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. 2004 Nov;24(21):9542–9556. doi: 10.1128/MCB.24.21.9542-9556.2004

FIG. 7.

FIG. 7.

Ste12 and Tec1 recruit the Swi/Snf complex to promote Flo8 and Mss11 binding to UAS1-2 in the endogenous STA1 promoter. (A) Interactions between activators and the endogenous STA1 promoter in a snf6Δ mutant. FLO8-HA wild-type (WT), FLO8-HA snf6Δ, MSS11-HA wild-type, MSS11-HA snf6Δ, STE12-HA wild-type, STE12-HA snf6Δ, TEC1-HA wild-type, TEC1-HA snf6Δ, and untagged strains were grown in synthetic medium containing 2% glycerol-ethanol to mid-log phase, and anti-HA (α-HA) ChIP assays were performed with 1 mg (for Flo8 and Mss11) and 500 μg (for Ste12 and Tec1) of total extracts to determine the binding of activators to UAS1-2 or UAS2-1. (B) Ste12 and Tec1 recruit the Swi/Snf complex to the STA1 promoter. SNF6-HA wild-type, SNF6-HA flo8Δ, SNF6-HA mss11Δ, SNF6-HA ste12Δ, SNF6-HA tec1Δ, and untagged strains were grown in synthetic medium containing 2% glycerol-ethanol to mid-log phase, and anti-HA ChIP assays were performed as described above with 500 μg of total extracts to determine Snf6 binding to the endogenous STA1 promoter. (C) Flo8 and Mss11 interact with a component of RNA polymerase II. Whole-cell extracts (500 μg) prepared from the integrated RPB3-HA strain were incubated with 5 μg (each) of GST, GST-Flo8, or GST-Mss11. The GST proteins and their interacting proteins were precipitated with glutathione-agarose beads. Fractions of input (1/10) and pellet (1/2) were analyzed by Western blot analysis with anti-HA antibody. (D) Flo8 and Mss11 are required for recruitment of RNA polymerase II. RPB3-HA wild-type, RPB3-HA flo8Δ, RPB3-HA mss11Δ, and untagged strains were grown in synthetic medium containing 2% glycerol-ethanol to mid-log phase, and anti-HA ChIP assays were performed as described above with 500 μg of total extracts to determine RNA polymerase II binding to the core promoter of the endogenous STA1 promoter.