Figure 3

Hot1p and Sko1p bind to the YHR087W promoter under high sugar stress in a Hog1p- dependent manner. Panel A1 shows the relative occupation of Hot1p over the region of the YHR087W promoter between -360 and -181, determined by chromatin immunoprecipitation (ChIP) followed by Real-Time PCR, in a Δ hog1 mutant and its corresponding wild type strain, both of which contained a TAP-tagged version of Hot1p. Data were obtained as described in the Materials and Methods section. Experiments were carried out in triplicate; the average and standard deviation data are indicated. Panel A2 contains the result of a representative ChIP experiment analyzed by semiquantitative PCR. In this case, a mix of three pairs of primers was used for each PCR reaction, which amplify the region corresponding to the YHR087W promoter described above and, those located between positions -750 and -420 of gene CLN2 and -822 to -567 of gene CLB2. In this panel WCE indicates the whole cell extract prior to the inmunoprecipitation. For these experiments, cells from the exponentially growing cultures were also incubated for 10 min in YP20 (YP20 in panel A.1, + in panel A.2) or not (Control in panel A.1, - in panel A.2) to determine the dependence of Hot1p binding on high glucose stress. Panels B1 and B2 correspond to the same experiments in the case of a Δ hog1 mutant and its corresponding wild type strain, both of which contained a TAP-tagged version of Sko1p.