESUMO
Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721-725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719-721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.
Assuntos
Heterocromatina/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Catálise/efeitos dos fármacos , Linhagem Celular Tumoral , Centrômero/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA/efeitos dos fármacos , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas Luminescentes/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Fenótipo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Chromosome ends are protected from degradation by the presence of the highly repetitive hexanucleotide sequence of TTAGGG and associated proteins. These so-called telomeric complexes are suggested to play an important role in establishing a functional nuclear chromatin organization. Using peptide nucleic acid (PNA) probes, we studied the dynamic behavior of telomeric DNA repeats in living human osteosarcoma U2OS cells. A fluorescent cy3-labeled PNA probe was introduced in living cells by glass bead loading and was shown to specifically associate with telomeric DNA shortly afterwards. Telomere dynamics were imaged for several hours using digital fluorescence microscopy. While the majority of telomeres revealed constrained diffusive movement, individual telomeres in a human cell nucleus showed significant directional movements. Also, a subfraction of telomeres were shown to associate and dissociate, suggesting that in vivo telomere clusters are not stable but dynamic structures. Furthermore, telomeres were shown to associate with promyelocytic leukemia (PML) bodies in a dynamic manner.