ADR1-mediated transcriptional activation requires the presence of an intact TFIID complex

Abstract

The yeast transcriptional activator ADR1, which is required for ADH2 and other genes' expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reduced ADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

FIG. 1

GST-TADI and GST-TADIV bind to TAF_II90 and TAF_II25. The expression of GST fusion proteins was induced as previously described (9). After binding to glutathione-agarose beads, the GST fusion protein was incubated with crude extracts (Ex.) from yeast strains yEK20 (containing TAF_II25-HA1₃), yBY40-8′ (containing TAF_II90-HA1₃), and yKG1794 (containing BRF1-HA1₃) as described in Materials and Methods. Proteins were eluted from glutathione-agarose beads by boiling and separated by SDS-PAGE. Western analysis was conducted by using mouse monoclonal antibody 12CA5 against the HA1 epitope. One hundred micrograms of GST fusion protein was loaded in each lane. GST-TADI contains residues 148 to 262 of ADR1; GST-TADII contains residues 262 to 359; GST-TADIII contains residues 420 to 462; and GST-TADIV contains residues 642 to 704. GST-TADI-7^c is the same as GST-TADI, except it contains an S230L mutation. Equivalent levels of each GST fusion were present as assayed by Coomassie blue staining.

FIG. 2

GST-TADIV binds TFIID. GST fusions were induced as previously described (9), bound to glutathione-agarose beads, and incubated with crude extract (Ex.) from yeast strain EGY188 as described in Materials and Methods. Proteins were eluted from glutathione-agarose beads by boiling and separated by SDS-PAGE. Proteins were then transferred to a PVDF membrane and probed with the appropriate antibodies. The amount of GST fusion protein per lane is the same as that described in the legend to Fig. 1. The GST fusions are the same as those described in the legend to Fig. 1. GST-TADIV-trunc. represents GST-TADIV-642-679 (see Fig. 3).

FIG. 3

TADIV binding to TAF_IIs correlates with TADIV activation ability. The GST-ADR1-TADIV derivatives indicated were expressed in E. coli and bound to glutathione-agarose beads as described in Materials and Methods. Extracts from yeast strain yBY40-8′ were bound to GST fusion proteins, and the resulting bound proteins were analyzed by Western analysis by using antibody 12CA5 directed against the HA1 tag, as described in the legend to Fig. 1. A 100-μg sample of each GST fusion protein was loaded on the gel. All of the GST fusion proteins were expressed at comparable levels (data not shown; 9). The β-galactosidase (β-gal) activities displayed at the bottom are for LexA-TADIV derivatives in their activation of LexA_op-lacZ and are taken from reference . The standard errors of the means in this case were less than 20% (9).

FIG. 4

ADR1 coimmunoprecipitates with TAF_II90. Extracts from strain yBY40-8′, containing triple HA1-tagged TAF_II90 and expressing ADR1 from a multicopy vector, and strain 938-9b, containing no HA1-tagged TAF_IIs and also expressing ADR1 from the same vector, were incubated with antibody (Ab) 12CA5 directed against the HA1 tag. The resulting immunoprecipitates were subjected to SDS–7.5% PAGE. ADR1 and TAF_II90-HA1₃ were detected by Western analysis. A polyclonal antibody raised against ADR1 residues 208 to 231 was used for ADR1 detection; antibody 12CA5 was used for TAF_II90-HA1₃ detection. A 100-μg protein sample was loaded in each crude extract lane. The level of ADR1 protein present in the cell was about 30- to 40-fold higher than that observed for ADR1 expressed at its normal dosage. IgG, immunoglobulin G.

FIG. 5

ADR1 at its physiological concentration coimmunoprecipitates with TBP. Extracts (Ex.) were prepared from yeast strains 500-16 (adr1-1), 411-40 (ADR1), and 411-33 (8-ADR1) following growth on YEP medium containing 3% ethanol. IPs were conducted with either an anti-TBP antibody (lanes 4 to 6) or an anti-CCR4 antibody (lanes 7 to 9). Lanes 1 to 3 display the ADR1 protein present in the crude extracts. Western analysis was conducted with an ADR1 antibody (upper panel) or with a TAF_II90 antibody (lower panel). None of the darkenings in the ADR1 region for lanes 7 to 9 correspond to ADR1 based on other repeats of this experiment and the fact that the darkenings did not comigrate with ADR1. IgG, immunoglobulin G.

FIG. 6

TAF_II90 is required for ADH2 derepression. (A) Total RNAs from strains ZMY60 (control) and ZMY68 were prepared, separated on an agarose gel, and analyzed by Northern hybridization as described in Materials and Methods. The time points shown are after both strains were shifted to SC-Ura–2% ethanol–500 μM CuSO₄ after 7 h of incubation in SC-Ura–4% glucose–500 μM CuSO₄. (Bottom) An identically loaded duplicate gel was stained with ethidium bromide to identify rRNA (25S and 18S) as described in reference . Previous experiments have determined that rRNA or CCR4 mRNA (13, 40) serves as a useful standard for quantitation of mRNA loadings. (B) A 100-μg sample of yeast crude extract protein per lane was resolved by SDS–8% PAGE, transferred to a PVDF membrane, and probed with an anti-TAF_II90 antibody (gift from M. Green). The time points shown are after strains were shifted to SC-Ura–2% ethanol–500 μM CuSO₄ after 7 h of incubation in SC-Ura–4% glucose–500 μM CuSO₄, with the exception of the no-Cu²⁺ lane, where the sample was taken prior to addition of 500 μM CuSO₄.