FIGURE 4.

Analysis of Paf1p recruitment to GAL1 in the presence and absence of Bre1p. A, analysis of Paf1p association with GAL1 under inducible and non-inducible conditions. Wild-type yeast strain expressing Myc-tagged Paf1p (RSY16) was grown, cross-linked, and immunoprecipitated as described in the legend to Fig. 3A. The maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100 (represented as normalized or relative occupancy). Error bars denote S.D. from three sets of biologically independent experiments. B, association of Paf1p with GAL1 is significantly decreased in the Δbre1 strain. Both the wild-type and Δbre1 strains expressing Myc-tagged Paf1p (RSY16 and RSY17) were grown, cross-linked, and immunoprecipitated as in Fig. 3B. C, Western blot analysis. Yeast cells were grown as described in the legend to Fig. 1B. D, Paf1p promotes the association of RNA polymerase II with GAL1. Both the wild-type and Δpaf1 strains (W303a and DY7014) were grown, cross-linked, and immunoprecipitated as described in the legend to Fig. 1B. E, analysis of GAL1 transcription in the Δpaf1 strain by RT-PCR. Yeast cells were grown as described in the legend to Fig. 1B. F, the RING domain of Bre1p does not regulate the recruitment of Paf1C to GAL1. Both the wild-type and mutant strains expressing Myc-tagged Paf1p (RSY21 and RSY22) were grown and cross-linked as described in the legend to Fig. 1B. Immunoprecipitation was performed as in panel A.