Nucleosome Dissociation Research Articles

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes.

Centromeres are specific segments of chromosomes comprising two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones and CENP-A nucleosomes in which H3 is replaced with its analogue CENP-A. This modification leads to a difference in DNA wrapping (∼121 bp), considerably less than 147 bp in canonical nucleosomes. We used atomic force microscopy (AFM) and high-speed AFM (HS-AFM) to characterize nanoscale features and dynamics for both types of nucleosomes. For both nucleosomes, spontaneous asymmetric unwrapping of DNA was observed, and this process occurs via a transient state with ∼100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. Additionally, HS-AFM revealed higher stability of CENP-A nucleosomes compared with H3 nucleosomes in which dissociation of the histone core occurs prior to the nucleosome dissociation. These results help elucidate the differences between these nucleosomes and the potential biological necessity for CENP-A nucleosomes.

Oncogenic changes in histones and their impact on nucleosome dissociation and H2B tail structure and dynamics

Oncogenic changes in histones and their impact on nucleosome dissociation and H2B tail structure and dynamics

Exploring Nucleosome Unwrapping Using DNA Origami.

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.

Nucleosome Kinetics Regulates the Binding Timescales of Non-Histone Proteins to DNA Sites

Nucleosome organization in the promoter region of genes is thought to be a crucial factor in determining the accessibility of target DNA sites of regulatory non-histone proteins (e.g. transcription factors (TF), TATA- binding proteins (TBP) and RNA polymerase). Binding of most of these regulatory proteins needs access to bare DNA which depends on the “kinetic” rearrangement of nucleosomes in that region. Most of the works done in the past, focus only on static nucleosome occupancy and not on its stochastic kinetics. We go beyond this static picture and using a simple model for nucleosome kinetics address the kinetic problem of TF/TBP binding by posing it as a “first passage time (FPT)” problem. We have solved this model analytically (with some approximations) and supported our theory numerically via Monte Carlo simulations. In our model, we account for binding and dissociation of nucleosomes, action of ATP-dependent remodelers, sequence dependent histone-DNA interaction potential, and explicit competition between TF/TBP and nucleosomes for site accessibility. We find that even if the nucleosome occupancy is the same, differences in kinetics of nucleosomes can cause differences in kinetic histories of TF/TBP binding. We show that the first passage time of TF/TBP binding could be very different, depending upon the initial nucleosome organization and the genomic sequence. The first passage timescales of TF/TBP binding (in competition with nucleosomes) may also be indirectly indicated from calculations of exposure timescales with kinetic nucleosomes alone. Our analytical method provides an easy way to compute the first passage time of TF/TBP binding. We apply this method to a genome-wide study of TBP binding timescales at TATA sites for the genes of Saccharomyces cerevisiae.

Nucleosome Kinetics and Accesibility of DNA

Crucial cellular processes like gene regulation, transcription, and replication require access to DNA that is covered with nucleosomes. Many experiments suggest that nucleosome organization and dynamics can significantly influence exposure and accessibility of various locations on the genome. In this work we investigate the kinetics of DNA exposure as a result of nucleosome dynamics. We consider binding and dissociation of nucleosomes taking into account both sequence specificity and ATP-dependent activity, and study accessibility of DNA near different kinds of barriers (e.g. a well-positioned protein or a nucleosome free region near transcription start site). Using analytical calculations and numerical simulations, we find the following results. We show that the timescale of exposure of a DNA site near a barrier can be very diverse and crucially depends on the DNA sequence and the initial nucleosome organization. We show how nucleosome-mediated cooperativity can emerge when multiple transcription factors are binding at nearby locations and we investigate how multi-nucleosome correlations influence the time scale of accessibility as a function of the distance from the barrier. We discuss ramifications of our findings in understanding gene regulation and stochasticity in gene expression.

Clearing the Way for Unpaused Polymerases

Heat shock loci in the polytene chromosomes of the fruit fly Drosophila undergo a characteristic change in appearance that coincides with the onset of gene expression. Petesch and Lis (2008) now show that nucleosomes are lost across the entire Hsp70 locus in an initial wave that precedes transcription by RNA polymerase II.

The histone chaperone Asf1 at the crossroads of chromatin and DNA checkpoint pathways

Nucleosome assembly involves deposition of a heterotetramer of histones H3/H4 onto DNA followed by two heterodimers of histones H2A/H2B. Cycles of nucleosome assembly and disassembly are essential to cellular events such as replication, transcription, and DNA repair. After synthesis in the cytoplasm, histones are shuttled into the nucleus where they are associated with chaperone proteins. Chaperones of histones H3/H4 include CAF-I, the Hir proteins, and Asf1. CAF-I and the Hir proteins function as replication-coupled and replication-independent deposition factors for H3/H4, respectively, whereas Asf1 may play a role in both pathways. In addition to acting as assembly factors, histone chaperones assist nucleosome dissociation from DNA and they may recruit other proteins to chromatin. The past few years have witnessed a notable accumulation of genetic, biochemical, and structural data on Asf1, which motivated this review. We discuss the sequence and structural features of Asf1 before considering its roles in nucleosome assembly/disassembly, the cellular response to DNA damage, and the regulation of gene expression. We emphasize the key role of Asf1 as a central node in a network of partners that place it at the crossroads of chromatin and DNA checkpoint pathways.

