Figure 1.
TAF8 is present in both TFIID and TFTC preparations while SPT7L is associated with TFTC.
(A) TFIID and TFTC complexes were purified [7], separated by SDS-PAGE and the presence of different subunits were analysed by Western blot using the indicated antibodies. (B) TAFs, TBP (left panels) and certain TFTC subunits (right panels) were individually expressed (In factor) or coexpressed with TAF8 (In factor+TAF8) in Sf9 cells as indicated and WCEs were made. TAF8-containing complexes were immunopurified (IPed) with an anti-TAF8 mAb (2TAU 2B8). Protein expression in the input fractions and TAF8-bound proteins were analysed by western blot with the indicated antibodies. In each experiment, the expression and the IP efficiency of TAF8 was the same as shown in a single representative experiment (lower panel).
Figure 2.
TAF8 interacts with TAF10 through its HF domain, while it interacts with SPT7L through its C-terminal region.
GST-TAF8 (A) and GST-TAF10 (B) full length (FL) or the corresponding deletion mutants were immobilised on glutathion-sepharose beads (as indicated), washed and the bound GST-fusion proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining (CBB). (*) labels premature termination products. Equal amounts of the indicated baculovirus infected Sf9 whole cell extracts (WCEs) were incubated with each resin, washed and the bound proteins analysed by western blot (WB). The positions of molecular weight markers are indicated in kDa. HFD: histone fold domain; PRD: proline rich domain; NLS: nuclear localization signal. (C) TAF8 was expressed alone (lane 1 and 3), coexpressed with TAF10 (lane 2) or with Flag-SPT7L (lane 4) in Sf9 cells using the baculovirus system. From these cells WCEs were prepared and proteins immunoprecipitated with the indicated antibodies. Bound proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining (CBB). The co-immunoprecipitation between TAF8 and TAF10 as well as TAF8 and SPT7L indicates strong stoichiometric interactions between these proteins.
Figure 3.
TAF8 and SPT7L interact in vivo (A)
The nuclear localization of TAF8 lacking the NLS [TAF8(1-294] depends on its in vivo interaction with SPT7L. HeLa cells were co-transfected with the indicated CFP- and YFP-containing expression vectors and localization of the expressed proteins were visualised by fluorescence microscopy. The images shown in each panel are representative of all the transfected cells. (B) Sensitized emission of YFP fusion proteins due to FRET was measured in two different experiments in the nucleus of 25 individual HeLa cells transfected with the indicated combinations of vectors expressing YFP and CFP fusion proteins. The mean value of FRET efficiency (in%) over the entire nucleus in each cell was calculated as described in the Materials and Methods. A threshold was set to 5%, above the highest value of the negative control CFP/YFP (see horizontal line in each graph), and for the other pairs only values above this level were averaged. The average value of the negative control is 1.03%. The average value for each pair is the following: CFP-TAF8/YFP-TAF10 = 16.9%; CFP-TAF8/YFP-SPT7L = 15.8%; CFP-TAF6/YFP-TAF9 = 27.1%. Note that the scale for the TAF6/TAF9 is different.
Figure 4.
The TAF8-TAF10 HF pair incorporates into a higher order TFIID subcomplex only if another five TAFs are simultaneously present.
(A) Either seven TAFs (TAF4, -5, -6, -8, -9, -10 and -12) or only six of them (each time only the omitted TAF is indicated compared to the seven-TAF complex) were coexpressed in Sf9 cells and WCEs made. Protein expression in the WCEs was verified by western blot analysis (WB). Note that in the seven-TAF complex each TAF is indicated with only its corresponding number according to the TAF nomenclature [3]. (B–C) TAF10-containing complexes were purified from the WCEs with an anti-TAF10 mAb (1H8). Bound complexes were eluted by peptide competition and analysed either by western blot (WB) with the indicated antibodies (B) or by silver nitrate staining (Silver) (C).
Table 1.
Figure 5.
TAF8 is not a subunit of TFTC/STAGA type complexes, but is present in TFIID and a novel TAF8-, TAF10- and SPT7L-containing complex that can also be formed in vitro.
(A) The indicated transcription factors were immunoprecipitated using specific mAbs (1H8 anti-TAF10; 2A10 anti-ATXN7 and 2C1 anti-TBP) from HeLa cell nuclear extract (NE) and eluted by an excess of peptides against which the mAbs were raised. Eluted protein complexes were separated by SDS-PAGE and analysed by western blot with the indicated antibodies. (B) TAF8-containing protein complexes were purified from HeLa NE according to the scheme shown on the left of the panel. The eluate obtained after the first anti-TAF8 IP using the 2TAU 2B8 mAb (anti-TAF8 IP E1; lane 3) and the supernatant obtained after the TAF8-containing complexes were depleted in TFIID using the anti-TBP 2C1 mAb (SN2; lane 4). They were separated along with complexes obtained after an anti-TAF10 IP (TFIID and TFTC together; lane 1) or with a highly purified TFIID fraction (lane 2) on SDS-PAGE and analysed with the indicated antibodies by western blot. (C) TAF8 containing complexes were purified from HeLa NE according to the scheme on the left of the panel. TAF10-containing complexes were re-precipitated from the eluate obtained after the first anti-TAF8 IP (E1, lane 1) extensively washed and loaded after boiling the beads (lane 2) with loading buffer on a SDS-PAGE. The migration of the antibody alone (-control) is shown in lane 3. Proteins were analyzed with the indicated antibodies by western blot. (D) TAF8, TAF10 and Flag-SPT7L were co-expressed in Sf9 cells. WCEs were made and proteins were subjected to two successive immunoprecipitations and elutions by peptide competition according to the schemes shown on the left of the panel. Input and eluted protein complexes were separated by SDS-PAGE and analysed with the indicated antibodies by western blot.
Figure 6.
The TAF8-, TAF10- and SPT7L-containing complex has a size of about 300–350 kDa and may exist in vivo in crude nuclear extracts.
Immunupurified and eluted TAF10-containing complexes (TAF10 IP) (A), or HeLa nuclear extract (NE) (B) (50 µl) were injected on a Superose 6 size exclusion chromatography column using the SMART FPLC system (Pharmacia) and separated. Portions of the input (in; 5 µl) and every fraction from 7 to 38 (20 µl) were analysed by western blot (as indicated). Fraction numbers are shown under each lane and the elution profile of known molecular mass markers is indicated above the panels. To detect the non-phosphorylated form of SPT7L, 20 µl from each fraction was first phosphatase treated for 30 min at 37°C, then separated by SDS-PAGE and analyzed by western blot with the anti-SPT7L mAb (panel SPT7L-PPase). (C) Baculovirus expressed SPT7L in WCE (lane 2) and purified TFTC (lane 4) were treated with phosphatase (PPase) for 30 min at 37°C, separated on SDS-PAGE and analysed by western blot with the anti-SPT7L mAb (15) (upper panel), and an anti-TAF10 mAb (lower panel). The corresponding mock treated fractions are shown in lanes 1 and 3, respectively.