Selective Recruitment of TAFs by Yeast Upstream Activating Sequences Xiao-Yong Li, Sukesh R Bhaumik, Xiaocun Zhu, Lei Li, Wu-Cheng Shen, Bharat L Dixit, Michael R Green  Current Biology  Volume 12, Issue 14, Pages 1240-1244 (July 2002) DOI: 10.1016/S0960-9822(02)00932-6

Figure 1 TAF Recruitment Is Dependent upon the UAS but Not the Core Promoter Plasmids containing the indicated promoter sequences cloned upstream of the lacZ open reading frame in pRS406 were integrated into the yeast strain W303a at the URA3 locus. Yeast cells were grown in either YPD (1% yeast extract containing 2% peptone plus 2% dextrose) or YPG (YP plus 2% galactose), as indicated. Formaldehyde-based in vivo crosslinking/immunoprecipitation (ChIP) analysis was performed as previously described [5] and was performed using anti-TBP and anti-TAF1 polyclonal antibodies. The promoter DNA was analyzed by quantitative PCR using the primers indicated in the schematic diagram, which allowed specific amplification of the integrated promoter sequences. In the diagram, open boxes represent the promoter sequences from each gene, and the numbers denote the nucleotide at the 5′ and 3′ ends relative to the transcription start site (indicated by the arrow). Current Biology 2002 12, 1240-1244DOI: (10.1016/S0960-9822(02)00932-6)

Figure 2 TAF6, but Not TAF1, Is Required for Transcription and TBP Recruitment (A) Transcription. The RPS5-LacZ and RPS5UAS/ADH1core-LacZ plasmids were integrated into taf1ts1, taf6-19, and isogenic wild-type strains at the URA3 locus. The taf1 and taf6 mutant strains, and their respective isogenic wild-type counterparts, have been described previously [30, 31]. The yeast strains were grown at 23°C. After shifting to the nonpermissive temperature (37°C) for 1 hr, cells were harvested, and total RNA was prepared. Promoter activity was analyzed by primer extension as previously described [32], using primers located in the lacZ sequence. The transcripts of the endogenous ADH1 promoter were analyzed in a similar manner using an ADH1-specific primer. (B) ChIP. Yeast strains were grown and shifted to the nonpermissive temperature as in (A), followed by formaldehyde crosslinking. The ChIP assay was carried out as in Figure 1 using polyclonal antibodies against TBP. Current Biology 2002 12, 1240-1244DOI: (10.1016/S0960-9822(02)00932-6)

Figure 3 A TAFind UAS Cannot Efficiently Activate Transcription through a TAFdep Core Promoter (A) The promoter sequences were cloned, in frame, upstream of the lacZ open reading frame in the LRΔ101(Δ2μ) plasmid (see the Experimental Procedures for details). The resulting plasmids were integrated into the yeast strain W303a at the URA3 locus and were grown in YPD. Cells were harvested in the exponential growth phase. Protein extracts were analyzed for β-galactosidase (β-gal) activity as described in [33]. The activity of each sample was normalized by the total amount of protein used in the β-gal reactions. (B) The chimeric promoter constructs were integrated into the yeast genome at the URA3 locus, and the promoter activities were analyzed as in (A). Current Biology 2002 12, 1240-1244DOI: (10.1016/S0960-9822(02)00932-6)

Figure 4 A Strong Transcriptional Activation Domain Can Relieve the TAF1 Requirement (A) Transcription activities of chimeric promoters containing the UASs and core regions of the RPS5 and GAL1 promoters were analyzed as in Figure 3. (B) (Top) A schematic representation of the LexAop-RPS5core-LacZ reporter construct, containing four LexA operator binding sites located upstream of the RPS5core sequence driving the expression of the lacZ gene. This plasmid was integrated into the yeast strain W303a at the URA3 locus. The Gal4 activator was expressed as a fusion to the LexA DNA binding domain. (Bottom) Northern blot analysis. The yeast strains were grown in synthetic selective media at 23°C and were shifted to the nonpermissive temperature for 1, 2, or 4 hr as indicated. Total RNA was prepared and probed for the lacZ transcript. Current Biology 2002 12, 1240-1244DOI: (10.1016/S0960-9822(02)00932-6)

Figure 5 Model for Transcription Activation at TAFdep and TAFind Promoters The results obtained with the four classes of promoters analyzed in this study are summarized. At natural TAFdep promoters, activators bound at the UAS target TAFs and recruit them along with TBP to the core promoter, and transcription is efficient. At natural TAFind promoters, activators bound at the UAS target some component other than TAFs (termed “target”, indicated in purple), TBP rather than TFIID is recruited to the core promoter, and transcription is efficient. In the experimental situation in which a TAFdep UAS is fused to a TAFind core promoter, activators bound at the TAFdep UAS target TAFs and recruit them along with TBP to the TAFind core promoter, and transcription is efficient. In the experimental situation in which a TAFind UAS is fused to a TAFdep core promoter, activators bound at the TAFind UAS do not target or recruit TAFs, and, as a result, TBP binds poorly to the TAFdep core promoter (indicated by the dotted lines) and transcription is inefficient. Current Biology 2002 12, 1240-1244DOI: (10.1016/S0960-9822(02)00932-6)