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. 2004 Sep;24(18):8312-21.
doi: 10.1128/MCB.24.18.8312-8321.2004.

Role for Nhp6, Gcn5, and the Swi/Snf complex in stimulating formation of the TATA-binding protein-TFIIA-DNA complex

Debabrata Biswas  1 , Anthony N Imbalzano, Peter Eriksson, Yaxin Yu, David J Stillman
Affiliations

Role for Nhp6, Gcn5, and the Swi/Snf complex in stimulating formation of the TATA-binding protein-TFIIA-DNA complex

Debabrata Biswas et al. Mol Cell Biol. 2004 Sep.

Abstract

The TATA-binding protein (TBP), TFIIA, and TFIIB interact with promoter DNA to form a complex required for transcriptional initiation, and many transcriptional regulators function by either stimulating or inhibiting formation of this complex. We have recently identified TBP mutants that are viable in wild-type cells but lethal in the absence of the Nhp6 architectural transcription factor. Here we show that many of these TBP mutants were also lethal in strains with disruptions of either GCN5, encoding the histone acetyltransferase in the SAGA complex, or SWI2, encoding the catalytic subunit of the Swi/Snf chromatin remodeling complex. These synthetic lethalities could be suppressed by overexpression of TOA1 and TOA2, the genes encoding TFIIA. We also used TFIIA mutants that eliminated in vitro interactions with TBP. These viable TFIIA mutants were lethal in strains lacking Gcn5, Swi2, or Nhp6. These lethalities could be suppressed by overexpression of TBP or Nhp6, suggesting that these coactivators stimulate formation of the TBP-TFIIA-DNA complex. In vitro studies have previously shown that TBP binds very poorly to a TATA sequence within a nucleosome but that Swi/Snf stimulates binding of TBP and TFIIA. In vitro binding experiments presented here show that histone acetylation facilitates TBP binding to a nucleosomal binding site and that Nhp6 stimulates formation of a TBP-TFIIA-DNA complex. Consistent with the idea that Nhp6, Gcn5, and Swi/Snf have overlapping functions in vivo, nhp6a nhp6b gcn5 mutants had a severe growth defect, and mutations in both nhp6a nhp6b swi2 and gcn5 swi2 strains were lethal.

