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. 2020 May 7;48(8):4244-4255.
doi: 10.1093/nar/gkaa165.

Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae

Elisabeth R Knoll  1 , Z Iris Zhu  2 , Debasish Sarkar  3 , David Landsman  2 , Randall H Morse  1   3
Affiliations

Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae

Elisabeth R Knoll et al. Nucleic Acids Res. .

Abstract

Transcription of eukaryotic mRNA-encoding genes by RNA polymerase II (Pol II) begins with assembly of the pre-initiation complex (PIC), comprising Pol II and the general transcription factors. Although the pathway of PIC assembly is well established, the mechanism of assembly and the dynamics of PIC components are not fully understood. For example, only recently has it been shown that in yeast, the Mediator complex normally occupies promoters only transiently, but shows increased association when Pol II promoter escape is inhibited. Here we show that two subunits of TFIID, Taf1 and Taf4, similarly show increased occupancy as measured by ChIP upon depletion or inactivation of Kin28. In contrast, TBP occupancy is unaffected by depletion of Kin28, thus revealing an uncoupling of Taf and TBP occupancy during the transcription cycle. Increased Taf1 occupancy upon Kin28 depletion is suppressed by depletion of TBP, while depletion of TBP in the presence of Kin28 has little effect on Taf1 occupancy. The increase in Taf occupancy upon depletion of Kin28 is more pronounced at TFIID-dominated promoters compared to SAGA-dominated promoters. Our results support the suggestion, based on recent structural studies, that TFIID may not remain bound to gene promoters through the transcription initiation cycle.

