The yeast FACT complex has a role in transcriptional initiation

Abstract

A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.

FIG. 1.

spt16 synthetic lethality with TBP mutants. A. Strain DY8552 (spt15Δ spt16Δ + YCp-URA3-TBP-Spt16) was transformed with two plasmids, a YCp-TRP1 plasmid with a TBP mutant and a YCp-LEU2 plasmid with either wild-type SPT16 or spt16-11, and dilutions were plated on complete or 5-FOA medium at 33°C for 3 days. B. Strains DY8969 and DY9452 were transformed with either YEp-TFIIA, YEp-TFIIB, YEp-NHP6A, or the vector and were plated on selective medium at 33°C for 3 days. C. Strain DY8969 was transformed with either YEp-TFIIB, YEp-NHP6A, or the vector, and dilutions were plated on selective medium at 30°C for 2 days.

FIG. 2.

spt16 synthetic lethality with TFIIA mutants. A. Strains DY8541 (toa2Δ) and DY8699 (spt16-11 toa2Δ) transformed with the indicated TFIIA mutant plasmids were plated on complete or 5-FOA plates and incubated at 33°C for 3 days. Two other toa2 mutants (Y69F and F71R) were viable in the spt16-11 mutant (data not shown). B. Strain DY8700 (spt16-11 toa2Δ) was transformed with a YCp-LEU2 plasmid with the indicated toa2 mutant and a multicopy URA3 plasmid with either TFIIA, NHP6A, or the vector, and dilutions were plated on complete or 5-FOA medium at 33°C [toa2(Y10G,R11Δ)] or 30°C [toa2(W76A)].

FIG. 3.

Suppression of spt16-11 by spt3. A. Dilutions of strains DY3398 (wild type), DY8788 (spt16-11), DY8980 (spt3), DY8981 (spt8), DY8977 (spt16-11 spt3), and DY8978 (spt16-11 spt8) were incubated on YEPD medium for 2 days at either 25°C or 35°C. B. Disruption of SPT3 suppresses the spt16-11 gcn5 synthetic lethality. Strains DY150 (wild type), DY6220 (spt3), DY8154 (spt16-11 gcn5), and DY9071 (spt16-11 gcn5 spt3) were grown on YEPD plates at 25°C for 4 days or at 30°C for 2 days. C. Disruption of SPT3 or SPT8 suppresses the spt16-11 rpd3 synthetic lethality. Strains DY8941 (spt16-11 rpd3), DY8946 (spt16-11 rpd3 spt3), DY8948 (spt16-11 rpd3 spt8), and DY8950 (spt16-11 rpd3 spt3 spt8) were grown on YEPD plates at 30°C for 3 days or at 34°C for 3 days. D. Disruption of SPT3 suppresses the spt16-11 nhp6ab synthetic lethality. Strains DY150 (wild type), DY8808 (spt16-11 nhp6ab), DY6220 (spt3), and DY8985 (spt16-11 nhp6ab spt3) were grown on YEPD plates at 25°C for 4 days or at 33°C for 2 days. E. spt16-11 is synthetic lethal with TBP(G174E).Strain DY8552 (spt15Δ spt16Δ + YCp-URA3-TBP-Spt16) was transformed with a YCp-LEU2-spt16-11 plasmid and a YCp-TRP1 plasmid with either TBP(wild type [wt]), TBP(G174E), or the empty YCp-TRP1 vector, and dilutions were plated on complete or 5-FOA medium at 33°C for 2 days. F. Spt3(E240K) suppresses spt16-11 TBP(G174E) synthetic lethality. Strains DY150 (wild type), DY6220 (spt3), DY8107 (spt16-11), DY8903 (spt16-11 spt3Δ), DY9036 [spt16-11 Spt3(E240K)], DY9038 [spt16-11 TBP(G174E)], and DY9040 [spt16-11 TBP(G174E) Spt3(E240K)] were plated on complete or 5-FOA medium at 25°C for 3 days or at 34°C for 4 days.

FIG. 4.

spt16 and spt3 mutations affect TBP binding. TBP occupancy at the ELP3 and SER3 promoters was determined by chromatin immunoprecipitation with polyclonal anti-TBP antisera and quantitative PCR, using cells that had been grown at 25°C and then shifted to 37°C for 3 h. Relative binding is shown, after normalization to an Intergenic V internal control. Error bars reflect variance among replicate PCRs. Strains DY3398 (wild type), DY8788 (spt16-11), DY8980 (spt3), and DY8977 (spt16-11 spt3) were used.

FIG. 5.

yFACT stimulates TBP and TFIIA binding to a nucleosomal TATA site. The PH-MLT(+3) template with a nucleosome positioning sequence and a TATA element near the dyad (24) was radiolabeled and assembled into nucleosomes, and the structure of these nucleosomes was assessed by partial DNase I digestion followed by electrophoresis and phosphorimager analysis. Each set of three lanes has twofold decreases in the amount of DNase I. Lanes 1 to 3 (nucleosome only), the DNase I digestion pattern shows the 10-bp periodicity of a rotationally phased nucleosome. Lanes 4 to 6, addition of yFACT to the binding reaction results in changes in the pattern of DNase I protection, particularly near the dyad (marked with an arrow), demonstrating that yFACT reorganizes the structure of the nucleosome. The changes in the DNase I digestion pattern of the PH-MLT(+3) sequence due to yFACT are different from those seen with either the 5S or 601 nucleosome positioning sequences previously examined with yFACT (20, 43), but as in those cases increased access to DNase I is observed near the dyad axis. This is consistent with the previous conclusion that the effects of nucleosome reorganization induced by yFACT are focused in specific regions of the nucleosome structure but the specific sites digested are strongly influenced by the DNA sequence. Lanes 7 to 18, with added TBP and/or TFIIA, as indicated. The position of the TATA element is indicated. The regions marked with single or double asterisks are discussed in the text. The single asterisk indicates the sequences that display constant accessibility to DNase I (showing that protection is specific to the TATA region). The double asterisks indicate a site within the nucleosomes in which the combination of TBP and TFIIA strongly enhances DNase I sensitivity.

FIG. 6.

TBP and TFIIA binding to a nucleosome is affected by the integrity and rotational phase of the TATA site. A. An intact TATA box is required for TBP-TFIIA binding. Nucleosomes were assembled onto either the PH-MLT(+3) template with a wild-type TATA or the PH-MLT(+3)-Mu template with a mutated TATA (24), and TBP-TFIIA binding was examined as described for Fig. 5. B. The position of the TATA sequence within the nucleosome affects binding. Nucleosomes were assembled onto either the PH-MLT(+3) template or the PH-MLT(0) template which has a 3-nucleotide change in the rotational position of the TATA sequence relative to the histone core (24), and TBP-TFIIA binding was examined as described for Fig. 5.