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

Abstract

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...

Nucleosome-Depleted Regions in Cell-Cycle-Regulated Promoters Ensure Reliable Gene Expression in Every Cell Cycle

“…TFBS localization in NDR is generally believed to be important in enhancing transcription factor binding and facilitating subsequent transcription based on several lines of evidence. Studies on many different factors suggested that nucleosomes limit their TFBS accessibility both in vitro (for review, see Owen-Hughes and Workman, 1994) and in vivo (Liu et al, 2006; Morohashi et al, 2007; Sekinger et al, 2005). Well-studied promoters, such as GAL1-10pr and PHO5pr , have their major TFBS positioned within constitutive NDR, regardless of the transcriptional status of the gene (Lohr, 1997; Svaren and Horz, 1997).…”

The Cyc8–Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein

“…Second, Cyc8-Tup1 can interact with multiple histone deacetylases in certain in vitro conditions (Watson et al 2000;Wu et al 2001;Davie et al 2003), and it has been proposed that repression is mediated by recruitment of the Hda1 histone deacetylase complex to target promoters (Wu et al 2001;Robyr et al 2002). Third, Tup1 interacts with hypoacetylated tails of histones H3 and H4 (Cooper et al 1994;Edmondson et al 1996;Huang et al 1997) and appears to influence deposition of the Htz1 histone variant (Green and Johnson 2004;Gligoris et al 2007;Morohashi et al 2007). Fourth, Tup1 appears to stabilize nucleosome positioning, perhaps through the Isw2 nucleosome remodeling complex, although Tup1 and Isw2 independently associate with promoters (Zhang and Reese 2004b;Rizzo et al 2011).…”

The Yeast Minichromosome System Consisting of Highly Positioned Nucleosomes <i>in Vivo</i>

“…2B, in good agreement with previous reports. 19,20,22,23,28,30) It is noteworthy that the tentatively assigned nucleosome between nucleosomes V and VI in the previous MNase mapping experiments (white dotted ellipse between lanes 4 and 5 in Fig. 2B) was absent in the H4 S47C-site directed chemical mapping experiments.…”

“…The TALS plasmid was constructed by Roth et al 19) TALS was inserted into the Hin-dIII site of a pBR322 derivative to manipulate plasmid construction in Escherichia coli, to form the TALS-pBR322ΔRI plasmid. 22) To eliminate the pUC19 DNA sequence from TALS, the 358 bp region between the two EcoRI sites was replaced by the 378 bp fragment containing the upstream sequence of STE6 (−457 to −83, the translation start site of the gene is defined as +1), forming spTALS1-pBR322ΔRI (spTALS is designated from superior-positioning-TALS). The spTALS2-pBR322ΔRI plasmid was constructed by inverse PCR, using spTALS1-pBR322ΔRI as the template and primers attached to the STE6 upstream sequences at the 5′-end, to extend the STE6 region from −83 to −66.…”

“…18) and its derivatives, including the TALS minichromosome, 19) have been utilized to examine the effects of DNA sequences on nucleosome formation and the binding of transcription factors to nucleosomal DNA. [20][21][22][23][24][25][26][27][28] Recently, we established a parallel mapping method that utilizes site-directed hydroxyl radical cleavage and micrococcal nuclease (MNase) digestion to characterize individual nucleosomes in more detail. 29) We found that the occupancy and stability of each nucleosome in the minichromosome were different.…”