Rad26p promotes the association of RNA polymerase II with the GAL1 coding sequence following transcriptional induction. (A) The schematic diagram of the GAL1 coding sequence or ORF with the PCR amplification regions (ORF, ORF1 and ORF2) in the ChIP assay. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). (B–D) Analysis of Rpb1p (the largest subunit of RNA polymerase II) association with the GAL1 coding sequence in the Δrad26 strain and its isogenic wild-type equivalent. The wild-type and Δrad26 strains were first grown in YPR up to an OD₆₀₀ of 0.9, and then shifted to YPG for different periods of induction prior to formaldehyde-based in vivo cross-linking. Immunoprecipitation was performed using the mouse monoclonal antibody 8WG16 (Covance) against Rpb1p-CTD (carboxy terminal domain). The specific primer pair targeted to the different locations in the GAL1 coding sequence was used for PCR analysis of the immunoprecipitated DNA samples. The ChIP signal is the ratio of immunoprecipitate over the input in the autoradiogram. The maximum ChIP signal in the wild-type strain was set to 100, and other ChIP signals in the wild-type and Δrad26 strains were normalized with respect to 100. The normalized ChIP signals (represented as normalized occupancy) were plotted as a function of induction times. (E) Primer extension analysis. Both the wild-type and Δrad26 strains were grown in YPR up to an OD₆₀₀ of 0.9, and then switched to YPG for 60 min. Total RNA was prepared and analyzed by primer extension. (F) RT-PCR analysis. Yeast strains were grown as in panel E. Total RNA was prepared and analyzed for transcription. Oligo(dT) primer was used for cDNA synthesis. (G) Rad26p does not regulate the formation of transcription complex at the GAL1 promoter. Both the wild-type and Δrad26 strains were grown as in panel E prior to formaldehyde-based in vivo cross-linking. Immunoprecipitations were performed using anti-Gal4p (RK5C1; Santa Cruz Biotechnology), anti-TAF12p (obtained from the laboratory of Michael R. Green) and anti-TBP (obtained from the laboratory of Michael R. Green) antibodies against Gal4p, TAF12p and TBP, respectively. (H) Regulation of Rpb1p occupancy at the GAL1 core promoter by Rad26p in the presence and absence of the coding sequence. Wild-type and mutant strains were grown as in panel E prior to cross-linking. +ORF, with open reading frame; and –ORF, without open reading frame. (I) Analysis of Rpb1p occupancy at different regions of the GAL1 coding sequence following 60 min transcriptional induction in YPG. The maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100.

Rad26p does not alter the eviction of histone H3 from the coding sequences of GAL1 (A), GAL7 (B) and GAL10 (C) upon transcriptional induction. Both the wild-type and Δrad26 strains were grown in YPR up to an OD₆₀₀ of 0.9, and then switched to YPG for 30 min prior to formaldehyde treatment. Immunoprecipitation was performed using an anti-histone H3 antibody (Abcam; Ab-1791) against histone H3. Immunoprecipitated DNA was analyzed by PCR, using the primer pairs targeted to the coding sequences of GAL1, GAL7 and GAL10.

Eviction of histone H3 from the GAL1 (A), GAL7 (B) and GAL10 (C) coding sequences is impaired immediately following transcriptional induction in the absence of Rad26p. Both the wild-type and Δrad26 strains were grown in YPR up to an OD₆₀₀ of 0.9, and then switched to YPG for 5 min prior to formaldehyde treatment. Immunoprecipitation was performed as in Figure 3.

Analysis of the roles of Rad26p's ATPase domain and Swi2p in regulation of the occupancy of histone H2B. (A) Schematic diagram of different mutant versions of Rad26p. (B) The Rad26p mutant without Swi2p/Snf2p domains IV, V and VI is defective in regulating the occupancy of histone H2B. Yeast strains bearing wild-type and mutant versions of Rad26p were grown in YPR up to an OD₆₀₀ of 0.9, and then switched to YPG for 30 min prior to cross-linking. Immunoprecipitation was performed as in Figure 4A. The primer pair targeted to the GAL1 coding sequence was used for PCR analysis of immunoprecipitated DNA samples. (C) Recruitment of TBP and Rpb1p to the GAL1 core promoter is impaired in the Δswi2 strain. Both the wild-type and mutant cells were grown and cross-linked as in panel B. Immunoprecipitation was performed as in Figure 1B and G. (D) Analysis of Gal4p recruitment to the GAL1 UAS in the Δswi2 and wild-type strains. Immunoprecipitation was performed as in Figure 1G. (E) Analysis of histone H2B occupancy at the GAL1 core promoter in the Δswi2 strain and its isogenic wild-type equivalent.

