1. Cdk1 Triggers Association of RNA Polymerase to Cell Cycle Promoters Only after Recruitment of the Mediator by SBF 
Maria Pia Cosma, Silvia Panizza, Kim Nasmyth 
Molecular Cell 
Volume 7, Issue 6, Pages (June 2001)
DOI: /S (01)

2. Figure 1
Association of PolII, Srb4, Srb5, Srb6, TFIIB, and Kin28 with HO Is Cell Cycle Regulated
Association of PolII, Srb4-myc, Srb5-myc, Srb6-myc, TFIIB-myc, and Kin28-myc with the HO TATA box was analyzed using SRB4-myc18 Δash1 GAL-CDC20 Δcdc20 (K8535), SRB5-myc18 Δash1 GAL-CDC20 Δcdc20 (K8534) and SRB6-myc18 Δash1 GAL-CDC20 Δcdc20 (K8537), TFIIB-myc18 Δash1 GAL-CDC20 Δcdc20 (K8538), and KIN28-myc18 Δash1 GAL-CDC20 Δcdc20 (K8539) strains. Cultures were arrested in metaphase by incubation in YEP raffinose for 2.5 hr and then released into galactose-containing medium at 25°C. Samples were taken each 5–10 min, cross-linked with formaldehyde, and analyzed by PCR after immunoprecipitation. DNA purified from the cross-linked whole-cell extract (WCE) was also analyzed by PCR. DNA content was determined by FACS analysis. The 0 time point corresponds to the metaphase-arrested cells after 2.5 hr of galactose depletion. Immunoprecipitations were performed with mouse anti-Myc monoclonal antibody or mouse monoclonal antibody 8WG16 (Covance) against the C-terminal domain of Rpb1. In all experiments, four pairs of oligonucleotides were used simultaneously in the same PCR reaction. Primers spanning the TATA box of HO were combined with three pairs of oligonucleotides spanning coding regions of GLT1, MSH6, and RAD1. To detect PolII association, the primers spanning the TATA box of HO were combined with three pairs of oligonucleotides spanning intergenic regions (I.R.)
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Association of Srb Proteins, TFIIB and Kin28 with HO's TATA Box Depends on Swi5 and SBF
(A) Association of Srb4, Srb5, and Srb6 depends on Swi5. SRB4-myc18 Δash1 (K8376), SRB4-myc18 Δash1 Δswi5 (K8471), SRB5-myc18 Δash1 (K8404), SRB5-myc18 Δash1 Δswi5 (K8472), SRB6-myc18 Δash1 (K8405), and SRB6-myc18 Δash1 Δswi5 (K8473) asynchronous cultures (lanes 1–6, respectively) were grown in YEP raffinose at 25°C. Association with HO's TATA box was analyzed by PCR on DNA isolated from the immunoprecipitates. Amplification of DNAs from whole-cell extracts (WCE) is shown in lanes 7 and 8.
(B and C) Binding of TFIIB and Kin28 depends on Swi5. Association of TFIIB-myc and Kin28-myc with HO's TATA box was investigated in TFIIB-myc18 Δash1 Δswi5 GAL-CDC20 Δcdc20 (K8622), KIN28-myc18 Δash1 Δswi5 GAL-CDC20 Δcdc20 (K8992), and corresponding SWI5+ strains (K8538 and K8539) after release from metaphase arrest by incubation in galactose-containing medium at 25°C.
(D–F) Association of Srb4, TFIIB, and Kin28 depends on Swi4. SRB4-myc18 Δash1 swi4-29 GAL-CDC20 Δcdc20 (K9098), TFIIB-myc18 Δash1 swi4-29 GAL-CDC20 Δcdc20 (K8993), KIN28-myc18 Δash1 swi4-29 GAL-CDC20 Δcdc20 (K8997), and corresponding SWI4+ strains (K8535, K8538, and K8539) were processed in parallel. Cultures were arrested in metaphase by incubation for 2.5 hr at 25°C, shifted for 20 min at 37°C to inactivate Swi4-29, and released at the same temperature in galactose-containing medium. PCR was performed on DNA obtained after immunoprecipitation and from the whole-cell extracts (WCE). FACS data are also shown
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Binding of Swi4 and Srb4 to Cell Cycle-Dependent Promoters Is Independent of Cdk1
(A) Swi4 associates with the URS2 region of HO promoter in the absence of Cdk1 activity. SWI4-myc18 Δash1 cdc28-13 GAL-CDC20 Δcdc20 (K8994) and the corresponding CDC28+ cells (K8995) were arrested in metaphase in YEP raffinose for 2.5 hr. Cdk1 was then inactivated by incubation at 37°C for 20 min, and the culture was released into galactose-containing medium at the same temperature. Association of Swi4-myc with URS2 was investigated by PCR on DNA samples purified after cross-linking and immunoprecipitation.
(B) Srb4-myc associates with the TATA box of HO, CLN1, CLN2, and PCL1 promoters independently of Cdk1. Association of Srb4-myc with the TATA boxes of HO, CLN1, CLN2, and PCL1 was investigated using SRB4-myc18 Δash1 cdc28-13 GAL-CDC20 Δcdc20 (K8991) and the corresponding CDC28+ strain (K8535). Cultures were released at 37°C after metaphase arrest
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Recruitment of PolII, TFIIB, and Kin28 to Promoters of SBF-Regulated Genes Depends on Cdk1
(A) PolII's association with HO, CLN1, CLN2, and PCL1 but not GAL10 TATA boxes depends on Cdk1. Δash1 cdc28-13 GAL-CDC20 Δcdc20 (K9428) and the corresponding CDC28+ strain (K8283) were analyzed in parallel. Immunoprecipitation was done using the mouse monoclonal antibody 8WG16 (Covance) against the C-terminal domain (CTD) of PolII largest subunit. PCR analysis was performed on DNA fragments purified from the cross-linked samples after release at 37°C from metaphase arrest at 25°C.
(B), (C) Recruitment of TFIIB and Kin28 to the HO TATA box depends on Cdk1. TFIIB-myc18 Δash1 cdc28-13 GAL-CDC20 Δcdc20 (K8996), KIN28-myc18 Δash1 cdc28-13 GAL-CDC20 Δcdc20 (K8998), and corresponding CDC28+ strains (K8538, K8539) were analyzed in parallel. PCR analysis was performed on DNA fragments purified from cross-linked samples after releasing cells at 37°C from metaphase arrest at 25°C. FACS profiles are also shown
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 A Model for the Stepwise Activation of the HO Promoter
Entry of Swi5 into nuclei during late anaphase is triggered by inactivation of Cdk1. Swi5's binding to HO is crucial for recruiting the Swi/Snf chromatin-remodeling complex and the SAGA histone acetylase. Nucleosome remodeling and modification by these two complexes promotes recruitment of a second sequence-specific transcription factor, SBF. SBF then recruits the Srb/mediator to the TATA box. Finally, activation of Cdk1 in late G1 leads to recruitment of PolII, TFIIB, and TFIIH
Molecular Cell 2001 7, DOI: ( /S (01) )