Nucleosome Disruption Research Articles

Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA.

The dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 bp of DNA released from the histone core at low stretching force. Subsequently, 80 bp are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. The kinetic parameters for nucleosome disassembly also have been determined.

CBF/NF-Y functions both in nucleosomal disruption and transcription activation of the chromatin-assembled topoisomerase IIalpha promoter. Transcription activation by CBF/NF-Y in chromatin is dependent on the promoter structure.

To understand the role of CCAAT-binding factor (CBF) in transcription in the context of chromatin-assembled DNA, we used regularly spaced nucleosomal DNA using topoisomerase IIalpha (topo IIalpha) and alpha2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIalpha promoter containing four CBF-binding sites disrupted the regular nucleosomal structure not only in the promoter region containing the CBF-binding sites but also in the downstream region over the transcription start site. In contrast, no nucleosome disruption was observed in a mutant topo IIalpha promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIalpha promoter. In this experiment, a promoter containing one wild-type CBF-binding site was activated very weakly, whereas the promoter containing mutations in all sites was not activated by CBF. A truncated CBF that lacked the glutamine-rich domains did not activate transcription from nucleosomal wild-type topo IIalpha promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse alpha2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in an in vitro transcription reaction. None of the nucleosomal collagen promoters was activated by CBF. However, both of these collagen promoters were activated by CBF when the transcription reaction was performed using naked DNA templates. Binding of CBF to the nucleosomal collagen promoter containing four binding sites disrupted the nucleosomal structure, similarly as observed in the topo IIalpha promoter. Altogether this study indicates that CBF-mediated nucleosomal disruption occurred independently of transcription activation. It also suggests that specific promoter structure may play a role in the CBF-mediated transcription activation of nucleosomal topo IIalpha promoter template.

Histone Acetyltransferase Complexes Stabilize SWI/SNF Binding to Promoter Nucleosomes

To investigate the function of SWI/SNF in site-specific chromatin remodeling at promoters, we have used a purified system to analyze its distribution, function, and retention following recruitment by a sequence-specific transcription activator. Activator recruitment of SWI/SNF bound the complex to promoter proximal nucleosomes and led to localized nucleosome disruption. However, retention of SWI/SNF on the promoter required either the continued binding of the transcription activator or acetylated histones. Histone acetylation by either the SAGA or NuA4 HAT complexes increased the retention of SWI/SNF on the promoter. These data illustrate a functional link between HAT complexes and the SWI/SNF chromatin remodeling complex and provide a mechanistic basis for the ordered recruitment of these complexes.

Comparison of Nucleosome Remodeling by the Yeast Transcription Factor Pho4 and the Glucocorticoid Receptor

Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively. In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR. Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity. However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter. Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site. In contrast, GR had little effect on the stability of the MMTV B nucleosome. A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome. Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure. Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.

Yeast chromatin structure and regulation of GAL gene expression

Yeast genomic DNA is covered by nucleosome cores spaced by short, discrete length linkers. The short linkers, reinforced by novel histone properties, create a number of unique and dynamic nucleosome structural features in vivo: permanent unpeeling of DNA from the ends of the core, an inability to bind even full 147 bp core DNA lengths, and facility to undergo a conformational transition that resembles the changes found in active chromatin. These features probably explain how yeast can maintain most of its genome in a transcribable state and avoid large-scale packaging away of inactive genes. The GAL genes provide a closely regulated system in which to study gene-specific chromatin structure. GAL structural genes are inactive without galactose but are highly transcribed in its presence; the expression patterns of the regulatory genes can account for many of the features of GAL structural gene control. In the inactive state, GAL genes demonstrate a characteristic promoter chromosomal organization; the major upstream activation sequence (UASG) elements lie in open, hypersensitive regions, whereas the TATA and transcription start sites are in nucleosomes. This organization helps implement gene regulation in this state and may benefit the organism. Induction of GAL expression triggers Gal4p-dependent upstream nucleosome disruption. Disruption is transient and can readily be reversed by a Gal80p-dependent nucleosome deposition process. Both are sensitive to the metabolic state of the cell. Induction triggers different kinds of nucleosome changes on the coding sequences, perhaps reflecting the differing roles of nucleosomes on coding versus promoter regions. GAL gene activation is a complex process involving multiple Gal4p activities, numerous positive and negative cofactors, and the histone tails. DNA bending and chromosomal architecture of the promoter regions may also play a role in GAL regulation. Regulator-mediated competition between nucleosomes and the TATA binding protein complex for the TATA region is probably a central aspect of GAL regulation and a focal point for the numerous factors and processes that contribute to it.

