Search tips
Search criteria

Results 1-25 (812442)

Clipboard (0)

Related Articles

1.  ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling 
eLife  2013;2:e00863.
Chromatin assembly involves the combined action of ATP-dependent motor proteins and histone chaperones. Because motor proteins in chromatin assembly also function as chromatin remodeling factors, we investigated the relationship between ATP-driven chromatin assembly and chromatin remodeling in the generation of periodic nucleosome arrays. We found that chromatin remodeling-defective Chd1 motor proteins are able to catalyze ATP-dependent chromatin assembly. The resulting nucleosomes are not, however, spaced in periodic arrays. Wild-type Chd1, but not chromatin remodeling-defective Chd1, can catalyze the conversion of randomly-distributed nucleosomes into periodic arrays. These results reveal a functional distinction between ATP-dependent nucleosome assembly and chromatin remodeling, and suggest a model for chromatin assembly in which randomly-distributed nucleosomes are formed by the nucleosome assembly function of Chd1, and then regularly-spaced nucleosome arrays are generated by the chromatin remodeling activity of Chd1. These findings uncover an unforeseen level of specificity in the role of motor proteins in chromatin assembly.
eLife digest
In many cells, genomic DNA is wrapped around proteins known as histones to produce particles called nucleosomes. These particles then join together—like beads on a string—to form a highly periodic structure called chromatin. In the nucleus, chromatin is further folded and condensed into chromosomes. However, many important processes, including the replication of DNA and the transcription of genes, require access to the DNA. The cell must therefore be able to disassemble chromatin and remove the histones, and then, once these processes are complete, to reassemble the chromatin. Enzymes known as chromatin assembly factors are responsible for the disassembly and reassembly of chromatin.
There are two main types of chromatin assembly factors in eukaryotic cells (i.e., cells with nuclei)—histone chaperones and motor proteins. The histone chaperones escort histones from the cytoplasm, where they are made, to the nucleus. The motor proteins—using energy supplied by ATP molecules—then catalyze the formation of nucleosomes. This involves two activities: the motor proteins assemble nucleosomes by helping the DNA to wrap around the histones, and they also remodel chromatin by altering the positions of nucleosomes along the DNA to ensure that they are periodic—that is, regularly spaced.
A conserved motor protein called Chd1 performs chromatin assembly and remodeling in eukaryotic cells. Chd1 works in conjunction with histone chaperones—both are needed for chromatin assembly, and so are DNA, histones and ATP. However, whether or not chromatin assembly and chromatin remodeling by Chd1 are identical or distinct processes is not well understood.
Torigoe et al. have now discovered a mutant Chd1 protein that has nucleosome assembly activity (i.e., it can make nucleosomes) but cannot remodel chromatin (i.e., it is unable to move nucleosomes), and thus have demonstrated that these two processes are functionally distinct. Torigoe et al. additionally have found that the mutant Chd1 proteins produce randomly distributed nucleosomes rather than the periodic arrays normally found in chromatin. Further analysis then revealed that the wild-type Chd1 protein, which can remodel chromatin, is able to convert randomly distributed nucleosomes into periodic arrays.
These findings have led to a new model for chromatin assembly in which Chd1 initially generates randomly distributed nucleosomes (via its assembly function), and then converts them into periodic arrays of nucleosomes (via its remodeling function). Together, these studies shed light on the mechanisms by which chromatin is created and manipulated in cells.
PMCID: PMC3748710  PMID: 23986862
chromatin assembly; chromatin remodeling; Chd1; D. melanogaster; S. cerevisiae
2.  SWI-SNF-Mediated Nucleosome Remodeling: Role of Histone Octamer Mobility in the Persistence of the Remodeled State 
Molecular and Cellular Biology  2000;20(9):3058-3068.
SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling–restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.
PMCID: PMC85587  PMID: 10757790
3.  Octamer Transfer and Creation of Stably Remodeled Nucleosomes by Human SWI-SNF and Its Isolated ATPases 
Molecular and Cellular Biology  2000;20(17):6380-6389.
Chromatin remodeling complexes help regulate the structure of chromatin to facilitate transcription. The multisubunit human (h) SWI-SNF complex has been shown to remodel mono- and polynucleosome templates in an ATP-dependent manner. The isolated hSWI-SNF ATPase subunits BRG1 and hBRM also have these activities. The intact complex has been shown to produce a stable remodeled dimer of mononucleosomes as a product. Here we show that the hSWI-SNF ATPases alone can also produce this product. In addition, we show that hSWI-SNF and its ATPases have the ability to transfer histone octamers from donor nucleosomes to acceptor DNA. These two reactions are characterized and compared. Our results are consistent with both products of SWI-SNF action being formed as alternative outcomes of a single remodeling mechanism. The ability of the isolated ATPase subunits to catalyze these reactions suggests that these subunits play a key role in determining the mechanistic capabilities of the SWI-SNF family of remodeling complexes.
