Diagram of the SUC2 insertion in the rDNA. The top line represents the rDNA array on the right arm of chromosome XII. A representative rDNA repeat, indicating the position of the integration of the SUC2 URA3 cassette (see text), is shown below. The thick gray line flanking SUC2 represents DNA that normally flanks the SUC2 gene.

Swi/Snf represses transcription of SUC2 in the rDNA. Northern analysis of SUC2 mRNA levels was performed on strains that contain SUC2 in its normal genomic location (lanes 1 to 5) or in the rDNA (lanes 6 to 10). Strains were grown in YPD (repressing conditions; lanes r) and then shifted to YEP plus 0.05% glucose (derepressing conditions; lanes d) for 2 h 45 min. ACT1 served as a loading control. The strains analyzed are as follows: lanes 1 and 2, FY78; lane 3, FY49; lane 4, FY328; lane 5, FY2084; lanes 6 and 7, FY2313; lane 8, FY2314; lane 9, FY2316; and lane 10, FY2321. wt, wild type.

MNase analysis of SUC2 chromatin structure. Strains were grown in YPD medium to 10⁷ cells/ml and then shifted to derepressing conditions as described in Materials and Methods. Spheroplasts were isolated and then incubated with increasing amounts of MNase as described in Materials and Methods. DNA was purified, digested with HinfI, and subjected to indirect end-labeling analysis using a probe that anneals to +140 to +296 (+1 = ATG) in the coding sequence of SUC2. The SUC2 genomic region is diagramed on the left. The positions of two prominent sites that differ in levels of MNase sensitivity for SUC2 in its wild-type location are marked with arrows. N denotes the naked DNA controls (lanes 1 and 26). The strains used were FY78 (SNF2; lanes 2 to 7), FY328 (snf2Δ; lanes 8 to 13), FY2313 (SNF2 rDNA::SUC2; lanes 14 to 19), and FY2316 (snf2Δ rDNA::SUC2; lanes 20 to 25).

Swi/Snf silences mURA3 and URA3 in the rDNA. (A) Swi/Snf silences expression of mURA3 in the rDNA. Tenfold serial dilutions of stationary-phase cultures of SNF2 (L1081 and L1084) or snf2Δ (L1079) strains containing the mURA3-LEU2 cassette at the rDNA and SNF2 (L1082 and L1083) or snf2Δ (L1085 and L1086) containing the mURA3-LEU2 cassette at leu2Δ1 were spotted onto 5-FOA and YPD media to monitor expression of mURA3. Loss of silencing is indicated by less growth on 5-FOA. (B) Swi/Snf represses the transcription of URA3 in the rDNA. URA3 mRNA levels were measured in four strains by Northern hybridization analysis. For URA3 at its natural location, FY78 (wild type; lanes 1 and 2) and FY328 (snf2Δ; lanes 3 and 4) were used. For URA3 in the rDNA, FY2313 (wild type; lanes 5 and 6) and FY2316 (snf2Δ; lanes 7 and 8) were used. Strains were grown under conditions both repressing (YPD) and depressing (shifted to 0.05% glucose) for SUC2 transcription (see Materials and Methods). In low glucose, URA3 at its natural location is transcribed at a low level whereas URA3 in the rDNA is not significantly affected. ACT1 served as a loading control.

Analysis of the effects of sir2Δ and set1Δ mutations on rDNA silencing of SUC2. Northern analysis of SUC2 when integrated in the rDNA. Strains were grown in YPD (repressing conditions; lanes r) and then shifted to YEP plus 0.05% glucose (derepressing conditions; lanes d) for 2 h and 45 min. ACT1 serves as a loading control. The strains analyzed are as follows: SNF2 (FY2312), snf2Δ (FY2315), sir2Δ (L1075), sir2Δ snf2Δ (L1076), set1Δ (FY2318), and set1Δ snf2Δ (FY2319).

Analysis of Sir2 and histone H3 methylation in snf2Δ mutants. (A) ChIP analysis of Sir2 in the rDNA. ChIP was performed across the rDNA repeat in both wild-type (FY2313) and snf2Δ (FY2316) strains. The positions of the regions analyzed by PCR in the ChIP experiments are shown at the top. The comparison between the wild-type and snf2Δ strains, normalized to an untranscribed region of the genome, is shown below (36). The PCR products correspond to those previously used (28). (B) ChIP analysis of histone H3 K4 methylation in wild-type and snf2Δ strains. ChIP was performed in three strains: the wild type (FY2313), snf2Δ (FY2316), and set1Δ (FY2318). Comparisons of snf2Δ and set1Δ to the wild type are shown.

SNF2 is required for telomeric silencing. (A) Telomeric silencing phenotypes determined using a telomeric URA3 reporter gene. Tenfold serial dilutions of stationary-phase cultures of wild-type (L1087 and L1088), sir2Δ (L1091 and L1092), or snf2Δ (L1089 and L1090) strains containing the URA3 at the right telomere of chromosome V were spotted onto 5-FOA and YPD medium to monitor expression of the URA3. Loss of silencing is indicated by reduced growth on 5-FOA. (B) Swi/Snf represses URA3 transcription at the telomere. Strains with URA3 at its normal genomic location (lane 1) or integrated at the right telomere of chromosome V (lanes 2 to 7) were grown in SC medium supplemented with 100 mg of uracil/liter. mRNA levels of URA3 and the loading control, ACT1, were measured by Northern analysis. The strains used in the experiment were as follows: lane 1, FY78; lane 2, L1091; lane 3, L1092; lane 4, L1087; lane 5, L1088; lane 6, L1089; and lane 7, L1090. The quantitation represents the relative level of URA3 mRNA normalized to the level of ACT1. The numbers represent the averages for the pairs of strains shown. wt, wild type. (C) Swi/Snf has no detectable effect on HMLα silencing. Three MATa strains in lanes 1 to 3 (wild type [wt], FY78; snf2Δ, FY328; snf5Δ, FY1658) and a MATα strain, FY1856, were grown in YPD to 2 × 10⁷ cells/ml. α1 and ACT1 mRNA levels were measured by Northern analysis.