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SWI/SNF complex and nucleosome remodeling

The human SWI/SNF complex remodels nucleosome structure in an ATP-dependent manner, although the nature of this change has not been determined. hSWI/SNF and ATP are shown to generate an altered nucleosomal structure that is stable in the absence of SWI/SNF. This product has an altered sensitivity to digestion by DNAse, restriction enzymes, and micrococcal nuclease, and an increased affinity for GAL4. It has the same protein composition but is approximately twice the size of a normal nucleosome. Incubation of the altered nucleosome with hSWI/SNF converts this structure back to a standard nucleosome in an ATP-dependent process. These results suggest that hSWI/ SNF acts by facilitating an exchange between normal and altered, more accessible, nucleosome conformations (Schnitzler, 1998).

To understand the mechanisms by which the chromatin-remodeling SWI/SNF complex interacts with DNA and alters nucleosome organization, the SWI/SNF complex has been imaged 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 (Bazett-Jones, 1999).

RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle (containing DNA) to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule (Lorch, 1999).

Thus RSC, and by inference, SWI/SNF, is able to transfer a histone octamer from a nucleosome to naked DNA. As one naked region becomes protected, another, previously covered by a nucleosome, is exposed. The capacity of a chromatin-remodeling complex to disrupt a nucleosome completely is thus established. All that is required beyond the remodeling complex, nucleosome, and ATP is an acceptor for the histone octamer released. In these experiments, the acceptor is DNA. In vivo, DNA may also serve as an acceptor, for example a neighboring region of the chromosome or a region of an adjacent chromosome. It remains to be seen whether other nucleic acid or protein molecules function as octamer acceptors for chromatin remodeling as well. Histone octamer transfer by RSC evidently proceeds through the activated RSC-nucleosome complex. The involvement of this complex as an intermediate has implications for both the structure of the complex and the mechanism of the chromatin-remodeling process. It points to the occurrence in the complex of a region(s) of complete separation between histones and DNA. An invading DNA molecule may interact with histones in this region and then expand its contact to adjacent regions, displacing the resident DNA (Lorch, 1999).

The possible involvement of the altered nucleosome in histone octamer transfer appears unlikely. The altered nucleosome is formed upon removal of RSC and differs in structure from the nucleosome in the activated complex. It shows less perturbation of the histone-DNA interaction and displays physical characteristics of a nucleosome dimer. It may be formed by DNA released from the histones in one activated RSC-nucleosome complex binding to the histones exposed in another activated complex. Perhaps the closest equivalent to the mechanism envisaged here for histone octamer transfer is that demonstrated for transcription through a nucleosome by T7 RNA polymerase in vitro (Studitsky, 1994). DNA displaced from the octamer by the transcribing polymerase loops out and rebinds to the histone surface, preserving the structure of the nucleosome, but with retograde transfer of the octamer along the DNA. Loop formation and movement are driven by the energy-dependent advance of the polymerase along the DNA. It remains to be seen whether the reaction catalyzed by RSC results from a similar ATP-dependent translocation on DNA (Lorch, 1999).

The SWI-SNF complex participates in transcriptional activation at a step subsequent to activator binding. The ability of the transcriptional activator GAL4 to bind to a site in a positioned nucleosome is not appreciably impaired in swi mutant yeast cells. However, chromatin remodeling, which depends on a transcriptional activation domain, shows a considerable, although not complete, SWI-SNF dependence, suggesting that the SWI-SNF complex exerts its major effect at a step subsequent to activator binding. This idea was tested further by comparing the SWI-SNF dependence of a reporter gene based on the GAL10 promoter, which has an accessible upstream activating sequence and a nucleosomal TATA element, with that of a CYC1-lacZ reporter, which has a relatively accessible TATA element. The GAL10-based reporter gene shows a much stronger SWI-SNF dependence than does the CYC1-lacZ reporter with several different activators. Remarkably, transcription of the GAL10-based reporter by a GAL4-GAL11 fusion protein shows a nearly complete requirement for the SWI-SNF complex, strongly suggesting that SWI-SNF is needed to allow access of TFIID or the RNA polymerase II holoenzyme (Ryan, 1998).

