RESUMO
The packaging of DNA into chromatin in eukaryotes regulates gene transcription, DNA replication and DNA repair. ATP-dependent chromatin remodelling enzymes (re)arrange nucleosomes at the first level of chromatin organization. Their Snf2-type motor ATPases alter histone-DNA interactions through a common DNA translocation mechanism. Whether remodeller activities mainly catalyse nucleosome dynamics or accurately co-determine nucleosome organization remained unclear. In this Review, we discuss the emerging mechanisms of chromatin remodelling: dynamic remodeller architectures and their interactions, the inner workings of the ATPase cycle, allosteric regulation and pathological dysregulation. Recent mechanistic insights argue for a decisive role of remodellers in the energy-driven self-organization of chromatin, which enables both stability and plasticity of genome regulation - for example, during development and stress. Different remodellers, such as members of the SWI/SNF, ISWI, CHD and INO80 families, process (epi)genetic information through specific mechanisms into distinct functional outputs. Combinatorial assembly of remodellers and their interplay with histone modifications, histone variants, DNA sequence or DNA-bound transcription factors regulate nucleosome mobilization or eviction or histone exchange. Such input-output relationships determine specific nucleosome positions and compositions with distinct DNA accessibilities and mediate differential genome regulation. Finally, remodeller genes are often mutated in diseases characterized by genome dysregulation, notably in cancer, and we discuss their physiological relevance.
Assuntos
Cromatina , Histonas , Humanos , Histonas/metabolismo , Nucleossomos , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , DNA , Trifosfato de Adenosina/metabolismoRESUMO
Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.
Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos/química , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Cromatina/química , Cromatina/genética , DNA Fúngico/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Genoma Fúngico , Histonas/química , Histonas/genética , Poli dA-dT/química , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The origin recognition complex (ORC) is essential for initiation of eukaryotic chromosome replication as it loads the replicative helicase-the minichromosome maintenance (MCM) complex-at replication origins1. Replication origins display a stereotypic nucleosome organization with nucleosome depletion at ORC-binding sites and flanking arrays of regularly spaced nucleosomes2-4. However, how this nucleosome organization is established and whether this organization is required for replication remain unknown. Here, using genome-scale biochemical reconstitution with approximately 300 replication origins, we screened 17 purified chromatin factors from budding yeast and found that the ORC established nucleosome depletion over replication origins and flanking nucleosome arrays by orchestrating the chromatin remodellers INO80, ISW1a, ISW2 and Chd1. The functional importance of the nucleosome-organizing activity of the ORC was demonstrated by orc1 mutations that maintained classical MCM-loader activity but abrogated the array-generation activity of ORC. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, in addition to its canonical role as the MCM loader, has a second crucial function as a master regulator of nucleosome organization at the replication origin, a crucial prerequisite for efficient chromosome replication.
Assuntos
Cromatina , Complexo de Reconhecimento de Origem , Origem de Replicação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Replicação do DNA , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic cells are thought to arrange nucleosomes into extended arrays with evenly spaced nucleosomes phased at genomic landmarks. Here we tested to what extent this stereotypic organization describes the nucleosome organization in Saccharomyces cerevisiae using Fiber-Seq, a long-read sequencing technique that maps entire nucleosome arrays on individual chromatin fibers in a high throughput manner. With each fiber coming from a different cell, Fiber-Seq uncovers cell-to-cell heterogeneity. The long reads reveal the nucleosome architecture even over repetitive DNA such as the ribosomal DNA repeats. The absolute nucleosome occupancy, a parameter that is difficult to obtain with conventional sequencing approaches, is a direct readout of Fiber-Seq. We document substantial deviations from the stereotypical nucleosome organization with unexpectedly long linker DNAs between nucleosomes, gene bodies missing entire nucleosomes, cell-to-cell heterogeneity in nucleosome occupancy, heterogeneous phasing of arrays and irregular nucleosome spacing. Nucleosome array structures are indistinguishable throughout the gene body and with respect to the direction of transcription arguing against transcription promoting array formation. Acute nucleosome depletion destroyed most of the array organization indicating that nucleosome remodelers cannot efficiently pack nucleosomes under those conditions. Given that nucleosomes are cis-regulatory elements, the cell-to-cell heterogeneity uncovered by Fiber-Seq provides much needed information to understand chromatin structure and function.
