FIG. 3.

Chromatin immunoprecipitation to detect Nrg1-HA and Nrg2-HA binding. Cells expressed Nrg1-HA or Nrg2-HA from the chromosomal locus (MCY4744 and MCY4751, respectively) or expressed native, untagged Nrg1 (MCY4702). (A to C) Cells were grown in YEP containing 2% glucose (Hi). Cells expressing HA-tagged Nrg proteins were also shifted to 0.05% glucose (Lo) for 3 h or were grown in 2% glycerol-2% ethanol (GE). (D) Cells were grown in YEP containing 2% glycerol-2%ethanol and were shifted to 2% glucose for 5 min. DNA was purified from total chromatin (input) and chromatin that was immunoprecipitated with anti-HA (IP). DNA from the input was diluted 1:1,000, and DNA from the immunoprecipitation was used undiluted or diluted 1:10, where indicated. DNA was also used at a 1:3 dilution to confirm linearity (data not shown). Five microliters was used as a template in PCRs with primers specific for the control POL1 coding sequence and two regions of the RPI1 promoter, spanning nucleotides −1610 to −1352 and −1137 to −697 (CCCCT sequences are at positions −1571, −1086, and −731) or the CYC7 promoter (−480 to −188).