The SWI-SNF complex is required for transcriptional activation in a step that depends on both the transcription factor activation domain and the promoter. One mechanism that could account for this dependency would be recruitment by activation domains, directly or indirectly, of an RNA polymerase II holoenzyme: this would include the SWI-SNF complex. However, the extent to which the SWI-SNF complex is associated with the RNA polymerase II holoenzyme in vivo remains to be resolved. If the role of the SWI-SNF complex is entirely to remodel chromatin in order to allow access to DNA by proteins involved in transcriptional activation, the nearly complete SWI-SNF dependence on activation also implies a strong requirement for chromatin remodeling for transcriptional activation at this promoter. Since GAL4 shows only partial SWI-SNF dependence at this promoter, it is inferred that GAL4 must be able to remodel chromatin by some alternative pathway. It is possible that activators are generally capable of recruiting more than one chromatin-remodeling activity via more than one pathway. For example, SWI-SNF might be recruited along with the RNA polymerase holoenzyme and the SAGA (a histone acetylating complex) via adaptor complexes. Other identified chromatin-remodeling activities might be recruited by pathways as yet undefined. Whether these many candidate activities are partly redundant or have evolved to operate at specific promoters or during specific steps of transcriptional activation or are involved in other processes which must contend with nucleosomal templates, such as replication and repair, remains to be determined. Taken together, these results demonstrate that chromatin remodeling in vivo can occur along avenues that are both SWI-SNF-dependent and -independent: these data suggest that the SWI-SNF complex exerts its major effect in transcriptional activation at a step subsequent to transcriptional activator-promoter recognition (Ryan, 1998).

Gene activation in eukaryotes requires chromatin remodeling complexes like Swi/Snf and histone acetylases like SAGA. How these factors are recruited to promoters is not yet understood. Using surface plasmon resonance technology (CHIP), recruitment of Swi/Snf, SAGA, the repressor Ash1p, and transcription factors Swi5p and the cell cycle-regulatory transcription factor SBF were all measured to the HO endonuclease promoter as cells progressed through the yeast cell cycle. Swi5p's entry into nuclei at the end of anaphase recruits Swi/Snf, which then recruits SAGA. These two factors then facilitate SBF's binding. Ash1p, which only accumulates in daughter cell nuclei, binds to HO soon after Swi5p and aborts recruitment of Swi/Snf, SAGA, and SBF. Swi5p remains at HO for only 5 min. Swi/Snf's and SAGA's subsequent persistence at HO is self sustaining and constitutes an 'epigenetic memory' of HO's transient interaction with Swi5p (Cosma, 1999).

Are transcription factors with diverse DNA binding domains able to exploit nucleosome disruption by SWI/SNF? To test this, an investigation was made into the possible mechanisms by which the SWI/SNF complex differentially regulates different genes. In addition to GAL4-VP16, the SWI/SNF complex stimulates nucleosome binding by the Zn2+ fingers of Sp1, the basic helix-loop-helix domain of USF, and the rel domain of NF-kappaB. In each case SWI/SNF action results in the formation of a stable factor-nucleosome complex that persists after the detachment of SWI/SNF from the nucleosome. Thus, stimulation of factor binding by SWI/SNF appears to be universal. The degree of SWI/SNF stimulation of nucleosome binding by a factor appears to be inversely related to the extent that binding is inhibited by the histone octamer. Cooperative binding of 5 GAL4-VP16 dimers to a 5-site nucleosome enhances GAL4 binding relative to a single-site nucleosome, but this also reduces the degree of stimulation by SWI/SNF. The SWI/SNF complex increases the affinity of 5 GAL4-VP16 dimers for nucleosomes equal to that of DNA but no further. Similarly, multimerized NF-kappaB sites enhance nucleosome binding by NF-kappaB and reduce the stimulatory effect of SWI/SNF. Thus, cooperative binding of factors to nucleosomes is partially redundant with the function of the SWI/SNF complex (Utley, 1997).

