TFIIE orchestrates the recruitment of the TFIIH kinase module at promoter before release during transcription

Abstract

In eukaryotes, the general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. However, the mechanism by which these transcription factors incorporate the preinitiation complex and coordinate their action during RNA polymerase II transcription remains elusive. Here we show that the TFIIEα and TFIIEβ subunits anchor the TFIIH kinase module (CAK) within the preinitiation complex. In addition, we show that while RNA polymerase II phosphorylation and DNA opening occur, CAK and TFIIEα are released from the promoter. This dissociation is impeded by either ATP-γS or CDK7 inhibitor THZ1, but still occurs when XPB activity is abrogated. Finally, we show that the Core-TFIIH and TFIIEβ are subsequently removed, while elongation factors such as DSIF are recruited. Remarkably, these early transcriptional events are affected by TFIIE and TFIIH mutations associated with the developmental disorder, trichothiodystrophy.

Fig. 1

Defective transcription in cells harboring TFIIE mutations. a Increasing amounts of whole-cell lysates isolated from fibroblasts of patients with TFIIEβ/A150P, /D187Y, and of unaffected parents were used for immunoblot analysis of TFIIE (IIEα and IIEβ), TFIIH (XPB, XPD, and CDK7), TFIIA (p35), TFIIB (p33), TFIID (TAF1, TAF4, and TBP), TFIIF (RAP74 and RAP30), and Pol II (the hypo IIA- phosphorylated and hyper IIO-phosphorylated forms of RPB1). β-actin was used as loading control. b, c Wild-type (gray boxes) and TTD fibroblasts (open boxes) with the mutation IIEβ/A150P (panel b) and IIEβ/D187Y (panel c) have been treated with t-RA (10 µM). Relative RARβ2 gene expression have been measured by RT-PCR after 0, 6, and 8 h of t-RA treatment (panels b and c). The mRNA levels were normalized to the 18S RNA amount. The results (n = 9, means ± s.d.) are presented as n-fold induction relative to non-treated cells. d–i: ChIP experiments have been done 0, 6, and 8 h post-t-RA treatment from wild-type (gray boxes) and TTD fibroblasts (open boxes) with the mutation IIEβ/A150P (panels d, f, and h) and IIEβ/D187Y (panels e, g, and i) to analyze the recruitment of Pol II (RPB1, d and e), TFIIH (CDK7, f and g) and TFIIE (TFIIEβ, h and i) at the RARB2 proximal promoter. The values (n = 8, means ± s.d.) are expressed as percentage of immunoprecipitated DNA relative to the input. j Relative RARβ2 gene expression after 0, 6, and 8 h of t-RA treatment in IIEβ/WT (gray boxes) and KI-IIEβ/A150P (open boxes) cells. The mRNA levels were normalized to the 18S RNA amount. The results (n = 9, means ± s.d.) are presented as n-fold induction relative to non-treated cells. k–x ChIP experiments have been done 0, 6, and 8 h post-t-RA treatment from IIEβ/WT (gray boxes) and KI-IIEβ/A150P (open boxes) cells. We analyzed at the RARB2 proximal promoter (panels k–q) and exon 4 (panels r–x) the recruitment of Pol II (RPB1, k, r), TFIIH (CDK7, l, s), TFIIB (p33, m, t), TFIIEβ (n, u), Ser5-P (o, v), Ser2-P (p, w), and DSIF (SPT5, q, x). The values (n = 8, means ± s.d.) are expressed as percentage of immunoprecipitated (IP) DNA relative to the input. Source data are provided as a Source Data file

Fig. 2

Defective TFIIE and TFIIH affect transcription initiation. a Production of the recombinant mutated forms of TFIIE (rIIE). Equal amounts of purified rIIE were resolved by SDS–PAGE. Coomassie blue staining gel (top panel) and western blot (WB, bottom panel) with antibodies raised against TFIIEα and TFIIEβ are shown. b Production of the recombinant mutated forms of TFIIH (rIIH). Equal amounts of purified rIIHs were resolved by SDS–PAGE and immunoblotted for XPB, XPD, p62, and CDK7. c, d Purified rIIEs (c) and rIIHs (d) were added to in vitro reconstituted transcription systems, as indicated. After addition of NTPs (including radiolabelled CTP), the transcription activity was assessed after 5, 10, and 20 min of incubation. The length of the corresponding transcript (309nt) is indicated on the right side. The signals were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. e, f Abortive transcription assays using rIIEs (e) and rIIHs (f). After preinitiation complex assembly using equivalent amounts of the different purified rIIEs and rIIHs (as revealed by western blots), phosphodiester bond synthesis was initiated by the addition of priming dinucleotides CpA and radiolabelled CTP. After 30 min of incubation, the radioactive trinucleotide (CpApC*) synthesis was stopped, quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of two independent experiments. Source data are provided as a Source Data file

