Transcription factors (TF) bind DNA-target sites within promoters to activate gene expression. TFs target their DNA-recognition sequences with high specificity by binding with resident times of up to hours in vitro. However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays with single molecule total internal reflection fluorescence (smTIRF) microscopy. We find that the rate of TF dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to duplex DNA. Our results suggest that TF binding within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these studies demonstrate that nucleosomes regulate DNA–protein interactions not only by preventing DNA–protein binding but by dramatically increasing the dissociation rate of protein complexes from their DNA-binding sites.
The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structure changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6 bp, 26 bp and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remains fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding, but it significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.
Expression of the POX1 gene, which encodes peroxisomal acyl coenzyme A oxidase in the yeast Saccharomyces cerevisiae, is tightly regulated and can be induced by fatty acids such as oleate. Previously we have shown that this regulation is brought about by interactions between trans-acting factor(s) and an upstream activating sequence (UAS1) in the POX1 promoter. We recently identified and isolated a transcription factor, Oaf1p, that binds to the UAS1 of POX1 and mediates its induction. A screening strategy has been developed and used to identify eight S. cerevisiae mutants, from three complementation groups, that are defective in the oleate induction of POX1. Characterization of one such mutant led to the identification of Oaf2p, a protein that is 39% identical to Oaf1p. Oaf1p and Oaf2p form a protein complex that is required for the activation of POX1 and FOX3 and for proliferation of peroxisomes. We propose a model in which these two transcription factors heterodimerize and mediate this activation process. The mutants that we have isolated, and further identification of the corresponding defective genes, provide us with an opportunity to characterize the mechanisms involved in the coordinate regulation of peroxisomal -oxidation enzymes.Peroxisomes are organelles that play important roles in cellular respiration and lipid metabolism. In most organisms, peroxisomes contain enzymes involved in fatty acid oxidation (-oxidation) and catalase which decomposes the hydrogen peroxide generated from this process (for a review, see reference 21). Peroxisomes are essential for human survival, as demonstrated by the fact that the genetic disorder Zellweger syndrome, in which there is a lack of functional peroxisomes, is lethal (15,22).The size, abundance, and enzyme content of peroxisomes vary according to the cell type, organism, and metabolic requirements. In the yeast Saccharomyces cerevisiae, levels of peroxisomal -oxidation enzymes are regulated by the available carbon source. The rate-limiting enzyme in the -oxidation pathway is acyl coenzyme A (acyl-CoA) oxidase, which is encoded by a single-copy gene, POX1 (9). We have previously shown that POX1 expression is induced when S. cerevisiae is grown in the presence of oleic acid and that this transcriptional regulation is brought about by a protein, or proteins, binding to a specific upstream activating sequence (UAS1) in the POX1 promoter (41, 42). UAS1-like sequences (also called oleate response elements [OREs]) (11,14) are present in genes encoding many peroxisomal proteins, including the other enzymes of the peroxisomal -oxidation cycle (10).We recently purified one protein, Oaf1p, that binds to UAS1 when cells are grown in oleate medium (25). Furthermore, by cloning and subsequently disrupting the gene encoding Oaf1p, we demonstrated that this protein is required for the oleate induction of POX1. The deduced amino acid sequence of Oaf1p reveals a C 6 zinc cluster motif, placing it in the same family of transcription factors as Gal4p and Cyp1p (7,20).The genes R...
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