PMCID: PMC86113  PMID: 10938115
4.  Chromatin Remodelers Act Globally Sequence Positions Nucleosomes Locally 
The precise placement of nucleosomes has large regulatory effects on gene expression. Recent work suggests that nucleosome placement is regulated in part by the affinity of the underlying DNA sequence for the histone octamer. Nucleosome locations are also regulated by several different ATP-dependent chromatin remodeling enzymes. This raises the question whether DNA sequence influences the activity of chromatin remodeling enzymes. DNA sequence could most simply regulate nucleosome remodeling through its effect on nucleosome stability. In such a model, unstable nucleosomes would be remodeled faster than stable nucleosomes. It is also possible that certain DNA elements could regulate remodeling by inhibiting the interaction of nucleosomes with the remodeling enzyme. A third possibility is that DNA sequence could regulate the outcome of remodeling by influencing how reaction intermediates collapse into a particular set of stable nucleosomal positions. Here we dissect the contribution from these potential mechanisms to the activities of yeast RSC and human ACF, which are representative members of two major classes of remodeling complexes. We find that varying the histone-DNA affinity over three orders of magnitude has negligible effects on the rates of nucleosome remodeling and ATP hydrolysis by these two enzymes. This suggests that the rate-limiting step for nucleosome remodeling may not involve the disruption of histone-DNA contacts. We further find that a specific curved DNA element previously hypothesized to inhibit ACF activity does not inhibit substrate binding or remodeling by ACF. The element however, does influence the distribution of nucleosome positions generated by ACF. Our data support a model in which remodeling enzymes move nucleosomes to new locations by a general sequence-independent mechanism. However, consequent to the rate-limiting remodeling step, the local DNA sequence promotes a collapse of remodeling intermediates into highly resolved positions that are dictated by thermodynamic differences between adjacent positions.
PMCID: PMC2813840  PMID: 19450608
nucleosome positioning; histone-DNA affinity; chromatin remodeling; human ACF; RSC
5.  Disparity in the DNA translocase domains of SWI/SNF and ISW2 
Nucleic Acids Research  2012;40(10):4412-4421.
An ATP-dependent DNA translocase domain consisting of seven conserved motifs is a general feature of all ATP-dependent chromatin remodelers. While motifs on the ATPase domains of the yeast SWI/SNF and ISWI families of remodelers are highly conserved, the ATPase domains of these complexes appear not to be functionally interchangeable. We found one reason that may account for this is the ATPase domains interact differently with nucleosomes even though both associate with nucleosomal DNA 17–18 bp from the dyad axis. The cleft formed between the two lobes of the ISW2 ATPase domain is bound to nucleosomal DNA and Isw2 associates with the side of nucleosomal DNA away from the histone octamer. The ATPase domain of SWI/SNF binds to the same region of nucleosomal DNA, but is bound outside of the cleft region. The catalytic subunit of SWI/SNF also appears to intercalate between the DNA gyre and histone octamer. The altered interactions of SWI/SNF with DNA are specific to nucleosomes and do not occur with free DNA. These differences are likely mediated through interactions with the histone surface. The placement of SWI/SNF between the octamer and DNA could make it easier to disrupt histone–DNA interactions.
PMCID: PMC3378860  PMID: 22298509
6.  Chromatin is an ancient innovation conserved between Archaea and Eukarya 
eLife  2012;1:e00078.
The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved −1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.
eLife digest
Single-celled microorganisms called archaea are one of the three domains of cellular life, along with bacteria and eukaryotes. Archaea are similar to bacteria in that they do not have nuclei, but genetically they have more in common with eukaryotes. Archaea are found in a wide range of habitats including the human colon, marshlands, the ocean and extreme environments such as hot springs and salt lakes.
It has been known since the 1990s that the DNA of archaea is wrapped around histones to form complexes that closely resemble the nucleosomes found in eukaryotes, albeit with four rather than eight histone subunits. Nucleosomes are the fundamental units of chromatin, the highly-ordered and compact structure that all the DNA in a cell is packed into. Now we know exactly how many nucleosomes are present in a given cell for some eukaryotes, notably yeast, and to a good approximation we know the position of each nucleosome during a variety of metabolic states and physiological conditions. We can also quantify the nucleosome occupancy, which is measure of the length of time that the nucleosomes spend in contact with the DNA: this is a critical piece of information because it determines the level of access that other proteins, including those that regulate gene expression, have to the DNA. These advances have been driven in large part by advances in technology, notably high-density microarrays for genome wide-studies of nucleosome occupancy, and massively parallel sequencing for direct nucleosome sequencing.