Chromatin remodeling directly activates V(D)J recombination.

V(D)J recombination substrate choice is regulated to ensure that the appropriate gene segments are rearranged during lymphocyte development. It has been proposed that regulation of substrate usage is determined by changes in accessibility of the DNA targets. We show that Rag-mediated recombination of an episomal substrate in cells is affected by its packaging into chromatin. Chromatinized substrates were inefficiently rearranged, and methylation further reduced recombination. Disruption of nucleosomes by using butyrate on methylated substrates was sufficient to activate recombination, and dexamethasone could activate recombination in the absence of detectable transcription. Therefore, chromatin structure, and its manipulation by altering nucleosome positioning, can directly affect recombination efficiencies.

Selective Nucleosome Disruption by Drugs That Bind in the Minor Groove of DNA

Previous studies have shown that drugs which bind in the DNA minor groove reduce the curvature of bent DNA. In this article, we examined the effects of these drugs on the nucleosome assembly of DNA molecules that display different degrees of intrinsic curvature. DAPI (4,6-diamidino-2-phenylindole) inhibited the assembly of a histone octamer onto a 192-base pair circular DNA fragment from Caenorhabditis elegans and destabilized a nucleosome that was previously assembled on this segment. The inhibitory effect was highly selective since it was not seen with nonbent molecules, bent molecules with noncircular shapes, or total genomic DNA. This marked template specificity was attributed to the binding of the ligand to multiple oligo A-tracts distributed over the length of the fragment. A likely mechanism for the effect is that the bound ligand prevents the further compression of the DNA into the minor groove which is required for assembly of DNA into nucleosomes. To further characterize the effects of the drug on chromatin formation, a nucleosome was assembled onto a 322-base pair DNA fragment that contained the circular element and a flanking nonbent segment of DNA. The position of the nucleosome along the fragment was then determined using a variety of nuclease probes including exonuclease III, micrococcal nuclease, DNase I, and restriction enzymes. The results of these studies revealed that the nucleosome was preferentially positioned along the circular element in the absence of DAPI but assembled onto the nonbent flanking sequence in the presence of the drug. DAPI also induced the directional movement of the nucleosome from the circular element onto the nonbent flanking sequence when a nucleosome preassembled onto this template was exposed to the drug under physiologically relevant conditions.

RNA Polymerase-specific Nucleosome Disruption by Transcription in Vivo

The nucleosomal chromatin structure within genes is disrupted upon transcription by RNA polymerase II. To determine whether this disruption is caused by transcription per se as opposed to the RNA polymerase source, we engineered the yeast chromosomal HSP82 gene to be exclusively transcribed by bacteriophage T7 RNA polymerase in vivo. Interestingly, we found that a fraction of the T7-generated transcripts were 3' end processed and polyadenylated at or near the 3' ends of the hsp82 and the immediately downstream CIN2 genes. Surprisingly, the nucleosomal structure of the T7-transcribed hsp82 gene remained intact, in marked contrast to the disrupted structure generated by much weaker, basal level transcription of the wild type gene by RNA polymerase II under non-heat shock conditions. Therefore, disruption of chromatin structure by transcription is dependent on the RNA polymerase source. We propose that the observed RNA polymerase dependence for transcription-induced nucleosome disruption may be related either to the differential recruitment of chromatin remodeling complexes, the rates of histone octamer translocation and nucleosome reformation during polymerase traversal, and/or the degree of transient torsional stress generated by the elongating polymerase.

Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC.

