SBF recruits Rpd3(L) to promoters. (A) DY12794 (GALp∷CDC20 SWI4-Myc), DY12247 (GALp∷CDC20 SDS3-Myc) and DY6669-Cdc28-HA (GALp∷CDC20 YCp:KanMX:CDC28-HA) cells were synchronized in mitosis by removing galactose, followed by release by addition of galactose (0 min). The DY6669-Cdc28-HA cells were grown in medium containing G418. The CDC20 arrest is at the G2/M transition, and CLN2 expression begins at 30 min after release corresponds to late G1 phase. Binding of the Swi4-Myc subunit of SBF and the Sds3-Myc subunit of Rpd3(L) were measured ChIP using samples taken at various times after the release. CLN2 RNA measurements were from synchronized DY12752 (GALp∷CDC20 GCN5-Myc). (B) Swi4-Myc binding at CLN1 (SBF regulated), CLN2 (SBF regulated) and CDC21 (MBF regulated) was assessed in DY12794 cells (GALp∷CDC20 SWI4-Myc) either in G1 (35 min after CDC20 arrest and release) or arrested in G2 (0 min). (C) Strains DY12247 (GALp∷CDC20 SDS3-Myc) and DY12828 (GALp∷CDC20 SDS3-Myc swi6) were synchronized and ChIP experiments preformed to measure Rpd3(L) binding at CLN1, CLN2, CDC21 and CTS1. A full-colour version of this figure is available at The EMBO Journal Online.

Rpd3(L) binding to SBF and MBF promoters requires Stb1 and Whi5. (A–C) Strains DY12248 (GALp∷CDC20 SDS3-Myc) and DY13502 (GALp∷CDC20 SDS3-Myc stb1 whi5) were synchronized and ChIP experiments preformed to measure Rpd3(L) binding at (A) SBF-dependent CLN1 and CLN2, (B) MBF-dependent CDC6 and CDC21 and (C) SBF- and MBF-independent CTS1. (D) CLN1 and CLN2 expression is reduced in stb1 mutants but not in stb1 whi5 double mutants. RNA was isolated from log phase wild-type (DY150), whi5 (DY10474), stb1 (DY13454) and stb1 whi5 (DY13494), strains and mRNA levels measured by RT–qPCR. (E) CLN1 and CLN2 expression is modestly increased in synchronized stb1 whi5 double mutants. Strains DY6669 (GALp∷CDC20) and DY13496 (GALp∷CDC20 stb1 whi5) were synchronized and mRNA levels measured by RT–qPCR.

FACT interacts directly with Swi6. Glutathione-agarose beads were loaded with either GST only, GST-Swi6 or GST-Swi5 and purified FACT was applied to the beads, washed and eluted. The eluted material is analysed on western blots with antibodies to GST, Spt16 and Pob3. There are significant degradation products, particularly for GST-Swi6. Lane 1 contains the input extract (corresponding to 50% of the amount of material applied to the beads.

FACT binding at SBF and MBF promoters precedes nucleosome eviction and gene expression. (A) Histone H3 ChIP was performed to measure nucleosome occupancy at the CLN2 promoter using synchronized strains DY6669 (GALp∷CDC20), DY12914 (GALp∷CDC20 asf1), DY11246 (GALp∷CDC20 pob3) and DY13373 (GALp∷CDC20 swi6). (B) Histone H3 ChIP measuring nucleosome occupancy at the PIR1 promoter, as in panel A. (C–H) Strain DY6669 (GALp∷CDC20) was synchronized and samples taken for FACT (Spt16) ChIP, histone ChIP and mRNA measurements for (C) HO URS2, (D) PIR1, (E) CLN1, (F) CLN2, (G) CDC21 and (H) POL1. shows the same experiment conducted with a swi6 mutant.

