Chd1 and yFACT act in opposition in regulating transcription

Abstract

CHD1 encodes an ATP-dependent chromatin remodeler with two chromodomains. Deletion of CHD1 suppresses the temperature-sensitive growth defect caused by mutations in either SPT16 or POB3, which encode subunits of the yFACT chromatin-reorganizing complex. chd1 also suppresses synthetic defects caused by combining an spt16 mutation with other transcription factor mutations, including the synthetic lethality caused by combining an spt16 mutation with TATA binding protein (TBP) or TFIIA defects. Binding of TBP and RNA polymerase II to the GAL1 promoter is reduced in a pob3 mutant, resulting in low levels of GAL1 expression, and all three defects are suppressed by removing Chd1. These results suggest that Chd1 and yFACT have opposing roles in regulating TBP binding at promoters. Additionally, overexpression of Chd1 is tolerated in wild-type cells but is toxic in spt16 mutants. Further, both the ATPase and chromodomain are required for Chd1 activity in opposing yFACT function. Similar to the suppression by chd1, mutations in the SET2 histone methyltransferase also suppress defects caused by yFACT mutations. chd1 and set2 are additive in suppressing pob3, suggesting that Chd1 and Set2 act in distinct pathways. Although human Chd1 has been shown to bind to H3-K4-Me, we discuss evidence arguing that yeast Chd1 binds to neither H3-K4-Me nor H3-K36-Me.

FIG. 1.

chd1 suppresses spt16 and pob3 phenotypes. (Top panels) Tenfold dilutions of strains DY150 (wild type), DY6957 (chd1), DY8107 (spt16), and DY9151 (spt16 chd1) were plated on complete medium at the indicted temperature for 2 days. (Bottom panels) Tenfold dilutions of strains DY150 (wild type), DY6957 (chd1), DY7379 (pob3), and DY9458 (pob3 chd1) were plated on complete medium at the indicted temperature for 3 days.

FIG. 2.

Both the chromodomain and the ATPase contribute to Chd1 toxicity in yFACT mutants. (A) Tenfold dilutions of strains DY7379 (pob3), DY9458 (pob3 chd1), DY11724 (pob3 chd1 CHD1-HA), DY11736 [pob3 chd1 CHD1(ΔCD)-HA], and DY11770 [pob3 chd1 CHD1(K407R)-HA] were plated on complete medium at 25°C or 30°C for 2 days. (B) Tenfold dilutions of strains DY8107 (spt16), DY9152 (spt16 chd1), DY11614 (spt16 chd1 CHD1-HA), DY11624 (spt16 chd1 CHD1(ΔCD)-HA], and DY11643 [spt16 chd1 CHD1(K407R)-HA] were plated on complete medium at 30°C or 35°C for 2 days.

FIG. 3.

chd1 suppresses spt16 synthetic growth defects. (A) Tenfold dilutions of strains DY150 (wild type), DY8107 (spt16), DY9816 (isw1), DY9809 (chd1), DY9827 (chd1 isw1), DY9055 (spt16 isw1), and DY9834 (spt16 isw1 chd1) were plated on complete medium at 33°C for 2 days. (B) Tenfold dilutions of strains DY150 (wild type), DY8107 (spt16), DY9809 (chd1), DY9152 (spt16 chd1), DY7656 (isw1 isw2), DY9823 (chd1 isw1 isw2), DY9820 (spt16 isw1 isw2), and DY9831 (spt16 isw1 isw2 chd1) were plated on complete medium at 33°C for 2 days. (C) Tenfold dilutions of strains DY150 (wild type), DY6612 (nhp6ab), DY8107 (spt16), DY8808 (spt16 nhp6ab), and DY9978 (spt16 nhp6ab chd1) were plated on complete medium at 33°C for 2 days. (D) Tenfold dilutions of strains DY150 (wild type), DY8156 (elp3), DY8107 (spt16), DY8185 (spt16 elp3), and DY9965 (spt16 elp3 chd1) were plated on complete medium at 33°C for 2 days. (E) Tenfold dilutions of strains DY150 (wild type), DY7836 (htz1), DY8107 (spt16), DY9808 (spt16 htz1), and DY9811 (spt16 htz1 chd1) were plated on complete medium at 33°C for 2 days.

FIG. 4.

chd1 and gcn5 show a synthetic growth defect. (A) Tenfold dilutions of strains DY150 (wild type), DY9809 (chd1), DY5926 (gcn5), and DY9873 (gcn5 chd1) were plated on complete medium at 25°C for 3 days or at 35°C for 2 days. (B) Tenfold dilutions of strains DY5925 (gcn5), DY6957 (chd1), DY11500 (gcn5 chd1), DY11716 (gcn5 chd1 CHD1-HA), DY11694 [gcn5 chd1 CHD1(ΔCD)-HA], and DY11703 [gcn5 chd1 CHD1(K407R)-HA] were plated on complete medium at 30°C for 2 days.

FIG. 5.