In eukaryotes, DNA is packaged into chromatin, a compact structure that must be disrupted when genes are transcribed by RNA polymerase II. For transcription to take place, chromatin is remodeled via nucleosome disruption or displacement, a fundamental transcriptional regulatory mechanism in eukaryotic organisms. Here we show that the yeast chromatin-remodeling complex, RSC (remodels the structure of chromatin), isolated on the basis of homology to the SWI/SNF complex, is required for proper transcriptional regulation and nucleosome positioning in the highly inducible CHA1 promoter. In the absence of Sth1p/Nps1p (a homolog of Swi2p/Snf2p) or of Swh3p (a homolog of Swi3p), expression of CHA1 in non-induced cells is increased to a level comparable with that of fully induced cells. Furthermore, in non-induced cells depleted for Sth1p/Nps1p or Swh3p, a nucleosome positioned over the TATA box of the CHA1 promoter is disrupted, an architectural change normally only observed during transcriptional induction. In addition, deletion of the gene-specific activator Cha4p did not affect derepression of CHA1 in cells depleted for Swh3p. Thus, CHA1 constitutes a target for the RSC complex, and we propose that RSC is essential for maintaining a repressive chromatin structure at the CHA1 promoter.

Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae.

We have identified and characterized two Imitation Switch genes in Saccharomyces cerevisiae, ISW1 and ISW2, which are highly related to Drosophila ISWI, encoding the putative ATPase subunit of three ATP-dependent chromatin remodeling factors. Purification of ISW1p reveals a four-subunit complex with nucleosome-stimulated ATPase activity, as well as ATP-dependent nucleosome disruption and spacing activities. Purification of ISW2p reveals a two-subunit complex also with nucleosome-stimulated ATPase and ATP-dependent nucleosome spacing activities but no detectable nucleosome disruption activity. Null mutations of ISW1, ISW2, and CHD1 genes cause synthetic lethality in various stress conditions in yeast cells, revealing the first in vivo functions of the ISWI subfamily of chromatin-remodeling complexes and demonstrating their genetic interactions. A single point mutation within the ATPase domain of both ISW1p and ISW2p inactivated all ATP-dependent biochemical activities of the complexes, as well as the ability of the genes to rescue the mutant phenotypes. This demonstrates that the ATP-dependent chromatin-remodeling activities are essential for the in vivo functions of both ISW1 and ISW2 complexes.

The SWI/SNF complex creates loop domains in DNA and polynucleosome arrays and can disrupt DNA-histone contacts within these domains.

To understand the mechanisms by which the chromatin-remodeling SWI/SNF complex interacts with DNA and alters nucleosome organization, we have imaged the SWI/SNF complex with both naked DNA and nucleosomal arrays by using energy-filtered microscopy. By making ATP-independent contacts with DNA at multiple sites on its surface, SWI/SNF creates loops, bringing otherwise-distant sites into close proximity. In the presence of ATP, SWI/SNF action leads to the disruption of nucleosomes within domains that appear to be topologically constrained by the complex. The data indicate that the action of one SWI/SNF complex on an array of nucleosomes can lead to the formation of a region where multiple nucleosomes are disrupted. Importantly, nucleosome disruption by SWI/SNF results in a loss of DNA content from the nucleosomes. This indicates a mechanism by which SWI/SNF unwraps part of the nucleosomal DNA.

Le rôle de la structure chromatinienne dans la régulation transcriptionnelle de l'expression des gènes

Le rôle de la structure chromatinienne dans la régulation transcriptionnelle de l'expression des gènes

Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes.

Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes.

NURD, a Novel Complex with Both ATP-Dependent Chromatin-Remodeling and Histone Deacetylase Activities

ATP-dependent chromatin-remodeling complexes are known to facilitate transcriptional activation by opening chromatin structures. We report a novel human complex, named NURD, which contains not only ATP-dependent nucleosome disruption activity, but also histone deacetylase activity, which usually associates with transcriptional repression. The deacetylation is stimulated by ATP on nucleosomal templates, suggesting that nucleosome disruption aids the deacetylase to access its substrates. One subunit of NURD was identified as MTA1, a metastasis-associated protein with a region similar to the nuclear receptor corepressor, N-CoR; and antibodies against NURD partially relieve transcriptional repression by thyroid hormone receptor. These results suggest that ATP-dependent chromatin remodeling can participate in transcriptional repression by assisting repressors in gaining access to chromatin.

