Genomic Footprinting of the Promoter Regions of STE2 and STE3 genes in the Yeast Saccharomyces cerevisiae

Ganter, Brigitte; Tan, Song; Richmond, Timothy J.

doi:10.1006/jmbi.1993.1652

Cited by 28 publications

(29 citation statements)

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“…3; VOL. 16,1996 ␣2 REPRESSION AND NUCLEOSOME POSITIONING 2867 STE2 (in accordance with the results described by Ganter et al [8]), again suggesting that the failure to observe positioned nucleosomes across the artificial promoters is not due to a problem in detecting nucleosomes (data not shown). We repeated nucleosome mapping with the additional promoters (pGAL int and pASG 2 ) and, in agreement with the results of Fig.…”

Section: Resultssupporting

confidence: 88%

Accessibility of α2-Repressed Promoters to the Activator Gal4

Redd

Stark

Johnson

1996

Molecular and Cellular Biology

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It has been proposed that eukaryotic repressors of transcription can act by organizing chromatin, thereby preventing the accessibility of nearby DNA to activator proteins required for transcription initiation. In this study, we test this idea for the yeast ␣2 repressor using a simple, artificial promoter that contains a single binding site for the activator protein Gal4 and a single binding site for the repressor ␣2. When both the repressor and the activator are expressed in the same cell, the artificial promoter is efficiently repressed. In vivo footprinting experiments demonstrate that Gal4 can occupy its binding site even when the promoter is repressed. This result indicates that ␣2-directed repression must result from interference with some stage in transcription initiation other than activator binding to DNA.Negative regulation of transcription in eukaryotes occurs by a variety of mechanisms. Some repressors act by preventing the DNA binding of activators, some bind DNA and interact with nearby activators, ''quenching'' their activation surface, and some communicate directly with the general transcription machinery, blocking its function or assembly (for reviews, see references 14, 16, 18, and 26). Still other repressors appear to organize repressive forms of chromatin that block the accessibility of proteins to DNA (for reviews, see references 31, 33, and 45). For some repressors, more than one of these mechanisms is thought to function simultaneously, resulting in a very low level of gene expression under repressing conditions.One case in which two mechanisms of repression have been proposed is that of the yeast ␣2 protein. This protein is responsible for repressing the expression of two sets of cell-typespecific genes, a-specific genes and haploid-specific genes (for reviews, see references 7, 15, and 17). To repress a-specific genes, ␣2 binds cooperatively with the Mcm1 protein to a 34-bp DNA sequence called the a-specific gene operator. ␣2/ Mcm1 binds a second protein complex composed of the Tup1 and Ssn6 proteins. Tup1 and Ssn6 are required for the repression of at least five sets of yeast genes and have been proposed to function as a general repression machine in Saccharomyces cerevisiae, recruited to DNA by a variety of sequence-specific DNA-binding proteins (21,24,41,42).The a-specific gene operator will bring about repression when placed in many positions upstream of a target gene, and models for repression by ␣2/Mcm1/Ssn6/Tup1 (referred to as the ␣2 repression complex) must account for this action at a distance (20,32). One model proposes that the ␣2 repression complex interacts directly with the general transcription machinery at the promoter, blocking its assembly or maturation (13,20). A second model proposes that the ␣2 repression complex positions nucleosomes over promoter elements, blocking the accessibility of nearby DNA to proteins (23,34,35,37). In this work, we wished to determine whether an ␣2-repressed promoter is accessible to Gal4, a yeast activator protein that binds DNA. MATERIALS...

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Section: Resultssupporting

confidence: 88%

Accessibility of α2-Repressed Promoters to the Activator Gal4

Redd

Stark

Johnson

1996

Molecular and Cellular Biology

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“…Previous studies revealed that the α2/Mcm1 repressor, in conjunction with the Ssn6-Tup1 corepressor and the Isw2 complexes establishes an array of positioned nucleosomes to form a repressive chromatin structure [15,17,20,21,[28][29][30]32]. Our results imply that the positioned nucleosomes in the repressive chromatin function to limit the accessibility of at least a certain class of activators in vivo, therefore supporting the idea that nucleosome positioning plays a vital role in repression by α2/Mcm1.…”

Section: Discussionsupporting

confidence: 86%

“…The hypersensitive site was detectable in a cells, in which nucleosomes were not positioned, but it was strongly diminished in α cells, indicating that Hap1 binding is severely limited at the dyad in the positioned nucleosome IV. Similar results for Hap1 binding were obtained for TALS-UAS1b (lanes [19][20][21][22][23][24] and -UAS1c (lanes [28][29][30][31][32][33], in which the UAS1 site was located in the peripheral region of the positioned nucleosome IV; that is, the hypersensitive site is present in a cells, but absent in α cells. It should be noted that the location of UAS1 in TALSUASc is shifted to 5 bp exterior to the nucleosome edge, as compared to that in TALS-UAS1b.…”