To investigate the mechanism of SWI/SNF action, the pathway by which SWI/SNF stimulates formation of transcription factor-bound nucleosome core complexes has been analyzed. The SWI/SNF complex binds directly to nucleosome cores and uses the energy of ATP hydrolysis to disrupt histone/DNA interactions, altering the preferred path of DNA bending around the histone octamer. This disruption occurs without dissociating the DNA from the surface of the histone octamer. ATP-dependent disruption of nucleosomal DNA by SWI/SNF generates an altered nucleosome core conformation that can persist for an extended period after detachment of the SWI/SNF complex. This disrupted conformation retains an enhanced affinity for the transcription factor GAL4-AH. Thus, ATP-dependent nucleosome core disruption and enhanced binding of the transcription factor can be temporally separated. These results indicate that SWI/SNF can act transiently in the remodeling of chromatin structure, even before interactions of transcription factors (Cote, 1998).

Rotational phasing of DNA sequences on nucleosome cores is thought to largely result from intrinsic curvatures or anisotrophic flexibility of DNA sequences, which enhances the affinity of DNA sequences for histone octamers. Thus, the action of SWI/SNF in stimulating transcription factor binding might be explained by the complex reducing of the curvature of DNA sequences around the nucleosome core, thereby, reducing the strength of DNA interactions with the histone octamer and enhancing the affinity of DNA sites for transcription factors. Such a model is consistent with the observation that the human SWI/SNF complex reduces the number of stable supercoils in nucleosome-assembled plasmid DNA and is likely related to the DNA binding properties of SWI/SNF, which resemble those of high mobility group-box proteins and are capable of inducing positive supercoils in naked DNA (in the presence of Escherichia coli topoisomerase I). A similar interaction of SWI/SNF with nucleosomal DNA might require the energy of ATP hydrolysis to effect an allosteric change in nucleosome core structure. Importantly, this does not seem to involve a helicase activity of the SWI/SNF complex. The purified SWI/SNF complex does not appear to contain helicase activity; single-stranded regions are not detectable in SWI/SNF disrupted nucleosome cores (Cote, 1998 and references).

The continued accumulation of mechanistic data prompts a revision of a previously proposed model for SWI/SNF disruption of nucleosome cores and stimulation of activator binding. Earlier it was suggested that SWI/SNF action might provoke the loss of one or two H2A/H2B dimers from the core particle, stabilizing Gal4-AH-nucleosome core interaction. However, the data presented here showing the reversibility of the disrupted nucleosome core conformation argues against a loss of histone components during SWI/SNF disruption. These data suggest that SWI/SNF disrupts nucleosome cores primarily by perturbing histone/DNA interactions altering the path of the DNA around the core particle, without the eviction of histones. In addition to SWI/SNF disruption, actual displacement of histones appears to require further nucleosome core destabilization, as provided by the binding of multiple GAL4-AH dimers (Cote, 1998).

The size and subunit complexity of the SWI/SNF complex suggest that it may perform multiple functions and that its in vivo activity may differ in important details from the activities observed in vitro thus far. For example, the mechanisms by which the SWI/SNF complex might be targeted to specific chromosomal loci remains a topic of intense interest. Likely candidates for involvement in targeting of the complex includes interactions that have been detected between the SWI/SNF complex and holo-RNA polymerase, the glucocorticoid receptor, the retinoblastoma protein, and HIV-1 integrase. The biochemical studies presented here and in earlier reports support the notion that once recruited to a promoter or enhancer, the SWI/SNF complex has the potential to enhance nucleosome binding by a wide range of transcription factors. Enhanced nucleosome binding by transcription factors can occur subsequent to SWI/SNF binding and nucleosome disruption because of the persistence of the disrupted state of the nucleosome core. This observation clearly illustrates that the stimulation of transcription factor binding is caused by the disrupted state of the nucleosome core and does not require transcription factor-SWI/SNF interactions. Thus, the ATP requirement for SWI/SNF function in vitro is to mediate disruption of the nucleosome cores and is only indirectly required to stimulate transcription factor binding. The persistence of the SWI/SNF-disrupted nucleosome core conformation can provide a window of opportunity for enhanced transcription factor binding that extends beyond the actual interaction of the SWI/SNF complex. In the in vitro system used here, the disrupted state of the nucleosome cores persists for up to 4 hr before reverting to the original conformation, with its low affinity for transcription factors. The persistence of the SWI/SNF-disrupted nucleosome conformation may be a regulated event in vivo. For example, the reversibility of the disrupted conformation might be enhanced by transcriptional repressors or nucleosome-nucleosome interaction. Moreover, histone modifications thought to be involved in transcriptional activity (i.e., histone acetylation) might extend the persistence of the SWI/SNF-disrupted nucleosome conformation (Cote, 1998).

