FIG. 1.

Induction of Gcn4p increases Cbp20p occupancy in the transcribed, but not UAS, region of ARG1, dependent on the TATA element. (A) ARG1 locus with names and positions of fragments (relative to the ATG) that were PCR amplified for ChIP analyses given below. Dashed lines depict the adjacent gene YOL057w. (B to D) ChIP analysis of Myc-Cbp20p or Rpb3p occupancy at ARG1 following induction of Gcn4p. CBP20-myc strains CMY007 (gcn4Δ), CMY008 (WT), and CMY009 (arg1-ΔTATA) were cultured in synthetic complete medium lacking Ile and Val and treated with sulfometuron (final concentration, 0.5 μg/ml) for 30 min to induce Gcn4p synthesis. Cells were cross-linked with formaldehyde and subjected to ChIP analysis with anti-Myc (B and C) or anti-Rpb3p (D) antibodies. DNA was extracted from immunoprecipitates (IP) and input chromatin (Input) samples and subjected to PCR in the presence of [³³P]dATP to amplify radiolabeled fragments from the UAS, TATA, 5′-ORF, middle-ORF, or 3′-ORF regions of ARG1 together with a control fragment from a nontranscribed region of HML. PCR products were resolved by 6% Tris-buffered EDTA polyacrylamide gel electrophoresis and visualized by autoradiography (B) or quantified with a phosphorimager, and the ratios of experimental to control signals in the immunoprecipitate samples were normalized for the corresponding ratios for input samples to yield the occupancy values plotted in histograms (C and D). (E) Strains with genes encoding the relevant Myc-tagged proteins HQY691 (SUA7-myc), CMY005 (CBP20-myc), and CMY006 (CBP80-myc) were cultured in synthetic complete medium lacking histidine with 2% raffinose to an A₆₀₀ of ≈0.6 and treated with galactose (final concentration, 2%) for 30 min to induce GAL1. Cells were cross-linked with formaldehyde and subjected to ChIP analysis with anti-Myc antibodies as described above, except with the use of primers to amplify the TATA, 5′-ORF, and 3′-ORF sequences at GAL1 corresponding to nucleotides −185 to −57, +422 to + 567, and +1233 to +1355, respectively. The average results obtained from two independent cultures and two PCR amplifications for each culture were plotted in the histograms with standard errors shown as error bars.