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. 2015 Jan 14:6:6011.
doi: 10.1038/ncomms7011.

Cytoplasmic TAF2-TAF8-TAF10 complex provides evidence for nuclear holo-TFIID assembly from preformed submodules

Simon Trowitzsch  1 , Cristina Viola  1 , Elisabeth Scheer  2 , Sascha Conic  2 , Virginie Chavant  3 , Marjorie Fournier  2 , Gabor Papai  4 , Ima-Obong Ebong  5 , Christiane Schaffitzel  1 , Juan Zou  6 , Matthias Haffke  1 , Juri Rappsilber  7 , Carol V Robinson  5 , Patrick Schultz  4 , Laszlo Tora  2 , Imre Berger  8
Affiliations

Cytoplasmic TAF2-TAF8-TAF10 complex provides evidence for nuclear holo-TFIID assembly from preformed submodules

Simon Trowitzsch et al. Nat Commun. .

Abstract

General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID-a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)-assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8-TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core-TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo-TFIID, regulated by nuclear import of preformed cytoplasmic submodules.

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Figures

Figure 1
Figure 1. A TAF2–8–10 complex exists in the cytoplasm.
(a) Purified, polyclonal anti-TAF2 antibodies specifically recognize recombinant and endogenous TAF2. Recombinant (rec.) purified TAF2 (10 ng, lane 1) and immunopurified TFIID (300 and 150 ng; lanes 2, 3) were loaded on an 8% SDS–PAGE, blotted and analysed by western blot assay. Protein size markers are indicated. (b) Abundances of individual proteins co-immunoprecipitated from nuclear or cytoplasmic HeLa cell extracts (grey or black bars, respectively) using purified polyclonal anti-TAF2 antibodies were compared in units of normalized spectral abundance factors (NSAFs). Each column is the average of two independent experiments and error bars represent range of the data. (c) Domain organization of TAF2, TAF8 and TAF10 in a schematic view. Grey rectangles indicate predicted, unstructured regions. The NLS of TAF8 is shown as a black bar. Numbers indicate first and last amino acids in each protein. (d) Immunofluorescence microscopy of HeLa cells. Nuclei are visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). TAF2 is displayed in green and TAF8 in red. The bottom panel shows images of control cells, which were treated with secondary antibodies only. Scale bar, 10 μm. (e) Immunofluorescence microscopy of HeLa cells as in d, but displaying TAF2 (green) and TAF10 (red).
Figure 2
Figure 2. Recombinant TAF2–8–10 complex.
(a) TAF2, the TAF8–10 pair and a mixture of TAF2–8–10 were analysed by SEC. Elution profiles of TAF2 (green), TAF8–10 (blue) and TAF2–8–10 (purple) are plotted in relative absorption units at 280 nm versus elution volume (top). Fractions are numbered (top of graph). SDS–PAGE analyses of the eluted samples are shown (below). Molecular masses of protein standards are indicated on the left of gel sections. Protein denominations are shown on the right. First lane shows the SEC input (IN). (b) Absorbance c(s) profiles from sedimentation velocity analytical ultracentrifugation experiments are plotted for TAF2 (green), TAF8–10 (blue) and TAF2–8–10 (purple). (c) Mass spectrum of TAF2–8–10 complex electrosprayed from an aqueous ammonium acetate solution under high collision energy for subunit dissociation. The MS spectrum reveals peaks with corresponding masses for a TAF8–10 dimer (blue dots), TAF2 subunit (green dots) and a predominant TAF2–8–10 complex (purple dots) centred at 4,000, 6,000 and 7,500 m/z, respectively. At 12,000 m/z is a TAF2–8 dimer (yellow dots) resulting from the dissociation of the TAF10 subunit (light blue dots) from the intact TAF2–8–10 complex. Proteins and protein complexes are shown schematically as coloured circles.
Figure 3
Figure 3. TAF8–TAF10 interactions.
(a) Crystal structure of human TAF8–10 complex is depicted in a cartoon representation. Two orientations related by a vertical rotation of 90° are shown. TAF8 is coloured in blue and TAF10 in green. The disordered L2 loop of TAF10 is represented by a dotted line. Secondary structure elements and loops are labelled. The TAF8–TAF10 complex adopts a non-canonical HFD pair. (b–e) Close-up views of the interactions between TAF8 and TAF10. Key interacting residues are highlighted. All structure drawings were generated with PyMOL ( http://www.pymol.org/). (f) Pull-down experiments of TAF2 fused to MBP analysing the interactions with TAF8–10, TAF8–TAF10ΔN and TAF8ΔC–TAF10 HFD pairs (see main text for details). Unfused MBP is included as a control. Input samples (top) and samples precipitated on amylose resin (bottom) were resolved on 4–12% gradient gels. Protein identities are shown on the right.