A chd1 mutation suppresses the synthetic growth defect of spt16 with set1 or histone H3(K4R) mutations. (A) A chd1 mutation suppresses the spt16 set1 synthetic growth defect. Tenfold dilutions of strains DY150 (wild type), DY8875 (set1), DY8107 (spt16), DY9206 (spt16 set1), and DY9271 (spt16 set1 chd1) were plated on complete medium at 30°C or 33°C for 2 days. (B) A chd1 mutation suppresses the spt16 histone H3(K4R) synthetic growth defect. Tenfold dilutions of strains DY8862 [hht1-hhf1 hht2-hhf2 + YCp-TRP1:H3(wild type)-H4(wild type)], DY8865 (spt16 hht1-hhf1 hht2-hhf2 + YCp-TRP1:H3(wild type)-H4(wild type)], DY8863 [hht1-hhf1 hht2-hhf2 + YCp-TRP1:H3(K4R)-H4(wild type)], DY8866 [spt16 hht1-hhf1 hht2-hhf2 + YCp-TRP1:H3(K4R)-H4(wild type)], and DY10472 [spt16 chd1 hht1-hhf1 hht2-hhf2 + YCp-TRP1:H3(K4R)-H4(wild type)] were plated on complete medium at the indicated temperature for 2 days.

FIG. 6.

CHD1 overexpression is toxic in an spt16 mutant. Strains DY150 (wild type), DY8117 (spt16), and DY8799 (spt16 set2) were transformed with either YEp-CHD1 or the empty YEp-URA3 vector and plated on complete medium at 25°C for 3 days or on medium lacking uracil at 30°C for 2 days.

FIG. 7.

A chd1 mutation suppresses defects in GAL1 induction and Pol II and TBP binding caused by a pob3 mutation. Strains DY9591 (GAL1-YLR454w), DY9959 (chd1 GAL1-YLR454w), DY9972 (pob3 GAL1-YLR454w), and DY10020 (pob3 chd1 GAL1-YLR454w) 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 regions amplified by at the promoter and within the gene. (B) YLR454w mRNA levels measured from the GAL1-YLR454w allele, quantified after phosphorimaging of the gels in panel C. WT, wild type. (C) S1 protection assays to measure YLR454w mRNA from the GAL1-YLR454w allele, using probes specific for YLR454w and a tRNA internal control. (D) Distribution of Pol II at 60 min following galactose induction at different GAL1-YLR454w regions in four different strains. Error bars show variance (standard deviations) among replicate PCRs. (E) TBP binding to the GAL1-YLR454w promoter following galactose induction in four different strains. ChIP values were normalized to binding at time zero. Error bars show variance among replicate PCRs. (F) Map of the native GAL1 gene showing the positions of regions amplified by at the upstream activation sequence (UAS) and TATA within the promoter. (G) Pol II binding to the TATA region (positions −190 to + 54) of the native GAL1 promoter at 30 min following galactose induction in four different strains. Error bars show variance among replicate PCRs. (H) SAGA binding to the native UAS region (positions −496 to −316) of the native GAL1 promoter at 30 min following galactose induction in four different strains. Error bars show variance among replicate PCRs.

FIG. 8.

A chd1 mutation suppresses the synthetic lethality of an spt16 mutation with either TBP or TFIIA mutations. (A) Strains DY8552 (spt15 spt16 + YCp-URA3-TBP-Spt16) (indicated as “CHD1”) and DY10141 (spt15 spt16 chd1 + YCp-URA3-TBP-Spt16) (indicated as “chd1”) were transformed with two plasmids, a YCp-TRP1 plasmid encoding a TBP mutant and a YCp-LEU2 plasmid with either wild-type SPT16 or spt16 mutations, and 10-fold dilutions were plated at 33°C either on complete medium for 2 days or on FOA medium for 3 days. (B) Strains DY8700 (spt16-11 toa2 + YCp-URA3-TOA2) (indicated as “CHD1”) and DY10214 (spt16-11 chd1 toa2 + YCp-URA3-TOA2) (indicated as “chd1”) were transformed with a YCp-LEU2 plasmid with the indicated toa2 mutant, and 10-fold dilutions were plated for 2 days on complete medium at 25°C and on FOA medium at 30°C.

FIG. 9.

Additive suppression of pob3 by chd1 and set2 mutations. (A) Tenfold dilutions of strains DY150 (wild type), DY9809 (chd1), DY8690 (set2), DY9838 (chd1 set2), DY7379 (pob3), DY9458 (pob3 chd1), DY8878 (pob3 set2), and DY9547 (pob3 chd1 set2) were plated on complete medium at 25°C or 32°C for 3 days. (B) Tenfold dilutions of strains DY150 (wild type), DY9809 (chd1), DY8690 (set2), DY9838 (chd1 set2), DY8107 (spt16), DY9152 (spt16 chd1), DY8777 (spt16 set2), and DY9153 (spt16 chd1 set2) were plated on complete medium at either 30°C or 35°C for 2 days. (C) Tenfold dilutions of strains DY8777 (spt16 set2), DY9153 (spt16 set2 chd1), DY11619 (spt16 set2 chd1 CHD1-HA), DY11626 [spt16 set2 chd1 CHD1(ΔCD)-HA], and DY11645 [spt16 set2 chd1 CHD1(K407R)-HA] were plated on complete medium at 30°C for 2 days or at 35°C for 3 days.