Fig. 2

Effects of internal deletions 2 (ID2) and 7 (ID7) on nucleosome occupancy across the 3’ region of PMA1. (A) Nucleosome occupancy across the 3’ region of PMA1 in wild-type, ID2, and ID7 strains was assessed using a previously published protocol [22]. Briefly, logarithmically grown cells were spheroplasted and exposed to micrococcal nuclease (MNase), and MNase-resistant DNA derived from single nucleosomes was isolated and amplified using qPCR. The regions quantified by qPCR are indicated in the figure and labeled NP (Nucleosome Positioning) amplicons – the ChIP amplicons used in Fig. 1 are also shown here to facilitate a comparison of the locations assayed in the ChIP and the nucleosome positioning experiments. In each case, protection levels are relative to the protection level measured for a “strong” nucleosome located at the 5’ region of the GAL1 open reading frame. For each of the three strains assayed, the locations of nucleosomes across the PMA1 3’ region shown in the diagrams below the graph are based on the peaks in MNase protection, with darker shades of gray corresponding to higher peaks, or more stable nucleosomes. In each case, the results are presented as mean ± S.E.M from three independent experiments. Since the internal deletions remove 68 base pairs from the PMA1’s 3’ region, the location of the nucleosome in ID2 and ID7 are shifted closer to the end of the PMA1 open reading frame compared to the nucleosomes in wild-type cells, making it difficult to compare MNase protection profiles across the strains. To facilitate comparison across the three strains, the data shown in (A) are reproduced in (B) but the length of the 3’ PMA1 region in the ID2 and ID7 strains is artificially maintained the same as that in wild-type cells by introducing a gap corresponding to 68 base pairs where the corresponding regions have been removed. Representation of the data in this fashion makes it clear that ID7 causes alterations in the occupancy levels of the two more downstream nucleosomes, with statistically significant differences relative to the wild-type strain indicated by asterisks (Student’s t-test, P < 0.05). The strains used in these experiments are yADP141, yADP143, and yADP148. Detailed information on the primers used in these experiments is provided on Table S1