Section: Effect Of the Uas1 Locations In A Positioned Nucleosome On Hsupporting

confidence: 80%

A nucleosome positioned by α2/Mcm1 prevents Hap1 activator binding in vivo

Morohashi

Nakajima

Kurihara

et al. 2007

Biochemical and Biophysical Research Communications

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Nucleosome positioning has been proposed as a mechanism of transcriptional repression. Here, we examined whether nucleosome positioning affects activator binding in living yeast cells. We introduced the cognate Hap1 binding site (UAS1) at a location 24-43 bp, 29-48 bp or 61-80 bp interior to the edge of a nucleosome positioned by α2/Mcm1 in yeast minichromosomes. Hap1 binding to the UAS1 was severely inhibited, not only at the pseudo-dyad but also in the peripheral region of the positioned nucleosome in α cells, while it was detectable in a cells, in which the nucleosomes were not positioned. Hap1 binding was restored in α cells with tup1 or isw2 mutations, which caused the loss of nucleosome positioning. These results support the mechanism in which α2/Mcm1-dependent nucleosome positioning has a regulatory function to limit the access of transcription factors.Keywords nucleosome positioning; chromatin; activator binding; in vivo footprinting; yeast In a general view of the process of transcriptional activation, activators have to gain access to an array of nucleosomes, and chromatin remodeling complexes and/or histone modification enzymes change the structural features of chromatin to recruit transcriptional machinery to the promoter regions [reviewed in 1,2]. Regarding activator binding, several regulatory factors such as steroid hormone receptor, Gal4-derivatives, USF and Sp1, bind to reconstituted nucleosomes in vitro under some circumstances, although their affinities for nucleosomal DNA decrease by one to three orders of magnitude, relative to those for naked DNA [see reviews, 2-4]. Positioned nucleosomes assembled with Drosophila embryo extracts preclude NF1 binding to its cognate site [5]. In contrast, NF-κB binds to its recognition sites within a positioned nucleosome with an affinity close to that for free DNA in vitro [6]. Yeast Gal4 is one of the best characterized activators binding to nucleosomes in vivo. Gal4 can bind to its cognate site within a positioned nucleosome to perturb the nucleosome in yeast cells [7][8][9][10][11]. Also, the Pho4 activator can reportedly bind its nucleosome-occupied site before nucleosome disassembly in vivo [12]. However, only a few activators have been examined for their access *Corresponding author. Mitsuhiro Shimizu, Department of Chemistry, Meisei University, 2-1-1 Hodokubo, Hino, Tokyo 191-8506, Japan, Phone: +81-42-591-7483, Fax: +81-42-591-7419, E-mail: shimizum@chem.meisei-u.ac.jp. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. to DNA within the nucleosome in vivo. Although nucleosome positioning has been observ...

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“…The assignment of a nucleosome and its position adjacent to the ␣2 operator in ␣-cells is based on a considerable body of earlier mapping studies utilizing MNase (21,46,55,56,59,76), MTases (Dam, Sau3A1, and SssI) (29)(30)(31), and DNase I (21,46,56,59). In sum, these studies demonstrate that, abutting the ␣2 operator, a nucleosome is positioned by Mcm1p-Mat␣2p over the promoters of a-cell-specific genes (21,56), the recombination enhancer (76), minichromosomes (29,55,59), and heterologous random sequence in a lacZ-containing reporter plasmid (46). Nucleosome positioning over such disparate sequences reinforces the likelihood that the chromatin structures of closely related minichromosomes would be the same.…”

Section: Resultsmentioning

confidence: 99%

Gal4p-Mediated Chromatin Remodeling Depends on Binding Site Position in Nucleosomes but Does Not Require DNA Replication

Simpson

Kladde

1998

Molecular and Cellular Biology

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Biochemical studies have demonstrated decreased binding of various proteins to DNA in nucleosome cores as their cognate sites are moved from the edge of the nucleosome to the pseudodyad (center). However, to date no study has addressed whether this structural characteristic of nucleosomes modulates the function of a transcription factor in living cells, where processes of DNA replication and chromatin modification or remodeling could significantly affect factor binding. Using a sensitive, high-resolution methyltransferase assay, we have monitored the ability of Gal4p in vivo to interact with a nucleosome at positions that are known to be inaccessible in nucleosome cores in vitro. Gal4p efficiently bound a single cognate site (UAS G ) centered at 41 bp from the edge of a positioned nucleosome, perturbing chromatin structure and inducing transcription. DNA binding and chromatin perturbation accompanying this interaction also occurred in the presence of hydroxyurea, indicating that DNA replication is not necessary for Gal4p-mediated nucleosome disruption. These data extend previous studies, which demonstrated DNA replication-independent chromatin remodeling, by showing that a single dimer of Gal4p, without the benefit of cooperative interactions that occur at complex wild-type promoters, is competent for invasion of a preestablished nucleosome. When the UAS G was localized at the nucleosomal pseudodyad, relative occupancy by Gal4p, nucleosome disruption, and transcriptional activation were substantially compromised. Therefore, despite the increased nucleosome binding capability of Gal4p in cells, the precise translational position of a factor binding site in one nucleosome in an array can affect the ability of a transcriptional regulator to overcome the repressive influence of chromatin.

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Genomic Footprinting of the Promoter Regions of STE2 and STE3 genes in the Yeast Saccharomyces cerevisiae

Cited by 28 publications

References 0 publications

Accessibility of α2-Repressed Promoters to the Activator Gal4

Accessibility of α2-Repressed Promoters to the Activator Gal4

A nucleosome positioned by α2/Mcm1 prevents Hap1 activator binding in vivo

Gal4p-Mediated Chromatin Remodeling Depends on Binding Site Position in Nucleosomes but Does Not Require DNA Replication

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