Opposing roles for Set2 and yFACT in regulating TBP binding at promoters

Abstract

Previous work links histone methylation by Set2 with transcriptional elongation. yFACT (Spt16-Pob3 and Nhp6) reorganizes nucleosomes and functions in both transcriptional initiation and elongation. We show that growth defects caused by spt16 or pob3 mutations can be suppressed by deleting SET2, suggesting that Set2 and yFACT have opposing roles. Set2 methylates K36 of histone H3, and K36 substitutions also suppress yFACT mutations. In contrast, set1 enhances yFACT mutations. Methylation at H3 K4 by Set1 is required for set2 to suppress yFACT defects. We did not detect an elongation defect at an 8 kb ORF in yFACT mutants. Instead, pob3 mutants displayed reduced binding of both pol II and TBP to the GAL1 promoter. Importantly, both GAL1 transcription and promoter binding of pol II and TBP are significantly restored in the pob3 set2 double mutant. Defects caused by an spt16 mutation are enhanced by either TBP or TFIIA mutants. These synthetic defects are suppressed by set2, demonstrating that yFACT and Set2 oppose one another during transcriptional initiation at a step involving DNA binding by TBP and TFIIA.

Figure 1

Histone H3(K4R) substitutions enhance the defects caused by spt16 and pob3 mutations. (A) Strain DY7803 was transformed with a YCp-TRP1 plasmid with wild-type histone H4 gene and the indicated histone H3 mutation, and dilutions were plated on the indicated medium for 2 days at 33°C. (B) As in panel (A), except the strain is DY7809. (C) As in (A) except the strain is DY7818 and dilutions were incubated for 3 days at 25°C. (D) Dilutions of strains DY150, DY8788, DY8875, and DY9206 were plated on complete medium at 25°C for 3 days or at 33°C for 2 days.

Figure 2

Histone H3(K36) substitutions and set2 mutations suppress spt16 and pob3 mutations. (A) Strain DY7809 was transformed with a YCp-TRP1 plasmid with wild-type histone H4 gene and the indicated histone H3 mutant, and dilutions were plated on complete medium (2 days) or FOA medium (3 days) at 35°C. (B) As in (A), except the strain is DY7818 and dilutions were plated on complete medium (3 days) or FOA medium (5 days) at 30°C. (C) Dilutions of Strains DY8862, DY8864, DY8865, and DY8867 were plated on complete medium at the indicated temperature for 3 days. (D) Dilutions of strains DY8862, DY8864, DY10468, and DY10469 were plated on complete medium at the indicated temperature for 3 days. (E) Dilutions of strains DY150, DY8690, DY8787, and DY8790 were plated on complete medium at the indicated temperature for 2 days. (F) Dilutions of strains DY150, DY8690, DY8881, and DY8878 were plated on complete medium at 25°C for 2 days or at 30°C for 3 days.

Figure 3

set1 is epistatic to set2 in genetic interactions with spt16. (A) Dilutions of strains DY150, DY8787, DY8690, DY8875, DY8777, DY9178, and DY9180 were plated on complete medium at 25°C for 2 days or at 35°C for 3 days. (B) Strains DY7809 and DY7818 were transformed with a YCp-TRP1 plasmid with wild-type histone H4 gene and the indicated histone H3 mutant, and dilutions were plated and incubated as follows: spt16 on complete, 2 days at 25°C, spt16 on FOA, 4 days at 35°C, pob3 on complete, 3 days at 25°C, and pob3 on FOA, 5 days at 30°C. (C) As in (B), except the strains are DY7803 and DY7142, and dilutions were plated on the indicated medium at 33°C for 4 days.

Figure 4

set2 suppress spt16 phenotypes. (A) set2 suppresses the 6-AU sensitivity caused by an spt16 mutation. Dilutions of strains DY8883, DY8884, DY8885, DY8886, DY8887, and DY8888 were plated at 25°C on complete medium for 2 days or on medium lacking uracil containing 50 μg/ml 6-azauracil (6-AU) for 4 days. (B) H3(K36A) suppresses the 6-AU sensitivity caused by an spt16 mutation. As in (A), except the strains are DY3398, DY8789, DY8788, and DY8790. (C) set2 suppresses the spt16 isw1 synthetic growth defect. Dilutions of strains DY150 (wild type), DY8107 (spt16), DY8690 (set2), DY8235 (isw1), DY9022 (spt16 isw1), and DY9029 (spt16 isw1 set2) were plated on complete medium at the indicted temperature for 3 days. (D) set2 suppresses the spt16 htz1 synthetic growth defect. Strains DY7836, DY9808, DY9805, and DY8107 were plated on complete medium at the indicated temperature for 2 days.

Figure 5

A set2 mutation reverses the poor induction of GAL1 caused by a pob3 mutation. Strains DY9591, DY9976, DY9972, and DY9974 were grown on YP medium with 2% raffinose. Galactose was added to 2% concentration, and samples were taken at 10 min intervals and mRNA measured by S1 nuclease protection. (A) YLR454w mRNA levels from the GAL1-YLR454w allele. (B) GAL1 mRNA levels. (C) YLR454w mRNA levels before galactose induction.

Figure 6

A pob3 mutation reduces pol II and TBP binding, and binding is restored in a pob3 set2 strain. Strains DY9591, DY9976, DY9972, and DY9974 were grown on YP medium with 2% raffinose. Galactose was added to 2%, and samples were taken at 10 min intervals and processed for ChIP analysis to measure pol II and TBP binding. (A) Map of the GAL1-YLR454w allele showing the positions of the PCR primers at the promoter and within the gene. (B) Kinetics of pol II binding following galactose induction at different GAL1-YLR454w regions in a wild-type strain. Error bars show variance among replicate PCRs. (C) Kinetics of pol II binding following galactose induction at different GAL1-YLR454w regions in a pob3 strain. (D) Distribution of pol II at 50 min following galactose induction at different GAL1-YLR454w regions in four different strains. Error bars show variance among replicate PCRs. (E) Pol II binding to the native GAL1 promoter at 30 min following galactose induction in four different strains. Error bars show variance among replicate PCRs. (F) TBP binding to the GAL1-YLR454w promoter following galactose induction in four different strains. ChIP values were normalized to binding at t=0. Error bars show variance among replicate PCRs.

Figure 7

A set2 mutation suppresses the synthetic lethality of an spt16 mutation with either TBP or TFIIA mutations. (A) Strains DY8552 (indicated as ‘SET2') and DY10065 (indicated as ‘set2') were transformed with YCp-TRP1 plasmid encoding a TBP mutant, and dilutions were plated on complete or FOA medium at 33°C for 3 days. (B) Strains DY8700 (indicated as ‘SET2') and DY10212 (indicated as ‘set2') were transformed with a YCp-LEU2 plasmid with the indicated toa2 mutant, and dilutions were plated on complete medium at 25°C for 2 days and on FOA medium at 30°C for 4 days, except the TOA2(Y10G,R11Δ) strains were incubated on FOA medium at 33°C.