TAF4 Nucleates a Core Subcomplex of TFIID and Mediates Activated Transcription from a TATA-Less Promoter

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

Kevin J. Wright, Michael T. Marr II, and Robert Tjian*

Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, 16 Barker Hall, CA 94720

Contributed by Robert Tjian, June 30, 2006 Activator-dependent recruitment of TFIID initiates formation of the resolution structure nor a clear idea of the interactions respon- transcriptional preinitiation complex. TFIID binds core promoter sible for coordinating the assembly and maintaining the stability DNA elements and directs the assembly of other general transcrip- of the complex in vivo, the mechanism of action and the tion factors, leading to binding of RNA polymerase II and activation structure–function relationship of the different subunits of the of RNA synthesis. How TATA box-binding protein (TBP) and the TBP⅐TAF complex have remained obscure. TBP-associated factors (TAFs) are assembled into a functional TFIID Here, we exploit the robust RNAi response in Drosophila complex with promoter recognition and coactivator activities in tissue culture cells to address the stability and potential vivo remains unknown. Here, we use RNAi to knock down speciﬁc assembly path of TFIID in vivo. We have also begun to TFIID subunits in Drosophila tissue culture cells to determine which characterize functionally the role of individual TAFs at an subunits are most critical for maintaining stability of TFIID in vivo. inducible promoter. In contrast to the previously reported Contrary to expectations, we ﬁnd that TAF4 rather than TBP or central roles of TBP and TAF1 in nucleating the assembly of TAF1 plays the most critical role in maintaining stability of the holo-TFIID, our studies revealed a stable core subcomplex complex. Our analysis also indicates that TAF5, TAF6, TAF9, and that is nucleated by the C terminus of TAF4 and the N TAF12 play key roles in stability of the complex, whereas TBP, TAF1, terminus of TAF6 and that includes TAF5, TAF9, and TAF12,