Histone-DNA binding free energy cannot be measured in dilution-driven dissociation experiments.

Despite decades of study on nucleosomes, there has been no experimental determination of the free energy of association between histones and DNA. Instead, only the relative free energy of association of the histone octamer for differing DNA sequences has been available. Recently, a method was developed based on quantitative analysis of nucleosome dissociation in dilution experiments that provides a simple practical measure of nucleosome stability. Solution conditions were found in which nucleosome dissociation driven by dilution fit well to a simple model involving a noncooperative nucleosome assembly/disassembly equilibrium, suggesting that this approach might allow absolute equilibrium affinity of the histone octamer for DNA to be measured. Here, we show that the nucleosome assembly/disassembly process is not strictly reversible in these solution conditions, implying that equilibrium affinities cannot be obtained from these measurements. Increases in [NaCl] or temperature, commonly employed to suppress kinetic bottlenecks in nucleosome assembly, lead to cooperative behavior that cannot be interpreted with the simple assembly/disassembly equilibrium model. We conclude that the dilution experiments provide useful measures of kinetic but not equilibrium stability. Kinetic stability is of practical importance: it may govern nucleosome function in vivo, and it may (but need not) parallel absolute thermodynamic stability.

Partial denaturation of small chromatin fragments: direct evidence for the radial distribution of nucleosomes in folded chromatin fibers.

To examine the internal structure of chromatin fibers, we have developed procedures for partial denaturation of small chromatin fragments (8-30 nucleosomes) from chicken erythrocytes. Electron micrographs of samples prepared under conditions that cause nucleosome dissociation show rods and loops projecting from short compact fibers fixed by glutaraldehyde in 1.7 mM Mg2+. According to previous studies in our laboratory, these images correspond to the top view of partially denatured fibers. Our results indicate that rods and loops consist of extended duplex DNA of different lengths. DNA in loops is nicked, as demonstrated by experiments performed in the presence of high concentrations of ethidium bromide. Length measurements indicate that the radial projections of DNA are produced by unfolding of nucleosomal units. Loops are formed by DNA from denatured nucleosomes in internal positions of the fiber; DNA from denatured nucleosomes in terminal positions form rods. Our micrographs show clearly a radial distribution of DNA loops and rods projecting from fibers. Rods are orthogonal to the surface of the chromatin fragments. Considering that the high ionic strength used in this study (0.8-2.0 M NaCl) neutralizes the electrostatic repulsions between rods and fiber, this observation suggests that rods are extensions of nucleosomes radially organized inside the fiber. The position of the entry points of DNA loops into the fiber could be influenced by constraint on loops, but our results showing that the arc that separates these points in dinucleosome loops is relatively short suggest that consecutive nucleosomes are relatively close to each other in the folded fiber.

Impairment of Proliferating Cell Nuclear Antigen-dependent Apurinic/Apyrimidinic Site Repair on Linear DNA

Repair of apurinic/apyrimidinic (AP) sites by mammalian cell extracts was compared using circular and linear DNA substrates. Extracts prepared from DNA polymerase beta (polbeta)-proficient mouse fibroblasts repaired AP sites on both circular and linear DNA. However, extracts from the isogenic polbeta-knockout cells repaired AP sites on circular DNA but not efficiently on linear DNA. The circularity-dependent repair by the polbeta-knockout cell extract was completely inhibited by anti-proliferating cell nuclear antigen (PCNA) antibody but fully restored by addition of purified PCNA. Pretreatment of the linear DNA with AP endonuclease did not improve repair, indicating that impairment of AP site repair on linear DNA by polbeta-knockout cell extracts is not due to inefficiency of damage incision but rather to deficiency at the subsequent steps. These results indicate that AP sites can be repaired on circular DNA by the PCNA-dependent pathway in addition to the polbeta-dependent pathway and that the PCNA-dependent repair mechanism is poorly functional on linear DNA in vitro.

