RESUMO
The human ALC1/CHD1L oncogene encodes an SNF2 family ATPase with a macrodomain that binds poly(ADP-ribose) (PAR). We and others previously showed that ALC1 possesses a cryptic ATP-dependent nucleosome remodeling activity that is potently activated in the presence of PARP1 and NAD(+), its substrate for PAR synthesis. In this work, we dissected the mechanism by which PARP1 and NAD(+) activate ALC1 nucleosome remodeling. We demonstrate that ALC1 activation depends on the formation of a stable ALC1·PARylated PARP1·nucleosome intermediate. In addition, by exploiting a novel PAR footprinting assay, we obtained evidence that the ALC1 macrodomain remains stably associated with PAR on autoPARylated PARP1 during the course of nucleosome remodeling reactions. Taken together, our findings are consistent with the model that PAR present on PARylated PARP1 acts as an allosteric effector of ALC1 nucleosome remodeling activity.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , NAD/metabolismo , Nucleossomos/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , NAD/genética , Nucleossomos/genética , Poli(ADP-Ribose) Polimerase-1 , Poli Adenosina Difosfato Ribose/genética , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
Ubiquitously expressed transcription factor Yin-Yang-1 (YY1) controls expression of many genes. YY1 is tightly associated with the human ATP-dependent INO80-like chromatin-remodeling complex (INO80). INO80 is recruited by YY1 to YY1-activated genes, where it functions as an essential coactivator. Binding of YY1 to its DNA sites in target genes requires INO80, suggesting that YY1 uses the INO80 complex not only to activate transcription but also to gain access to target promoters.
Assuntos
DNA Helicases/fisiologia , Ativação Transcricional/fisiologia , Fator de Transcrição YY1/fisiologia , ATPases Associadas a Diversas Atividades Celulares , Linhagem Celular , Proteínas de Ligação a DNA , HumanosRESUMO
Posttranslational modifications play a key role in recruiting chromatin remodeling and modifying enzymes to specific regions of chromosomes to modulate chromatin structure. Alc1 (amplified in liver cancer 1), a member of the SNF2 ATPase superfamily with a carboxy-terminal macrodomain, is encoded by an oncogene implicated in the pathogenesis of hepatocellular carcinoma. Here we show that Alc1 interacts transiently with chromatin-associated proteins, including histones and the poly(ADP-ribose) polymerase Parp1. Alc1 ATPase and chromatin remodeling activities are strongly activated by Parp1 and its substrate NAD and require an intact macrodomain capable of binding poly(ADP-ribose). Alc1 is rapidly recruited to nucleosomes in vitro and to chromatin in cells when Parp1 catalyzes PAR synthesis. We propose that poly(ADP-ribosyl)ation of chromatin-associated Parp1 serves as a mechanism for targeting a SNF2 family remodeler to chromatin.
Assuntos
Trifosfato de Adenosina/química , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Poli(ADP-Ribose) Polimerases/química , Adenosina Trifosfatases/química , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/química , Montagem e Desmontagem da Cromatina , DNA Helicases/química , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Neoplasias Hepáticas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Fatores de Transcrição/químicaRESUMO
Chromatin modifying and remodeling enzymes play critical roles in many aspects of chromosome biology including transcription, replication, recombination, and repair. Our laboratory recently identified and characterized two multisubunit human chromatin remodeling enzymes designated the INO80 and SRCAP complexes. Mechanistic studies revealed that the human INO80 complex catalyzes nucleosome sliding and the SRCAP complex catalyzes ATP-dependent exchange of histone H2A/H2B dimers containing the histone variant H2A.Z into nucleosomes. Here we describe methods for purification and assay of the INO80 and SRCAP chromatin remodeling complexes.
Assuntos
Adenosina Trifosfatases/isolamento & purificação , Montagem e Desmontagem da Cromatina/fisiologia , Cromatografia de Afinidade/métodos , Proteínas Cromossômicas não Histona/isolamento & purificação , Complexos Multiproteicos/isolamento & purificação , Fatores de Transcrição/isolamento & purificação , Humanos , Nucleossomos/metabolismoRESUMO
The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665-13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding.