RESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Histonas/genética , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Clonagem Molecular , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
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.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/química , Trifosfato de Adenosina/genética , Montagem e Desmontagem da Cromatina/genética , Cromossomos/genética , Proteínas de Ligação a DNA/química , Histonas/química , Histonas/genética , Hidrólise , Nucleossomos/química , Ligação Proteica/genética , Domínios Proteicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
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.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/genética , Transferência Ressonante de Energia de Fluorescência , Nucleossomos/química , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Chromatin remodelers are essential for establishing and maintaining the placement of nucleosomes along genomic DNA. Yet how chromatin remodelers recognize and respond to distinct chromatin environments surrounding nucleosomes is poorly understood. Here, we use Lac repressor as a tool to probe how a DNA-bound factor influences action of the Chd1 remodeler. We show that Chd1 preferentially shifts nucleosomes away from Lac repressor, demonstrating that a DNA-bound factor defines a barrier for nucleosome positioning. Rather than an absolute block in sliding, the barrier effect was achieved by altered rates of nucleosome sliding that biased redistribution of nucleosomes away from the bound Lac repressor site. Remarkably, in addition to slower sliding toward the LacO site, the presence of Lac repressor also stimulated sliding in the opposite direction. These experiments therefore demonstrate that Chd1 responds to the presence of a bound protein on both entry and exit sides of the nucleosome. This sensitivity to both sides of the nucleosome allows for a faster and sharper response than would be possible by responding to only the entry side, and we speculate that dual entry/exit sensitivity is also important for regularly spaced nucleosome arrays generated by Chd1 and the related ISWI remodelers.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , DNA/metabolismo , Proteínas de Ligação a DNA/química , Repressores Lac/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Xenopus laevisRESUMO
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.
Assuntos
Fracionamento Celular/métodos , DNA , Histonas , Nucleossomos , DNA/química , DNA/isolamento & purificação , Nucleossomos/química , Nucleossomos/metabolismo , Proteínas Recombinantes , Frações SubcelularesRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Animais , Drosophila , Humanos , Nucleossomos/química , Saccharomyces cerevisiaeRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/análise , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Animais , Soluções Tampão , DNA Helicases/química , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Humanos , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , XenopusRESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , DNA Fúngico/metabolismo , DNA Super-Helicoidal/metabolismo , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.