Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation

Abstract

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

Keywords: TBP-associated factors; TFIID; Taf1; bromodomain; histone acetylation; transcription activation.

Figure 1.

Transcription-mediated TAF association with downstream DNA. (A) Schematic of the transcription template used to isolate RNAPII ECs as described in the Materials and Methods. Downstream bound proteins are eluted using Ssp I endonuclease, which cuts 35 nucleotides downstream from the TATA box but upstream of the first TSS. Total proteins are eluted with Pst I (see Supplemental Fig. S1D). (B) Scatter plot comparing NTP-dependent protein binding to downstream DNA (log₂ of +NTP/−NTP ratio) in the presence (Y-axis) or absence (X-axis) of α-amanitin. Each circle represents one protein (1511 total), with the value calculated from the sum of all peptide signals identified for that protein. Proteins in specific subgroups are color-coded as noted. Proteins specifically dependent on transcription are defined as those at the right of the vertical orange line (95% confidence level for enrichment with NTPs) on the X-axis and below the diagonal orange line (95% confidence level cutoff for reduction of NTP enrichment by α-amanitin) (see also Supplemental Fig. S1A–C; Supplemental Table S1). (C) Immunoblot of proteins eluted from immobilized templates with Ssp I before treatment with NTPs or after 15 min of incubation with A, C, and UTP plus 3′O-meGTP (to stall ECs) or all four NTPs to allow RNAPII runoff (see also Supplemental Fig. S2B). (D) TBP and Taf1 binding on immobilized templates of different lengths. The top panel shows a schematic of the template series analyzed as in B. The middle panels show immunoblots of total bound proteins eluted with Pst I, while the bottom panels show downstream binding proteins eluted with Ssp I (see also Supplemental Fig. S2A,C).

Figure 2.

Taf1 downstream binding correlates with its transcription function. (A) Scatter plot of Taf1 transcription dependence versus total occupancy. For 4348 scored mRNA genes, the taf1/TAF1 mRNA expression level ratios from Huisinga and Pugh (2004) were plotted in log₂ scale on the X-axis. For each promoter, total Taf1 occupancy was plotted by summing ChIP-exo counts from −100 bp to +300 bp relative to the TSS (Rhee and Pugh 2012), dividing by the average for all mRNA gene promoters, and plotting in log₂ scale on the Y-axis. Excluding the 137 RPGs, high TAF dependence genes (825) were defined as those with taf1/TAF1 expression ratios below −1 SD (SD = 0.57) from the mean of all gene ratios (−0.95). Medium dependence genes (4327) had ratios between −1 and +1 SD, and low dependence genes (729) had more than +1 SD from the mean of ratios. Average ratios in high, medium, low, and RPG groups were −1.73, −0.92, −0.08, and −1.82, respectively (see also Supplemental Fig. S3A). (B) Averaged Taf1 occupancy for the four groups was plotted relative to the TSS (high [red], medium [orange], low [blue], and RPG [black]) using ChIP-exo data from Rhee and Pugh (2012) (see also Supplemental Fig S3B,C). (C) Occupancies for TBP, Taf1, and nucleosomes were plotted for the four groups separately to compare peak positions. (Gray) TBP; (orange) Taf1; (green) histone H4. For each curve, data were normalized by setting the maximum value to 1.0. (D) Individual gene examples of Taf1 (TAF), Spt15 (TBP), and Sua7 (TFIIB) profiles at EFB1 (high TAF1 dependence, −2.02), YEF3 (medium TAF1 dependence, −1.21), PYK1 (low TAF1 dependence, 0.02), ADH1 (low TAF1 dependence, 0.35), and RPS13 (RPG; TAF1 dependence, −1.00).

Figure 3.

Downstream Taf1 binding correlates with +1 nucleosome H4ac. (A) ChIP patterns of TFIIB, TBP, Taf1, Bdf1, Swr1, H4ac, Htz1, and H3 at individual genes were plotted as horizontal lines to generate heat maps, with darker color signifying higher levels. Genes were sorted into the four TAF1 dependence groups and then within each group by Taf1 downstream occupancy (ChIP-exo reads from +20 bp to +160 bp relative to the TSS). Together with our newly generated data, raw data for TFIIB (Wong et al. 2014), Bdf1 (Rhee and Pugh 2012), Swr1 (Yen et al. 2013), and Htz1 (Gu et al. 2015) were reanalyzed as in the Materials and Methods (see also Supplemental Fig. S4A). (B) H3 and H4ac levels were plotted versus TFIIB and Taf1 binding. Occupancies of H3 and H4ac were determined as the sum of ChIP-seq reads from +30 bp to +170 bp relative to the TSS, encompassing the +1 nucleosome. Occupancies of Taf1 and TFIIB were determined as total ChIP-exo reads (Rhee and Pugh 2012) from −140 bp to −1 bp for the upstream core promoter and +20 bp to +160 bp for the downstream promoter. Each dot represents one mRNA gene (total 4720). For each graph, Spearman's rank correlation coefficient (ρ) is shown (see also Supplemental Fig. S4B).