Rad26p is predominantly associated with the coding sequences of CTT1 (A) and STL1 (B) following transcriptional induction. Yeast cells were grown in synthetic complete medium up to an OD₆₀₀ of 0.9, and then induced by 0.45 M NaCl for 7 min prior to cross-linking. Immunoprecipitation was performed as described previously (13). Rad26p facilitates the loss of histone H2B from the INO1 (C), CTT1 (D), and STL1 (E) coding sequences following transcriptional induction. Immunoprecipitation was performed as in Figure 4.

Rad26p promotes the association of RNA polymerase II with the GAL7 and GAL10 coding sequence following transcriptional induction. (A and B) Analysis of Rpb1p association with the GAL7 and GAL10 coding sequences in the Δrad26 strain and its isogenic wild-type equivalent. The yeast strains were grown and cross-linked as in Figure 1B. The specific primer pairs targeted to the GAL7 and GAL10 coding sequences were used for PCR analysis of the immunoprecipitated DNA samples. (C) RT-PCR analysis. Both the wild-type and Δrad26 strains were grown as in Figure 1E. (D) Rad26p does not regulate the recruitment of TBP to the GAL7, GAL10 and RPS5 core promoters. Both the wild-type and Δrad26 strains were grown as in Figure 1E prior to cross-linking. Immunoprecipitation was performed as in Figure 1G. The specific primers targeted to the core promoters were used for PCR analysis of the immunoprecipitated DNA samples. (E) The steady-state levels of RNA polymerase II association with the coding sequences of GAL1, GAL7 and GAL10 are not altered in the absence of Rad26p. Both the wild-type and Δrad26 strains were continuously grown in YPG prior to cross-linking. (F) Rad26p does not alter the association of RNA polymerase II with the coding sequence of a constitutively active gene, RPS5. Yeast strains were grown in YPD up to an OD₆₀₀ of 1.0 prior to cross-linking. Immunoprecipitated DNA was analyzed by PCR, using the primer pair targeted to the RPS5 coding sequence.

Rad26p promotes the loss of histone H2B from the coding sequences of GAL1, GAL7 and GAL10 following transcriptional induction. (A) Regulation of histone H2B occupancy at the GAL1 coding sequence by Rad26p. Both the wild-type and Δrad26 strains carrying Flag-tagged histone H2B were grown in YPR up to an OD₆₀₀ of 0.9, and then switched to YPG for different periods of transcriptional induction prior to formaldehyde treatment. Immunoprecipitation was performed using an anti-Flag antibody (Sigma; F1804) against Flag-tagged histone H2B. (B and C) Analysis of the occupancies of histone H3 and Flag-tagged histone H2B at the GAL1 coding sequence following 30 and 60 min transcriptional induction in YPG in the Δrad26 and wild-type strains. (D and E) Regulation of histone H2B occupancy at the GAL7 and GAL10 coding sequences by Rad26p following transcriptional induction.

Analysis of histone H3 eviction from the GAL1 (A), GAL7 (B) and GAL10 (C) coding sequences following 30 min transcriptional induction in the Δrad26, Δasf1, Δrad26Δasf1 and wild-type strains.

Growth analysis of the Δswi2, Δrad26, Δswi2Δrad26 and wild-type strains in the solid YPD, YPG, and YPG plus antimycin growth media (A), and liquid YPG medium (B). Fold increase of Rpb1p association with the GAL1 coding sequence following 90 min induction in the Δrad26, Δswi2, Δswi2Δrad26 and wild-type strains (C). Fold increase is the ratio of ChIP signal following 90 min induction to that prior to induction. (D) RT-PCR analysis of GAL1 mRNA following 90 min induction in YPG. (E) Analysis of histone occupancy at the GAL1 coding sequence in the Δrad26, Δswi2, Δswi2Δrad26 and wild-type strains following 90 min transcriptional induction in YPG.

Rad26p does not alter the eviction of histone H3 from the INO1 (A), CTT1 (B) and STL1 (C) coding sequences following transcriptional induction. Both the wild-type and mutant strains were grown and cross-linked as in Figure 9. Immunoprecipitation was performed as in Figure 3. (D) Rad26p facilitates the association of RNA polymerase II with the STL1, CTT1 and INO1 coding sequences. Immunoprecipitation was performed as in Figure 1B.