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.Eukaryotic chromatin has seen a rebirth of intense study over the past few years. Foremost among the biochemical reactions impinging on chromatin structure is ATP-dependent chromatin remodeling, which leads to an enhanced accessibility of nucleosomal DNA (for recent reviews, see references 18, 19, and 48). This reaction plays a key role in the regulation of transcription by RNA polymerase II, and it has been proposed to be a prerequisite for a variety of other cellular processes that require access to the chromatin template (for reviews, see references 35 and 48). In addition to ATP-dependent nucleosome remodeling, multisubunit complexes that can acetylate (12, 31, 39, 41) or methylate (6) histone and nonhistone proteins have the potential to directly modify chromatin structure and function.A host of ATP-dependent chromatin remodeling complexes have been identified via biochemical fractionation of cell extracts, yeast genetics, or genome database mining (2,5,7,17,20,23,33,44,46,47,49,50,53,55). A hallmark of these multisubunit complexes is that they contain a member of the SWI2/SNF2 subfamily of DNA-stimulated ATPases. Seventeen members of the SWI2/SNF2 family have been identified in the yeast genome (10, 38), and to date, four of these ATPases ha...

Transcriptional regulation in yeast involves a number of general trans-acting factors affecting chromatin structure. The Swi-Snf complex is required for expression of a large number of genes and has the ability to remodel chromatin in vitro. The Ssn6p-Tup1p repressor complex may be involved in chromatin organization through the interaction with pathwayspecific DNA-binding proteins. To study the interplay of these factors and their effect on chromatin we have analyzed SUC2 chromatin structure in wild-type cells and in strains bearing combinations of ssn6/tup1 and swi1 mutations. We have mapped nucleosome positioning of the repressed gene in wild-type cells using primer extension methodology, allowing base pair resolution, and have analyzed details of chromatin remodeling in the derepressed state. In ssn6 or tup1 mutants under repressing conditions the observed changes in SUC2 chromatin structure may be suppressed by the swi1 mutation, suggesting that Ssn6p-Tup1p is not required for the establishment of nucleosome positioning at the SUC2 promoter. Our data indicate the involvement of chromatin remodeling factors distinct from the Swi-Snf complex in SUC2 transcriptional regulation and suggest that Swi-Snf may antagonize Ssn6p-Tup1p by controlling remodeling activity. We also show that a relatively high level of SUC2 transcription can coexist with positioned nucleosomes.

ySWI/SNF complex belongs to a family of enzymes that use the energy of ATP hydrolysis to remodel chromatin structure. Here we examine the role of DNA topology in the mechanism of ySWI/SNF remodeling. We find that the ability of ySWI/SNF to enhance accessibility of nucleosomal DNA is nearly eliminated when DNA topology is constrained in small circular nucleosomal arrays and that this inhibition can be alleviated by topoisomerases. Furthermore, we demonstrate that remodeling of these substrates does not require dramatic histone octamer movements or displacement. Our results suggest a model in which ySWI/SNF remodels nucleosomes by using the energy of ATP hydrolysis to drive local changes in DNA twist.

Using zero-length covalent protein-DNA crossinking, we have mapp the hstone-DNA cts In nucleosome core particles from which the C-and N-terminal domains of histone H2A were selectively tim by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axds, whereas its glbular domain contacts the end of DNA in the nceme core particle. The appearance of the histoe H2A contact at the dyad axis occurs only in the absence oflinker DNA and does not depend on the absence of linker hit . Our results show the ability of the histone H2A C-terminal domain to rearrange.This rearrangement might play a biological role in nuce disassembly and reassembly and the retention ofthe HZ2A-H2B dimer (or the whole octamer) during the passing ofpolymerases through the nucleosome.The nucleosome as a basic repeating subunit ofchromatin has been studied by various methods and many details of its structure are known (1). However, the questions of the arrangements and behaviors of the flexible histone terminal domains in nucleosomes are not resolved.Although histone tails (exposed and trypsin-sensitive terminal domains) do not affect the conformational saltdependent stability of core particles, they play a significant role in their thermal stability (2). Histone tails do not play a role in determining nucleosome positioning (3) and do not affect the helical periodicity of DNA in isolated nucleosomes (4). However, histone tails have been shown to participate in the folding of oligonucleosomes (5) and in the stabilization of higher-order chromatin structure (6). In addition, they contain sites for reversible post-translational modifications that can modulate chromatin structure (for review, see ref. 7). How histone terminal domains are involved in these interactions is still not clear.Current information about the structure of the histone octamer and the nucleosome is based on the arrangement of histone globular domains or whole histone molecules (8-10). There is little direct data concerning the localization of the flexible histone terminal domains in the nucleosome. Protein-DNA crosslinking experiments have revealed the binding ofthe histone H4 N-terminal domain to DNA at a distance of 1.5 helical turns from either side of the nucleosomal dyad axis (11). In the present study, using zero-length covalent histone-DNA crosslinking, we demonstrate that in isolated core particles the flexible trypsin-sensitive C-terminal domain of histone H2A is bound to the dyad axis, whereas the globular domain is bound to the end ofthe nucleosomal DNA. By taking into consideration the results of our previous histone-DNA crosslinking experiments (10,29,31) and the results of other investigators (26,30,32,34), we discuss the possible arrangement of the histone H2A C-terminal domain in chromatin and in intact nuclei and its rearrangement after the removal of linker DNA. MATERIALS AND METHODSPreparation of Hl-Depleted Chromatinad Core Particles. Soluble chromatin from chicken erythrocyte nuclei wa...

BackgroundFibromyalgia (FM) is a clinical syndrome characterized by chronic pain and allodynia. The diagnosis of FM has been one of exclusion as a test to confirm the diagnosis is lacking. Recent data highlight the role of the immune system in FM. Aberrant expressions of immune mediators, such as cytokines, have been linked to the pathogenesis and traits of FM. We therefore determined whether cytokine production by immune cells is altered in FM patients by comparing the cellular responses to mitogenic activators of stimulated blood mononuclear cells of a large number of patients with FM to those of healthy matched individuals.MethodsPlasma and peripheral blood mononuclear cells (PBMC) were collected from 110 patients with the clinical diagnosis of FM and 91 healthy donors. Parallel samples of PBMC were cultured overnight in medium alone or in the presence of mitogenic activators; PHA or PMA in combination with ionomycin. The cytokine concentrations of IFN-γ, IL-5, IL-6, IL-8, IL-10, MIP-1β , MCP-1, and MIP1-α in plasma as well as in cultured supernatants were determined using a multiplex immunoassay using bead array technology.ResultsCytokine levels of stimulated PBMC cultures of healthy control subjects were significantly increased as compared to matched non-stimulated PBMC cultures. In contrast, the concentrations of most cytokines were lower in stimulated samples from patients with FM compared to controls. The decreases of cytokine concentrations in patients samples ranged from 1.5-fold for MIP-1β to 10.2-fold for IL-6 in PHA challenges. In PMA challenges, we observed 1.8 to 4-fold decreases in the concentrations of cytokines in patient samples.ConclusionThe cytokine responses to mitogenic activators of PBMC isolated from patients with FM were significantly lower than those of healthy individuals, implying that cell-mediated immunity is impaired in FM patients. This novel cytokine assay reveals unique and valuable immunologic traits, which, when combined with clinical patterns, can offer a diagnostic methodology in FM.