Fig. 4. Matα2p occupies the STE6 α2 operator in the absence of Tup1p. (A) The Matα2p–operator interaction in haploid cells of each mating type (a or α) was footprinted in vivo in WT (TUP1) or mutant (tup1) by M. Sss I. Expression of the single-copy methyltransferase under control of the GAL1 promoter was achieved by growth in galactose-containing media. Following rapid isolation of DNA, cytosines methylated in CpG target sites (indicated by filled circles) were identified by genomic bisulfite sequencing as described previously (Kladde et al., 1996). Band intensity is directly proportional to the degree of cytosine methylation at each CpG sequence. In vitro methylation of protein-free DNA (D) is shown in lane 1. The α2 operator is demarcated by the open bar and a CpG site within the α2 operator is marked by the asterisk. Note protection of three methylation sites adjacent to the α2 operator in α TUP1 cells (lane 3) due to the presence of a positioned nucleosome (marked by open ellipse). Increased methylation at these three CpG sites in a cells (lanes 2 and 4) or when TUP1 was deleted (lanes 4 and 5) indicates disruption of the nucleosome. (B) Operator binding by Matα2p in the same samples as in (A) was analyzed with a primer that anneals closer to the operator. Note protection of the operator CpG site (asterisk) in α cells (lanes 3 and 5) in the presence (lane 3) and absence (lane 5) of TUP1 as compared with a cells (lanes 2 and 4).