FIG. 8.
The intranucleosomal location of a UASp element in the −2 nucleosome is important but not essential for PHO5 promoter chromatin opening in vivo. DNase I indirect end-labeling analysis of the PHO5 promoter region in the (A) wild type (wt) (CY337), ΔUASp2 (CY341), ΔUASp2-Bst-hi (CY337 FE1600), and H1 (CY337 EB1615) (35) configurations and in the (B) UASp2-5 Δ2 (CY339 ura3 pCB-UASp2-5 Δ2), and UASp2-8 Δ2 (CY339 ura3 pCB-UASp2-8 Δ2) configurations after overnight (o/n) incubation in phosphate-free (−Pi) medium. For the UASp2-5 Δ2 mutant, an experiment with incubation in phosphate-free medium for 40 h, i.e., two times overnight, and one experiment with overexpression (o/x) of PHO4 are shown. Small dots between lanes mark the bands corresponding to the linker regions flanking the positioned −1 nucleosome as shown in the schematic on the left, and vertical bars between the lanes highlight the extent of a hypersensitive region, stippled if less extensive. For UASp2-5 Δ2, the stippled vertical line denotes less-pronounced hypersensitivity in the region of the −3 and −4 nucleosomes and the large dot marks the increased hypersensitivity of the linker between the −2 and −3 nucleosomes. Samples from the same chromatin preparation but from two different gels are shown for the H1 mutant promoter. Schematics above the panels are as in Table 1. Markers, ramps, and schematics next to the gels are as in Fig. 2.