Figure 1.

Recruitment of Sgf29p to the GAL1 UAS or minimal Gal4p binding site. (A) A schematic diagram showing the PCR amplification regions at the UAS and core promoter of the GAL1 gene. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). (B) Recruitment of Sgf29p to the UAS, but not core promoter, of the GAL1 gene. Yeast strain expressing myc-epitope tagged Sgf29p was grown at 30°C in 1% yeast extract containing 2% peptone plus 2% galactose (YPG) up to an OD₆₀₀ of 1.0 prior to formaldehyde-based in vivo crosslinking. The ChIP assay was performed as described previously (37, 57, 58). Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz Biotechnology, Inc.) or a polyclonal antibody against TAF12p. A mouse monoclonal antibody against the DNA binding domain of Gal4p (RK5C1; Santa Cruz Biotechnology, Inc.) was used. Primer-pair targeted to the UAS or core promoter of the GAL1 gene was used for PCR analysis of the immunoprecipitated DNA samples. (C) Recruitment of Sgf29p to the minimal Gal4p-binding sites. The plasmid containing minimal Gal4p-binding sites was transformed into the yeast strain expressing myc-epitope tagged Sgf29p. Yeast strain thus generated, was grown at 30°C in 1% yeast extract containing 2% peptone plus 2% raffinose (YPR) or YPG. The ChIP analysis was performed as described in panel B. The primers used for the PCR analysis are adjacent to the Gal4p-binding sites in the plasmid. The growth media are indicated on the left. (D) Recruitment of Sgf73p and Ubp8p to the minimal Gal4p-binding sites. The plasmid containing minimal Gal4p-binding sites was transformed into the yeast strain expressing myc-epitope tagged Sgf73p or Ubp8p. Yeast strains thus generated, was grown at 30°C in YPG or YPR. The ChIP analysis was performed as described in panel B.