RESUMEN
Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers exhibit a sequence-dependent bias in nucleosome positioning, which presumably reflects properties of the DNA duplex. Here, we show how nucleosome positioning by the Chd1 remodeler is influenced by local DNA perturbations throughout the nucleosome footprint. Using site-specific DNA cleavage coupled with next-generation sequencing, we show that nucleosomes shifted by Chd1 can preferentially localize DNA perturbations - poly(dA:dT) tracts, DNA mismatches, and single-nucleotide insertions - about a helical turn outside the Chd1 motor domain binding site, super helix location 2 (SHL2). This phenomenon occurs with both the Widom 601 positioning sequence and the natural +1 nucleosome sequence from the Saccharomyces cerevisiae SWH1 gene. Our modeling indicates that localization of DNA perturbations about a helical turn outward from SHL2 results from back-and-forth sliding due to remodeler action on both sides of the nucleosome. Our results also show that barrier effects from DNA perturbations can be extended by the strong phasing of nucleosome positioning sequences.
Asunto(s)
Proteínas de Unión al ADN , Nucleosomas , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfato/química , Ensamble y Desensamble de Cromatina , Nucleosomas/química , Nucleosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismoRESUMEN
The eukaryotic chromatin landscape plays important roles in DNA metabolism and is characterized by positioned nucleosomes near regulatory DNA, nucleosome-depleted regions and supranucleosomal organization. Nucleosome core histones limit DNA accessibility by structurally blocking half of the DNA surface and altering its topology, but how nucleosomes affect target search by sequence-specific transcription factors (TFs) remains enigmatic. Here, we used multi-color smFRET to investigate how Drosophila GAGA Factor (GAF) locates its targets. On free DNA, GAF rapidly diffuses in 1D to a single cognate motif but escapes after subsecond transient association. Nucleosomes effectively block 1D diffusion into its core, but GAF can bind, with surprisingly prolonged residence, at internal cognate sites by direct association from 3D. Our findings demonstrate the occlusive power of nucleosomes to 1D sliding and reveal that a combination of 1D and 3D diffusion by a zinc finger TF enables efficient target search on chromatin.
RESUMEN
The positions of nucleosomes along genomic DNA play a role in defining patterns of gene expression and chromatin organization. Determination of nucleosome positions in vivo and in vitro, as revealed by the locations of histones on DNA, has provided insight into mechanisms of nucleosome sliding, spacing, assembly, and disassembly. Here, we describe methods for the in vitro determination of histone-DNA contacts at base-pair (bp) resolution. The protocol involves the labeling of histones with ortho-phenanthroline (OP), site-specific cleavage of nucleosomal DNA, and processing and analysis of the resulting DNA fragments. This methodology provides an efficient and high-resolution means for studying kinetics and behavior of enzymes that alter nucleosome structure and/or positioning, and can be used to identify preferred distributions of nucleosomes on natural DNA sequences. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cysteine-specific chemical modification of folded histones with ortho-phenanthroline (OP) Basic Protocol 2: Nucleosome sliding assay adapted for OP mapping of histone-DNA contacts Basic Protocol 3: OP-mediated cleavage, processing, and analysis of DNA fragments using a sequencing gel Support Protocol 1: Preparation of dideoxy sequencing ladders Support Protocol 2: Preparation and running of a denaturing DNA sequencing gel.
Asunto(s)
Histonas , Nucleosomas , ADN/química , Histonas/genética , FenantrolinasRESUMEN
Chromatin remodelers are ATP-dependent enzymes that reorganize nucleosomes within all eukaryotic genomes. Here we report a complex of the Chd1 remodeler bound to a nucleosome in a nucleotide-free state, determined by cryo-EM to 2.3 Å resolution. The remodeler stimulates the nucleosome to absorb an additional nucleotide on each strand at two different locations: on the tracking strand within the ATPase binding site and on the guide strand one helical turn from the ATPase motor. Remarkably, the additional nucleotide on the tracking strand is associated with a local transformation toward an A-form geometry, explaining how sequential ratcheting of each DNA strand occurs. The structure also reveals a histone-binding motif, ChEx, which can block opposing remodelers on the nucleosome and may allow Chd1 to participate in histone reorganization during transcription.
Asunto(s)
ADN de Hongos/química , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Ensamble y Desensamble de Cromatina/fisiología , Microscopía por Crioelectrón , Proteínas de Unión al ADN/genética , Modelos Biológicos , Modelos Moleculares , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Nucleosomas/química , Nucleótidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In transcriptionally active genes, nucleosome positions in promoters are regulated by nucleosome-displacing factors (NDFs) and chromatin-remodeling enzymes. Depletion of NDFs or the RSC chromatin remodeler shrinks or abolishes the nucleosome-depleted regions (NDRs) in promoters, which can suppress gene activation and result in cryptic transcription. Despite their vital cellular functions, how the action of chromatin remodelers may be directly affected by site-specific binding factors like NDFs is poorly understood. Here, we demonstrate that two NDFs, Reb1 and Cbf1, can direct both Chd1 and RSC chromatin-remodeling enzymes in vitro, stimulating repositioning of the histone core away from their binding sites. Interestingly, although the Pho4 transcription factor had a much weaker effect on nucleosome positioning, both NDFs and Pho4 were able to similarly redirect positioning of hexasomes. In chaperone-mediated nucleosome assembly assays, Reb1 but not Pho4 showed an ability to block deposition of the histone H3/H4 tetramer, but Reb1 did not block addition of the H2A/H2B dimer to hexasomes. Our in vitro results show that NDFs bias the action of remodelers to increase the length of the free DNA in the vicinity of their binding sites. These results suggest that NDFs could directly affect NDR architecture through chromatin remodelers.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Regulación Fúngica de la Expresión Génica/genética , Nucleosomas/metabolismo , Unión Proteica/fisiología , Saccharomyces cerevisiae/metabolismoRESUMEN
Chromatin remodelers are ATP (adenosine triphosphate)-powered motors that reposition nucleosomes throughout eukaryotic chromosomes. Remodelers possess autoinhibitory elements that control the direction of nucleosome sliding, but underlying mechanisms of inhibition have been unclear. Here, we show that autoinhibitory elements of the yeast Chd1 remodeler block nucleosome sliding by preventing initiation of twist defects. We show that two autoinhibitory elements-the chromodomains and bridge-reinforce each other to block sliding when the DNA-binding domain is not bound to entry-side DNA. Our data support a model where the chromodomains and bridge target nucleotide-free and ADP-bound states of the ATPase motor, favoring a partially disengaged state of the ATPase motor on the nucleosome. By bypassing distortions of nucleosomal DNA prior to ATP binding, we propose that autoinhibitory elements uncouple the ATP binding/hydrolysis cycle from DNA translocation around the histone core.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Unión al ADN/genética , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfato/genética , Ensamble y Desensamble de Cromatina/genética , Cromosomas/genética , Proteínas de Unión al ADN/química , Histonas/química , Histonas/genética , Hidrólisis , Nucleosomas/química , Unión Proteica/genética , Dominios Proteicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
As primary carriers of epigenetic information and gatekeepers of genomic DNA, nucleosomes are essential for proper growth and development of all eukaryotic cells. Although they are intrinsically dynamic, nucleosomes are actively reorganized by ATP-dependent chromatin remodelers. Chromatin remodelers contain helicase-like ATPase motor domains that can translocate along DNA, and a long-standing question in the field is how this activity is used to reposition or slide nucleosomes. In addition to ratcheting along DNA like their helicase ancestors, remodeler ATPases appear to dictate specific alternating geometries of the DNA duplex, providing an unexpected means for moving DNA past the histone core. Emerging evidence supports twist-based mechanisms for ATP-driven repositioning of nucleosomes along DNA. In this review, we discuss core experimental findings and ideas that have shaped the view of how nucleosome sliding may be achieved.
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Ensamble y Desensamble de Cromatina , Adenosina Trifosfatasas/metabolismo , Fenómenos Biofísicos , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismoRESUMEN
Nucleosomes are substrates for a broad range of factors, including those involved in transcription or chromosome maintenance/reorganization and enzymes that covalently modify histones. Given the heterogeneous nature of nucleosomes in vivo (i.e., varying histone composition, post-translational modifications, DNA sequence register), understanding the specificity and activities of chromatin-interacting factors has required in vitro studies using well-defined nucleosome substrates. Here, we provide detailed methods for large-scale PCR preparation of DNA, assembly of nucleosomes from purified DNA and histones, and purification of DNA and mononucleosomes. Such production of well-defined nucleosomes for biochemical and biophysical studies is key for studying numerous proteins and protein complexes that bind and/or alter nucleosomes and for revealing inherent characteristics of nucleosomes. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Large-scale PCR amplification of DNA Basic Protocol 2: DNA and nucleosome purification using a Bio-Rad Mini Prep Cell/Prep Cell Basic Protocol 3: Nucleosome reconstitution via linear gradient salt dialysis.
