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1.
Cell Rep ; 18(9): 2135-2147, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28249160

RESUMO

The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/metabolismo , Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica/fisiologia , Nucleossomos/metabolismo , Subunidades Proteicas/metabolismo , Leveduras/metabolismo
2.
Mol Cell Biol ; 35(23): 4083-92, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26416878

RESUMO

Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetylation. Lysines are acetylated in histone tails and the core domain/lateral surface of histone octamers. While acetylated lysines in histone tails are frequently recognized by other factors referred to as "readers," which promote transcription, the mechanistic role of the modifications in the lateral surface of the histone octamer remains unclear. By using X-ray crystallography, we found that acetylated lysines 115 and 122 in histone H3 are solvent accessible, but in biochemical assays they appear not to interact with the bromodomains of SWI/SNF and RSC to enhance recruitment or nucleosome mobilization, as previously shown for acetylated lysines in H3 histone tails. Instead, we found that acetylation of lysines 115 and 122 increases the predisposition of nucleosomes for disassembly by SWI/SNF and RSC up to 7-fold, independent of bromodomains, and only in conjunction with contiguous nucleosomes. Thus, in combination with SWI/SNF and RSC, acetylation of lateral surface lysines in the histone octamer serves as a crucial regulator of nucleosomal dynamics distinct from the histone code readers and writers.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Adenosina Trifosfatases/metabolismo , Animais , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Histonas/química , Lisina/análise , Lisina/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Nucleossomos/química , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Xenopus
3.
Mol Cell Biol ; 33(2): 360-70, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23149935

RESUMO

The SWI/SNF chromatin remodeling complex changes the positions where nucleosomes are bound to DNA, exchanges out histone dimers, and disassembles nucleosomes. All of these activities depend on ATP hydrolysis by the catalytic subunit Snf2, containing a DNA-dependent ATPase domain. Here we examine the role of another domain in Snf2 called SnAC (Snf2 ATP coupling) that was shown previously to regulate the ATPase activity of SWI/SNF. We have found that SnAC has another function besides regulation of ATPase activity that is even more critical for nucleosome remodeling by SWI/SNF. We have found that deletion of the SnAC domain strongly uncouples ATP hydrolysis from nucleosome movement. Deletion of SnAC does not adversely affect the rate, processivity, or pulling force of SWI/SNF to translocate along free DNA in an ATP-dependent manner. The uncoupling of ATP hydrolysis from nucleosome movement is shown to be due to loss of SnAC binding to the histone surface of nucleosomes. While the SnAC domain targets both the ATPase domain and histones, the SnAC domain as a histone anchor plays a more critical role in remodeling because it is required to convert DNA translocation into nucleosome movement.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/genética , Histonas/metabolismo , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Mapeamento Cromossômico , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Hidrólise , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Xenopus laevis
4.
Nucleic Acids Res ; 40(10): 4412-21, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22298509

RESUMO

An ATP-dependent DNA translocase domain consisting of seven conserved motifs is a general feature of all ATP-dependent chromatin remodelers. While motifs on the ATPase domains of the yeast SWI/SNF and ISWI families of remodelers are highly conserved, the ATPase domains of these complexes appear not to be functionally interchangeable. We found one reason that may account for this is the ATPase domains interact differently with nucleosomes even though both associate with nucleosomal DNA 17-18 bp from the dyad axis. The cleft formed between the two lobes of the ISW2 ATPase domain is bound to nucleosomal DNA and Isw2 associates with the side of nucleosomal DNA away from the histone octamer. The ATPase domain of SWI/SNF binds to the same region of nucleosomal DNA, but is bound outside of the cleft region. The catalytic subunit of SWI/SNF also appears to intercalate between the DNA gyre and histone octamer. The altered interactions of SWI/SNF with DNA are specific to nucleosomes and do not occur with free DNA. These differences are likely mediated through interactions with the histone surface. The placement of SWI/SNF between the octamer and DNA could make it easier to disrupt histone-DNA interactions.


Assuntos
Adenosina Trifosfatases/química , Fatores de Transcrição/química , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Histonas/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Estrutura Terciária de Proteína , Fatores de Transcrição/metabolismo
5.
Methods Mol Biol ; 809: 381-409, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22113290

RESUMO

Chromatin plays a key regulatory role in several DNA-dependent processes as it regulates DNA access to different protein factors. Several multisubunit protein complexes interact, modify, or mobilize nucleosomes: the basic unit of chromatin, from its original location in an ATP-dependent manner to facilitate processes, such as transcription, replication, repair, and recombination. Knowledge of the interactions of chromatin remodelers with nucleosomes is a crucial requirement to understand the mechanism of chromatin remodeling. Here, we describe several methods to analyze the interactions of multisubunit chromatin-remodeling enzymes with nucleosomes.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Pegada de DNA , Nucleossomos/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Mol Cell Biol ; 28(19): 6010-21, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18644858

RESUMO

The SWI/SNF complex disrupts and mobilizes chromatin in an ATP-dependent manner. SWI/SNF interactions with nucleosomes were mapped by DNA footprinting and site-directed DNA and protein cross-linking when SWI/SNF was recruited by a transcription activator. SWI/SNF was found by DNA footprinting to contact tightly around one gyre of DNA spanning approximately 50 bp from the nucleosomal entry site to near the dyad axis. The DNA footprint is consistent with nucleosomes binding to an asymmetric trough of SWI/SNF that was revealed by the improved imaging of free SWI/SNF. The DNA site-directed cross-linking revealed that the catalytic subunit Swi2/Snf2 is associated with nucleosomes two helical turns from the dyad axis and that the Snf6 subunit is proximal to the transcription factor recruiting SWI/SNF. The highly conserved Snf5 subunit associates with the histone octamer and not with nucleosomal DNA. The model of the binding trough of SWI/SNF illustrates how nucleosomal DNA can be mobilized while SWI/SNF remains bound.


Assuntos
Proteínas Cromossômicas não Histona/química , Adenosina Trifosfatases , Animais , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Mutação , Nucleossomos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Xenopus laevis
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