Assuntos
Cromatina , Nucleossomos , Cromatina/genética , Nucleossomos/genética , DNA/genética , Genoma , Saccharomyces cerevisiae/genéticaRESUMO
Chromatin remodeling by ATP-dependent remodeling enzymes is crucial for all genomic processes, like transcription or replication. Eukaryotes harbor many remodeler types, and it is unclear why a given chromatin transition requires more or less stringently one or several remodelers. As a classical example, removal of budding yeast PHO8 and PHO84 promoter nucleosomes upon physiological gene induction by phosphate starvation essentially requires the SWI/SNF remodeling complex. This dependency on SWI/SNF may indicate specificity in remodeler recruitment, in recognition of nucleosomes as remodeling substrate or in remodeling outcome. By in vivo chromatin analyses of wild type and mutant yeast under various PHO regulon induction conditions, we found that overexpression of the remodeler-recruiting transactivator Pho4 allowed removal of PHO8 promoter nucleosomes without SWI/SNF. For PHO84 promoter nucleosome removal in the absence of SWI/SNF, an intranucleosomal Pho4 site, which likely altered the remodeling outcome via factor binding competition, was required in addition to such overexpression. Therefore, an essential remodeler requirement under physiological conditions need not reflect substrate specificity, but may reflect specific recruitment and/or remodeling outcomes.
Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. Analyzing genome-wide data from 733 human cell and tissue samples, we identified 2 prototypical regions that exhibit high or absent hypersensitivity to deoxyribonuclease I, respectively. These regulatory active or inactive regions were examined in the lymphoblast cell line K562 by using high-throughput super-resolution microscopy. In both regions, we systematically measured the physical distance of 2 fluorescence in situ hybridization spots spaced by only 5 kb of DNA. Unexpectedly, the resulting distance distributions range from very compact to almost elongated configurations of more than 200-nm length for both the active and inactive regions. Monte Carlo simulations of a coarse-grained model of these chromatin regions based on published data of nucleosome occupancy in K562 cells were performed to understand the underlying mechanisms. There was no parameter set for the simulation model that can explain the microscopically measured distance distributions. Obviously, the chromatin state given by the strength of internucleosomal interaction, nucleosome occupancy, or amount of histone H1 differs from cell to cell, which results in the observed broad distance distributions. This large variability was not expected, especially in inactive regions. The results for the mechanisms for different distance distributions on this scale are important for understanding the contacts that mediate gene regulation. Microscopic measurements show that the inactive region investigated here is expected to be embedded in a more compact chromatin environment. The simulation results of this region require an increase in the strength of internucleosomal interactions. It may be speculated that the higher density of chromatin is caused by the increased internucleosomal interaction strength.
Assuntos
Cromatina , Nucleossomos , DNA/genética , Humanos , Hibridização in Situ Fluorescente/métodos , Conformação MolecularRESUMO
Mapping of nucleosomes, the basic DNA packaging unit in eukaryotes, is fundamental for understanding genome regulation because nucleosomes modulate DNA access by their positioning along the genome. A cell-population nucleosome map requires two observables: nucleosome positions along the DNA ("Where?") and nucleosome occupancies across the population ("In how many cells?"). All available genome-wide nucleosome mapping techniques are yield methods because they score either nucleosomal (e.g., MNase-seq, chemical cleavage-seq) or nonnucleosomal (e.g., ATAC-seq) DNA but lose track of the total DNA population for each genomic region. Therefore, they only provide nucleosome positions and maybe compare relative occupancies between positions, but cannot measure absolute nucleosome occupancy, which is the fraction of all DNA molecules occupied at a given position and time by a nucleosome. Here, we established two orthogonal and thereby cross-validating approaches to measure absolute nucleosome occupancy across the Saccharomyces cerevisiae genome via restriction enzymes and DNA methyltransferases. The resulting high-resolution (9-bp) map shows uniform absolute occupancies. Most nucleosome positions are occupied in most cells: 97% of all nucleosomes called by chemical cleavage-seq have a mean absolute occupancy of 90 ± 6% (±SD). Depending on nucleosome position calling procedures, there are 57,000 to 60,000 nucleosomes per yeast cell. The few low absolute occupancy nucleosomes do not correlate with highly transcribed gene bodies, but correlate with increased presence of the nucleosome-evicting chromatin structure remodeling (RSC) complex, and are enriched upstream of highly transcribed or regulated genes. Our work provides a quantitative method and reference frame in absolute terms for future chromatin studies.
