TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

Abstract

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNAi to knock down specific TFIID subunits in Drosophila tissue culture cells to determine which subunits are most critical for maintaining stability of TFIID in vivo. Contrary to expectations, we find that TAF4 rather than TBP or TAF1 plays the most critical role in maintaining stability of the complex. Our analysis also indicates that TAF5, TAF6, TAF9, and TAF12 play key roles in stability of the complex, whereas TBP, TAF1, TAF2, and TAF11 contribute very little to complex stability. Based on our results, we propose that holo-TFIID comprises a stable core subcomplex containing TAF4, TAF5, TAF6, TAF9, and TAF12 decorated with peripheral subunits TAF1, TAF2, TAF11, and TBP. Our initial functional studies indicate a specific and significant role for TAF1 and TAF4 in mediating transcription from a TATA-less, downstream core promoter element (DPE)-containing promoter, whereas a TATA-containing, DPE-less promoter was far less dependent on these subunits. In contrast to both TAF1 and TAF4, RNAi knockdown of TAF5 had little effect on transcription from either class of promoter. These studies significantly alter previous models for the assembly, structure, and function of TFIID.

Fig. 1.

Analysis of TFIID stability in vivo. (A) S2 cells were either left untreated (NT) or treated with dsRNA targeting the TFIID subunit indicated at the top of the panel for 3 days. Whole-cell lysates were then subjected to Western blot analysis with antibodies directed against the subunits indicated at the left of the panel. β-Tubulin served as a loading control. (B) A TAF4 monoclonal antibody was used to immunoprecipitate TAF4-containing complexes from nuclear extracts prepared from either untreated S2 cells (NT) or cells treated with TAF1 dsRNA. The precipitated proteins were eluted and subjected to Western blot analysis with antibodies against the proteins indicated at the left of the panel. (Left) Input. (Right) Eluted coprecipitating (IP) proteins. Protein G–Sepharose beads were used as a nonspecific control.

Fig. 2.

TAF4 CTR rescues TFIID stability. (A) Schematic of the Drosophila TAF4 protein indicating the glutamine-rich region, the ETO domain, the histone fold, and the CTR construct. Also noted is the region of TAF4 targeted by RNAi. (B) A stable cell line expressing the TAF4 CTR under the control of the copper-inducible MtnA promoter was either left untreated or treated with TAF4 dsRNA. These cells were then either left untreated or treated with copper, as indicated. Whole-cell lysates were then immunoblotted for the TFIID subunits indicated on the left. β-Tubulin was included as a loading control. (C) The M2 anti-FLAG monoclonal antibody was used to immunoprecipitate (IP) 3× FLAG-tagged TAF4 CTR-containing complexes from nuclear extracts prepared from either WT control S2 cells or the TAF4 CTR cell line treated with TAF4 dsRNA and copper. The input and immunoprecipitated proteins were probed with antibodies directed against the subunits indicated on the left. (Left) Input. (Right) Immunoprecipitated eluates.

Fig. 3.

The TAF6 NTR is sufficient for TFIID stability. (A) Schematic of the Drosophila TAF6 protein showing the histone fold, the NTR and CTR constructs, and the regions of TAF6 targeted by RNAi. (B) Copper-inducible stable S2 cell lines were generated expressing either 3× FLAG-tagged TAF6 NTR or 3× FLAG-tagged TAF6 CTR. These cells were treated with dsRNA against TAF6, and copper and nuclear extracts were prepared. The M2 anti-FLAG antibody was used to precipitate truncated TAF6-containing complexes, and Western blotting with antibodies against the proteins indicated at the left of the panel was used to detect the coprecipitating proteins. (Left) Input. (Right) Immunoprecipitated (IP) proteins.

Fig. 4.

Transcription from a TATA-less, DPE-containing promoter requires TAF1 and TAF4. (A) Diagrams of the reporter constructs used to transfect S2 cells. (Upper) WT MtnA promoter driving luciferase expression. It contains a TATA box (TATAAAA) and an initiator (TCAGTT), but no DPE (AATCATC starting at position +28). (Lower) WT MtnA promoter with a mutated TATA box (GCGCCCC) and a DPE (AGACGTG) inserted at position +28 from the transcription start site. (B) S2 cells were either left untreated (NT) or treated with dsRNA directed against TAF1, TAF4, or TAF5 and transfected with either the WT MtnA-luc reporter or the mMtnA+DPE-luc reporter and actin-Renilla to control for transfection efficiency. After 3 days, the transfected cells were treated with copper to induce transcription. Six hours later, luciferase expression was determined, normalized to Renilla expression, and plotted as fraction of untreated luminescence.

Fig. 5.

Model of TFIID assembly in vivo. TFIID consists of a stable core subcomplex made up of TAF4, TAF5, TAF6, TAF9, and TAF12, which becomes decorated with TBP, TAF1, TAF2, and TAF11. Subunit stoichiometry is adapted from Sanders et al. (7).