Figure 4
Figure 4. TAF8–TAF2 interactions.
(a) His-tagged TAF2 binding to overlapping peptides of the TAF8 C-terminal region (residues 105–310) spotted onto nitrocellulose membranes (spots A2-G4, left) was analysed by utilizing a peptide array. Bound TAF2 was visualized by luminol reaction and signal intensities were plotted for each spot after background subtraction (right). Spots A1, G5 and G6 served as positive controls. TAF2 protein was omitted for the control membrane. The four major binding regions (I–IV) are indicated above the histogram. (b) SPR experiments with immobilized full-length TAF2 as ligand and MBP (control) as well as MBP fusions of TAF8 fragments 105–310, 141–310, 200–310 and 105–260 as analytes. TAF8 deletion constructs are schematically shown as bar diagrams (left). TAF2-interacting regions on TAF8 as identified in a are highlighted. SPR sensorgrams at identical analyte concentrations of 500 nM are plotted as RU versus time (right). (c) SEC analyses assessing the influence of TAF8 point mutations on TAF2 binding. Elution profiles for the indicated proteins and protein complexes are plotted on the left and SDS–PAGE analyses of each run are shown on the right. Molecular masses of protein standards are denoted on the left of the gels and protein names on the right.
Figure 5
Figure 5. TAF8 promotes TAF2 incorporation in TFIID.
(a) TFIID components studied are shown as bar diagrams. Predicted low-complexity regions of the proteins are coloured in grey. A black bar denotes the TAF8 NLS. C-terminally truncated TAF8 (TAF8ΔC), which was used to reconstitute the 7TAFΔ complex, is depicted on the right. Numbers denote first and last amino acids for each protein. (b) Impact of the TAF8 truncation on TAF2 binding to 7TAF complexes. SEC elution profiles for indicated proteins and protein complexes are shown (top). Corresponding SDS–PAGE gel sections of peak fractions of each run are shown (bottom). Protein size markers are shown on the left; protein identities on the right. (c) Three-dimensional single-particle EM reconstruction of negatively stained 8TAF complex (grey mesh) superimposed on 7TAF complex (yellow, from ref. 12) is shown in two views related by a 90° rotation as indicated (arrow). Difference density attributed to bound TAF2 is highlighted in blue. (e) Protein–protein crosslink maps for the 7TAF complex (left) and the 8TAF complex (right) are shown. Circle sizes represent relative molecular weights of each protein. Black lines connect crosslinked proteins. Grey bars superimposed on black lines indicate crosslink frequencies ( www.crosslinkviewer.org). Original images corresponding to the gel sections shown in Fig. 5b (bottom panel) are provided in Supplementary Figure 8.
Figure 6
Figure 6. Nuclear TFIID assembly from preformed submodules.
(a) A complex consisting of TAF2, TAF8, TAF10 and Importin α1ΔIBB was formed from highly purified components. Importin α1ΔIBB was mixed in a twofold molar excess with purified TAF2–8–10 and the mixture purified by SEC. SDS–PAGE analysis of the peak fraction is shown. (b) Importin α1-TAF8 complex crystal structure. Magnified view of interacting residues of the major binding site of Importin α1 (grey) with residues of the NLS of TAF8 (blue). Importin α1 is shown in ribbon representation and the TAF8–NLS as a stick model. TAF8 residues R303 and R304 are not involved in contacting Importin α1 and are omitted for clarity. (c) Immunofluorescence microscopy of HeLa cells depleted of TAF8 (TAF8 siRNA) by RNAi or control cells (Control siRNA). Nuclei are visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). TAF2 is displayed in green and TAF8 in red. Arrows point to a non-transfected cell. Scale bar, 10 μm. (d) Cartoon model of cytoplasmic TAF2–8–10 complex and nuclear holo–TFIID assembly. The NLS of TAF8 is filled in black. The TAF2-interaction domain within TAF8 is highlighted by shading. TAF2, blue; TAF8, green; TAF10, orange. The TAF2–8–10 complex resides in the cytoplasm, whereas the physiological symmetric core–TFIID complex is found in the nucleus. The cryo-electron microscopy density envelope of core–TFIID complex is shown (adapted from ref. 12). On binding of Importin α1 (grey) to the TAF8 NLS, TAF2–8–10 translocates into the nucleus through a nuclear pore (arrow). In the nucleus, Importin α1 is released and TAF2, 8 and 10 associate with core–TFIID, to form intermediates including the asymmetric 8TAF complex along the holo–TFIID assembly pathway.
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