Assuntos
Mapeamento Cromossômico , DNA Fúngico/genética , Genoma Fúngico , Nucleossomos/genética , Saccharomyces cerevisiae/genética , DNA Fúngico/metabolismo , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Poly(dA:dT) tracts cause nucleosome depletion in many species, e.g., at promoters and replication origins. Their intrinsic biophysical sequence properties make them stiff and unfavorable for nucleosome assembly, as probed by in vitro nucleosome reconstitution. The mere correlation between nucleosome depletion over poly(dA:dT) tracts in in vitro reconstituted and in in vivo chromatin inspired an intrinsic nucleosome exclusion mechanism in vivo that is based only on DNA and histone properties. However, we compile here published and new evidence that this correlation does not reflect mechanistic causation. (1) Nucleosome depletion over poly(dA:dT) in vivo is not universal, e.g., very weak in S. pombe. (2) The energy penalty for incorporating poly(dA:dT) tracts into nucleosomes is modest (<10%) relative to ATP hydrolysis energy abundantly invested by chromatin remodelers. (3) Nucleosome depletion over poly(dA:dT) is much stronger in vivo than in vitro if monitored without MNase and (4) actively maintained in vivo. (5) S. cerevisiae promoters evolved a strand-biased poly(dA) versus poly(dT) distribution. (6) Nucleosome depletion over poly(dA) is directional in vivo. (7) The ATP dependent chromatin remodeler RSC preferentially and directionally displaces nucleosomes towards 5' of poly(dA). Especially distribution strand bias and displacement directionality would not be expected for an intrinsic mechanism. Together, this argues for an in vivo mechanism where active and species-specific read out of intrinsic sequence properties, e.g., by remodelers, shapes nucleosome organization.
Assuntos
Sequência Rica em At , Montagem e Desmontagem da Cromatina , Nucleossomos/genética , Regulação Fúngica da Expressão Gênica , Nucleossomos/química , Nucleossomos/metabolismo , Saccharomyces cerevisiae , SchizosaccharomycesRESUMO
Long noncoding RNAs (lncRNAs), which are longer than 200 nucleotides but often unstable, contribute a substantial and diverse portion to pervasive noncoding transcriptomes. Most lncRNAs are poorly annotated and understood, although several play important roles in gene regulation and diseases. Here we systematically uncover and analyze lncRNAs in Schizosaccharomyces pombe. Based on RNA-seq data from twelve RNA-processing mutants and nine physiological conditions, we identify 5775 novel lncRNAs, nearly 4× the previously annotated lncRNAs. The expression of most lncRNAs becomes strongly induced under the genetic and physiological perturbations, most notably during late meiosis. Most lncRNAs are cryptic and suppressed by three RNA-processing pathways: the nuclear exosome, cytoplasmic exonuclease, and RNAi. Double-mutant analyses reveal substantial coordination and redundancy among these pathways. We classify lncRNAs by their dominant pathway into cryptic unstable transcripts (CUTs), Xrn1-sensitive unstable transcripts (XUTs), and Dicer-sensitive unstable transcripts (DUTs). XUTs and DUTs are enriched for antisense lncRNAs, while CUTs are often bidirectional and actively translated. The cytoplasmic exonuclease, along with RNAi, dampens the expression of thousands of lncRNAs and mRNAs that become induced during meiosis. Antisense lncRNA expression mostly negatively correlates with sense mRNA expression in the physiological, but not the genetic conditions. Intergenic and bidirectional lncRNAs emerge from nucleosome-depleted regions, upstream of positioned nucleosomes. Our results highlight both similarities and differences to lncRNA regulation in budding yeast. This broad survey of the lncRNA repertoire and characteristics in S. pombe, and the interwoven regulatory pathways that target lncRNAs, provides a rich framework for their further functional analyses.
