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.

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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.

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HDAC1 and -2 are highly conserved enzymes and often coexist in the same coregulator complexes. Understanding the regulation of histone deacetylase activities is extremely important because these enzymes play key roles in epigenetic regulation in normal and cancer cells. We previously showed that HDAC1 is required for glucocorticoid receptor-mediated transcription activation and that its activity is regulated through acetylation by p300 during the induction cycle. Here, we showed that HDAC2 is also required for glucocorticoid receptor-mediated gene activation. HDAC2, however, is regulated through a different mechanism from that of HDAC1. HDAC2 is not acetylated by p300, although 5 of 6 acetylated lysine residues in HDAC1 are also present in HDAC2. More importantly, the activity of HDAC2 is inhibited by acetylated HDAC1. Additionally, we showed that acetylated HDAC1 can trans-regulate HDAC2 through heterodimerization. Thus, this study uncovered fundamental differences between HDAC1 and HDAC2. It also unveiled a new mechanism of collaborative regulation by HDAC1/2 containing coregulator complexes.Histone deacetylases are responsible for the deacetylation of lysine residues at the N-terminal part of the core histones as well as at non-histone proteins. Histone deacetylation plays an important role in transcriptional regulation, cell cycle progression, and developmental events and is often linked to epigenetic repression (1-4). However, emerging evidence also suggests that histone deacetylase activity may be required for transcriptional activation (5-10) and for preventing the cryptic initiation of transcription (11). HDAC1 and HDAC2 belong to class I histone deacetylases and often coexist in multicomponent protein complexes (12). They are closely related enzymes with an 82% overall sequence identity and can partially compensate for each others' functions (13-17). HDAC1 and -2 can also undergo differential posttranslational modification, such as phosphorylation and sumoylation (18 -20).We recently reported that HDAC1 is required for glucocorticoid receptor (GR) 2 -mediated transcription activation. Furthermore, we showed that HDAC1 is acetylated after its association with the GR, and this acetylation event correlates with a change in promoter activity (8). The acetylated HDAC1 loses its deacetylase activity. The majority of acetylated lysine residues of HDAC1 are present in the C-terminal regulatory domain, which reveals only partial homology to the corresponding region of HDAC2. The C-terminal domain of HDAC1 does not have any catalytic activity. The deletion of this region, however, greatly reduces deacetylase activity (21), indicating that the C-terminal domain plays an indispensable role in the regulation of deacetylase activity. Therefore, it is important to compare the C-terminal regions of HDAC1 and HDAC2 functionally and structurally and to determine how these domains collaborate in the regulation of transcription. In this study, we show that HDAC2 is also important for GR-mediated gene activation; however, i...

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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Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids. Peroxisomes are the sole site for the ␤-oxidation of fatty acids in yeast. The first and rate-limiting enzyme of this cycle is fatty acyl-CoA oxidase. The gene encoding this enzyme in Saccharomyces cerevisiae (POX1) undergoes a complex regulation that is dependent on the growth environment. When this yeast is grown in medium containing oleic acid as the main carbon source, peroxisomes are induced and POX1 expression is activated. When cells are grown in the presence of glucose, the expression of POX1 mRNA is repressed, whereas growth on a carbon source such as glycerol or raffinose causes derepression. This rigorous regulation is brought about by the complex interactions between trans-acting factors and cis-elements in the POX1 promoter. Previously, we characterized regulatory elements in the promoter region of POX1 that are involved in the repression and activation of this gene (Wang, T., Luo, Y., and Small, G. M. (1994) J. Biol. Chem. 269, 24480 -24485). In this study we have purified and identified an oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene. The protein has a predicted molecular mass of approximately 118 kDa.Peroxisomal ␤-oxidation is an important pathway in mammalian metabolism for catabolizing long and very long chain fatty acids. In many organisms, including yeasts, peroxisomal ␤-oxidation is the sole mechanism for the breakdown of fatty acids (1). The enzymes involved in this pathway are regulated according to the growth environment. Expression of genes encoding peroxisomal proteins in the yeast Saccharomyces cerevisiae is repressed when the yeast cells are grown in the presence of glucose, derepressed during growth on a nonfermentable carbon source, and activated when a fatty acid such as oleate is supplied for growth (2). This control is achieved through stringent transcriptional regulation of the genes encoding these proteins (3-8).Over the past several years we have focused our attentions toward understanding the mechanisms that regulate genes encoding peroxisomal ␤-oxidation enzymes in S. cerevisiae. In order to address this question, we have concentrated on the regulation of POX1, the gene encoding acyl-CoA oxidase, the rate-limiting enzyme of this cycle. Previously we characterized two upstream repression sequences (URS1 and URS2) 1 and one upstream activating sequence (UAS1) in the promoter region of POX1 (7, 9). We demonstrated that a protein or protein complex binds to UAS1 in an oleate-dependent fashion, and this brings about the activation of POX1. A similar UAS sequence (termed oleate response element) was identified in the upstream regions of genes encoding some of the other peroxisomal proteins (3, 4, 10).Several factors have been shown to be involved in the glucose repression of thiolase, the last enzyme in the peroxisomal ␤-oxidation cycle, which in S. cerevisiae ...