Ammar et al. have used these techniques to explore how the DNA of Haloferax volcanii, a species of archaea that thrives in the hyper-salty waters of the Dead Sea, is organized on a genome-wide basis. Despite some clear differences between the genomes of archaea and eukaryotes—for example, genomic DNA is typically circular in archaea and linear in eukaryotes—they found that the genome of Hfx. volcanii is organized into chromatin in a way that is remarkably similar to that seen in all eukaryotic genomes studied to date. This is surprising given that the chromatin in eukaryotes is confined to the nucleus, whereas there are no such constraints in archaea. In particular, Ammar et al. found that those regions of the DNA near the ends of genes that mark where the transcription of the DNA into RNA should begin and end contain have lower nucleosome occupancy than other regions. Moreover, the overall level of occupancy in Hfx. volcanii was twice that of eukaryotes, which is what one would expect given that nucleosomes in archaea contain half as many histone subunits as nucleosomes in eukaryotes. Ammar et al. also confirmed that that the degree of nucleosome occupancy is correlated with gene expression.
These two findings—the similarities between the chromatin in archaea and eukaryotes, and the correlation between nucleosome occupancy and gene expression in archaea—raise an interesting evolutionary possibility: the initial function of nucleosomes and chromatin formation might have been for the regulation of gene expression rather than the packaging of DNA. This is consistent with two decades of research that has shown that there is an extraordinary and complex relationship between the structure of chromatin and the process of gene expression. It is possible, therefore, that as the early eukaryotes evolved, nucleosomes and chromatin started to package DNA into compact structures that, among other things, helped to prevent DNA damage, and that this subsequently enabled the early eukaryotes to flourish.
PMCID: PMC3510453  PMID: 23240084
Haloferax volcanii; Nucleosome; Chromatin; Transcriptome; RNA-seq; Archaea; Other
7.  The Mi-2 Homolog Mit1 Actively Positions Nucleosomes within Heterochromatin To Suppress Transcription 
Molecular and Cellular Biology  2014;34(11):2046-2061.
Mit1 is the putative chromatin remodeling subunit of the fission yeast Snf2/histone deacetylase (HDAC) repressor complex (SHREC) and is known to repress transcription at regions of heterochromatin. However, how Mit1 modifies chromatin to silence transcription is largely unknown. Here we report that Mit1 mobilizes histone octamers in vitro and requires ATP hydrolysis and conserved chromatin tethering domains, including a previously unrecognized chromodomain, to remodel nucleosomes and silence transcription. Loss of Mit1 remodeling activity results in nucleosome depletion at specific DNA sequences that display low intrinsic affinity for the histone octamer, but its contribution to antagonizing RNA polymerase II (Pol II) access and transcription is not restricted to these sites. Genetic epistasis analyses demonstrate that SHREC subunits and the transcription-coupled Set2 histone methyltransferase, which is involved in suppression of cryptic transcription at actively transcribed regions, cooperate to silence heterochromatic transcripts. In addition, we have demonstrated that Mit1's remodeling activity contributes to SHREC function independently of Clr3's histone deacetylase activity on histone H3 K14. We propose that Mit1 is a chromatin remodeling factor that cooperates with the Clr3 histone deacetylase of SHREC and other chromatin modifiers to stabilize heterochromatin structure and to prevent access to the transcriptional machinery.
PMCID: PMC4019058  PMID: 24662054
8.  Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes 
Nature  2012;492(7428):280-284.
Chromatin remodeling complexes (CRCs) mobilize nucleosomes to mediate the access of DNA-binding factors to their sites in vivo. These CRCs contain a catalytic subunit that bears an ATPase/DNA translocase domain, and flanking regions that bind nucleosomal epitopes1. A central question is whether and how these flanking regions regulate ATP hydrolysis or the coupling of hydrolysis to DNA translocation, to affect nucleosome sliding efficiency. ISWIfamily CRCs contain ISWI2, which utilizes its ATPase/DNA translocase domain to pump DNA around the histone octamer to enable sliding3-7_ENREF_13. ISWI is positively regulated by two ‘activating’ nucleosomal epitopes: the ‘basic patch’ on the H4 tail, and extranucleosomal (linker) DNA8-13. Previous work defined the HSS domain in the ISWI C-terminus that binds linker DNA, needed for ISWI activity14,15. Here, we define two new, conserved, and separate regulatory regions on Drosophila ISWI, AutoN and NegC, that negatively regulate ATP hydrolysis (AutoN) or the coupling of ATP hydrolysis to productive DNA translocation (NegC). Rather than ‘activating’, the two aforementioned nucleosomal epitopes actually inhibit the negative regulation of AutoN and NegC. Remarkably, mutation/removal of AutoN and NegC enables significant nucleosome sliding without the H4 ‘basic patch’ or extranucleosomal DNA, or the HSS domain – converting ISWI to biochemical attributes of SWI/SNF-family ATPases. Thus, the ISWI ATPase catalytic core is an intrinsically-active DNA translocase which conducts nucleosome sliding, onto which selective ‘inhibition-of-inhibition’ modules are placed, to help ensure that remodeling occurs only in the presence of proper nucleosomal epitopes. This supports a general concept for the specialization of chromatin remodeling ATPases, where specific regulatory modules adapt an ancient active DNA translocase to conduct particular tasks only on the appropriate chromatin landscape.