Assuntos
Exonucleases/metabolismo , Exossomos/metabolismo , RNA Longo não Codificante/genética , Schizosaccharomyces/genética , Análise de Sequência de RNA/métodos , Núcleo Celular/metabolismo , Citoplasma/enzimologia , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica , Meiose , Anotação de Sequência Molecular , Mutação , Interferência de RNA , Estabilidade de RNA , RNA Fúngico/genética , RNA Longo não Codificante/química , Schizosaccharomyces/química , Schizosaccharomyces/enzimologiaRESUMO
We establish a DNA origami based tool for quantifying conformational equilibria of biomolecular assemblies as a function of environmental conditions. As first application, we employed the tool to study the salt-induced disassembly of nucleosome core particles. To extract binding constants and energetic penalties, we integrated nucleosomes in the spectrometer such that unwrapping of the nucleosomal template DNA, leading from bent to more extended states was directly coupled to the conformation of the spectrometer. Nucleosome unwrapping was induced by increasing the ionic strength. The corresponding shifts in conformation equilibrium of the spectrometer were followed by direct conformation imaging using negative staining TEM and by FRET read out after gel electrophoretic separation of conformations. We find nucleosome dissociation constants in the picomolar range at low ionic strength (11 mM MgCl2), in the nanomolar range at intermediate ionic strength (11 mM MgCl2 with 0.5-1 M NaCl) and in the micromolar range at larger ionic strength (11 mM MgCl2 with ≥1.5 M NaCl). Integration of up to four nucleosomes stacked side-by-side, as it might occur within chromatin fibers, did not appear to affect the salt-induced unwrapping of nucleosomes. Presumably, such stacking interactions are already effectively screened at the nucleosome unwrapping conditions. Our spectrometer provides a modular platform with a direct read out to study conformational equilibria for targets from small biomolecules up to large macromolecular assemblies.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Nucleossomos/química , Animais , Drosophila melanogaster , Embrião não Mamífero , Transferência Ressonante de Energia de Fluorescência , Histonas , Substâncias Macromoleculares , Concentração OsmolarRESUMO
Eukaryotic nuclear DNA is packaged into nucleosomes. During the past decade, genome-wide nucleosome mapping across species revealed the high degree of order in nucleosome positioning. There is a conserved stereotypical nucleosome organization around transcription start sites (TSSs) with a nucleosome-depleted region (NDR) upstream of the TSS and a TSS-aligned regular array of evenly spaced nucleosomes downstream over the gene body. As nucleosomes largely impede access to DNA and thereby provide an important level of genome regulation, it is of general interest to understand the mechanisms generating nucleosome positioning and especially the stereotypical NDR-array pattern. We focus here on the most advanced models, unicellular yeasts, and review the progress in mapping nucleosomes and which nucleosome positioning mechanisms are discussed. There are four mechanistic aspects: How are NDRs generated? How are individual nucleosomes positioned, especially those flanking the NDRs? How are nucleosomes evenly spaced leading to regular arrays? How are regular arrays aligned at TSSs? The main candidates for nucleosome positioning determinants are intrinsic DNA binding preferences of the histone octamer, specific DNA binding factors, nucleosome remodeling enzymes, transcription, and statistical positioning. We summarize the state of the art in an integrative model where nucleosomes are positioned by a combination of all these candidate determinants. We highlight the predominance of active mechanisms involving nucleosome remodeling enzymes which may be recruited by DNA binding factors and the transcription machinery. While this mechanistic framework emerged clearly during recent years, the involved factors and their mechanisms are still poorly understood and require future efforts combining in vivo and in vitro approaches.