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Figures

FIG. 1.
FIG. 1.
Genetic interactions among SWI2, NHP6, TBP, and TFIIA. (A) nhp6ab is synthetically lethal with swi2. Dilutions of strains DY8660 (nhp6ab swi2 strain with a YCp-URA3-SWI2 plasmid) and DY8668 (nhp6ab swi2 strain with a YCp-URA3-NHP6A plasmid) were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 30°C for 3 days. (B) Examples of synthetic lethality of TBP mutants and swi2. Dilutions of strains DY8712 (swi2 spt15) and DY7472 (spt15) transformed with the indicated TBP mutation plasmids were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 30°C for 3 days. SPT15(wt), wild-type SPT15. (C) Multicopy TFIIA suppresses the TBP mutant E93G-swi2 and TBP mutant G147W-swi2 synthetic lethalities, and multicopy NHP6A suppresses the TBP mutant R220H-swi2 and TBP mutant E186L-swi2 synthetic lethalities. Strain DY8783 (swi2 spt15) was transformed with two plasmids, a TRP1 plasmid corresponding to the indicated TBP mutant and either pRS327 (YEp-LYS2 vector), M4793 (YEp-TFIIA), or M4797 (YEp-NHP6A), dilutions were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 33°C for either 2 (complete medium) or 3 (5-FOA) days.
FIG. 2.
FIG. 2.
Genetic interactions among GCN5, TBP, and TFIIA. (A) Examples of synthetic lethality of TBP mutants and gcn5. Dilutions of strain DY7514 (gcn5 spt15) transformed with the indicated TBP mutation plasmids were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 25°C for 4 days. SPT15(wt), wild-type SPT15. (B) Multicopy TFIIA suppresses the TBP-gcn5 synthetic lethality for certain TBP mutants. Strain DY8158 (gcn5 spt15) was transformed with two plasmids, a TRP1 plasmid corresponding to the indicated TBP mutant and either YEp351 (YEp-LEU2 vector) or pSH346 (YEp-TFIIA), and was plated onto 5-FOA-containing plates, and the plates were incubated at 30°C for 4 days.
FIG. 3.
FIG. 3.
TFIIA mutants are lethal in gcn5 or swi2 mutant strains. (A) Dilutions of strain DY8541 (toa2) transformed with the indicated TFIIA mutant plasmids were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 33°C for 3 days. (B) Dilutions of strain DY8709 (gcn5 toa2) transformed with the indicated TFIIA mutant plasmids were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 33°C for 3 days. (C) Dilutions of strain DY8811 (swi2 toa2) transformed with the indicated TFIIA mutant plasmids were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 33°C for 2 days. Note that the TFIIA mutants designated in the figure correspond to substitutions in the Toa2 subunit of TFIIA.
FIG. 4.
FIG. 4.
Overexpression of TBP suppresses gcn5-TFIIA and swi2-TFIIA lethalities. (A) Strain DY8709 (gcn5 toa2) was transformed with two plasmids, a LEU2 plasmid corresponding to the TFIIA W76F mutant and either pRS327 (YEp-LYS2 vector) or M4533 (YEp-TBP), dilutions were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 33°C for either 2 (complete medium) or 3 (5-FOA) days. (B) Strain DY8811 (swi2 toa2) was transformed with two plasmids, a LEU2 plasmid corresponding to the indicated TFIIA mutant and either YEplac112 (YEp-TRP1 vector) or M4827 (YEp-TBP), dilutions were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated as follows: plates with TFIIA mutant F71E or W76F, 34°C for 2 (complete medium) or 3 (5-FOA) days, and those with TFIIA mutant Y10G R11Δ, 25°C for 2 (complete medium) or 4 (5-FOA) days. Note that the incubation of the TFIIA Y10G R11Δ mutant on 5-FOA was considerably longer in this experiment than in the one described in the legend to Fig. 3C, and thus tiny colonies are visible when the vector control is grown at 25°C. (C) gcn5 is synthetically lethal with swi2. Dilutions of strains DY8827 (swi2 gcn5 strain with a YCp-URA3-SWI2 plasmid) or DY8664 (swi2 strain with a YCp-URA3-SWI2 plasmid) were plated at 25°C onto complete medium-containing plates for 3 days or onto FOA-containing plates for 5 days. Note that the TFIIA mutants designated in the figure correspond to substitutions in the Toa2 subunit of TFIIA.
FIG. 5.
FIG. 5.
TBP binds to acetylated nucleosomes. (A) Twenty micrograms of HeLa histones or hyperacetylated HeLa histones were loaded onto a 15% TAU gel and stained with Coomassie brilliant blue following electrophoresis. (B) TBP binding was assessed by DNase I digestions by using free DNA (lanes 2, 3, 7, 8, 13, and 14) or nucleosomes (nuc.) assembled with regular histones (lanes 4 and 5) or hyperacetylated histones (lanes 9 to 12). The DNA template for lanes 11 to 14 has mutations at the TATA sequence. Lanes 1 and 6 contain G+A sequencing ladders. Addition of TBP to the binding reaction mixtures is indicated by +. The data in lanes 1 to 5 are reprinted from Nature (24) with permission of the publisher. The arrows indicate hypersensitive DNase I cleavages 5′ to the TATA sequence.
FIG. 6.
FIG. 6.
Nhp6 interacts with TBP and TFIIA. (A) nhp6ab is synthetically lethal with TFIIA mutants. Dilutions of strain DY8510 (nhp6ab toa2) carrying the YCp-URA3-TFIIA (wild type) plasmid and transformed with the indicated TFIIA mutant plasmids were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 25°C for 4 days. (B) Dilutions of strain DY 8541 (NHP6A NHP6B toa2) transformed with the indicated TFIIA mutant plasmids were plated onto complete medium- or 5-FOA-containing plates, and the plates were incubated at 25°C for 3 days. Note that the TFIIA mutants designated in the figure correspond to substitutions in the Toa2 subunit of TFIIA. (C) Nhp6 stimulates formation of the TBP-TFIIA-DNA complex. TBP (144 nM) was added to lanes 3 to 7 and 10 to 14, and Nhp6 (70 nM) was added to lanes 8 to 14. TFIIA was added to reaction mixtures in the following amounts: 0.15 nM, lanes 4 and 11; 0.3 nM, lanes 5 and 12; 0.6 nM, lanes 6 and 13; and 1.2 nM, lanes 2, 7, 9, and 14. +, present; −, absent. (D) Nhp6 stimulates formation of the TBP-DNA complex. Nhp6 (70 nM) was added to lanes 6 to 10, and TBP was added to lanes 2 to 4 and 7 to 10 in the following amounts: 96 nM, lanes 2 and 7; 192 nM, lanes 3 and 8; 288 nM, lanes 4 and 9; and 384 nM, lanes 5 and 10.
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