PMCID: PMC3631562  PMID: 23143334
9.  Divergent human remodeling complexes remove nucleosomes from strong positioning sequences 
Nucleic Acids Research  2009;38(2):400-413.
Nucleosome positioning plays a major role in controlling the accessibility of DNA to transcription factors and other nuclear processes. Nucleosome positions after assembly are at least partially determined by the relative affinity of DNA sequences for the histone octamer. Nucleosomes can be moved, however, by a class of ATP dependent chromatin remodeling complexes. We recently showed that the human SWI/SNF remodeling complex moves nucleosomes in a sequence specific manner, away from nucleosome positioning sequences (NPSes). Here, we compare the repositioning specificity of five remodelers of diverse biological functions (hSWI/SNF, the SNF2h ATPase and the hACF, CHRAC and WICH complexes than each contain SNF2h) on 5S rDNA, MMTV and 601 NPS polynucleosomal templates. We find that all five remodelers act similarly to reduce nucleosome occupancy over the strongest NPSes, an effect that could directly contribute to the function of WICH in activating 5S rDNA transcription. While some differences were observed between complexes, all five remodelers were found to result in surprisingly similar nucleosome distributions. This suggests that remodeling complexes may share a conserved repositioning specificity, and that their divergent biological functions may largely arise from other properties conferred by complex-specific subunits.
PMCID: PMC2811002  PMID: 19906705
10.  ATP-Dependent Chromatin Remodeling by Cockayne Syndrome Protein B and NAP1-Like Histone Chaperones Is Required for Efficient Transcription-Coupled DNA Repair 
PLoS Genetics  2013;9(4):e1003407.
The Cockayne syndrome complementation group B (CSB) protein is essential for transcription-coupled DNA repair, and mutations in CSB are associated with Cockayne syndrome—a devastating disease with complex clinical features, including the appearance of premature aging, sun sensitivity, and numerous neurological and developmental defects. CSB belongs to the SWI2/SNF2 ATP–dependent chromatin remodeler family, but the extent to which CSB remodels chromatin and whether this activity is utilized in DNA repair is unknown. Here, we show that CSB repositions nucleosomes in an ATP–dependent manner in vitro and that this activity is greatly enhanced by the NAP1-like histone chaperones, which we identify as new CSB–binding partners. By mapping functional domains and analyzing CSB derivatives, we demonstrate that chromatin remodeling by the combined activities of CSB and the NAP1-like chaperones is required for efficient transcription-coupled DNA repair. Moreover, we show that chromatin remodeling and repair protein recruitment mediated by CSB are separable activities. The collaboration that we observed between CSB and the NAP1-like histone chaperones adds a new dimension to our understanding of the ways in which ATP–dependent chromatin remodelers and histone chaperones can regulate chromatin structure. Taken together, the results of this study offer new insights into the functions of chromatin remodeling by CSB in transcription-coupled DNA repair as well as the underlying mechanisms of Cockayne syndrome.
Author Summary
Cockayne syndrome is a devastating inherited disease; the average life span of those afflicted is 12 years. Cockayne syndrome patients have features of premature aging, are highly sensitive to sunlight, and suffer from numerous developmental and neurological disorders. The majority of Cockayne syndrome patients have mutations in the CSB protein; however, how these mutations can lead to Cockayne syndrome is largely unknown. CSB is essential for transcription-coupled DNA repair—a process that preferentially removes bulky DNA lesions that stall transcription, such as those created by ultraviolet light. In eukaryotes, DNA is packaged into nucleosomes, which consists of DNA wrapped around a set of core histone proteins, and nucleosomes can create barriers to the DNA repair process. In this study, we found that CSB can slide histones along DNA. We also found that histone chaperones, proteins that accept and donate histones, greatly facilitate this process. Importantly, we show that CSB derivatives that are unable to move nucleosomes or collaborate with histone chaperones cannot repair UV-induced DNA lesions. Our study reveals that nucleosome remodeling by CSB is important for transcription-coupled DNA repair and suggests that an inability to efficiently mobilize nucleosomes might contribute to the underlying mechanism of Cockayne syndrome.
PMCID: PMC3630089  PMID: 23637612
11.  Histone Modifications Influence the Action of Snf2 Family Remodelling Enzymes by Different Mechanisms 
Journal of Molecular Biology  2007;374(3):563-579.
Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.
PMCID: PMC2279226  PMID: 17949749
Histone; Acetylation; Snf2; Nucleosome; Chromatin
12.  Nuclear receptors and chromatin remodeling machinery 
Molecular and cellular endocrinology  2007;265-266:162-167.
Eukaryotic genetic information is stored within the association of DNA and histone proteins resulting in a dynamic polymer called chromatin. The fundamental structural unit of chromatin is the nucleosome which consists of ~146 bp of DNA wrapped around an octamer of histones containing two copies each of four core histones, H2A, H2B, H3 and H4. It is this DNA/protein fiber that transcription factors and other agents of chromatin metabolism must access and regulate. We have developed model systems to study the mechanisms by which steroid receptors control physiological activities by regulating gene expression within a higher order chromatin organization. Our studies have focused on the glucocorticoid receptor and its ability to remodel chromatin which is mediated by the BRG1 complex. Using novel cell systems, we demonstrate that GR-mediated transactivation from chromatin templates requires BRG1 remodeling activity and that other ATP-dependent remodeling proteins cannot substitute for this activity.