Assuntos
Mapeamento Cromossômico , DNA Fúngico/genética , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Animais , Montagem e Desmontagem da Cromatina , Estudos de Associação Genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Alinhamento de Sequência , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Nucleosome positioning governs access to eukaryotic genomes. Many genes show a stereotypic organisation at their 5'end: a nucleosome free region just upstream of the transcription start site (TSS) followed by a regular nucleosomal array over the coding region. The determinants for this pattern are unclear, but nucleosome remodelers are likely critical. Here we study the role of remodelers in global nucleosome positioning in S. pombe and the corresponding changes in expression. We find a striking evolutionary shift in remodeler usage between budding and fission yeast. The S. pombe RSC complex does not seem to be involved in nucleosome positioning, despite its prominent role in S. cerevisiae. While S. pombe lacks ISWI-type remodelers, it has two CHD1-type ATPases, Hrp1 and Hrp3. We demonstrate nucleosome spacing activity for Hrp1 and Hrp3 in vitro, and that together they are essential for linking regular genic arrays to most TSSs in vivo. Impaired arrays in the absence of either or both remodelers may lead to increased cryptic antisense transcription, but overall gene expression levels are only mildly affected.
Assuntos
Adenosina Trifosfatases/fisiologia , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Fúngica da Expressão Gênica , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA Helicases/química , Proteínas de Ligação a DNA/química , Dactinomicina/farmacologia , Deleção de Genes , Histonas/química , Modelos Biológicos , Mutação , Oligonucleotídeos Antissenso/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Transcrição Gênica , TranscriptomaRESUMO
Chromatin dynamics crucially contributes to gene regulation. Studies of the yeast PHO5 promoter were key to establish this nowadays accepted view and continuously provide mechanistic insight in chromatin remodeling and promoter regulation, both on single locus as well as on systems level. The PHO5 promoter is a context independent chromatin switch module where in the repressed state positioned nucleosomes occlude transcription factor sites such that nucleosome remodeling is a prerequisite for and not consequence of induced gene transcription. This massive chromatin transition from positioned nucleosomes to an extensive hypersensitive site, together with respective transitions at the co-regulated PHO8 and PHO84 promoters, became a prime model for dissecting how remodelers, histone modifiers and chaperones co-operate in nucleosome remodeling upon gene induction. This revealed a surprisingly complex cofactor network at the PHO5 promoter, including five remodeler ATPases (SWI/SNF, RSC, INO80, Isw1, Chd1), and demonstrated for the first time histone eviction in trans as remodeling mode in vivo. Recently, the PHO5 promoter and the whole PHO regulon were harnessed for quantitative analyses and computational modeling of remodeling, transcription factor binding and promoter input-output relations such that this rewarding single-locus model becomes a paradigm also for theoretical and systems approaches to gene regulatory networks.
Assuntos
Fosfatase Ácida/genética , Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Cromatina/química , Redes Reguladoras de Genes , Modelos Genéticos , RegulonRESUMO
Although yeast PHO5 promoter chromatin opening is a founding model for chromatin remodeling, the complete set of involved remodelers remained unknown for a long time. The SWI/SNF and INO80 remodelers cooperate here, but nonessentially, and none of the many tested single or combined remodeler gene mutations could prevent PHO5 promoter opening. RSC, the most abundant and only remodeler essential for viability, was a controversial candidate for the unrecognized remodeling activity but unassessed in vivo. Now we show that remodels the structure of chromatin (RSC) is crucially involved in PHO5 promoter opening. Further, the isw1 chd1 double deletion also delayed chromatin remodeling. Strikingly, combined absence of RSC and Isw1/Chd1 or Snf2 abolished for the first time promoter opening on otherwise sufficient induction in vivo. Together with previous findings, we recognize now a surprisingly complex network of five remodelers (RSC, SWI/SNF, INO80, Isw1 and Chd1) from four subfamilies (SWI/SNF, INO80, ISWI and CHD) as involved in PHO5 promoter chromatin remodeling. This is likely the first described complete remodeler set for a physiological chromatin transition. RSC was hardly involved at the coregulated PHO8 or PHO84 promoters despite cofactor recruitment by the same transactivator and RSC's presence at all three promoters. Therefore, promoter-specific chromatin rather than transactivators determine remodeler requirements.
