Analysis of the recruitment of CBC to the GAL1 and ADH1 genes. (A) Both the Cbp20p and Cbp80p components of CBC are predominantly recruited to the GAL1 ORF. Yeast strains expressing myc-tagged Cbp20p or Cbp80p were grown at 30°C in YPG. The ChIP assay was performed as previously described (62–65). Primer-pairs [(69), ‘Materials and Methods’ section] located in the UAS, core promoter and two different locations of ORF (ORF1 and ORF2) of GAL1 were used for PCR analysis of the immunoprecipitated DNA samples. Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz Biotechnology Inc.). The ratio of immunoprecipite over the input in the autoradiogram is indicated below each band. IP, immunoprecipitate. (B) Recruitment of CBC to the GAL1 ORF is dependent on TBP. Wild-type and ts mutant strains of TBP (spt15-ts) expressing myc-tagged Cbp80p were first grown in YPG at 23°C to an OD₆₀₀ of 0.85, and then transferred to 37°C for 1 h prior to formaldehyde-based in vivo cross-linking. The mouse monoclonal antibody 8WG16 (Covance) against Rpb1p-CTD (carboxy terminal domain) was used. (C) Cbp20p and Cbp80p are mutually dependent for their recruitment to GAL1. Wild-type and mutant strains were grown in YPR up to an OD₆₀₀ of 0.9, and then switched to YPG for 90 minutes at 30°C prior to cross-linking. (D) Western blot analysis. The wild-type and deletion mutant strains expressing myc-tagged Cbp20p/Cbp80p were grown as in panel C. The whole-cell extracts from the wild-type and deletion mutant strains were prepared as in the ChIP assay (62–65), and were analyzed by western blot using anti-myc and anti-TBP antibodies against myc-tagged Cbp20p/Cbp80p and TBP, respectively. (E) CBC is predominantly recruited to the ADH1 ORF. Primer pairs [(69), ‘Materials and Methods’ section] located in the UAS, core promoter and two different locations of ORF (ORF1 and ORF2) of ADH1 were used for PCR analysis of the immunoprecipitated DNA samples. (F) Recruitment of CBC to the ADH1 ORF is dependent on RNAPII as well as TBP. Wild-type and ts mutant strains of TBP (spt15-ts) and RPB1 expressing myc-tagged Cbp80p were first grown in YPD at 23°C to an OD₆₀₀ of 0.85, and then transferred to 37°C for 1 h prior to formaldehyde cross-linking. Immunoprecipitations were performed as described in panels A and B. (G) Recruitments of Cbp20p and Cbp80p to ADH1 are dependent on each other.

The rate of association of RNAPII with the GAL1 coding sequence is significantly reduced in the absence of CBC. (A) Analysis of the rate of association of Rpb3p with the GAL1 coding sequence in Δcbp80 and its isogenic wild-type equivalent. The wild-type and CBP80 deletion mutant strains expressing myc epitope-tagged Rpb3p were first grown in raffinose-containing growth media (YPR) up to an OD₆₀₀ of 0.9, and then shifted to galactose-containing growth media (YPG) for different time periods before treatment with formaldehyde. Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag. The specific primer pair targeted to the GAL1 ORF2 (Figure 1A) was used for PCR analysis of the immunoprecipitated DNA samples. The ratio of immunoprecipite over the input in the autoradiogram was measured. The maximum ratio was normalized to 100. The normalized ratio (represented as normalized occupancy) was plotted as a function of induction time. (B) Analysis of the rate of association of Rpb1p with the GAL1 coding sequence in Δcbp80 and its isogenic wild-type equivalent. The wild-type and deletion mutant strains were grown, cross-linked and immunoprecipitated as in panel A. (C) Growth analysis of the CBP80 wild-type and deletion mutant strains following induction in YPG. Both the wild-type and mutant strains were grown in raffinose-containing growth medium up to an OD₆₀₀ of 0.9, and then switched to galactose-containing growth medium for 90 min. The OD₆₀₀ of both the wild-type and mutant strains were measured in the galactose-containing growth medium within 90 min. (D) Western blot analysis. The wild-type and CBP80 deletion mutant strains were grown as in panel A, but induced for 60 min in YPG. The whole-cell extracts from both the wild-type and deletion mutant strains were prepared as in the ChIP assay (62–65). The whole-cell extracts were analyzed by western blot using anti-myc, anti-Rpb1 and anti-TBP antibodies against myc-tagged Rpb3p, Rpb1p and TBP, respectively.