PMCID: PMC3582388  PMID: 17240047
GR; Chromatin; Epigenetics; SWI/SNF; BRG1; Transcriptional activation
13.  FRET-based methods to study ATP-dependent changes in chromatin structure 
Methods (San Diego, Calif.)  2007;41(3):291-295.
DNA packaging into chromatin imposes several levels of regulation on the central nuclear processes of DNA replication, recombination, repair and transcription. ATP-dependent chromatin remodeling enzymes play a critical role in this regulation by altering the accessibility of nucleosomal DNA. Remodeling can result in large-scale changes in chromatin, such as the formation of heterochromatin, or smaller changes in exposure or occlusion of specific DNA regions. To understand the mechanisms of chromatin remodeling, we report a FRET-based method to follow remodeling of a single histone octamer on DNA. This technique provides a non-perturbing, solution-based approach to quantitatively track the movement of DNA with respect to the octamer in real-time. The method can easily be altered to examine other conformational changes within the nucleosome, and is applicable to study the enzymatic activity of several classes of chromatin-remodeling complexes.
PMCID: PMC1941662  PMID: 17309839
chromatin; ATP-dependent chromatin-remodeling; nucleosome; FRET; histone; real-time; SNF2h
14.  Critical Role for the Histone H4 N Terminus in Nucleosome Remodeling by ISWI 
Molecular and Cellular Biology  2001;21(3):875-883.
The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.
PMCID: PMC86678  PMID: 11154274
15.  Structure of a RSC–nucleosome complex and insights into chromatin remodeling 
Nature structural & molecular biology  2008;15(12):1272-1277.
ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict or restructure nucleosomes. A structure of a RSC–nucleosome complex with a nucleosome determined by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into solution as required for remodeling, but the structure of the RSC–nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC–nucleosome structure and published biochemical information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.
PMCID: PMC2659406  PMID: 19029894
16.  Repressive LTR Nucleosome Positioning by the BAF Complex Is Required for HIV Latency 
PLoS Biology  2011;9(11):e1001206.
The SWI/SNF BAF chromatin remodeling complex generates a repressive nucleosome structure at the HIV LTR conducive to establishment and maintenance of HIV latency, while PBAF augments HIV transcription.
Persistence of a reservoir of latently infected memory T cells provides a barrier to HIV eradication in treated patients. Several reports have implicated the involvement of SWI/SNF chromatin remodeling complexes in restricting early steps in HIV infection, in coupling the processes of integration and remodeling, and in promoter/LTR transcription activation and repression. However, the mechanism behind the seemingly contradictory involvement of SWI/SNF in the HIV life cycle remains unclear. Here we addressed the role of SWI/SNF in regulation of the latent HIV LTR before and after transcriptional activation. We determined the predicted nucleosome affinity of the LTR sequence and found a striking reverse correlation when compared to the strictly positioned in vivo LTR nucleosomal structure; sequences encompassing the DNase hypersensitive regions displayed the highest nucleosome affinity, while the strictly positioned nucleosomes displayed lower affinity for nucleosome formation. To examine the mechanism behind this reverse correlation, we used a combinatorial approach to determine DNA accessibility, histone occupancy, and the unique recruitment and requirement of BAF and PBAF, two functionally distinct subclasses of SWI/SNF at the LTR of HIV-infected cells before and after activation. We find that establishment and maintenance of HIV latency requires BAF, which removes a preferred nucleosome from DHS1 to position the repressive nucleosome-1 over energetically sub-optimal sequences. Depletion of BAF resulted in de-repression of HIV latency concomitant with a dramatic alteration in the LTR nucleosome profile as determined by high resolution MNase nucleosomal mapping. Upon activation, BAF was lost from the HIV promoter, while PBAF was selectively recruited by acetylated Tat to facilitate LTR transcription. Thus BAF and PBAF, recruited during different stages of the HIV life cycle, display opposing function on the HIV promoter. Our data point to the ATP-dependent BRG1 component of BAF as a putative therapeutic target to deplete the latent reservoir in patients.
Author Summary
Despite the effectiveness of antiretroviral medication, the HIV virus persists in resting memory T cells of infected patients in a latent state, providing the main impediment to eradication of the virus. In this article, we examined the molecular mechanism responsible for the establishment and maintenance of HIV latency and its re-activation, and uncovered the role played in this process by the SWI/SNF class of chromatin remodeling complexes, which use energy from ATP to alter the structure of chromatin. We show that two distinct sub-classes of SWI/SNF, BAF and PBAF, play functionally opposing roles in distinct steps of the HIV promoter (or long terminal repeat, LTR) transcription cycle. The PBAF complex augments transcription of the LTR by the viral transactivator Tat. In contrast, the distinct BAF complex generates a chromatin structure at the LTR that is energetically unfavorable with respect to the intrinsic histone-DNA sequence preferences. Specifically, we find that BAF positions a repressive nucleosome immediately downstream of the HIV transcription start site, abrogating transcription, and in this way contributes to the establishment and maintenance of HIV latency. Our data describe a novel molecular mechanism for the establishment and maintenance of HIV latency, and we identify the catalytic subunit of BAF, the enzyme BRG1, as a putative molecular target to deplete the latent reservoir in infected patients.