SWI/SNF Complexes and Facilitation of TATA Binding Protein:Nucleosome Interactions

It has become increasingly apparent that eukaryotic cells possess machinery that modifies chromatin structure and that this machinery contributes to the regulation of gene expression. Identification of factors that alter chromatin structure has made possible biochemical analyses that have begun to define what structural changes each factor can cause as well as what consequences these changes have on transcription factor function. Here, a protocol that has facilitated study of energy-dependent chromatin remodeling complexes containing SWI/SNF proteins is described. Rotationally phased mononucleosome particles were assembledin vitroand used to demonstrate that human SWI/SNF complexes and the yeast RNA polymerase II holoenzyme, which contains yeast SWI/SNF proteins, can directly alter nucleosome structure in an ATP-dependent manner. A functional consequence of this nucleosome disruption is that the pol II general transcription factor, TATA binding protein (TBP), which cannot bind to unaltered nucleosomal DNA, can bind to its site on the altered nucleosome. This experimental system has been invaluable for characterization of both nucleosome alteration and facilitated transcription factor binding mediated by SWI/SNF complexes. These procedures should also be useful to examine other factors that interact with or structurally affect nucleosome particles.

Perturbation of nucleosome structure by the erythroid transcription factor GATA-1

The ability of transcription factors to gain access to their sites in chromatin requires the disruption or displacement of nucleosomes covering the promoter, signalled by the generation of a nuclease hypersensitive site. We characterise here the alterations in nucleosome structure caused by binding of the erythroid factor GATA-1 to a nucleosome carrying GATA-1 sites. DNase I and micrococcal nuclease probes show that GATA-1 binding causes extensive, cooperative breakage of the histone/DNA contacts to generate a complex very similar to that formed by the factor with free DNA. The only region which differs is confined to about 50 bp surrounding the nucleosome dyad axis which appears to be the domain of residual contact between the DNA and histone octamer. Despite considerable breakage of the histone/DNA contacts, the complex is completely stable in solution, and disruption of the nucleosome is entirely reversible: it is regenerated quantitatively upon removal of the transcription factor. Moreover, the histone 2A/2B component of the octamer does not exchange to external competitor. We suggest that formation of this complex may be a step in the generation of a fully hypersensitive site in vivo over regulatory elements containing GATA family binding sites.

Promoter regions of four Balbiani ring genes in Chironomus tentans exhibit a common salivary gland-specific chromatin organisation, which is independent of the rate of transcriptional initiation.

The Balbiani ring (BR) gene family consists of four closely related genes which show some differences in transcriptional regulation. Here we analyse the chromatin structure of the promoter regions of the four BR genes. In cells in which the genes are inactive, digestion of chromatin with micrococcal nuclease suggests for the BR1 gene that precisely positioned nucleosomes are present from position +200 to -600. In salivary gland cells, a distinctly different organisation of the promoter chromatin leads to the appearance of two sites that are hypersensitive to both DNase I and micrococcal nuclease, located at the approximate positions -50 and -240. Nuclease digestion experiments suggest the presence of a nucleosome in the region between the two hypersensitive sites, and nucleosome disruption at the transcription initiation site. The positions of these hypersensitive sites in all BR genes is unaffected by extensive modulation of the rate of transcriptional initiation, although some alterations in the intensity of the hypersensitive sites were observed. We also show that total nucleosome displacement from the transcribing template is seen only when the RNA polymerase density exceeds one polymerase molecule per 100 bp.

Association of the human papillomavirus type 11 E1 protein with histone H1.