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Fraccionamiento Celular/métodos , ADN , Histonas , Nucleosomas , ADN/química , ADN/aislamiento & purificación , Nucleosomas/química , Nucleosomas/metabolismo , Proteínas Recombinantes , Fracciones SubcelularesAsunto(s)
Adenosina Trifosfatasas/metabolismo , ADN/química , Nucleosomas/fisiología , Nucleosomas/ultraestructura , Adenosina Trifosfato/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , ADN/metabolismo , Regulación de la Expresión Génica , Genoma , Histonas/química , Histonas/metabolismo , Histonas/ultraestructura , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleosomas/genéticaRESUMEN
Chromatin remodelers use helicase-like ATPase domains to reorganize histone-DNA contacts within the nucleosome. Like other remodelers, the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler repositions nucleosomes by altering DNA topology at its internal binding site on the nucleosome, coupling different degrees of DNA twist and DNA movement to distinct nucleotide-bound states of the ATPase motor. In this work, we used a competition assay to study how variations in the bound nucleotide, Chd1, and the nucleosome substrate affect stability of Chd1-nucleosome complexes. We found that Chd1-nucleosome complexes formed in nucleotide-free or ADP conditions were relatively unstable and dissociated within 30 s, whereas those with the nonhydrolyzable ATP analog AMP-PNP had a mean lifetime of 4.8 ± 0.7 min. Chd1-nucleosome complexes were remarkably stable with ADP·BeF3- and the transition state analogs ADP·AlFX and ADP·MgFX, being resistant to competitor nucleosome over a 24-h period. For the tight ADP·BeF3--stabilized complex, Mg2+ was a critical component that did not freely exchange, and formation of these long-lived complexes had a slow, concentration-dependent step. The ADP·BeF3--stabilized complex did not require the Chd1 DNA-binding domain nor the histone H4 tail and appeared relatively insensitive to sequence differences on either side of the Widom 601 sequence. Interestingly, the complex remained stable in ADP·BeF3- even when nucleosomes contained single-stranded gaps that disrupted most DNA contacts with the guide strand. This finding suggests that binding via the tracking strand alone is sufficient for stabilizing the complex in a hydrolysis-competent state.
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Adenosina Difosfato/química , ADN de Hongos/química , Proteínas de Unión al ADN/química , Fluoruros/química , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Difosfato/genética , Adenosina Difosfato/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Magnesio/química , Nucleosomas/genética , Nucleosomas/metabolismo , Dominios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Chromatin remodelers are ATP-driven motors that pump double-stranded DNA around the histone core of the nucleosome. Recent work by Chen and coworkers (Li et al., Nature, 2019 and Yan et al., Nat. Struct. Mol. Biol., 2019) has revealed an unexpected intermediate where initial translocation involves only one of the two DNA strands.
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Ensamble y Desensamble de Cromatina , Nucleosomas , Cromatina , ADN , HistonasRESUMEN
The acidic patch is a functionally important epitope on each face of the nucleosome that affects chromatin remodeling. Although related by 2-fold symmetry of the nucleosome, each acidic patch is uniquely positioned relative to a bound remodeler. An open question is whether remodelers are distinctly responsive to each acidic patch. Previously we reported a method for homogeneously producing asymmetric nucleosomes with distinct H2A/H2B dimers (Levendosky et al., 2016). Here, we use this methodology to show that the Chd1 remodeler from Saccharomyces cerevisiae and ISWI remodelers from human and Drosophila have distinct spatial requirements for the acidic patch. Unlike Chd1, which is equally affected by entry- and exit-side mutations, ISWI remodelers strongly depend on the entry-side acidic patch. Remarkably, asymmetry in the two acidic patches stimulates ISWI to slide mononucleosomes off DNA ends, overriding the remodeler's preference to shift the histone core toward longer flanking DNA.
