FIG. 4.

Sfl1 inhibits access of Ste12 and Tec1 to UAS2-1. (A) Cells containing a control, multicopy NRG1, or multicopy SFL1 plasmid were grown in 2% glycerol-ethanol medium to mid-log phase and subjected to a glucoamylase activity assay. (B) pRS323 (−), pRS323-NRG1 (+), or pRS323-SFL1 (+) was transformed into integrated HA-tagged strains. Transformants were grown to mid-log phase in synthetic medium containing 2% glycerol-ethanol and were fixed with formaldehyde. After α-HA ChIP, UAS1-2 and UAS2-1 were PCR amplified by using purified DNA. (C) Cells containing the multicopy SFL1 plasmid (+) or controls (−) were grown in 2% glycerol-ethanol medium to mid-log phase, and total proteins were extracted from these cells. Levels of HA-tagged transcriptional activators from 50 or 100 μg of protein extract were determined. (D) pLG-UAS1 (lane 1), pLG-UAS1-NRG1 (lane 2), pLG-UAS2 (lane 3), or pLG-UAS2-SFL1 (lane 4) was introduced into wild-type cells. The resulting transformants were grown in synthetic medium containing 2% glycerol-ethanol before being subjected to a β-galactosidase activity assay. (E) pLG-UAS2 (−) or pLG-UAS2-SFL1 (+) was transformed into wild-type, STE12-HA, or TEC1-HA cells. The resulting transformants were cultured and subjected to an α-HA ChIP assay as in panel B. (F) pLG-UAS2a (−) and pLG-UAS2a-SFL1 (+) were transformed into the same strains. A ChIP assay was performed with an α-HA antibody as above, and Ste12 or Tec1 binding to UAS2-1 was detected by PCR.