FACT binding to SBF-regulated genes increases in stb1 whi5 mutant. (A) FACT binding to SBF promoters increases in a stb1 whi5 mutant. Strains DY6669 (GALp∷CDC20) and DY13496 (GALp∷CDC20 swi6) were synchronized and FACT binding to SBF promoters HO URS2, CLN1 and CLN2 was measured. (B) FACT binding to MBF promoters does not change in a stb1 whi5 mutant. The ChIP material used in panel A was analysed for FACT binding to MBF promoters CDC21, POL1 and CDC6. A full-colour version of this figure is available at The EMBO Journal Online.

stb1 and whi5 suppress cdc28 and FACT mutations. (A) Serial dilutions of strains DY150 (wild type), DY13454 (stb1), DY9558 (whi5), DY13640 (stb1 whi5), DY7815 (spt16-11), DY13714 (spt16-11 stb1), DY13716 (spt16-11 whi5) and DY13718 (spt16-11 stb1 whi5) were incubated on medium at 30°C for 2 days, at 35°C for 2 days, at 36°C for 3 days, or on medium containing 50 mM HU at 25°C for 5 days. (B) Serial dilutions of strains DY150 (wild type), DY13454 (stb1), DY9558 (whi5), DY13640 (stb1 whi5), DY12236 (pob3(Q308K)), DY13696 (pob3(Q308K) stb1), DY13698 (pob3(Q308K) whi5) and DY13700 (pob3(Q308K) stb1 whi5) were incubated on medium at 30°C for 2 days, at 36°C for 3 days, at 37°C for 3 days, or on medium containing 25 mM HU at 25°C for 3 days. (C) Serial dilutions of strains DY150 (wild type), DY13454 (stb1), DY9558 (whi5), DY13640 (stb1 whi5), DY7379 (pob3(L78R)), DY13704 (pob3(L78R) stb1), DY13702 (pob3(L78R) whi5) and DY13706 (pob3(L78R) stb1 whi5) were incubated on medium at 25°C for 3 days, at 26.5°C for 4 days, at 30°C for 2 days, or on medium containing 100 mM HU at 25°C for 5 days. (D) Serial dilutions of strains DY150 (wild type), DY10474 (whi5), DY13454 (stb1), DY13494 (stb1 whi5), DY12836 (cdc28-13), DY11982 (cdc28-13 whi5), DY13541 cdc28-13 stb1) and DY13543 (cdc28-13 stb1 whi5) were incubated on medium at 30°C for 3 days, at 35°C for 3 days, at 36°C for 3 days or at 37°C for 5 days. (E) Serial dilutions of strains DY150 (wild type), DY7379 (pob3(L78R)), DY13357 (swi6) and DY14024 (pob3(L78R) swi6), each transformed with the YCp(URA3):POB3 plasmid, were incubated on either complete medium or 5-FOA plates at room temperature for 4 days. (F) Strain DY14024 (pob3(L78R) swi6) with the YCp(URA3):POB3 plasmid was transformed with one of four multicopy plasmids, the empty YEp-LEU2 vector, YEp:CLN1, YEp:CLN2, or YEp:SPT15, and the transformants tested for the ability to lose the YCp(URA3):POB3 plasmid and grow on 5-FOA plates. In the left two panels, four transformants were patched on medium lacking leucine and uracil, and replica plated and grown at room temperature for 4 days. In the right two panels, serial dilutions of a single transformant were incubated at room temperature for 5 days.

SBF recruits multiple components to promoters. (A) Swi6 enters the nucleus in late M/early G1, interacts with Swi4 to form SBF, and binds to promoters. Whi5 is recruited through interaction with Swi4, and Stb1 and FACT are recruited through interaction with Swi6. Whi5 and Stb1 recruit the Rpd3(L) HDAC. As cells pass START in the cell cycle, the Cdc28 kinase is active and phosphorylates Whi5 and Stb1. Phosphorylated Whi5 leaves the nucleus. We suggest phosphorylated Stb1 remains at the promoter and stimulates gene activation. There appears to be two pulses of FACT binding to CLN1 and CLN2 promoters. (B) Mbp1 and Swi6 form the MBF factor, which binds to MBF-dependent promoters. Whi5 is not recruited. Stb1 and FACT are recruited through interaction with Swi6, and Stb1 recruits Rpd3. We suggest that Stb1 phosphorylation by Cdc28 results in disassociation of Rpd3(L). The kinetics of FACT binding is different at distinct MBF-dependent promoters. A full-colour version of this figure is available at The EMBO Journal Online.