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Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Animales , Drosophila , Humanos , Nucleosomas/química , Saccharomyces cerevisiaeRESUMEN
ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET. During sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. The temporal delay between these movements implies a single rate-limiting step dependent on ATP binding and transient absorption or buffering of at least one base pair. High-resolution cross-linking experiments show that sliding can be achieved by buffering as few as 3 bp between entry and exit sides of the nucleosome. We propose that DNA buffering ensures nucleosome stability during ATP-dependent remodelling, and provides a means for communication between remodellers acting on opposite sides of the nucleosome.
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Adenosina Trifosfatasas/metabolismo , Cromatina/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/análisis , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/química , Animales , Tampones (Química) , ADN Helicasas/química , Transferencia Resonante de Energía de Fluorescencia , Histonas/química , Humanos , Unión Proteica , Saccharomyces cerevisiae/metabolismo , XenopusRESUMEN
Chromatin remodelers are ATP-dependent motors that reorganize DNA packaging by disrupting canonical histone-DNA contacts within the nucleosome. Here, we show that the Chd1 chromatin remodeler stimulates DNA unwrapping from the edge of the nucleosome in a nucleotide-dependent and DNA sequence-sensitive fashion. Nucleosome binding, monitored by stopped flow, was complex and sensitive to nucleotide, with AMP-PNP promoting faster binding than ADP·BeF3-. Nucleosome unwrapping by Chd1, examined by bulk FRET, occurred in the presence and absence of nucleotide and did not require the Chd1 DNA-binding domain. In AMP-PNP conditions, Chd1 unwrapped one side of the Widom 601 DNA more easily than the other, consistent with previous observations of 601 asymmetry and indicating that Chd1 amplifies intrinsic sequence properties of nucleosomal DNA. Using small angle X-ray scattering (SAXS) with contrast variation, we found distinct DNA conformations depending on the nucleotide analog bound to Chd1: with AMP-PNP, DNA primarily unwrapped in-plane with the nucleosomal disk, whereas with ADP·BeF3-, a significant fraction showed distinctive out-of-plane unwrapping as well. Taken together, our findings show tight coupling between entry/exit DNA of the nucleosome and the Chd1 ATPase motor, suggesting that dynamic nucleosome unwrapping is coupled to nucleosome binding and remodeling by Chd1.
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Ensamble y Desensamble de Cromatina/fisiología , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/genética , Transferencia Resonante de Energía de Fluorescencia , Nucleosomas/química , Nucleosomas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
As superfamily 2 (SF2)-type translocases, chromatin remodelers are expected to use an inchworm-type mechanism to walk along DNA. Yet how they move DNA around the histone core has not been clear. Here we show that a remodeler ATPase motor can shift large segments of DNA by changing the twist and length of nucleosomal DNA at superhelix location 2 (SHL2). Using canonical and variant 601 nucleosomes, we find that the Saccharomyces cerevisiae Chd1 remodeler decreased DNA twist at SHL2 in nucleotide-free and ADP-bound states, and increased twist with transition state analogs. These differences in DNA twist allow the open state of the ATPase to pull in ~1 base pair (bp) by stabilizing a small DNA bulge, and closure of the ATPase to shift the DNA bulge toward the dyad. We propose that such formation and elimination of twist defects underlie the mechanism of nucleosome sliding by CHD-, ISWI-, and SWI/SNF-type remodelers.
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Adenosina Trifosfato/metabolismo , ADN de Hongos/metabolismo , ADN Superhelicoidal/metabolismo , Nucleosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , ADN de Hongos/química , ADN de Hongos/genética , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Conformación de Ácido Nucleico , Nucleosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: The list of Mendelian disorders of the epigenetic machinery has expanded rapidly during the last 5 years. A few missense variants in the chromatin remodeler CHD1 have been found in several large-scale sequencing efforts focused on uncovering the genetic aetiology of autism. OBJECTIVES: To explore whether variants in CHD1 are associated with a human phenotype. METHODS: We used GeneMatcher to identify other physicians caring for patients with variants in CHD1. We also explored the epigenetic consequences of one of these variants in cultured fibroblasts. RESULTS: Here we describe six CHD1 heterozygous missense variants in a cohort of patients with autism, speech apraxia, developmental delay and facial dysmorphic features. Importantly, three of these variants occurred de novo. We also report on a subject with a de novo deletion covering a large fraction of the CHD1 gene without any obvious neurological phenotype. Finally, we demonstrate increased levels of the closed chromatin modification H3K27me3 in fibroblasts from a subject carrying a de novo variant in CHD1. CONCLUSIONS: Our results suggest that variants in CHD1 can lead to diverse phenotypic outcomes; however, the neurodevelopmental phenotype appears to be limited to patients with missense variants, which is compatible with a dominant negative mechanism of disease.