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Figures

Figure 1.
Figure 1.
Increased occupancy of Taf1 at gene promoters upon inactivation of Kin28. (A) Normalized Taf1 occupancy at promoter regions of indicated genes was determined by ChIP followed by qPCR. ChIP was performed in wild type (WT) yeast (EPY4706) and kin28-as yeast (YFR763) grown in CSM-ura with or without 1 h treatment with NaPP1 as indicated. Error bars reflect s.d.; n = 3. Analysis of the four sets of data for each gene promoter by Anova followed by Tukey's posthoc test showed that only occupancy at RPL12A in kin28-as yeast in the presence of NaPP1 varied significantly (P < 0.05) from occupancy in the other strains (indicated by **P < 0.01). (B) Normalized Taf1 occupancy (log2 ratio of IP reads to input reads) in WT and kin28-as yeast after 1 h treatment with NaPP1. Reads were mapped to all SAGA-dominated (567 genes), non-RP TFIID-dominated (4896 genes) and RP genes (137 genes). Genes were normalized for length and aligned by start and end of coding sequence (CDS) and sorted according to average signal intensity. Each horizontal line in the heat maps represents a gene, and the line graphs depict averages over all genes in the heat maps. (C) Browser scans showing Taf1 occupancy in kin28-as yeast with and without NaPP1 treatment, and in WT cells treated with NaPP1. Peaks at SAGA-dominated and TFIID-dominated gene promoters are indicated by ‘S’ and ‘T’, respectively. Scales, in reads, are shown in brackets, and have been adjusted relative to the total number of reads in each sample to allow direct comparison of occupancies. For example, the kin28-as sample yielded 19.3 million mapped reads, while the kin28-as + NaPP1 sample yielded 12.7 million mapped reads; the scales have therefore been adjusted to reflect the ratio of 0.66 in reads.
Figure 2.
Figure 2.
Increased occupancy of Taf1 and Taf4 at gene promoters upon depletion of Kin28. (A) Normalized occupancy of Taf1 plotted against Taf4 occupancy for the ∼1000 genes (see Methods) most highly occupied by Pol II. (B, C) Normalized occupancy (log2 ratio of IP reads to input reads) is depicted for Taf1 (B) and Taf4 (C) in kin28-AA yeast, without and with 1 h rapamycin treatment, mapped to all SAGA-dominated (567 genes), non-RP TFIID-dominated (4896 genes), and RP genes (137 genes). Genes were normalized for length, aligned by coding sequence (CDS) start and stop, and sorted according to average signal intensity. The 19 SAGA-dominated genes at the bottom of the Taf4 heat maps were removed before calculating averages used in the line graphs, as these were almost all Ty1 elements that had higher intensity in the input control than in the Taf4 ChIP sample, and therefore yielded negative values in the heat map. Note that a different scale is shown for the RP gene line graphs than for SAGA- and TFIID-dominated genes. (D) Ratios of Taf1 (top) and Taf4 (bottom) occupancy in kin28-AA yeast in the presence and absence of rapamycin are shown in box and whisker plots for the ∼1000 genes having highest occupancy by Pol II, sorted into SAGA-dominated (154 genes), non-RP TFIID-dominated (534 genes), and RP genes (136 genes) (see Materials and Methods). The boxes show the second and third quartiles, and the whiskers indicate the first and fourth quartiles; median values are indicated by the horizontal lines in the boxes separating second and third quartiles, and outliers are depicted as points above or below the whiskers. (E) Browser scans showing Taf1 and Taf4 occupancy in kin28-AA yeast with and without rapamycin treatment. Peaks at SAGA-dominated and TFIID-dominated gene promoters are indicated by ‘S’ and ‘T’, respectively. Scales, in reads, are shown in brackets, and have been adjusted relative to the total number of reads in each sample to allow direct comparison of occupancies within a given IP group (e.g. Taf1 with and without rapamycin).
Figure 3.
Figure 3.
TBP occupancy is not affected by depletion of Kin28. (A) Normalized TBP occupancy (log2 ratio of IP reads to input reads) in kin28-AA yeast, without and with 1 h rapamycin treatment, mapped to all SAGA-dominated, non-RP TFIID-dominated, and RP genes. Genes were normalized for length, aligned by coding sequence (CDS) start and stop, and sorted according to average signal intensity. (B) Ratios of TBP occupancy in kin28-AA yeast in the presence and absence of rapamycin are shown in box and whisker plots, as in Figure 2, for the ∼1000 genes having highest occupancy by Pol II, sorted into SAGA-dominated (151 genes), non-RP TFIID-dominated (530 genes) and RP genes (136 genes) (see Materials and Methods). (C) Browser scan showing Taf1, Taf4 and TBP occupancy in kin28-AA yeast with and without rapamycin treatment. Scales, in reads, are shown in brackets, and have been adjusted relative to the total number of reads in each sample to allow direct comparison of occupancies within a given IP group.
Figure 4.
Figure 4.
Depletion of TBP suppresses increased occupancy of Taf1 seen upon depletion of Kin28. (A) Normalized TBP occupancy (log2 ratio of IP reads to input reads) in kin28-tbp-AA yeast, without and with 1 h rapamycin treatment, mapped to all SAGA-dominated, non-RP TFIID-dominated, and RP genes. Genes were normalized for length, aligned by coding sequence (CDS) start and stop, and sorted according to average signal intensity. The 21 SAGA-dominated genes at the bottom of the heat maps were removed before calculating averages used in the line graphs, as these were almost Ty1 elements that had higher intensity in the input control than in the Taf1 ChIP sample, and therefore yielded negative values in the heat map. (B) Ratios of Taf1 and Taf4 occupancy in kin28-AA yeast in the presence and absence of rapamycin (same data as in Figure 2) and of Taf1 occupancy in kin28-tbp-AA yeast in the presence and absence of rapamycin are shown in box and whisker plots, as in Figure 2, for the ∼1000 genes having highest occupancy by Pol II (see Materials and Methods), sorted into SAGA-dominated (151 genes), non-RP TFIID-dominated (530 genes) and RP genes (136 genes).
Figure 5.
Figure 5.
Depletion of TBP has little effect on Taf1 occupancy. (A) Normalized Taf1 occupancy (log2 ratio of IP reads to input reads) in tbp-AA yeast, without and with 1 h rapamycin treatment, mapped to all SAGA-dominated, non-RP TFIID-dominated, and RP genes. Genes were normalized for length, aligned by coding sequence (CDS) start and stop, and sorted according to average signal intensity. The 22 SAGA-dominated genes at the bottom of the heat maps were removed before calculating averages used in the line graphs, as these were almost Ty1 elements that had higher intensity in the input control than in the Taf1 ChIP sample, and therefore yielded negative values in the heat map. (B) Ratios of Taf1 occupancy in tbp-AA yeast in the presence and absence of rapamycin are shown in box and whisker plots, as in Figure 2, for the ∼1000 genes having highest occupancy by Pol II (see Materials and Methods), sorted into SAGA-dominated (151 genes), non-RP TFIID-dominated (530 genes) and RP genes (136 genes). (C) Browser scans showing normalized Taf1 occupancy in tbp-AA yeast with and without rapamycin treatment. Scales, in reads, are shown in brackets, and have been adjusted relative to the total number of reads in each sample to allow direct comparison of occupancies.
Figure 6.
Figure 6.
Differential TBP/Taf1 ratios at SAGA- and TFIID-dominated genes, and at gene promoters having and lacking consensus TATA elements. (A) Normalized occupancy of TBP plotted against Taf1 occupancy for the ∼1000 genes (see Materials and Methods) most highly occupied by Pol II, separated into SAGA-dominated (151 genes), non-RP TFIID-dominated (530 genes), and RP genes (136 genes). (B) Ratios of TBP to Taf1 occupancy in kin28-AA yeast in the absence of rapamycin are shown in box and whisker plots, as in Figure 2, for the ∼1000 genes having highest occupancy by Pol II, sorted into SAGA-dominated, non-RP TFIID-dominated, and RP genes. (C) As in (B), with genes sorted by the presence or absence of a consensus TATA element and enrichment or depletion of Taf1 (28). The designation ‘±’ indicates that both Taf1 enriched and Taf1 depleted genes are included. RP genes were removed from these categories and are shown separately. (D) Ratios of Pol II, normalized for gene length, to TBP occupancy in kin28-AA yeast in the absence of rapamycin are shown in box and whisker plots, as in Figure 2, for the ∼1000 genes having highest occupancy by Pol II (see Materials and Methods), sorted into SAGA-dominated, non-RP TFIID-dominated and RP genes.
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