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. 2012 Aug 10;287(33):27580-92.
doi: 10.1074/jbc.M112.379206. Epub 2012 Jun 13.

TFIID TAF6-TAF9 complex formation involves the HEAT repeat-containing C-terminal domain of TAF6 and is modulated by TAF5 protein

Elisabeth Scheer  1 , Frédéric Delbac, Laszlo Tora, Dino Moras, Christophe Romier
Affiliations

TFIID TAF6-TAF9 complex formation involves the HEAT repeat-containing C-terminal domain of TAF6 and is modulated by TAF5 protein

Elisabeth Scheer et al. J Biol Chem. .

Abstract

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes.

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Figures

FIGURE 1.
FIGURE 1.
Domain organization of TAF6 and sequence alignment of the TAF6C domain. A, schematic representation of the putative domain organization of TAF6 from E. cuniculi, A. locustae, S. cerevisiae, and Homo sapiens with their corresponding amino acid numbering. The colored boxes indicate the evolutionary conserved regions. HFD, histone fold domain; TAF6M, middle domain; TAF6C, C-terminal domain. B, multiple sequence alignment of the evolutionary conserved TAF6C domain from different species, as indicated. Human TAF6L was also included in the alignment. Different levels of gray shading indicate distinct levels of conservation. The numbering above and in the middle of the alignment corresponds to the TAF6 proteins from A. locustae and H. sapiens, respectively. The numbers at the end of each row correspond to the sequence displayed on the row. The red cylinders depict the α-helices observed in the structure of A. locustae TAF6C. + symbols above the sequences mark residues that are participating in the A. locustae TAF6C evolutionary conserved positive electrostatic patch. Symbols underneath the sequences indicate the residues mutated: m1 (*), m2 (YY), m3 (&), m4 (#), m5 (§), m6 (@), m7 (P), and m8 (A). Symbols that are circled indicate the residues that have been used to creates single point mutants m9 to m16 (see also Tables 2 and 3). The alignment was created with Aline (53). S. pombe, Schizosaccharomyces pombe.
FIGURE 2.
FIGURE 2.
Structure of TAF6C domain. A, ribbon representations of the A. locustae TAF6C domain using three different views. α-Helices are colored red, and the loops connecting them are colored yellow. The five HEAT repeats composing the domain are labeled (HEAT1 to HEAT5). The different helices composing these HEAT repeats are labeled in the middle view according to the nomenclature used in Fig. 1B. B, electrostatic potential at the surface of the A. locustae TAF6C domain. The electrostatic potentials −7 and +7 kBT (kB, Boltzmann constant; T, temperature) are colored red and blue, respectively. The orientation of the protein in each view corresponds to the one showed in panel A. A clear positive electrostatic patch is observed in the surface represented in the lower view. C, detail of the residues participating in the positive electrostatic patch (A. locustae numbering). For clarity, the orientation of the structure is slightly tilted from the one used in the views of the lower panels in A and B. The figure was made by using GRASP (54) and PyMOL (55).
FIGURE 3.
FIGURE 3.
Electrostatic potential at the surface of A. locustae, H. sapiens, and S. cerevisiae TAF6C domains. The electrostatic potentials −7 and +7 kBT (kB, Boltzmann constant; T, temperature) are colored red and blue, respectively. The structures used for the calculations are those of A. locustae TAF6C crystallographic structure and the modeled structures of H. sapiens and S. cerevisiae TAF6C domains built by homology using the A. locustae structure. The two orientations showed correspond to the upper and lower orientations of A. locustae TAF6C displayed in Fig. 2B. A clear conserved positive patch is observed in the three proteins in panel B. In contrast, the other side of the protein (panel A) does not seem to have conserved electrostatic features.
FIGURE 4.
FIGURE 4.
Effect of mutants with multiple amino acid changes in H. sapiens TAF6 on TAF6-TAF9 and TAF5-TAF6-TAF9 complex formation. A, Coomassie Blue staining of TAF6 and Western blotting of TAF6 and TAF9 from baculovirus-expressed and immunopurified FLAG-TAF6-TAF9 human complexes, isolated upon FLAG tag purification. The different lanes correspond to complexes incorporating full-length human TAF6, either wild type (WT) or with various mutations (m1 to m8). Each mutant harbors multiple amino acid changes (except for m7 and m8) described in Table 2. Inputs are displayed on the right side of the panel. B, graph displaying the amount of TAF9 (assessed upon quantification of the TAF9 Western blotting) bound to TAF6 upon complex formation in the presence of the WT or different m1–m8 mutants of TAF6. C, same as in panel A in the case of baculovirus-expressed and immunoprecipitated TAF5/FLAG-TAF6-TAF9 complexes isolated upon FLAG tag purification. Inputs are displayed on the right side of the panel. D, graph displaying the different amounts of TAF5 and TAF9 (assessed upon quantification of the TAF5 and TAF9 Western blots) bound to TAF6 upon complex formation in the presence of the WT or different m1–m8 mutants of TAF6. Error bars in panels B and D represent ± S.D.
FIGURE 5.
FIGURE 5.
Effect of mutants with single amino acid changes in H. sapiens TAF6 on TAF6-TAF9 and TAF5-TAF6-TAF9 complex formation. A, Coomassie Blue staining of TAF6 and Western blotting of TAF6 and TAF9 from baculovirus-expressed and immunopurified human TAF6-TAF9 human complexes. The different lanes correspond to complexes incorporating full-length human TAF6 either with WT or with single mutations (m9 to m16). Each mutant is described in Table 3. Inputs are displayed on the right side of the panel. B, graph displaying the amount of TAF9 (assessed upon quantification of the TAF9 Western blotting) bound to TAF6 upon complex formation in the presence of the WT or different m9–m16 mutants of TAF6. C, same as in panel A in the case of baculovirus-expressed TAF5-TAF6-TAF9 complexes isolated upon FLAG tag purification of TAF6. Inputs are displayed on the right side of the panel. D, graph displaying different amounts of TAF5 and TAF9 (assessed upon quantification of the TAF5 and TAF9 Western blots) bound to TAF6 upon complex formation in the presence of the WT or different m9–m16 mutants of TAF6. Error bars in panels B and D represent ± S.D.
FIGURE 6.
FIGURE 6.
Multiple and single amino acid mutations in the H. sapiens TAF6C domain affect TAF6 stability and endogenous TFIID assembly in HeLa cells. A, eukaryotic expression vectors expressing the indicated FLAG-tagged TAF6 wild type and different mutant proteins (see also Tables 2 and 3) were transfected into HeLa cells. 48 h following transfection, cells were collected, and WCEs were made. WCEs were normalized by Western blot analysis for equal TBP amounts (lower panel), and then the expression of wild type and mutant FLAG-tagged TAF6 proteins was analyzed by Western blot using an anti-FLAG antibody. Ctrl, control. B, several representative blots were scanned, and the quantities of FLAG-tagged wild type and mutant TAF6 proteins are represented in the graphs. The y axis shows arbitrary units. Error bars represent ± S.D. C, FLAG-TAF6-containing TFIID complexes were purified from HeLa WCEs by an anti-FLAG immunoprecipitation and analyzed by Western blotting for the presence of various subunits (as indicated). Wild type FLAG-TAF6 and the tested mutants are able to incorporate in TFIID complexes. D, model summarizing the influence of the TAF6C domain and the TAF5 protein on TAF6-TAF9 assembly. The model has been established with the structure of the Drosophila TAF6-TAF9 histone-like pair (Protein Data Bank (PDB) 1TAF) and the A. locustae TAF6C structure (this study; PDB 4ATG). HFD, histone fold domain.
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