Figure 4.

Taf1 promotes activator-independent multiround transcription. In vitro transcription was performed with TAF1 (YF157/YSW87; black) or taf1 (YF158/YSW90; gray) yeast nuclear extracts. NTPs were added for 3 min in single-round reactions (A) or 45 min for multiround transcription (B) in the presence or absence of transcription activator Gal4-vp16, as indicated. Transcripts were quantified, normalized, and plotted as the average value from three independent reactions. (C) The ratio of multiround transcripts in taf1 versus TAF1 extracts was divided by the same ratio in single-round reactions (see also Supplemental Fig. S5). Error bars show SD. (**) P = 0.014; (***) P = 0.006; (unmarked pairs) P > 0.01.

Figure 5.

Taf1 promotes transcription reinitiation. (A) A schematic of two-step sequential reactions used to analyze transcription reinitiation is at the left. Note that no transcription activator was used in these experiments. Yeast nuclear extract was preincubated with immobilized template 1 (as in Fig. 1A) in the presence or absence of NTPs (400 µM each). After three washes, nuclear extract, ³²P-labeled NTPs, and the longer G-less cassette template 2 (pG5CG-D2/F916) were added. (Top right panel) After 2, 4, 8, and 16 min, labeled transcripts were isolated and analyzed by gel electrophoresis and autoradiography. (Bottom right panel) Quantitation from three independent reactions plots transcript 1 levels over time, normalized by setting the 16-min maximum to 100 and using transcript 2 to correct for variations in recovery or reaction conditions. Error bars indicate SD (see also Supplemental Fig. S6A). (**) P = 0.01; (***) P = 7.5 × 10⁻⁵. (B) The sequential transcription assay was performed with NTPs and 10 µg/mL α-amanitin in the preincubation, as indicated. (C) Sequential transcription assay comparing wild-type (YSW87) with mutant taf1 (YSW90) or taf11 (YSB1732) nuclear extracts in the first preincubation reaction. After washes, the second transcription reaction was performed with wild-type nuclear extract prepared from strain BJ2168. Quantitation from three (taf11) or five (taf1) independent replicates is shown. (**) P = 0.08; (***) P = 5.2 × 10⁻⁵. All other differences were not statistically significant (see also Supplemental Fig. S6C). (D) Immobilized template reactions as in Figure 1 were performed with elution by SspI digestion and immunoblotting for Taf1 and TBP, comparing wild-type (YSW87) with mutant taf1 (YSW90) or taf11 (YSB1732) nuclear extracts. Note that Taf1 levels were roughly similar in all three extracts, as can be seen from the basal signal in the −NTP lanes.

Figure 6.

Depletion of Rpb1 or Bdf1 alters Taf1 binding in vivo. (A) ChIP-seq for Taf1 or TBP was performed from cells before or after a 60-min depletion of Rpb1 or Bdf1 by Anchor Away. Normalized reads (reads per million) were mapped, and differences were plotted as heat maps, showing individual genes sorted by the classes defined in Figure 2 (see also Supplemental Fig. S7A,B). (B) The ratios of total TBP and Taf1 reads for each promoter were calculated and plotted as quartile box and whisker plots.

Figure 7.

Mechanism of TAF-mediated RNAPII transcription. (A) Model for how TAFs may promote reinitiation (see the Discussion for details). TFIID is shown as TBP (dark green), TAFs (three flexibly linked triangles in medium green), and Bdf1 (light-green oval). (Black line) DNA; (beige circles) nucleosomes; (red rectangle) transcription activators; (gray cloud) basal factors; (yellow oval) RNAPII; (blue hexagon) SwrC; (red line) the RNA transcript. The lighter version of TBP after step 2 is to signify that it may be partially or completely dissociated after initiation. (B) A reinitiation model of how two promoters of similar strength can exhibit differential responses to TAF or activator/SAGA mutants (Huisinga and Pugh 2004; de Jonge et al. 2017). Each horizontal trace represents behavior over time. An initial activator- and SAGA-stimulated round of transcription is represented in red, while subsequent TAF-stimulated reinitiations are represented in green.