FIGURE 7.

Met32-1 prevents recruitment of Met4 to MET gene promoters. A, met30-6 met31 met32 mutants expressing TAP-tagged versions of either Met31, Met32, or Met32-1 under control of the GAL1 promoter were analyzed by chromatin immunoprecipitation. Binding of ^TAPMet31, ^TAPMet32, and ^TAPMet32-1 to the promoter regions indicated was analyzed in cells grown at 25 °C and after MET gene expression was induced by inactivation of Met30-6 for 1 h at 37 °C. Cells were grown in YEP-galactose medium to continuously express the TAP-tagged proteins. TAP-tagged proteins were purified from formaldehyde cross-linked samples on IgG beads, and promoter fragments were detected by qPCR. Promoter binding is represented on the y axis as the relative signal obtained in the ChIP compared with the input from whole cell lysates. The highest value was arbitrarily set to 100%. The dotted line represents the background-binding signal obtained by analysis of binding to the ADH1 promoter region. B, to test whether Met32 and Cbf1 are found in the same Met4-containing transcription complex, met31 met32 double and met31 met32 met4 triple mutants expressing endogenous HA₃-tagged Cbf1 and TAP-tagged Met32 under control of the GAL1 promoter were analyzed. Cells were grown in SD-Gal/+Met, and MET gene expression was induced for 30 min by shifting cells to SD-Gal/-Met medium. ^TAPMet32 was purified on IgG beads, and the purified protein complexes were separated by SDS-PAGE and analyzed by immunoblotting using antibodies directed against the HA tag or peroxidase-conjugated anti-peroxidase antibody to detect the TAP tag. Cbf1 binding was induced by methionine depletion and depended on Met4. C, promoter association of Met31, Met32, and Met32-1 was analyzed by ChIP. met31 met32 double and met31 met32 cbf1 triple mutants expressing TAP-tagged Met31, Met32, or Met32-1 under the control of the GAL1 promoter were grown in SD-Gal/+Met, and MET gene expression was induced by shifting cells to SD-Gal/-Met for 30 min. ChIP and data representation are as described for A. D, to test the effect of Met32-1 on recruitment of Met4 to MET gene promoters, cells expressing endogenous 18-Myc-tagged Met4 and either Met31, Met32, or Met32-1 under control of the GAL1 promoter were analyzed by ChIP. Cell growth and MET gene induction were as described for C. Met4 was immunopurified with anti-Myc antibodies and ChIP analyses, and presentation of binding data is as described for A. Note that cells express endogenous levels of Met31 and Met32 in addition to the proteins expressed under GAL1 control. WCL, whole cell lysates.