The E1 and E2 proteins are the only virus-encoded factors required for human papillomavirus (HPV) DNA replication. The E1 protein is a DNA helicase responsible for initiation of DNA replication at the viral origin. Its recruitment to the origin is facilitated by binding to E2, for which specific recognition elements are located at the origin. The remaining replication functions for the virus, provided by the host cell's replication machinery, may be mediated by further interactions with E1 and E2. Histone H1 was identified as an HPV type 11 (HPV-11) E1-binding protein by far-Western blotting and by microsequence analyses of a 34-kDa protein purified by E1 affinity chromatography. E1 also bound in vitro to H1 isolated under native conditions in association with intact nucleosomes. In addition, E1 and H1 were coimmunoprecipitated by an E1 antiserum from a nuclear extract prepared from cells expressing recombinant E1. Bound H1 was displaced from HPV-11 DNA by the addition of E1, suggesting that E1 can promote replication initiation and elongation by alteration of viral chromatin structure and disruption of nucleosomes at the replication fork. Furthermore, a region of the HPV-11 genome containing the origin of replication was identified which had weaker affinity for H1 than that of the remaining genome. This result suggests that the presence of a DNA structure at or near the HPV origin facilitates initiation of DNA replication by exclusion of H1. These results are similar to those of studies of simian virus 40 DNA replication, in which a large T antigen-H1 interaction and an H1-resistant region at the origin of DNA replication have also been demonstrated.

Gal4p-mediated chromatin remodeling depends on binding site position in nucleosomes but does not require DNA replication.

Biochemical studies have demonstrated decreased binding of various proteins to DNA in nucleosome cores as their cognate sites are moved from the edge of the nucleosome to the pseudodyad (center). However, to date no study has addressed whether this structural characteristic of nucleosomes modulates the function of a transcription factor in living cells, where processes of DNA replication and chromatin modification or remodeling could significantly affect factor binding. Using a sensitive, high-resolution methyltransferase assay, we have monitored the ability of Gal4p in vivo to interact with a nucleosome at positions that are known to be inaccessible in nucleosome cores in vitro. Gal4p efficiently bound a single cognate site (UASG) centered at 41 bp from the edge of a positioned nucleosome, perturbing chromatin structure and inducing transcription. DNA binding and chromatin perturbation accompanying this interaction also occurred in the presence of hydroxyurea, indicating that DNA replication is not necessary for Gal4p-mediated nucleosome disruption. These data extend previous studies, which demonstrated DNA replication-independent chromatin remodeling, by showing that a single dimer of Gal4p, without the benefit of cooperative interactions that occur at complex wild-type promoters, is competent for invasion of a preestablished nucleosome. When the UASG was localized at the nucleosomal pseudodyad, relative occupancy by Gal4p, nucleosome disruption, and transcriptional activation were substantially compromised. Therefore, despite the increased nucleosome binding capability of Gal4p in cells, the precise translational position of a factor binding site in one nucleosome in an array can affect the ability of a transcriptional regulator to overcome the repressive influence of chromatin.

Retinoid receptor-induced alteration of the chromatin assembled on a ligand-responsive promoter in Xenopus oocytes.

Retinoic acid (RA) stimulates transcription from the retinoic acid receptor beta2 (RARbeta2) promoter in mammalian embryonal cells. Evidence by in vivo deoxyribonuclease I (DNase I) hypersensitivity assay indicates that RA treatment of these cells results in an alteration of chromatin structure in and near the promoter. To study the role of chromatin in RA-activated transcription, we assembled the RARbeta2 promoter into chromatin in Xenopus oocytes. Ectopic expression of RAR and retinoid X receptor (RXR) enhanced transcription without ligand, irrespective of whether chromatin was assembled in a replication-dependent or -independent manner, although ligand addition led to a further, marked increase in transcription. Moreover, expression of RAR and RXR, without ligand addition, induced DNase I-hypersensitive sites in the chromatin-assembled promoter. Furthermore, expression of RAR and RXR in oocytes led to local disruption of chromatin assembled over the promoter without ligand. Similar ligand-independent, but RXR/RAR-dependent nucleosomal disruption was observed in an in vitro chromatin reconstitution system using Drosophila embryonic extracts. Thus, unliganded receptors expressed in oocytes are capable of accessing to the chromatin-assembled promoter and activating transcription without ligand, indicating that chromatin assembly per se is not sufficient to reproduce ligand-dependent chromatin changes and promoter activation seen in mammalian cells. The oocyte system may serve as a model to study mechanisms of RA-dependent alterations of chromatin structure.