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.

BackgroundLead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity.ResultsWe analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high zinc protoporphyrin blood levels.ConclusionsThe results of our study defined specific changes in gene and protein expression in response to lead challenges and determined the injurious effects of exposures to lead on a cellular level. This information can be used for documenting the health effects of exposures to lead which will facilitate identifying and monitoring efficacious treatments for lead-related maladies.

Benzene is a common air pollutant and confirmed carcinogen, especially in reference to the hematopoietic system. In the present study we analyzed cytokine/chemokine production by, and gene expression induction in, human peripheral blood mononuclear cells upon their exposure to the benzene metabolites catechol, hydroquinone, 1,2,4-benzenetriol, and p-benzoquinone. Protein profiling showed that benzene metabolites can stimulate the production of chemokines, the proinflammatory cytokines TNF-alpha and IL-6, and the Th2 cytokines IL-4 and IL-5. Activated cells showed concurrent suppression of anti-inflammatory cytokine IL-10 expression. We also identified changes in global gene expression patterns in response to benzene metabolite challenges by using high-density oligonucleotide microarrays. Treatment with 1,2,4-benzenetriol resulted in the suppression of genes related to the regulation of protein expression and a concomitant activation of genes that encode heat shock proteins and cytochrome P450 family members. Protein and gene expression profiling identified unique human cellular responses upon exposure to benzene and benzene metabolites.

Making a correct diagnosis is pivotal in the practice of clinical rheumatology. Occasionally, the consultation fails to provide desired clarity in making labeling an individual as having fibromyalgia (FM), systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). A chemokine and cytokine multiplex assay was developed and tested with the goal of improving and achieving an accurate differential diagnosis. 160 patients with FM, 98 with RA and 100 with SLE fulfilling accepted criteria were recruited and compared to 119 controls. Supernatant cytokine concentrations for IL-6, IL-8, MIP-1 alpha and MIP-1 beta were determined using the Luminex multiplex immunoassay bead array technology after mitogenic stimulation of cultured peripheral blood mononuclear cells. Each patient’s profile was scored using a logistical regression model to achieve statistically determined weighting for each chemokine and cytokine. Among the 477 patients evaluated, the mean scores for FM (1.7 ± 1.2; 1.52–1.89), controls (−3.56 ± 5.7; −4.59 to −2.54), RA (−0.68 ± 2.26; −1.12 to −0.23) and SLE (−1.45 ± 3.34, −2.1 to −0.79). Ninety-three percent with FM scored positive compared to only 11 % of healthy controls, 69 % RA or 71 % SLE patients had negative scores. The sensitivity, specificity, positive predictive and negative predictive value for having FM compared to controls was 93, 89, 92 and 91 %, respectively (p < 2.2 × 10−16). Evaluating cytokine and chemokine profiles in stimulated cells reveals patterns that are uniquely present in patients with FM. This assay can be a useful tool in assisting clinicians in differentiating systemic inflammatory autoimmune processes from FM and its related syndromes and healthy individuals.