RESUMO
Drosophila NURF is an ATP-dependent chromatin remodeling complex that contains ISWI, a member of the SWI2/SNF2 family of ATPases. We demonstrate that NURF catalyzes the bidirectional redistribution of mononucleosomes reconstituted on hsp70 promoter DNA. In the presence of NURF, nucleosomes adopt one predominant position from an ensemble of possible locations within minutes. Movements occur in cis, with no transfer to competing DNA. Migrating intermediates trapped by Exo III digestion reveal progressive nucleosome motion in increments of several base pairs. All four core histones are retained quantitatively during this process, indicating that the general integrity of the histone octamer is maintained. We suggest that NURF remodels nucleosomes by transiently decreasing the activation energy for short-range sliding of the histone octamer.
Assuntos
Trifosfato de Adenosina/metabolismo , Cromatina/fisiologia , Histonas/metabolismo , Proteínas de Insetos/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Pareamento de Bases , Drosophila melanogaster , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Nucleossomos/metabolismo , Nucleossomos/fisiologia , Regiões Promotoras Genéticas , RNA Ribossômico 5S , Fatores de Transcrição/metabolismoAssuntos
Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Proteínas de Drosophila , Proteínas de Insetos/isolamento & purificação , Chaperonas Moleculares , Proteínas Nucleares , Pirofosfatases , Animais , Fracionamento Celular/métodos , Núcleo Celular/ultraestrutura , Centrifugação com Gradiente de Concentração/métodos , Cromatografia/métodos , Cromatografia DEAE-Celulose/métodos , Cromatografia em Gel/métodos , Drosophila/embriologia , Embrião não Mamífero , Proteínas de Insetos/metabolismo , Proteína 4 de Ligação ao RetinoblastomaRESUMO
The Drosophila nucleosome remodeling factor (NURF) is a protein complex consisting of four polypeptides that facilitates the perturbation of chromatin structure in vitro in an ATP-dependent manner. The 140-kD NURF subunit, imitation switch (ISWI), is related to the SWI2/SNF2 ATPase. Another subunit, NURF-55, is a 55-kD WD repeat protein homologous to the human retinoblastoma-associated protein RbAp48. Here, we report the cloning and characterization of the smallest (38 kD) component of NURF. NURF-38 is strikingly homologous to known inorganic pyrophosphatases. Both recombinant NURF-38 alone and the purified NURF complex are shown to have inorganic pyrophosphatase activity. Inhibition of the pyrophosphatase activity of NURF with sodium fluoride has no significant effect on chromatin remodeling, indicating that these two activities may be biochemically uncoupled. Our results suggest that NURF-38 may serve a structural or regulatory role in the complex. Alternatively, because accumulation of unhydrolyzed pyrophosphate during nucleotide incorporation inhibits polymerization, NURF may also have been adapted to deliver pyrophosphatase to chromatin to assist in replication or transcription by efficient removal of the inhibitory metabolite.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/enzimologia , Proteínas de Insetos/metabolismo , Nucleossomos/enzimologia , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/enzimologia , Clonagem Molecular , Drosophila melanogaster/genética , Ativação Enzimática , Pirofosfatase Inorgânica , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Substâncias Macromoleculares , Dados de Sequência Molecular , Nucleossomos/genética , Pirofosfatases/genética , Pirofosfatases/isolamento & purificação , Frações Subcelulares/enzimologiaRESUMO
The suppressor of Hairy-wing [su(Hw)] protein represses enhancer function in a unidirectional fashion: enhancers segregated from the promoter by the su(Hw) binding region are rendered inactive. whereas those in the same domain are unaffected. In the case of the gypsy-induced y2 allele, the repressive effect of su(Hw) is rendered bidirectional in mod(mdg4) mutant flies, and all enhancers of the affected gene become inactive. This silencing of enhancer elements might be due to exposure of specific domains of su(Hw) when the mod(mdg4) protein is absent. Two of three regions of su(Hw) that are located adjacent to the leucine zipper motif and are conserved across Drosophila species are necessary for both the unidirectional and bidirectional repression of transcription by su(Hw). In contrast, two acidic domains that are dispensable for the unidirectional repression of enhancer elements are critical for the bidirectional silencing of enhancer activity observed in mutants lacking functional mod(mdg4) protein.
Assuntos
Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Supressão Genética , Fatores de Transcrição/genética , Animais , Deleção de Genes , Hormônios de Inseto/genética , Masculino , Mutação , Proteínas RepressorasRESUMO
Boundary or insulator elements set up independent territories of gene activity by establishing higher order domains of chromatin structure. The gypsy retrotransposon of Drosophila contains an insulator element that represses enhancer-promoter interactions and is responsible for the mutant phenotypes caused by insertion of this element. The gypsy insulator inhibits the interaction of promoter-distal enhancers with the transcription complex without affecting the functionality of promoter-proximal enhancers; in addition, these sequences can buffer a transgene from chromosomal position effects. Two proteins have been identified that bind gypsy insulator sequences and are responsible for their effects on transcription. The suppressor of Hairy-wing [su(Hw)] protein affects enhancer function both upstream and downstream of its binding site by causing a silencing effect similar to that of heterochromatin. The modifier of mdg4 [mod(mdg4)] protein interacts with su(Hw) to transform this bi-directional repression into the polar effect characteristic of insulators. These effects seem to be modulated by changes in chromatin structure.
Assuntos
Cromatina , Proteínas de Drosophila , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Retroelementos , Animais , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Repressoras , Fatores de Transcrição/metabolismoRESUMO
The suppressor of Hairy wing (su(Hw)) protein inhibits the function of transcriptional enhancers located distally from the promoter with respect to the location of su(Hw)-binding sites. This polarity is due to the ability of the su(Hw)-binding region to form a chromatin insulator. Mutations in modifier of mdg4 (mod(mdg4)) enhance the effect of su(Hw) by inhibiting the function of enhancers located on both sides of the su(Hw)-binding region. This inhibition results in a variegated expression pattern, and mutations in mod(mdg4) act as classical enhancers of position-effect variegation. The mod(mdg4) and su(Hw) proteins interact with each other. The mod(mdg4) protein controls the nature of the repressive effect of su(Hw): in the absence of mod(mdg4) protein, su(Hw) exerts a bidirectional silencing effect, whereas in the presence of mod(mdg4), the silencing effect is transformed into unidirectional repression.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas Repressoras/genética , Alelos , Sequência de Aminoácidos , Animais , Cromatina/genética , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genes de Insetos , Genes Supressores , Zíper de Leucina/genética , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Fenótipo , Fatores de Transcrição/genéticaRESUMO
The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers controlling the expression of the mutant gene. A structural and functional analysis of su(Hw) was carried out to identify domains of the protein responsible for its negative effect on enhancer action. Sequence comparison among the su(Hw) proteins from three different species allows the identification of evolutionarily conserved domains with possible functional significance. An acidic domain located in the carboxy-terminal end of the Drosophila melanogaster protein is not present in su(Hw) from other species, suggesting a nonessential role for this part of the protein. A second acidic domain located in the amino-terminal region of su(Hw) is present in all species analyzed. This domain is dispensable in the D. melanogaster protein when the carboxy-terminal acidic domain is present, but the protein is nonfunctional when both regions are simultaneously deleted. Mutations in the zinc fingers result in su(Hw) protein unable to interact with DNA in vivo, indicating a functional role for this region of the protein in DNA binding. Finally, a region of su(Hw) homologous to the leucine zipper motif is necessary for the negative effect of this protein on enhancer function, suggesting that su(Hw) might exert this effect by interacting, directly or indirectly, with transcription factors bound to these enhancers.