The stimulation of the PIC formation by CBC in vivo. (A) Analysis of the rate of recruitment of Rpb1p at the GAL1 core promoter in Δcbp80 and its isogenic wild-type equivalent. The wild-type and CBP80 deletion mutant strains were grown, cross-linked and immunoprecipitated as in Figure 2A and B. The specific primer pair targeted to the GAL1 core promoter was used for PCR analysis of the immunoprecipitated DNA samples. (B) Analysis of the rate of recruitment of Rpb3p at the GAL1 core promoter in Δcbp80 and its isogenic wild-type equivalent. (C) Analysis of the rate of recruitment of TBP at the GAL1 core promoter in Δcbp80 and its isogenic wild-type equivalent. Immunoprecipitations were performed using an anti-TBP antibody. (D) Analysis of recruitment of TFIIB at the GAL1 core promoter following 60 min induction in YPG. The wild-type and CBP80 deletion mutant strains were first grown in YPR up to an OD₆₀₀ of 0.9 at 30°C, and then shifted to YPG for 60 min before treatment with formaldehyde. The mouse monoclonal antibody against TFIIB (obtained from Danny Reinberg, UMDNJ) was used. (E) TAF14p is predominantly recruited to the GAL1 core promoter as a component of TFIIF. Immunoprecipitation was carried out using a polyclonal antibody against TAF14p (obtained from Michael R. Green, UMass Medical School). (F) Recruitment of TAF14p (TFIIF) to the GAL1 core promoter is significantly decreased in the CBP80 deletion mutant strain. Both the wild-type and CBP80 deletion mutant strains were grown and cross-linked as in panel D. (G) Recruitment of Rad3p, a component of TFIIH, to the GAL1 core promoter is significantly decreased in the CBP80 deletion mutant strain. Both the wild-type and CBP80 deletion mutant strains were grown and cross-linked as in panel D.

CBC does not alter the recruitment of Gal4p, SAGA, Mediator and SWI/SNF to the GAL1 UAS. (A) Analysis of the rate of recruitment of Gal4p to the GAL1 UAS in Δcbp80 and its isogenic wild-type equivalent. The wild-type and CBP80 deletion mutant strains were grown, cross-linked and immunoprecipitated as in Figure 2A. A mouse monoclonal antibody against the DNA binding domain of Gal4p (RK5C1; Santa Cruz Biotechnology Inc.) was used. The specific primer-pair targeted to the GAL1 UAS was used for PCR analysis of the immunoprecipitated DNA samples. (B) Analysis of the rate of recruitment of SAGA (Spt20p-myc) to the GAL1 UAS in Δcbp80 and its isogenic wild-type equivalent. (C) Analysis of the rate of recruitment of Mediator (Srb4p-myc) to the GAL1 UAS in Δcbp80 and its isogenic wild-type equivalent. (D) Snf2p, a component of the SWI/SNF complex, is predominantly recruited to the GAL1 UAS. The yeast strain expressing myc epitope-tagged Snf2p was grown and cross-linked as in Figure 1A. Immunoprecipitation was performed using a modified ChIP protocol as described in the ‘Materials and Methods’ section. (E) The recruitment of Snf2p at the GAL1 UAS was not altered in the CBP80 deletion mutant strain. Both the wild-type and CBP80 deletion mutant strains expressing myc epitope-tagged Rad3p were grown and cross-linked as in Figure 3D.

The ChIP analysis for the recruitment of Gal4p, Mediator (Srb4p), TBP and RNAPII (Rpb1p) to the GAL1 promoter in the raffinose, galactose and dextrose-containing growth media. (A and B) Disassembly of PIC in the absence of active Gal4p. Yeast cells were first grown in raffinose-containing growth medium (Raf.) up to an OD₆₀₀ of 0.8, and then transferred to galactose-containing growth medium (Gal.) for 90 min induction, and finally transferred to dextrose-containing growth medium (Dex.) for different time periods as mentioned in the left panel. The recruitment of TBP, RNAPII (Rpb1p), Mediator (Srb4p) and Gal4p was analyzed in raffinose, galactose and dextrose-containing growth media. The input DNA was diluted by 5-folds for the analysis of Mediator (Srb4p) recruitment. (C) The ChIP assay to analyze the recruitment of RNAPII (Rpb3p-myc) under the activated and reactivated conditions in Δcbp80 and its wild-type equivalent. The yeast cells were first grown in raffinose-containing growth medium, and then switched to galactose-containing growth medium for 90 min (activated state). These cells were then transferred to dextrose-containing growth medium for 60 min (repressed state), and finally switched to galactose-containing growth medium for 90 min (reactivated state). The recruitment of RNAPII was analyzed at the GAL1 core promoter under activated and reactivated conditions. (D) Primer extension analysis. Both the wild-type and CBP80 deletion mutant strains were grown under activated and reactivated conditions as in panel C. The total mRNA was isolated and analyzed by primer extension assay. (E) The recruitment of TBP and RNAPII to the GAL1 core promoter is significantly impaired in the cet1-ts mutant strain. (F) The recruitment of TBP and RNAPII to the GAL1 core promoter is significantly impaired in the ceg1-ts mutant strain. (G) The steady-state level of RNAPII at the core promoter is not altered in the absence of CBC. Analysis of recruitment of RNAPII to the GAL1 core promoter in the CBP80 wild-type and deletion mutant strains following a long period of induction in YPG. Both the wild-type and deletion mutant strains were grown as in Figure 2A, but induced for 1.5 and 3 h in YPG or continuously in YPG.