The assembly of transcriptional regulatory DNA sequences into chromatin plays a fundamental role in modulating gene expression. The promoter of the mouse mammary-tumour virus (MMTV) is packaged into a regular array of nucleosomes when it becomes stably integrated into mammalian chromosomes, and has been used to investigate the relationship between chromatin architecture and transcriptional activation by the hormone-bound glucocorticoid and progesterone receptors. In mammalian cells that express both of these receptors, the progesterone receptor activates transcription from transiently transfected MMTV DNA but not from organized chromatin templates. The activated progesterone receptor inhibits the chromatin remodelling and consequent transcriptional stimulation that is mediated by the glucocorticoid receptor. The mechanism of this inhibition was investigated by characterizing the interaction of the glucocorticoid receptor with transcriptional co-activator and chromatin remodelling protein complexes. When this receptor is prevented from interacting with the hBRG1/BAF chromatin remodelling complex, it can activate transcription from transiently transfected DNA but not from organized chromatin templates. T results indicate that it may be possible to separate the transcriptional activation and chromatin remodelling activities of proteins that interact with hormone receptors (Fryer, 1998).

The dynamic assembly and remodeling of eukaryotic chromosomes facilitate fundamental cellular processes such as DNA replication and gene transcription. The repeating unit of eukaryotic chromosomes is the nucleosome core, consisting of DNA wound about a defined octamer of histone proteins. Two enzymatic processes that regulate transcription by targeting elements of the nucleosome include ATP-dependent nucleosome remodeling and reversible histone acetylation. The histone deacetylases, however, are unable to deacetylate oligonucleosomal histones in vitro. The protein complexes that mediate ATP-dependent nucleosome remodeling and histone acetylation/deacetylation in the regulation of transcription have been considered to be different, although it has recently been suggested that these activities might be coupled. A novel ATP-dependent nucleosome remodeling activity has been identified and functionally characterized that is part of an endogenous human histone deacetylase complex. This activity is derived from the CHD3 and CHD4 proteins that contain helicase/ATPase domains found in SWI2-related chromatin remodeling factors, and facilitates the deacetylation of oligonucleosomal histones in vitro. This complex is referred to as the nucleosome remodeling and deacetylating (NRD) complex. These results establish a physical and functional link between the distinct chromatin-modifying activities of histone deacetylases and nucleosome remodeling proteins (Tong, 1998).

The structure of the SWI/SNF-remodeled nucleosome was characterized with single base-pair resolution by mapping the contacts of specific histone fold residues with nucleosomal DNA. SWI/SNF peels up to 50 bp of DNA from the edge of the nucleosome, translocates the histone octamer beyond the DNA ends via a DNA bulge propagation mechanism, and promotes the formation of an intramolecular DNA loop between the nucleosomal entry and exit sites. This stable altered nucleosome conformation also exhibits alterations in the distance between contacts of specific histone residues with DNA and higher electrophoretic and sedimentation mobility, consistent with a more compact molecular shape. SWI/SNF converts a nucleosome to the altered state in less than one second, hydrolyzing fewer than 10 ATPs per event (Kassabov, 2003).