PMCID: PMC3226458  PMID: 22140357
17.  Genetic Identification of a Network of Factors that Functionally Interact with the Nucleosome Remodeling ATPase ISWI 
PLoS Genetics  2008;4(6):e1000089.
Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.
Author Summary
The eukaryotic genome is organized in a highly dynamic structure called chromatin. Access to DNA in the context of chromatin is granted by enzymatic activities that use the energy of hydrolysis of ATP to slide or covalently modify nucleosomes. ISWI is an evolutionarily conserved nucleosome-sliding factor that plays essential roles in transcription, DNA replication, and chromosome organization. Despite the wealth of data on ISWI function, little is known about how its activity is regulated and integrated in different physiological contexts in vivo. Using D. melanogaster as a model system, we conducted a genetic screen for factors regulating ISWI activity. One of the genes identified in our screen, Sin3A, encodes a subunit of a histone deacetylase complex that may regulate ISWI function by modifying its nucleosome substrate. Our genetic screen revealed that ISWI interacts with a network of cellular and nuclear factors that escaped previous biochemical analyses, indicating the participation of ISWI in a variety of biological processes not linked to date with known ISWI functions.
PMCID: PMC2390755  PMID: 18535655
18.  ATP-dependent Chromatin Remodeling Enzymes: Two Heads are not Better, Just Different 
ATP-dependent chromatin remodeling complexes enable rapid rearrangements in chromatin structure in response to developmental cues. The ATPase subunits of remodeling complexes share homology with the helicase motifs of DExx box helicases. Recent single-molecule experiments indicate that, like helicases, many of these complexes use ATP to translocate on DNA. Despite sharing this fundamental property, two key classes of remodeling complexes, the ISWI class and the SWI/SNF class, generate distinct remodeled products. SWI/SNF complexes generate nucleosomes with altered positions, nucleosomes with DNA loops and nucleosomes that are capable of exchanging histone dimers or octamers. In contrast, ISWI complexes generate nucleosomes with altered positions but in standard structures. Here we draw analogies to monomeric and dimeric helicases and propose that ISWI and SWI/SNF complexes catalyze different outcomes in part because some ISWI complexes function as dimers while SWI/SNF complexes function as monomers.
PMCID: PMC2494867  PMID: 18339542
19.  The ATP-dependent remodeler RSC transfers histone dimers and octamers through the rapid formation of an unstable encounter intermediate 
Biochemistry  2010;49(45):9882-9890.
RSC, an essential chromatin remodeling complex in budding yeast, is involved in a variety of biological processes including transcription, recombination, repair and replication. How RSC participates in such diverse processes is not fully understood. In vitro, RSC uses ATP to carry out several seemingly distinct reactions: it repositions nucleosomes, transfers H2A/H2B dimers between nucleosomes and transfers histone octamers between pieces of DNA. This raises the intriguing mechanistic question of how this molecular machine can use a single ATPase subunit to create these varied products. Here, we use a FRET-based approach to kinetically order the products of the RSC reaction. Surprisingly, transfer of H2A/H2B dimers and histone octamers is initiated on a time scale of seconds when assayed by FRET, but formation of stable nucleosomal products occurs on a time scale of minutes when assayed by native gel. These results suggest a model in which RSC action rapidly generates an unstable encounter intermediate that contains the two exchange substrates in close proximity. This intermediate then collapses more slowly to form the stable transfer products seen on native gels. The rapid, biologically relevant time scale on which the transfer products are generated implies that such products can play key roles in vivo.
PMCID: PMC2976819  PMID: 20853842
20.  The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways 
PLoS Pathogens  2008;4(12):e1000237.
Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks.
Author Summary
In eukaryotes, genomic DNA is organized into a complex DNA-protein structure termed chromatin. The organization of chromatin serves to compact DNA within the nucleus and plays a central role in regulating transcription by controlling the access of DNA to transcriptional machinery. Chromatin structure can be altered through several mechanisms, one of which is chromatin remodeling, a process that disrupts DNA–protein interactions resulting in altered accessibility of specific DNA regions to regulatory proteins in the transcriptional machinery. In this study, we investigated the biological role of chromatin remodeling in defense responses to biotic stresses using the model plant Arabidopsis. We found that a chromatin remodeling protein, SPLAYED, is required for gene expression within specific biotic stress signaling networks. Consistent with this observation, loss of SPLAYED chromatin-remodeling activity resulted in increased susceptibility to a fungal pathogen, Botrytis cinerea, but not to a bacterial pathogen, Pseudomonas syringae. These results demonstrate that reduced stress tolerance in a chromatin-remodeling mutant plant can be stress specific, and is not simply due to a decrease in overall fitness as a result of non-specific global mis-regulation of gene expression.
