Nucleosomes are positioned with base pair precision adjacent to the alpha 2 operator in Saccharomyces cerevisiae.

Abstract

Analysis of the chromatin structure of minichromosomes containing the binding site for the yeast alpha 2 repressor protein by indirect end‐labeling has previously indicated that nucleosomes are stably positioned over sequences adjacent to the alpha 2 operator in the presence of the repressor. Development of a primer extension assay for nucleosome position now allows a more detailed examination of the location of these nucleosomes relative to the operator sequence, and indicates that nucleosomes are precisely and stably positioned both translationally and rotationally over sequences adjoining the operator. In addition, this assay enables analysis of the chromatin structure of single copy, genomic sequences. Chromatin structures determined for two genes regulated by alpha 2, STE6 and BAR1, are consistent with nucleosomes precisely positioned downstream of the operator sequence, incorporating promoter elements, in alpha cells but not in a‐cells. The location of these nucleosomes relative to the operator sequence is highly analogous to that observed in the minichromosome. The stability of the nucleosomes adjacent to the operator together with the precision of their location suggests that they may play a role in repression of a specific gene expression by alpha 2. Further, the primer extension assay allows a comparison of the structure of these positioned nucleosomes formed in vivo to that previously described for core particles reconstituted in vitro.

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

“…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.…”

“…The α2 operator is located in the region of 1535-1565 nucleotides (nt) in TALS. Nucleosome IV is positioned both translationally and rotationally adjacent to the α2 operator in the region of 1376-1522 nt in TALS in α cells, but not in a cells, as shown previously [17,23]. These minichromosomes have been utilized to examine the binding of Gal4 [7][8][9][10][11] (Fig.…”

“…Isolation and digestion of nuclei with micrococcal nuclease (MNase) were described previously [13,17]. MNase cleavage sites were analyzed by primer extension mapping, using the IRDye800-labeled MS-2 primer (1735-1701 nt in TALS) [17] with the LI-COR 4200L DNA sequencer.…”

“…MNase cleavage sites were analyzed by primer extension mapping, using the IRDye800-labeled MS-2 primer (1735-1701 nt in TALS) [17] with the LI-COR 4200L DNA sequencer.…”

Nucleosome positioning and gene regulation

“…A translational signal for nucleosome positioning generated by specific protein binding is also observed on studying a2-mediated repression of a-cell-type specific gene expression in haploid a Levinger 119851 Ambrose et al [1990] cells and in diploid a / a cells in yeast [reviewed by Herskowitz, 19891. The a2-repressor, in association with another promoter-specific protein MCM1, represses gene expression apparently by placing nucleosomes next to the a,-MCM1 complex, resulting in nucleosome occupancy of the TATA box of the adjacent genes [Shimizu et al, 1991;Roth et al, 19921. Five similar cases have been studied; in three of these, nucleosomes have been carefully mapped.…”