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Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Discapacidades del Desarrollo/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Mutación Missense , Niño , Preescolar , ADN Helicasas/química , Proteínas de Unión al ADN/química , Discapacidades del Desarrollo/diagnóstico , Facies , Femenino , Fibroblastos/metabolismo , Estudios de Asociación Genética/métodos , Histonas/metabolismo , Humanos , Lactante , Modelos Moleculares , Fenotipo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Chromatin remodelers catalyze dynamic packaging of the genome by carrying out nucleosome assembly/disassembly, histone exchange, and nucleosome repositioning. Remodeling results in evenly spaced nucleosomes, which requires probing both sides of the nucleosome, yet the way remodelers organize sliding activity to achieve this task is not understood. Here, we show that the monomeric Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome. During sliding, Chd1 generates unstable remodeling intermediates that spontaneously relax to a pre-remodeled position. We demonstrate that nucleosome sliding is tightly controlled by two regulatory domains: the DNA-binding domain, which interferes with sliding when its range is limited by a truncated linking segment, and the chromodomains, which play a key role in substrate discrimination. We propose that active interplay of the ATPase motor with the regulatory domains may promote dynamic nucleosome structures uniquely suited for histone exchange and chromatin reorganization during transcription.
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Proteínas de Unión al ADN/genética , ADN/genética , Histonas/genética , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ensamble y Desensamble de Cromatina , Clonación Molecular , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Nucleosomas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Chromatin remodelers are ATP-dependent enzymes that are critical for reorganizing and repositioning nucleosomes in concert with many basic cellular processes. For the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler, nucleosome sliding has been shown to depend on the DNA flanking the nucleosome, transcription factor binding at the nucleosome edge, and the presence of the histone H2A/H2B dimer on the entry side. Here, we report that Chd1 is also sensitive to the sequence of DNA within the nucleosome and slides nucleosomes made with the 601 Widom positioning sequence asymmetrically. Kinetic and equilibrium experiments show that poly(dA:dT) tracts perturb remodeling reactions if within one and a half helical turns of superhelix location 2 (SHL2), where the Chd1 ATPase engages nucleosomal DNA. These sequence-dependent effects do not rely on the Chd1 DNA-binding domain and are not due to differences in nucleosome affinity. Using site-specific cross-linking, we show that internal poly(dA:dT) tracts do not block the engagement of the ATPase motor with SHL2, yet they promote multiple translational positions of DNA with respect to both Chd1 and the histone core. We speculate that Chd1 senses the sequence-dependent response of DNA as the remodeler ATPase perturbs the duplex at SHL2. These results suggest that the sequence sensitivity of histones and remodelers occur at unique segments of DNA on the nucleosome, allowing them to work together or in opposition to determine nucleosome positions throughout the genome.
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Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Chromatin remodelers use a helicase-like ATPase motor to reposition and reorganize nucleosomes along genomic DNA. Yet, how the ATPase motor communicates with other remodeler domains in the context of the nucleosome has so far been elusive. Here, we report for the Chd1 remodeler a unique organization of domains on the nucleosome that reveals direct domain-domain communication. Site-specific cross-linking shows that the chromodomains and ATPase motor bind to adjacent SHL1 and SHL2 sites, respectively, on nucleosomal DNA and pack against the DNA-binding domain on DNA exiting the nucleosome. This domain arrangement spans the two DNA gyres of the nucleosome and bridges both ends of a wrapped, â¼90-bp nucleosomal loop of DNA, suggesting a means for nucleosome assembly. This architecture illustrates how Chd1 senses DNA outside the nucleosome core and provides a basis for nucleosome spacing and directional sliding away from transcription factor barriers.