Fig. 3

Defective TFIIE and TFIIH alter the preinitiation complex. a Biotinylated AdMLP bound to streptavidin magnetic beads was incubated with Pol II, TFIIA, TFIIB, TFIID (TBP), TFIIF, and TFIIH, in the presence (+) of either rIIEαβ/WT, rIIEαβ/A150P, or rIIEαβ/D187Y. After washes, the binding of different factors was evaluated by immunoblotting. The loading proteins used for PIC formation and the different rIIEs (in the following order rIIEαβ/WT, rIIEαβ/A150P, and rIIEαβ/D187Y) are indicated at the right part of the panel (load). The signals for IIEα, IIEβ, XPB, XPD, and CDK7 were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. b Immunoprecipitated rIIH/WT (using anti-p62) was incubated with rIIEs at 150 and 500 mM KCl. After washes, the coimmunoprecipitated proteins were resolved by SDS–PAGE and blotted with anti-XPB, anti-IIEα, and anti-IIEβ. Graph shows in arbitrary units (a.u.) the ratio IIEα/IIEβ for each rIIE. The results are representative of three independent experiments. c Pol II, TFIIA, TFIIB, TFIID (TBP), and TFIIF were incubated with biotinylated AdMLP bound to streptavidin beads in presence (+) of rIIEα/WT, rIIEβ/WT, and TFIIH. Immunoblot analysis has been done with different antibodies, as indicated. d TFIIH was immunoprecipitated with anti-p62 and incubated (+) with rIIEα/WT and/or rIIEβ/WT. After washes, immunoblot analysis of TFIIH (XPD, p52, CDK7) and TFIIE (IIEα and IIEβ) has been done. e Recombinant rIIEα/WT, co-produced with either rIIEβ/WT (lanes 1 and 2), rIIEβ/A150P (lanes 3 and 4) or rIIEβ/D187Y (lanes 5 and 6), was immunoprecipitated at 150 and 500 mM KCl. After washes, the proteins were resolved by SDS–PAGE and blotted with antibodies against IIEα and IIEβ. Graph shows the ratio IIEβ/IIEα (n = 3, means ± s.d.) in arbitrary units (a.u.). The results are representative of three independent experiments. f rIIH containing either XPD/WT (lanes 1 and 2), XPD/R112H (lanes 3 and 4), or XPD/R722W (lanes 5 and 6) were immunoprecipitated with anti-XPD and incubated at 150 and 500 mM KCl. After washes, the bound proteins were resolved by SDS–PAGE. The immunoprecipitated signals (IP) for XPB, XPD, and CDK7 were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. g Streptavidin magnetic beads were incubated (when indicated, +) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, the Core-TFIIH, the CAK and either XPD/WT, /R112H, or /R722W. After washes, immunoblot analysis of different factors has been done. The results (n = 3, means ± s.d.) are representative of three independent experiments. Source data are provided as a Source Data file

Fig. 4

TFIIEα and CAK releases and Pol II phosphorylation occur before DNA opening. a Streptavidin magnetic beads were incubated (+) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF and rIIH/WT, in presence of either ATP (200 μM) or ATPγS (200 μM). After washes, the binding of various factors has been evaluated by immunoblotting. The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. b Streptavidin magnetic beads were incubated (when indicated, +) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, the Core-TFIIH, XPD/WT, and the CAK, in presence of ATP (200 μM). After washes, immunoblot analysis was done for different proteins. The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. c Streptavidin magnetic beads were incubated (+) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, and rIIH/WT, in presence of ATP (200 μM) and THZ1 (1 and 10 μM). The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. d When indicated (+), biotinylated AdMLP bound to streptavidin magnetic beads were preincubated 20min (at 25 °C) with the general transcription machinery (Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, and rIIH/WT) in presence of Triptolide (TPL, 10 μM) before addition of ATP (200 μM). The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. e Streptavidin magnetic beads were incubated (+) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, and TFIIF, in presence of ATP (200 μM) and either rIIH-XPB/WT or /Fs740. The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. f Streptavidin magnetic beads were incubated (+) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, and TFIIF, in presence of ATP (200 μM) and either rIIH-XPB/WT or /K346R. The immunoblot signals for TFIIEα, CyclinH, and Ser5-P were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. Source data are provided as a Source Data file

