Volume 7, Issue 4, Pages 741-751 (April 2001) A Rsc3/Rsc30 Zinc Cluster Dimer Reveals Novel Roles for the Chromatin Remodeler RSC in Gene Expression and Cell Cycle Control  Melinda L. Angus-Hill, Alisha Schlichter, Douglas Roberts, Hediye Erdjument-Bromage, Paul Tempst, Bradley R. Cairns  Molecular Cell  Volume 7, Issue 4, Pages 741-751 (April 2001) DOI: 10.1016/S1097-2765(01)00219-2

Figure 1 Identification of Rsc3 and Rsc30 (A) Purified RSC and RSCa complexes (5 μg) stained with Coomassie dye (from Cairns et al., 1996). (B) Domain structure of Rsc3 and Rsc30. Endpoints for the predicted coils are as follows: for Rsc3, 75–130, C1; 176–195, C2; 465–479, C3; 599–613, C4; 760–805, C5; and for Rsc30, 108–123, C1; 154–195, C2; 364–407, C3; 500–542, C4; 699–721, C5. (C) Alignment of zinc cluster proteins is shown Molecular Cell 2001 7, 741-751DOI: (10.1016/S1097-2765(01)00219-2)

Figure 2 Compositional Analysis of RSC Complexes (A) Polyclonal antibodies raised against Rsc3 or Rsc30 are specific for their respective antigen. Purified RSC (2 μg) was separated in three lanes of a 7.5% acrylamide SDS–PAGE gel and transferred to PVDF membrane. Lanes were probed separately with anti-Sth1 and either anti-Rsc3 (lane 1), anti-Rsc30 (lane 3), or both (lane 2). (B) Anti-Rsc3 antibodies immunodeplete Rsc3 and Rsc30. Purified RSC (5 μg) was incubated with affinity-purified Rsc3 antibodies conjugated to protein A-Sepharose beads or with beads lacking antibodies (control). Following extensive washing, immune complexes were eluted with 5 M urea. Samples were separated on a 7.5% acrylamide SDS–PAGE gel, and proteins were revealed by staining with silver. Samples: load (RSC, 1 μg, lane 1), supernatant (20%, lane 2), eluates (50%, lanes 3 and 5), and supernatants (20%, lanes 4 and 5). (C) Rsc3 and Rsc30 form a stable complex outside of RSC. Rsc3 and Rsc30 were over-produced in YBC693, bearing the plasmids pGCT.Flag.RSC3.Flag (CEN6 TRP1) and pYep24.RSC30 (2u URA3). Immunoprecipitation was performed by incubating 600 μg with anti-Flag M2 agarose beads, and samples were separated on a 7.5% acrylamide SDS–PAGE gel and immunoblotted with Sth1 or Rsc30 antisera. Samples: load, extract (200 μg, lane1); supernatant (20%, lane 2); and eluate (50%, lane 3). No precipitation is observed with control beads (data not shown). (D) Quantitative analysis of Rsc3/Rsc30 complexes. Eluates derived in (C) along with a titration series of pure RSC were separated on a 7.5% acrylamide SDS–PAGE gel, immunoblotted to PVDF membrane, and probed with anti-Rsc30 and anti-Sth1 antibodies. As shown, the anti-Sth1 is much more immunoreactive Molecular Cell 2001 7, 741-751DOI: (10.1016/S1097-2765(01)00219-2)

Figure 3 Phenotypes of rsc3 and rsc30 Mutants (A) RSC3 is an essential gene. Dissections of the rsc3Δ::HIS3/RSC3 heterozygous diploid YBC583 show 2:2 segregation for viability. All viable spores are His− and RSC3+. (B) Null phenotypes conferred by rsc30Δ or zinc cluster mutations. Strains were incubated for 3–5 days at 30°C. Strains: YBC693, rsc30Δ (p316.RSC30); YBC693, rsc30Δ (p316); and YBC693, rsc30Δ (p316.rsc30.C15G). (C) Rsc3 function requires the zinc cluster and C terminus. Strains (YBC628) harboring a RSC3+ URA3-marked plasmid and a TRP1-marked rsc3 derivative (left panel, pGCT.Flag.RSC3.Flag; pGCT.Flag.rsc3.1–360; and pGCT.Flag.rsc3-VP16) or LEU2-marked rsc3 derivative (right panel, pGCL.rsc3.C14G) were grown as patches and replica plated to selective media with or without 5-FOA to select for the loss of the RSC3+ URA3-marked plasmid Molecular Cell 2001 7, 741-751DOI: (10.1016/S1097-2765(01)00219-2)