High-resolution mapping of histone-DNA contacts of the stable remodeled nucleosome provided significant insight into the mechanism of SWI/SNF remodeling by demonstrating that DNA is directionally unraveled from the edge of the nucleosome up to points of stronger histone-DNA contacts in the interior of the core particle between SHL ± 4.5 and the dyad axis. The peeling of DNA from the surface of the octamer stalls or pauses at these positions so that these species become the principal remodeled products. The data support a model in which DNA peeled from the nucleosomal edge by SWI/SNF is bound to new sites closer to the dyad axis and the resulting DNA bulge propagates around the surface of the nucleosome until it reaches the opposite end of the nucleosome. Sliding of the nucleosome off the end of DNA leaves a portion of the surface of the nucleosome exposed that is rapidly rebound by noncontiguous DNA to create an entry/exit DNA loop traversing the dyad axis of the nucleosome. It is proposed that SWI/SNF remodeling is, thus, a highly coordinated process entailing the sequential unwrapping of DNA from the nucleosomal edge, sliding, and the reassociation of the spooled out DNA with the newly exposed octamer surface. Nucleosomal DNA is rendered accessible in this process in two ways: (1) DNA is made transiently accessible as it is initially peeled off the surface of the nucleosome and propagates as a bulge on the octamer surface; (2) particular DNA regions are rendered more stably accessible by being located within the entry/exit-site DNA loop that persists after removal of SWI/SNF (Kassabov, 2003).

Consistent with this model, it has been found that SWI/SNF remodeling proceeds significantly faster and more efficiently than previously thought. It has been suggested that a single remodeling event could occur in 0.5-3 min and require the hydrolysis of 50-3000 ATPs. In contrast, less than 10 ATP molecules are hydrolyzed per remodeling/conversion event, which can be completed within one second and entails the unraveling of up to 50 bp of DNA from the histone octamer (Kassabov, 2003).

Although it is formally possible that the initial peeling of nucleosomal DNA could be caused by superhelical tension due to overtwisting of DNA, a model is favored in which an ATP-dependent conformational change in the SWI/SNF complex bound to the dyad and entry/exit nucleosomal sites peels DNA from the nucleosomal edge and pushes it into the interior of the particle. The results argue against models in which the remodeling complex tracks along linker DNA and pushes it into the nucleosome, since such a mechanism could explain octamer sliding along DNA but not the extensive nucleosome unraveling and octamer repositioning far outside the DNA ends observed in this study (Kassabov, 2003).

p63 and Brg1 control developmentally regulated higher-order chromatin remodelling

Chromatin structural states and their remodelling, including higher-order chromatin folding and three-dimensional (3D) genome organisation, play an important role in the control of gene expression. The role of 3D genome organisation in the control and execution of lineage-specific transcription programmes during the development and differentiation of multipotent stem cells into specialised cell types remains poorly understood. This study shows that substantial remodelling of the higher-order chromatin structure of the epidermal differentiation complex (EDC), a keratinocyte lineage-specific gene locus on mouse chromosome 3, occurs during epidermal morphogenesis. During epidermal development, the locus relocates away from the nuclear periphery towards the nuclear interior into a compartment enriched in SC35-positive nuclear speckles. Relocation of the EDC locus occurs prior to the full activation of EDC genes involved in controlling terminal keratinocyte differentiation and is a lineage-specific, developmentally regulated event controlled by transcription factor p63, a master regulator of epidermal development. It was also shown that, in epidermal progenitor cells, p63 directly regulates the expression of the ATP-dependent chromatin remodeller Brg1, which binds to distinct domains within the EDC and is required for relocation of the EDC towards the nuclear interior. Furthermore, Brg1 also regulates gene expression within the EDC locus during epidermal morphogenesis. Thus, p63 and its direct target Brg1 play an essential role in remodelling the higher-order chromatin structure of the EDC and in the specific positioning of this locus within the landscape of the 3D nuclear space, as required for the efficient expression of EDC genes in epidermal progenitor cells during skin development (Mardaryev, 2014).

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brahma: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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