The H3/H4 Tetramer Blocks Transcript Elongation by RNA Polymerase II in Vitro

We have investigated transcript elongation efficiency by RNA polymerase II on chromatin templates in vitro. Circular plasmid DNAs bearing purified RNA polymerase II transcription complexes were assembled into nucleosomes using purified histones and transient exposure to high salt, followed by dilution and dialysis. This approach resulted in nucleosome assembly beginning immediately downstream of the transcription complexes. RNA polymerases on these nucleosomal templates could extend their 15- or 35-nucleotide nascent RNAs by only about 10 nucleotides in 15 min, even in the presence of elongation factors TFIIF and SII. Efficient transcript elongation did occur upon dissociation of nucleosomes with 1% sarkosyl, indicating that the RNA polymerases were not damaged by the high salt reconstitution procedure. Since the elongation complexes were released by sarkosyl but not by SII, these complexes apparently did not enter the arrested conformation when they encountered nucleosomes. Surprisingly, elongation was no more efficient on nucleosomal templates reconstituted only with H3/H4 tetramers, even in the presence of elongation factors and/or competitor DNA at high concentration. Thus, in a purified system lacking nucleosome remodeling factors, not only the core histone octamer but also the H3/H4 tetramer provide an nearly absolute block to transcript elongation by RNA polymerase II, even in the presence of elongation factors.

Mechanism of Nucleosome Dissociation Produced by Transcription Elongation in a Short Chromatin Template

We have used a linear DNA template (239 bp) containing a nucleosome positioning sequence (NX1) downstream of the T7 RNA polymerase promoter to study the mechanism of transcription elongation through a nucleosome. Under ionic strength approaching physiological conditions we have observed that transcription causes nucleosome dissociation and histone redistribution within the template. We have examined the role of the different elements that, in principle, could induce nucleosome dissociation during transcription. The high affinity of histones for single-stranded DNA observed in titration experiments performed using the purified (+) and (-) strands of the NX1 fragment suggests that nucleosome dissociation is not due to the formation of segments of single-stranded DNA by RNA polymerase in the elongation process. Furthermore, our results show that although RNA can interact with core histones, the synthesized RNA is not bound to the histones dissociated by transcription. Our results indicate that core histones released during transcription can be bound to naked DNA and chromatin (with or without histones H1-H5). From the dynamic properties of excess histones bound to chromatin, we suggest a nucleosome transcription mechanism in which displaced histones are transiently bound to chromatin and finally are reassembled with DNA after the passage of the polymerase.

Chromatin transitions during activation and repression of galactose-regulated genes in yeast.

To study the fate of nucleosomes during transcription, a yeast gene 'GAL-URARIB' was constructed which is tightly regulated by the GAL1 promoter and shows in its inactive state a series of positioned nucleosomes that are sensitive for monitoring structural changes by micrococcal nuclease. Upon transcriptional activation, nucleosome positions were lost, but a residual nucleosomal repeat with an altered repeat length and no changes in psoralen accessibility measured by a band shift assay indicated that nucleosomes were present but rearranged on the transcribed gene. When chromatin was prepared 10 or 50 min after glucose repression, nucleosomes were repositioned in a large fraction of the population by a rapid process which most likely did not depend on histone synthesis or DNA replication. However, complete regeneration of the inactive structure and repeat length was observed after one cell generation (2.5 h) suggesting that in this step some missing histones were replaced. The results are consistent with a local dissociation of nucleosomes at the site of the polymerase followed by a rapid reassembly into nucleosomes behind it. The data are further supported by analysis of the chromosomal GAL1, GAL7 and GAL10 genes.

Nucleosome dissociation at physiological ionic strengths.

Monomer nucleosomes purified on isokinetic sucrose gradients are shown to dissociate into component DNA and histones at physiological ionic strength upon dilution to a DNA concentration below 20 microgram/ml. The starting material is 11S, contains 145-190 BP DNA, and equimolar amounts of the four core histones with slightly less H1. Dilution of monomers in the presence of 0.14 M NaCl results in the rapid conversion of 10-40% of the 3H thymidine labeled material from 11S to 5S (5S is coincident with the S value of monomer length DNA). The proportion of nucleosomes which dissociate increases with increasing NaCl concentration between 0.15 M and 0.35 M and decreases with increasing DNA concentration above 1 microgram/ml. Recycling 11S monomers, which remain after dissociation, through a second dilution in salt generates an equivalent proportion of 5S material as seen after the initial dilution. Thus, the dissociation does not result from special properties of a subset of nucleosomes. An equilibrium between intact monomer and free DNA and histones appears to be rapidly established under the conditions described and the dissociated DNA will reassociate with histones to form 11S monomers if conditions of high DNA concentration and low ionic strength are established.

Nucleosome dissociation and transfer in concentrated salt solutions.

We have examined the dissociation of nucleosomes into histones and free, 4.5S DNA over a range of sodium chloride concentrations between 0.25 and 1 M. We have also studied this dissociation as a function of nucleosome concentration at two salt concentrations, 0.8 M and 0.9 M. In addition, we have measured the kinetics of transfer of histone cores from nucleosomes onto recipient bacteriophage T7 DNA in 0.6, 0.7 and 0.8 M NaCl solutions. Although the mechanism of nucleosome transfer is unknown the data presented here are consistent with either a reversible dissociation of the nucleosome or DNA strand displacement by another DNA.