In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.
The nature of the interaction of coactivator proteins with transcriptionally active promoters in chromatin is a fundamental question in transcriptional regulation by RNA polymerase II. In this study, we used a biochemical approach to examine the functional association of the coactivator p300 with chromatin templates. Using in vitro transcription template competition assays, we observed that p300 forms a stable, templatecommitted complex with chromatin during the transcription process. The template commitment is dependent on the time of incubation of p300 with the chromatin template and occurs independently of the presence of a transcriptional activator protein. In studies examining interactions between p300 and chromatin, we found that p300 binds directly to chromatin and that the binding requires the p300 bromodomain, a conserved 110-amino-acid sequence found in many chromatin-associated proteins. Furthermore, we observed that the isolated p300 bromodomain binds directly to histones, preferentially to histone H3. However, the isolated p300 bromodomain does not bind to nucleosomal histones under the same assay conditions, suggesting that free histones and nucleosomal histones are not equivalent as binding substrates. Collectively, our results suggest that the stable association of p300 with chromatin is mediated, at least in part, by the bromodomain and is critically important for p300 function. Furthermore, our results suggest a model for p300 function that involves distinct activator-dependent targeting and activator-independent chromatin binding activities.Activated transcription of genes by RNA polymerase II (RNA Pol II) requires the concerted actions of sequencespecific DNA-binding transcriptional activators, chromatin remodeling complexes, histone acetyltransferases (HATs), and coactivators. Together, these factors function to overcome the transcriptional repression caused by the packaging of genes into chromatin and to stimulate the activity of RNA Pol II. The initiating event in the process of activated transcription is the binding of transcriptional activator proteins to binding sites in the promoters of target genes. In many cases, the activities of the activator proteins are regulated by input from cellular signaling pathways (e.g., steroid hormones and mitogen-activated protein kinase pathways). Once bound to DNA, activators can recruit chromatin remodeling complexes, HATs, and coactivators to the promoter, leading to the formation of a stable RNA Pol II preinitiation complex and, subsequently, transcription initiation (reviewed in references 3, 19, 23, 30, 34, 50, and 67).Transcriptional coactivators are a diverse group of factors and multipolypeptide complexes, some of which possess intrinsic HAT activity (16,42,43,45,63,68). Coactivators with HAT activity include p300 and the closely related CREB binding protein (CBP; often referred to with p300 as p300/CBP) (4, 20), as well as the p300/CBP-associated factor (PCAF), which functions as part of a multipolypeptide complex (54, 69). In general, coact...
Ciguatera fish poisoning is the most extensive and difficult to control of the seafood poisonings. To facilitate monitoring of fish toxicity, toxin profiles were investigated by an LC/MS/MS method using 14 reference toxins on eight representative species of fish collected in four different areas of the Pacific. Snappers and groupers from Okinawa contained ciguatoxin-1B (CTX1B) and two deoxy congeners at variable but species-specific ratios, while red snapper, Lutjanus bohar, from Minamitorishima, and amberjack, Seriola dumerili, from Hawaii, contained both CTX1B-type and CTX3C-type toxins. Spotted knifejaw, Oplegnathus punctatus, from Okinawan waters, contained mainly CTX4A and CTX4B, but the same species caught at Miyazaki was contaminated primarily with the CTX3C-type toxins. Otherwise, the toxin profiles were consistently species-specific in fish collected from various locations around Okinawa over 20 years. The LC/MS/MS and mouse bioassay results agreed well, indicating the LC/MS/MS method is a promising alternative to the mouse bioassay. Pure CTX1B and CTX3C were prepared for use in future LC/MS/MS analysis.
Ciguatoxins (CTXs) contaminate fish worldwide and cause the foodborne illness ciguatera. In the Pacific, these toxins are produced by the dinoflagellate Gambierdiscus toxicus, which accumulates in fish through the food chain and undergoes oxidative modification, giving rise to numerous analogs. In this study, we examined the oxidation of CTXs in vitro with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using reference toxins, and found that CTX4A, CTX4B, and CTX3C, which are produced by the alga, are oxidized to the analogs found in fish, namely CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, 2-hydroxyCTX3C, and 2,3-dihydroxyCTX3C. This oxidation was catalyzed by human CYP3A4, fish liver S9 fractions, and microsomal fractions prepared from representative ciguateric fishes (Lutjanus bohar, L. monostigumus, and Oplegnathus punctatus). In addition, fish liver S9 fractions prepared from non-ciguateric fishes (L. gibbus and L. fulviflamma) in Okinawa also converted CTX4A and CTX4B to CTX1B, 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B in vitro. This is the first study to demonstrate the enzymatic oxidation of these toxins, and provides insight into the mechanism underlying the development of species-specific toxin profiles and the fate of these toxins in humans and fish.
Detection of volatile organic compounds (VOCs) using weight-detectable quartz microbalance and silicon-based microcantilever sensors coated with crystalline metal-organic framework (MOF) thin films is described in this paper. The thin films of two MOFs were grown from COOH-terminated self-assembled monolayers onto the gold electrodes of sensor platforms. The MOF layers worked as the effective concentrators of VOC gases, and the adsorption/desorption processes of the VOCs could be monitored by the frequency changes of weight-detectable sensors. Moreover, the MOF layers provided VOC sensing selectivity to the weight-detectable sensors through the size-selective adsorption of the VOCs within the regulated nanospace of the MOFs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.