Fig. 5

The CAK is released during RNA synthesis. a Streptavidin magnetic beads were incubated (as depicted in the scheme, left part) with biotinylated AdMLP, Pol II, and the GTFs (TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, and TFIIH/WT), in presence of ATP (200 μM). After 15 min of incubation with ATP, the beads were washed and NTPs (200 μM) were added (lane 4). Immunoblot analysis (for TFIIEα, TFIIEβ, XPB, CyclinH, RPB1) and the transcription activity (309nt run-off transcript) were assessed after 45 min of incubation. The results are representative of three independent experiments. b Streptavidin magnetic beads with biotinylated AdMLP, Pol II, and the GTFs (TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, and TFIIH/WT) were pre incubated 15 min (as depicted in the scheme) in presence (lane 2) or absence (lane 1) of ATP (200 μM). The beads were washed and NTPs (200 μM) were added. Transcription activity (309nt run-off transcript) were assessed after 45 min of incubation. The results are representative of three independent experiments. c, d Streptavidin magnetic beads were pre incubated (when indicated, +) with biotinylated AdMLP, Pol II and the GTFs (TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, and TFIIH/WT) in presence of ATP (200 μM). After washes, NTP (200 μM) and increasing amounts of rIIEα/WT were added. Immunoblot analysis (c) and transcription activity (d) were assessed after 45 min of incubation. The transcription activities were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of two independent experiments. e, f Streptavidin magnetic beads were incubated (when indicated, +) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID (TBP), rIIEαβ/WT, TFIIF, and TFIIH/WT, in presence of whole cell extracts (WCE), ATP (200 μM) (f) and NTP (200 μM) (e, f). Immunoblot analysis (for TFIIEα, TFIIEβ, XPB, CyclinH, Ser5-P, and SPT5) and the transcription activity (309nt run-off transcript) were assessed after 45 min of incubation. The results are representative of three independent experiments. Source data are provided as a Source Data file

Fig. 6

TTD mutations alter CAK release and Pol II phosphorylation. a Biotinylated AdMLP bound to streptavidin magnetic beads was incubated with Pol II, TFIIA, TFIIB, TFIID (TBP), TFIIF, and TFIIH/WT, in presence (when indicated, +) of ATP (200 μM) and either rIIEαβ/WT, /A150P, or /D187Y. The immunoblot signals for TFIIEα, CDK7, and CyclinH were quantified and plotted in arbitrary units (a.u.). The results (n = 3, means ± s.d.) are representative of three independent experiments. b Streptavidin magnetic beads were incubated (when indicated, +) with biotinylated AdMLP, Pol II, TFIIA, TFIIB, TFIID, rIIEαβ/WT, TFIIF, the Core-TFIIH, and the CAK, in presence of ATP (200 μM) and either XPD/WT, /R112H or /R722W. The immunoblot signals for TFIIEα, CDK7, and CyclinH were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. c, d In vitro reconstituted transcription systems with rIIEs (c) and rIIHs (d) have been performed in presence of cold NTPs. Immunoblot analysis next has been done using specific antibody against Ser5-P of Pol II. Signals were quantified (n = 3, means ± s.d.) and plotted in arbitrary units (a.u.). The results are representative of three independent experiments. Source data are provided as a Source Data file

Fig. 7

Sequential association/dissociation of TFIIE and TFIIH. Once a stable TFIID–TFIIA–TFIIB–Pol II–TFIIF promoter complex is assembled, TFIIE and TFIIH can be recruited. The TFIIEβ subunit is required to anchor TFIIEα. Altgough the Core-TFIIH sub-complex can incorporate the complex in absence of TFIIE, TFIIEα, and TFIIEβ are both required to anchor the CAK within the PIC. In presence of ATP, Pol II phosphorylation is accompanied by the release of the CAK and TFIIEα from the promoter, a process that takes place before DNA opening. In presence of NTP, RNA synthesis is initiated, the Core-TFIIH and TFIIEβ are removed while DSIF and other elongation factors are recruited