PMCID: PMC2588541  PMID: 19079584
21.  The SWI/SNF tumor suppressor complex 
Nucleus  2013;4(5):374-378.
Nucleosomes, octamers of histones wrapped in 147 bp of DNA, are the basic unit of chromatin. In eukaryotic cells, the placement of nucleosomes along the genome is highly organized, and modulation of this ordered arrangement contributes to regulation of gene expression. The SWI/SNF complex utilizes the energy of ATP hydrolysis to mobilize nucleosomes and remodel chromatin structure. Recently, the complex has also been implicated in oncogenesis as genes encoding multiple SWI/SNF subunits have been found mutated at high frequency across a wide spectrum of cancers. Given that epigenetic aberrations are now characterized as a hallmark of human cancer, hypotheses have been put forth that the SWI/SNF complex inhibits tumor formation by regulating key chromatin functions. To understand how the SWI/SNF complex contributes to nucleosome organization in vivo we performed a genome-wide study in mammalian cells. We found that inactivation of SWI/SNF subunits leads to disruptions of specific nucleosome patterning and a loss of nucleosome occupancy at a large number of promoters. These findings define a direct relationship between the SWI/SNF complex, chromatin structure, and transcriptional regulation. In this extra view, we discuss our findings, their relevance to gene regulation, and possible links to the tumor suppression activities of the SWI/SNF complex.
PMCID: PMC3899127  PMID: 24145903
nucleosome; chromatin remodeling; epigenetic instability; SWI/SNF; SNF5; BAF47; SMARCB1; Brg1; SMARCA4
22.  The Nucleosome-Remodeling ATPase ISWI Is Regulated by Poly-ADP-Ribosylation 
PLoS Biology  2008;6(10):1-14.
ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatin set the functional state of chromatin. However, how these enzymatic activities are coordinated in the nucleus is largely unknown. We found that the evolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribose polymerase PARP genetically interact. We present evidence showing that ISWI is target of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI function in vitro and in vivo. Our work suggests that ISWI is a physiological target of PARP and that poly-ADP-ribosylation can be a new, important post-translational modification regulating the activity of ATP-dependent nucleosome remodelers.
Author Summary
The ISWI protein is a highly conserved nucleosome remodeler that plays essential roles in regulating chromosome structure, DNA replication, and gene expression. The variety of functions associated with ISWI activity are probably connected to the ability of other cellular factors to regulate its ATP-dependent nucleosome-remodeling activity. We identified one factor—the poly-ADP-ribose polymerase, PARP—that can counteract ISWI function. PARP is an abundant nuclear protein that catalyzes the transfer of ADP-ribose units to specific proteins involved in DNA repair, transcription, and chromatin structure. Our work suggests that the activity of an ATP-dependent remodeler can be modulated by poly-ADP-ribosylation in order to regulate chromatin function in vivo.
Enzymes that mediate nucleosome remodeling and poly-ADP-ribosylation play essential roles in the eukaryotic cell. A new study suggests a mechanism to explain how two nuclear enzymes can coordinate their activities to regulate chromatin structure and function.
PMCID: PMC2567001  PMID: 18922045
23.  Histone Chaperone FACT Coordinates Nucleosome Interaction through Multiple Synergistic Binding Events* 
The Journal of Biological Chemistry  2011;286(48):41883-41892.
Background: The histone chaperone FACT binds and reorganizes nucleosomes during critical cellular processes.
Results: FACT binds histones, DNA, and mono- and tri-nucleosomes with high affinity. FACT reduces non-nucleosomal histone/DNA interactions.
Conclusion: Multiple regions of FACT strategically bind target sites on nucleosomes to coordinate (dis)assembly.
Significance: The thermodynamic parameters guiding multiple FACT/nucleosome interaction(s) coincide with reorganization events.
In eukaryotic cells, DNA maintenance requires ordered disassembly and re-assembly of chromatin templates. These processes are highly regulated and require extrinsic factors such as chromatin remodelers and histone chaperones. The histone chaperone FACT (facilitates chromatin transcription) is a large heterodimeric complex with roles in transcription, replication, and repair. FACT promotes and subsequently restricts access to DNA as a result of dynamic nucleosome reorganization. However, until now, there lacked a truly quantitative assessment of the critical contacts mediating FACT function. Here, we demonstrate that FACT binds histones, DNA, and intact nucleosomes at nanomolar concentrations. We also determine roles for the histone tails in free histone and nucleosome binding by FACT. Furthermore, we propose that the conserved acidic C-terminal domain of the FACT subunit Spt16 actively displaces nucleosomal DNA to provide access to the histone octamer. Experiments with tri-nucleosome arrays indicate a possible mode for FACT binding within chromatin. Together, the data reveal that specific FACT subunits synchronize interactions with various target sites on individual nucleosomes to generate a high affinity binding event and promote reorganization.
