A nucleosome positioned by alpha2/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 alpha2/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 alpha cells, while it was detectable in a cells, in which the nucleosomes were not positioned. Hap1 binding was restored in alpha cells with tup1 or isw2 mutations, which caused the loss of nucleosome positioning. These results support the mechanism in which alpha2/Mcm1-dependent nucleosome positioning has a regulatory function to limit the access of transcription factors.

Fig. 1

Experimental design in this study. Chromatin structures of the TALS minichromosome in MATα cells and the TRP1ARS1 minichromosome. The UAS1 site was introduced into the TALS and TRP1ARS1 minichromosomes, and its location is indicated by the shaded boxes in TALS-UAS1a, -UAS1b and UAS1c and TRP1ARS1-UAS1. The α2 operator and the nucleosomes are shown by the hatched boxes and the gray ellipses, respectively. The asterisk indicates the dyad of the positioned nucleosome IV.

Fig. 2

High resolution mapping of MNase cleavage sites in TALS and TRP1ARS1 derivatives containing the UAS1 site. aWT and αWT are wild-type strains. The locations of the α2 operator (α2 op, the hatched box), UAS1 (the shaded box) and the nucleosome IV (the gray ellipse) are shown on the left side of the gel. In each set of data, lanes labeled C indicate MNase digestion of isolated nuclei (chromatin) at two nuclease levels, and lanes labeled D indicate MNase digestion of the naked DNA as a control. The degree and integrity of MNase digestion in each sample were judged from nucleosome ladders of genomic DNA analyzed by agarose gel electrophoresis.

Fig. 3

In vivo UV-photo footprints of Hap1 in TALS and TRP1ARS1 derivatives containing the UAS1 site. Lanes marked C are samples from intact cells irradiated with UV light (500 and 750mJ/cm²); lanes marked D are samples from purified DNA irradiated with UV light (240 mJ/cm²). Arrows indicate sites of enhancements of UV photoproducts in irradiated cells, as compared with irradiated purified DNA (lane D).