Figure 4 Properties of rsc3 Mutants (A) Temperature-dependent growth of rsc3 mutants. Strains were grown on rich media plates (YPD) for three days. Strains: YBC843 (RSC3), YBC840 (rsc3-1), YBC842 (rsc3-2), and YBC906 (rsc3-3). (B) rsc3-1 and rsc3-2 require both substitutions to confer temperature sensitivity. Strains were grown on rich media plates (YPD) for three days. (C) rsc3 mutations confer a G2/M cell cycle arrest. Strains YBC843 (RSC3) or YBC842 (rsc3-2) were shifted to 38°C for 8 hr and analyzed by FACS and fluorescence microscopy. Propidium iodide staining is color enhanced, with bright staining in blue and lighter staining in red. (D) Loss of the spindle pole checkpoint greatly improves the growth of rsc3-3 mutants. Strains were grown on rich complete medium (YPD) at the indicated temperatures for three days. Strains: WT, YBC843; mad1Δ, YBC936; rsc3-1, YBC840; rsc3-1 mad1Δ, YBC950 (p315.rsc3-1, LEU2 CEN6); rsc3-2, YBC842; rsc3-2 mad1Δ, YBC950 (p315.rsc3-2, LEU2 CEN6); rsc3-3, YBC906; and rsc3-3 mad1Δ, YBC950 (p315.rsc3-3, LEU2 CEN6) Molecular Cell 2001 7, 741-751DOI: (10.1016/S1097-2765(01)00219-2)

Figure 5 Genetic Interactions between RSC3 and RSC30 (A) rsc3 mutants are suppressed by increased RSC30 dosage. Strains were grown on selective media lacking uracil for three days. Strains: YBC843 (RSC3), YBC840 (rsc3-1), YBC842 (rsc3-2), and YBC906 (rsc3-3). (B) rsc3 mutations are lethal in combination with rsc30Δ mutations. Strains were grown as patches on YPD, replica plated to synthetic media lacking tryptophan or like media containing 5-FOA, and grown three days at 30°C. Strains: YBC984 harboring the URA3-marked RSC3+ plasmid (pNCU.RSC3) was transformed with rsc3 Ts− plasmids. (C) rsc30Δ mutants are suppressed by increased RSC3 dosage. YBC693 derivatives were grown three days at 30°C. (D) Rsc3 does not require Rsc30 to assemble into RSC complex. Extracts were prepared from WT (YBC605) or rsc30Δ (YBC973) bearing pGal.RSC3.Flag. Immunoprecipitation was performed with anti-Flag M2 beads, whereas the control is YBC973 lacking plasmid. Samples were separated on a 7.5% acrylamide SDS–PAGE gel and immunoblotted with Sth1 antibodies. All loads (L) contain 20% of input, whereas all IP pellets (P) represent 50% of total Molecular Cell 2001 7, 741-751DOI: (10.1016/S1097-2765(01)00219-2)

Figure 6 Impact of rsc3 or rsc30 Mutations on Gene Expression Gene expression in rsc3 and rsc30 mutants was determined by DNA microarray, S1 nuclease, and Northern analysis. Strains YBC843 (WT; RSC3) and YBC842 (Ts; rsc3-2) were initially grown at 28°C in YPD, then collected after 8 hr of growth at 38°C. Strains rsc30Δ (YBC693 [p316]) and RSC30 (YBC693 [p316.RSC30]) were grown in media lacking uracil. (A) Comparison expression profiles of several RP genes and cell wall genes. The magnitude of changes in expression for a subset of genes upregulated (red) or downregulated (blue) in the mutant compared to the wild type is illustrated. Data are the average of four replicates. (B) Comparison of the expression profile of a rsc3-2 strain at 38°C (compared to WT at 38°C) to a rsc30Δ strain (compared to WT) at 28°C. Crosshairs represent individual genes. The yellow lines are simply for orientation and locating the zero change point. (C) S1 nuclease analysis comparing RNA levels in the rsc3 Ts− mutant (YBC842; rsc3-2) to WT (YBC843). (D) Northern analysis of mRNA levels of rsc30Δ. (YBC693 [p316]), lanes 1 and 3; WT RSC30 (YBC693 [p316.RSC30]), lanes 2 and 4; YBC843 (WT; RSC3), lanes 5 and 7; YBC906 (Ts; rsc3-3), lanes 6 and 8; and YBC842 (Ts; rsc3-2), lane 9. Experimental RNA or mRNA levels were normalized to ACT1 RNA levels ([C]) or CMD1 mRNA ([D]) Molecular Cell 2001 7, 741-751DOI: (10.1016/S1097-2765(01)00219-2)