RESUMO
The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica , Multimerização ProteicaRESUMO
The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.
Assuntos
Histona Desacetilases/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Proteínas Repressoras/química , Proteína 4 de Ligação ao Retinoblastoma/química , Sequência de Aminoácidos , Animais , Montagem e Desmontagem da Cromatina , Sequência Conservada , Cristalografia por Raios X , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/química , Proteína 7 de Ligação ao Retinoblastoma/genética , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Homologia de Sequência de Aminoácidos , Transativadores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The mechanisms by which histones are disassembled and reassembled into nucleosomes and chromatin structure during DNA replication, repair and transcription are poorly understood. A better understanding of the processes involved is, however, crucial if we are to understand whether and how histone variants and post-translationally modified histones are inherited in an epigenetic manner. To this end we have studied the interaction of the histone H3-H4 complex with the human retinoblastoma-associated protein RbAp48 and their exchange with a second histone chaperone, anti-silencing function protein 1 (ASF1). Exchange of histones H3-H4 between these two histone chaperones has a central role in the assembly of new nucleosomes, and we show here that the H3-H4 complex has an unexpected structural plasticity, which is important for this exchange.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Chaperonas de Histonas/metabolismo , Histonas/química , Histonas/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Chaperonas de Histonas/química , Histonas/genética , Humanos , Nucleossomos/metabolismo , Ligação Proteica , Multimerização Proteica , Proteína 4 de Ligação ao Retinoblastoma/químicaRESUMO
Chromatin-modifying complexes such as the NuRD complex are recruited to particular genomic sites by gene-specific nuclear factors. Overall, however, little is known about the molecular basis for these interactions. Here, we present the 1.9 Å resolution crystal structure of the NuRD subunit RbAp48 bound to the 15 N-terminal amino acids of the GATA-1 cofactor FOG-1. The FOG-1 peptide contacts a negatively charged binding pocket on top of the RbAp48 ß-propeller that is distinct from the binding surface used by RpAp48 to contact histone H4. We further show that RbAp48 interacts with the NuRD subunit MTA-1 via a surface that is distinct from its FOG-binding pocket, providing a first glimpse into the way in which NuRD assembly facilitates interactions with cofactors. Our RbAp48·FOG-1 structure provides insight into the molecular determinants of FOG-1-dependent association with the NuRD complex and into the links between transcription regulation and nucleosome remodeling.