Assuntos
Fosfatase Ácida/genética , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/fisiologia , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/fisiologia , Fosfatase Ácida/biossíntese , Adenosina Trifosfatases/genética , Fosfatase Alcalina/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Ciclinas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the 'softness' of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to 'hard' particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.
Assuntos
Replicação do DNA , Nucleossomos/metabolismo , Cinética , Modelos GenéticosRESUMO
Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction.
Assuntos
Histonas/metabolismo , Complexo Mediador/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Northern Blotting , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexo Mediador/genética , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Iniciação da Transcrição Genética , Ativação TranscricionalRESUMO
Nucleosomes impede access to DNA. Therefore, nucleosome positioning is fundamental to genome regulation. Nevertheless, the molecular nucleosome positioning mechanisms are poorly understood. This is partly because in vitro reconstitution of in vivo-like nucleosome positions from purified components is mostly lacking, barring biochemical studies. Using a yeast extract in vitro reconstitution system that generates in vivo-like nucleosome patterns at S. cerevisiae loci, we find that the RSC chromatin remodelling enzyme is necessary for nucleosome positioning. This was previously suggested by genome-wide in vivo studies and is confirmed here in vivo for individual loci. Beyond the limitations of conditional mutants, we show biochemically that RSC functions directly, can be sufficient, but mostly relies on other factors to properly position nucleosomes. Strikingly, RSC could not be replaced by either the closely related SWI/SNF or the Isw2 remodelling enzyme. Thus, we pinpoint that nucleosome positioning specifically depends on the unique properties of the RSC complex.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Montagem e Desmontagem da CromatinaRESUMO
Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1 - accessibility) in absolute terms as the percentage of cut or uncut molecules, respectively, out of all molecules. The protocol presented here takes this classical approach to the genome-wide level. After exhaustive restriction enzyme digestion of chromatin, DNA is purified, sheared, and converted into libraries for high-throughput sequencing. Bioinformatic analysis counts uncut DNA fragments as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straightforward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments. Our protocol includes two orthogonal approaches to correct for this bias, the "corrected cut-uncut" and the "cut-all cut" method, so that accurate measurements of absolute accessibility or occupancy at restriction sites throughout a genome are possible. The protocol is presented for the example of S. cerevisiae chromatin but may be adapted for any other species.
Assuntos
Cromatina , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , DNA/genética , Genoma , Enzimas de Restrição do DNA/genética , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Genome-wide nucleosome maps revealed well-positioned nucleosomes as a major theme in eukaryotic genome organization. Promoter regions often show a conserved pattern with an NDR (nucleosome-depleted region) from which regular nucleosomal arrays emanate. Three mechanistic contributions to such NDR-array-organization and nucleosome positioning in general are discussed: DNA sequence, DNA binders and DNA-templated processes. Especially, intrinsic biophysics of DNA sequence preferences for nucleosome formation was prominently suggested to explain the majority of nucleosome positions ('genomic code for nucleosome positioning'). Nonetheless, non-histone factors that bind DNA with high or low specificity, such as transcription factors or remodelling enzymes respectively and processes such as replication, transcription and the so-called 'statistical positioning' may be involved too. Recently, these models were tested for yeast by genome-wide reconstitution. DNA sequence preferences as probed by SGD (salt gradient dialysis) reconstitution generated many NDRs, but only few individual nucleosomes, at their proper positions, and no arrays. Addition of a yeast extract and ATP led to dramatically more in vivo-like nucleosome positioning, including regular arrays for the first time. This improvement depended essentially on the extract and ATP but not on transcription or replication. Nucleosome occupancy and close spacing were maintained around promoters, even at lower histone density, arguing for active packing of nucleosomes against the 5' ends of genes rather than statistical positioning. A first extract fractionation identified a direct, specific, necessary, but not sufficient role for the RSC (remodels the structure of chromatin) remodelling enzyme. Collectively, nucleosome positioning in yeast is actively determined by factors beyond intrinsic biophysics, and in steady-state rather than at equilibrium.