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. 2020 May 21;78(4):785-793.e8.
doi: 10.1016/j.molcel.2020.03.008. Epub 2020 Mar 30.

TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

Charli B Fant  1 , Cecilia B Levandowski  1 , Kapil Gupta  2 , Zachary L Maas  1 , John Moir  1 , Jonathan D Rubin  1 , Andrew Sawyer  3 , Meagan N Esbin  1 , Jenna K Rimel  1 , Olivia Luyties  1 , Michael T Marr  3 , Imre Berger  2 , Robin D Dowell  4 , Dylan J Taatjes  5
Affiliations

TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

Charli B Fant et al. Mol Cell. .

Abstract

RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.

Keywords: DSIF; NELF; P-TEFb; PRO-seq; RNA polymerase II; TAF1; TBP; TFIID; TRIM-Away; pausing.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Biochemical reconstitution of promoter-proximal RNAPII pausing with purified human factors.
(A) Overview of in vitro transcription assay. (B) Representative data from in vitro transcription reactions with the complete PIC (lane 1) or supplemented with NELF/DSIF (lane 2) and P-TEFb (lane 3). At left are approximate lengths of the RNA transcripts. Note increased transcripts in paused region and reduced transcripts in elongation region upon addition of NELF and DSIF (lane 1 vs. lane 2); addition of P-TEFb reverses this trend (lane 2 vs. lane 3). (C) A time course showing maximum paused transcripts at 5 minutes and maximum elongated transcripts at 10 minutes; the elongated transcripts increased in equal proportion to the decrease in paused transcripts (5 min vs. 10 min), indicating that paused complexes can ultimately generate elongated products. (D) Calculation of an in vitro pause index (PI) at the HSP70 promoter, using paused and elongated regions defined in B. A NELF/DSIF outlier (PI=18) is not shown. Across replicate experiments (n=8) PI almost exclusively increased with NELF/DSIF and decreased with added P-TEFb. The spread in the data is consistent with replicate-to-replicate PI variability observed in human cells (see Methods). Bars represent mean ± standard error. See also Figure S2E.
Figure 2.
Figure 2.. TFIID is required to establish RNAPII promoter-proximal pausing in vitro.
(A) Reconstituted transcription reactions with PICs containing TFIID or TBP (i.e. reactions contain TFIIA, IIB, IIE, IIF, IIH, Mediator, and RNAPII, plus either TFIID or TBP). PICs with TBP still support transcription, but paused products were not observed (data from same gel). Reduced [ATP] was used in these experiments compared to Figure 1, causing an upstream shift in pausing, similar to results seen in Drosophila (Li et al., 2013). (B) Coomassie-stained gel of the complete human TFIID complex, generated by recombinant expression (Fitzgerald et al., 2006); *core TBP (residues 155– 335). (C) As with endogenously purified human TFIID, PICs with recombinant TFIID support transcription and RNAPII promoter-proximal pausing. Relative to A, increased [ATP] caused a downstream shift in paused products.
Figure 3.
Figure 3.. PRO-seq data suggest increased pause release upon TAF1/2 knockdown in human cells.
(A) Workflow for TRIM-Away (Clift et al., 2017) and PRO-seq (Kwak et al., 2013). (B) Representative western blots and quantitation (at right) for TFIID subunits. Bar plots represent mean and standard error, with actin as a loading control. (C) Normalization of PRO-seq data based upon 3’-end reads of long genes (Mahat et al., 2016)(see Methods) or based upon total run-on signal comparing control vs. TAF1/2-knockdown samples (inset; bar = S.E.M). (D) Genome browser view of PRO-seq reads at JUN locus (control vs. TAF1/2-depleted), which reflects genome-wide trends shown in MA plots for gene bodies (+500 to −500 from TES; E) and gene 5’-ends (−500 to +500; F).
Figure 4.
Figure 4.. Depletion of TFIID lobe C subunits increases pause release; TFIID function is conserved in Drosophila cells.
(A) Metagene plot (all genes, n = 18687) of promoter-proximal region that shows increased 5’-end reads in TAF1/2 knockdown cells, which extend beyond the pause site. These data are consistent with increases in pause release and re-initiation, which are coupled events in metazoan cells (Gressel et al., 2017; Shao and Zeitlinger, 2017). A sharp reduction in reads typically occurs around +300 from the TSS (e.g. HSPA1B locus, inset; vertical dashed line +60 from TSS) which suggests downstream auxiliary factors that terminate or arrest RNAPII (see Discussion). (B) Metagene plot and MA plot (inset) of non-annotated regions, showing that eRNA transcription is reduced in TAF1/2 knockdown cells. (C) Metagene plot of expressed genes (n = 10995) in Drosophila, comparing control vs. Taf1 knockdown S2 cells. As in TAF1/2 knockdown HCT116 cells, transcription increased at gene 5’-ends. Inset: Representative western blots showing Taf1 knockdown in S2 cells. Taf1 knockdown was determined to be 88% (± 6.7%) from 3 biological replicates. (D) Model. TFIID is required to establish RNAPII promoter-proximal pausing. Disruption of TFIID lobe C correlates with increased RNAPII pause release, which enables other RNAPII complexes to re-initiate transcription (Gressel et al., 2017; Shao and Zeitlinger, 2017).
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