CBC interacts with Mot1p. (A) The formaldehyde-based in vivo cross-linking and co-immunoprecipitation assay as discussed in the ‘Materials and Methods’ section; WB, western blot. (B) The schematic outlines of the experimental strategy to analyze protein–protein interaction in vitro. (C) The analysis of interaction of Cbp20p with Cbp80p, Mot1p and Spt20p. (D) The analysis of interaction of Cbp80p with Mot1p.

CBC stimulates the recruitment of Mot1p to the core promoter of GAL1. (A) Mot1p is predominantly recruited to the GAL1 core promoter. The yeast strain expressing myc epitope-tagged Mot1p was grown and cross-linked as in Figure 1A. Immunoprecipitation was performed following the modified ChIP protocol as described in ‘Materials and Methods’ section. The anti-HA antibody was used as a non-specific antibody. (B) CBC promotes Mot1p recruitment to the GAL1 core promoter. Both the wild-type and CBP80 deletion mutant strains carrying myc-tagged Mot1p were grown in raffinose-containing growth medium up to OD₆₀₀ of 0.9, and then shifted to galactose-containing growth medium for 90 min prior to formaldehyde-based in vivo cross-linking. Immunoprecipitation was performed as in panel A. (C) The results at the GAL1 core promoter in panel B were presented in the form of a histogram. (D) Mot1p is essential for recruitment of TBP and RNAPII. Both the wild-type and ts mutant strains were grown in galactose-containing growth medium up to OD₆₀₀ of 0.85 at 23°C, and then switched to 37°C for 1 h prior to cross-linking. Immunoprecipitations were performed as in Figures 2B and 3C. (E) Mot1p is not essential for recruitment of Gal4p and SAGA to the GAL1 UAS. The MOT1 wild-type and ts mutant strains were grown and cross-linked as in panel D. Immunoprecipitation was performed using the polyclonal antibodies against TAF10p and TAF12p (obtained from Michael R. Green, UMass Medical School). (F) Analysis of the roles of SAGA and TBP for recruitment of Mot1p to the GAL1 core promoter. The wild-type and ts mutant strain of TBP (spt15-ts) were grown and cross-linked as in panel D. The SPT20 deletion mutant and its isogenic wild-type strains were grown and cross-linked as in panel B. Immunoprecipitation was performed as in panel A.

CBC stimulates the PIC formation at the core promoters of the GAL7 and GAL10 genes. (A) Mot1p is essential for recruitment of TBP and RNA polymerase II at the GAL7 and GAL10 core promoters. Yeast cells were grown, cross-linked and immunoprecipitated as in Figure 7D. (B and C) Recruitment of TBP and RNA polymerase II at the GAL7 and GAL10 core promoters is significantly decreased in the CBP80 deletion mutant strain. Both the wild-type and CBP80 deletion mutant strains were grown, cross-linked and immunoprecipitated as in Figures 2A and B and 3C. (D) Mot1p is recruited to the GAL7 and GAL10 core promoters. Yeast cells were grown, cross-linked and immunoprecipitated as in Figure 7A. (E) The recruitment of Mot1p to the GAL7 and GAL10 core promoters is significantly decreased in the CBP80 deletion mutant strain. Both the wild-type and CBP80 deletion mutant strains were grown, cross-linked and immunoprecipitated as in Figure 7B.

CBC represses the PIC formation at the core promoter of HSP26, but does not alter the PIC formation at the core promoter of CUP1. (A) CBC represses the PIC formation at the HSP26 core promoter. Immunoprecipitation was performed as in Figures 2B and 3C. (B and C) Mot1p is recruited to the HSP26 core promoter under inducible conditions. Immunoprecipitation was performed as in Figure 7A. (D) The recruitment of Mot1p at the HSP26 core promoter is significantly decreased in the CBP80 deletion mutant strain. (E) Mot1p represses the PIC formation at the HSP26 core promoter. Both the wild-type and ts mutant strains of MOT1 were grown in YPD at 23°C to 0.9, and then switched to 39°C for 30 min prior to cross-linking. (F) Mot1p does not alter the recruitment of TBP and RNA polymerase II at the CUP1 core promoter. Yeast cells were grown in synthetic complete medium at 23°C up to OD₆₀₀ of 0.85, and then switched to 37°C for 45 min followed by 15 min induction using 1 mM CuSO₄ before cross-linking. (G) CBC does not alter the recruitment of TBP and RNA polymerase II at the CUP1 core promoter.

A schematic model for the stimulatory role of CBC in formation of PIC (and hence transcriptional initiation) at the Gal4p-regulated genes. Gal4p is required for recruitment of Mot1p which is essential for the PIC formation at the Gal4p-regulated genes. Following PIC formation, mRNA is synthesized, and then CBC binds to the cap structure at the 5′-end of newly synthesized mRNA. The turnover of Gal4p destabilizes PIC. The fresh Gal4p is recruited to form new PIC in the subsequent round of transcription. CBC appears to promote the recruitment of Mot1p to the core promoter through its interaction with Mot1p in the subsequent rounds of transcription. Such stimulation of Mot1p recruitment by Gal4 as well as CBC enhances the rate of the PIC formation (and hence transcriptional initiation) in the subsequent rounds of transcription.