See Introduction for description.

(A) Genetic interaction of the bre1Δ and htb1-K123R htb2-K123R mutations with the lte1Δ mutation. Shown is the growth on 5-FOA and SD –Ura minimal media of five-fold serial dilutions of strains of the indicated genotype that initially harbored an URA3-marked centromeric plasmid encoding LTE1. Plates were incubated for 3 days at 30°C. See Materials and Methods for details. (B) Representative images from Cdc14 release assay. Cells are assayed for spindle morphology and Cdc14 release phenotype by indirect immunofluorescence with anti-tubulin or anti-HA, which recognizes the HA-tagged version of Cdc14 in all strains. Cells are visualized by nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and by differential interference contrast (DIC) optics for comparison. No release refers to dense nucleolar staining of Cdc14-HA, full release refers to cells with uniform Cdc14-HA localization throughout the nucleus, while partial release refers to cells with both nuclear and residual nucleolar staining. (C) Time course analysis. The temporal profile of Cdc14 release from nucleolar sequestration is displayed for cdc15-2 mad1Δ mutants harboring either no additional mutation, bre1Δ, htb1-K123R htb2-K123R, or spo12Δ bns1Δ mutations. Alpha-factor arrested cells are released into YPD media at 37°C to inactivate the cdc15-2 mutant gene product and timepoints are processed for indirect immunofluorescence as described in Figure 2B. Approximately 200 cells were scored for each timepoint for metaphase or anaphase spindles and for full or partial release of Cdc14-HA. (D) Time course analysis. Cell cycle progression of mad1Δ, mad1Δ bre1Δ, and mad1Δ slk19Δ mutants arrested and released from alpha-factor arrest was measured by scoring ∼200 cells from each timepoint for anaphase spindles by indirect immunofluorescence.

(A) Reverse transcription coupled to quantitative PCR analysis (RT–QPCR) of known mitotic exit regulators. Levels of cDNA generated from RT reactions of wildtype and bre1Δ total RNA were measured by QPCR. The mean values and standard deviations presented represent triplicate samples for each strain and are normalized to ACT1 levels. Reactions were performed without RT (−RT) to establish background DNA contamination. BFA1, the lowest level transcript, was enriched 15 fold over the −RT control (data not shown). (B) Western blots of epitope-tagged regulators of Cdc14 release. Cdc15, Fob1, Cfi1/Net1, Tpd3 and Cdc55 were tagged with 3xHA while Slk19 and Spo12 were tagged with 4xmyc and 13xmyc, respectively. The blots were cut into two parts to simultaneously probe for the epitope tag on the upper part and with anti-dimeH3K4 on the lower to demonstrate loss of BRE1. The upper blots were stripped and reprobed with anti-Pgk1 to control for loading. (C) Chromatin immunoprecipitation of HA tagged Cdc14, 3xHA tagged Cfi1/Net1, and 3xHA tagged Fob1 in wild type and bre1Δ mutant backgrounds. The numbers on the x-axis refer to primer pairs used to amplify the immunoprecipitated samples corresponding to locations at the rDNA locus as depicted in the schematic below and from [62]. Quantitative PCR mean values and standard deviations presented represent triplicate samples for each rDNA primer set and are normalized to values from a control primer set located within the open reading frame (ORF) of BUD3. Dotted lines separate the primer pairs corresponding to different regions of the rDNA locus. Bottom panel: NTS 1 and 2 = non-transcribed spacer regions 1 and 2. RFB = replication fork block. 5S, 5.8S, 18S, 25S = rDNA open reading frames.

(A) Genetic interactions of set1Δ, dot1Δ, set2Δ, set1Δ dot1Δ, and set1Δ set2Δ mutations with the lte1Δ mutation. Strains were assayed as in Figure 2A. (B,C) The temporal profile of Cdc14 release from nucleolar sequestration is displayed for strains of the indicated genotypes. Time courses were performed as described in Figure 2C.

(A) Genetic interactions of the sir2Δ, hda1Δ, and rpd3Δ mutations with the lte1Δ mutation. Strains were assayed as in Figure 2A. (B) Time course analysis. The temporal profile of Cdc14 release from nucleolar sequestration is displayed for strains of the indicated genotypes. Time courses were performed as described in Figure 2C.

(A,B) Time course analysis. The temporal profile of Cdc14 release from nucleolar sequestration is displayed for strains of the indicated genotypes. Time courses were performed as described in Figure 2C.

This model proposes that the three known histone methylations present in S. cerevisiae function nonredundantly to change nucleolar chromatin structure and permit FEAR pathway signaling.