FIGURE 1.

Analysis of the association of RNA polymerase II with GAL1. A, a schematic diagram showing the locations of the primer pairs (ORF1 and ORF2) at the GAL1 coding sequence for the ChIP analysis. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). B, Rad6p promotes the association of RNA polymerase II with GAL1. Yeast strains (STY1 and STY2) were grown in YPR at 30 °C up to an A₆₀₀ of 0.9, and then switched to YPG for 60 min prior to formaldehyde-based in vivo cross-linking. The ChIP assay was performed as described under ”Experimental Procedures.“ Immunoprecipitation was performed using 8WG16 antibody (Covance, Inc.) against the carboxyl-terminal domain of the largest subunit (Rpb1p) of RNA polymerase II. Immunoprecipitated DNA was analyzed by PCR using the primer pairs located at two different locations of the GAL1 coding sequence (ORF1 and ORF2). The ratio of the immunoprecipitate over the input in the autoradiogram (termed as a ChIP signal) was measured. The ChIP signal of the wild-type strain was set to 100, and the ChIP signal of the mutant strain was normalized with respect to 100 (represented as normalized or relative occupancy). Error bars denote S.D. from three sets of biologically independent experiments. p values were calculated using the Student's t test. C, association of RNA polymerase II with GAL1 is significantly decreased in the histone H2B-K123R point mutant strain. Yeast strains (YKH045 and YKH046) were grown, cross-linked, and immunoprecipitated as in panel B. D, association of RNA polymerase II with GAL1 is not impaired in the absence of Bre1p. Yeast strains (YTT31 and SLY1a) were grown, cross-linked, and immunoprecipitated as in panel B. E, analysis of the GAL1 transcription by RT-PCR. The wild-type and mutant strains were grown as in panel B. The transcript level of the wild-type strain was set to 100, and the mRNA level in the mutant strain was normalized with respect to 100. F and G, analysis of RNA polymerase II association with GAL1 and transcription in the commercial Δbre1 and isogenic wild-type (BY4741) strains (from Open Biosystems). Both wild-type and mutant strains were grown as in panel B. H, Western blot analysis for histone H2B ubiquitylation in the presence and absence of Bre1p or its RING domain, using an anti-FLAG antibody against FLAG-tagged histone H2B. The bre1Δ50 strain (RSY13) represents Bre1p without its RING domain. UbH2B, ubiquitylated histone H2B; and Flag-H2B, FLAG-tagged histone H2B. I, growth analysis of the Δrad6, Δbre1, and isogenic wild-type strains in solid YPD containing 100 mm HU or 0.026% MMS.