PMCID: PMC3308894  PMID: 21969370
Chromatin Remodeling; Chromatin Structure; Chromosomes/Non-histone Chromosomal Proteins; Histone Chaperone; Nucleosome; FACT
24.  In Vivo Effects of Histone H3 Depletion on Nucleosome Occupancy and Position in Saccharomyces cerevisiae 
PLoS Genetics  2012;8(6):e1002771.
Previous studies in Saccharomyces cerevisiae established that depletion of histone H4 results in the genome-wide transcriptional de-repression of hundreds of genes. To probe the mechanism of this transcriptional de-repression, we depleted nucleosomes in vivo by conditional repression of histone H3 transcription. We then measured the resulting changes in transcription by RNA–seq and in chromatin organization by MNase–seq. This experiment also bears on the degree to which trans-acting factors and DNA–encoded elements affect nucleosome position and occupancy in vivo. We identified ∼60,000 nucleosomes genome wide, and we classified ∼2,000 as having preferentially reduced occupancy following H3 depletion and ∼350 as being preferentially retained. We found that the in vivo influence of DNA sequences that favor or disfavor nucleosome occupancy increases following histone H3 depletion, demonstrating that nucleosome density contributes to moderating the influence of DNA sequence on nucleosome formation in vivo. To identify factors important for influencing nucleosome occupancy and position, we compared our data to 40 existing whole-genome data sets. Factors associated with promoters, such as histone acetylation and H2A.z incorporation, were enriched at sites of nucleosome loss. Nucleosome retention was linked to stabilizing marks such as H3K36me2. Notably, the chromatin remodeler Isw2 was uniquely associated with retained occupancy and altered positioning, consistent with Isw2 stabilizing histone–DNA contacts and centering nucleosomes on available DNA in vivo. RNA–seq revealed a greater number of de-repressed genes (∼2,500) than previous studies, and these genes exhibited reduced nucleosome occupancy in their promoters. In summary, we identify factors likely to influence nucleosome stability under normal growth conditions and the specific genomic locations at which they act. We find that DNA–encoded nucleosome stability and chromatin composition dictate which nucleosomes will be lost under conditions of limiting histone protein and that this, in turn, governs which genes are susceptible to a loss of regulatory fidelity.
Author Summary
Chromatin is formed by wrapping 146 bp of DNA around a disc-shaped complex of proteins called histones. These protein–DNA structures are known as nucleosomes. Nucleosomes help to regulate gene transcription, because nucleosomes compete with transcription factors for access to DNA. The precise positioning and level of nucleosome occupancy are known to be vital for transcriptional regulation, but the mechanisms that regulate the position and occupancy of nucleosomes are not fully understood. Recently, many studies have focused on the role of DNA sequence and chromatin remodeling proteins. Here, we manipulate the concentration of histone proteins in the cell to determine which nucleosomes are most susceptible to changes in occupancy and position. We find that the chromatin-associated proteins Sir2 and Tup1, and the chromatin remodelers Isw2 and Rsc8, are associated with stabilized nucleosomes. Histone acetylation and incorporation of the histone variant H2A.z are the factors most highly associated with destabilized nucleosomes. Certain DNA sequence properties also contribute to stability. The data identify factors likely to influence nucleosome stability and show a direct link between changes in chromatin and changes in transcription upon histone depletion.
PMCID: PMC3380831  PMID: 22737086
25.  Mouse BAZ1A (ACF1) Is Dispensable for Double-Strand Break Repair but Is Essential for Averting Improper Gene Expression during Spermatogenesis 
PLoS Genetics  2013;9(11):e1003945.
ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations.
Author Summary
The eukaryotic genome is packaged into a periodic nucleoprotein complex known as chromatin. Wrapping of DNA around nucleosomes, the basic repeat unit of chromatin, enables packing of long stretches of DNA into a compact nucleus but also impedes access by protein factors involved in essential cellular processes such as transcription, replication, recombination and repair. Chromatin remodeling factors are multi-protein complexes that utilize the energy released during ATP-hydrolysis to assemble, reposition, restructure and disassemble nucleosomes. These complexes disrupt histone-DNA contacts to ‘remodel’ the chromatin and grant access to the genome. Alternatively, access can also be denied to repress transcription, for example. Spermatogenesis, the developmental program that produces sperm, comprises a dramatic chromatin makeover and the induction of a transcriptional program that engages nearly one-third of the genome. Here we provide evidence suggesting that these large-scale alterations leave the genomic material vulnerable to spurious transcriptional changes which are normally repressed by ACF1 (BAZ1A in mammals), the defining member of the well-studied ACF/CHRAC chromatin remodeling complex. These findings indicate that Baz1a plays a previously unrealized role in male fertility and may represent a novel target for male contraceptive development.
PMCID: PMC3820798  PMID: 24244200

Results 1-25 (812442)