TAF2, and TAF11 contribute very little to complex stability. Based which then becomes decorated with peripheral subunits TBP, BIOCHEMISTRY on our results, we propose that holo-TFIID comprises a stable core TAF1, TAF2, and TAF11 to form holo-TFIID. We also found subcomplex containing TAF4, TAF5, TAF6, TAF9, and TAF12 deco- that both TAF1 and TAF4 play a critical role in mediating rated with peripheral subunits TAF1, TAF2, TAF11, and TBP. Our activated transcription from a TATA-less, downstream core initial functional studies indicate a specific and significant role for promoter element (DPE)-containing promoter but not from a TAF1 and TAF4 in mediating transcription from a TATA-less, down- TATA-containing, DPE-less promoter, whereas knockdown of stream core promoter element (DPE)-containing promoter, TAF5 has little effect on transcription from either promoter. whereas a TATA-containing, DPE-less promoter was far less de- Our results shed light on the in vivo assembly of TFIID and pendent on these subunits. In contrast to both TAF1 and TAF4, identify key roles for TAF1 and TAF4 in mediating transcrip- RNAi knockdown of TAF5 had little effect on transcription from tion from a specific core promoter architecture. either class of promoter. These studies significantly alter previous models for the assembly, structure, and function of TFIID. Results TFIID Integrity Is Differentially Dependent on TBP⅐TAFs. Current RNA interference ͉ TATA box-binding protein ͉ S2 cells models of metazoan TFIID suggest that TAF1 and TBP serve as central scaffold subunits on which the other subunits assemble. egulated initiation of transcription to produce mRNA in However, these early studies were largely based on in vitro assembly Reukaryotes requires the stepwise assembly of an elaborate reactions using tagged recombinant proteins. To address the con- multiprotein preinitiation complex consisting of the general tribution of individual subunits to the integrity of TFIID in vivo and transcription factors, various coactivators, and RNA polymerase perhaps gain some insight into the order of assembly in cells, we II (for review, see ref. 1). The core promoter-recognition treated Drosophila Schneider Line 2 (S2) cells with dsRNA directed complex, TFIID, consists of the TATA box-binding protein against specific TAF subunits or TBP, and we immunoblotted (TBP) and 8–12 TBP-associated factors (TAFs). In addition to whole-cell lysates to monitor the disappearance of TBP⅐TAFs. As binding core promoter elements and initiating formation of the ⅐ shown in Fig. 1A, the targeted reduction of certain TAFs results in preinitiation complex, this TBP TAF multisubunit transcription a dramatic depletion͞degradation of other TAFs and to a lesser factor also serves as a coactivator by transmitting signals from extent TBP, whereas a reduction of other individual TAFs has sequence-specific activators to other components of the basal minimal or no effect on the nontargeted subunits. RNA analysis machinery (for review, see ref. 2). confirmed that the mRNA of the RNAi-targeted TAF subunit was Critical to dissecting the diverse functions of TFIID in both degraded. In contrast, mRNA corresponding to each of the non- promoter recognition and coactivation is an understanding of targeted TAFs continued to be expressed, suggesting that the how the complex is assembled and maintained in cells. Initial in observed depletion of nontargeted subunits is most likely due to a vitro assembly reactions suggested that TBP and the largest TAF loss of protein stability (8). Strikingly, RNAi-mediated depletion of subunit (TAF1) may form a scaffold on which the other TAFs TAF4 had the most dramatic global effects on overall Drosophila bind to form holo-TFIID (3). Subsequent studies have proposed that TAF5 may dimerize and also help coordinate complex ͞ assembly (4). Recent low-resolution electron microscopy single- Conflict of interest statement: No conflicts declared. particle reconstruction models of TFIID have revealed a trilobed Abbreviations: CTR, C-terminal region; DPE, downstream core promoter element; luc, architecture containing a large central cavity that has been luciferase; MtnA, metallothionein A; NTR, N-terminal region; S2 cells, Drosophila Schneider conserved from yeast to humans, with TBP situated at the 2 cells; SAGA, Spt-Ada-Gin5-acetyltransferase; TAF, TBP-associated factor; TBP, TATA box- junction of the three lobes (5, 6). The TAFs, some of which are binding protein; TFTC, TBP-free TAF-containing complex. present in multiple copies per TFIID complex (7), interact to *To whom correspondence should be addressed. E-mail: [email protected]. form the separate lobes. However, with neither an atomic © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605499103 PNAS ͉ August 15, 2006 ͉ vol. 103 ͉ no. 33 ͉ 12347–12352 Downloaded by guest on October 2, 2021 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. ␤-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.

TFIID integrity and stability. When TAF4 (formerly dTAFII110) is TAF9 (i.e., loss of TAF6 but preservation of TAF9) does not lost, all of the other TAF subunits tested except TAF2 (formerly hold true. dTAFII150) become severely depleted, presumably because of Treating S2 cells with TAF2 dsRNA has minimal impact on destabilization of the TFIID complex followed by degradation. This the stability of the core complex, reducing primarily the levels of finding suggests that TAF4 likely acts as a keystone subunit, TAF2 itself and to some extent TAF6. Likewise, TAF2 is the lone nucleating and maintaining TFIID stability. Furthermore, although subunit that remains largely intact when the other TAF subunits nearly every subunit is degraded when directly targeting TAF4 for are targeted for RNAi knockdown. Depleting TAF11 (formerly RNAi knockdown, the levels of this core component become dTAFII30␤) by RNAi modestly reduces the levels of TBP while depleted only when its histone-fold partner, TAF12 (formerly leaving all of the other TAFs unaffected. Surprisingly, depleting dTAFII30␣), is targeted for RNAi knockdown. As might be ex- TBP has little effect on the stability of the core TAF subcomplex. pected, RNAi depletion of TAF12 has a drastic effect on the other Also unexpected was the only modest effect on overall TAF TFIID subunits, reducing the levels of TBP and all of the TAFs levels seen when TAF1 levels were decreased, indicating that this except TAF2 and TAF6 (formerly dTAFII60). These results suggest largest of the subunits does not serve as a critical scaffold. By that TAF12 may also be a key component of a core subcomplex. contrast, TAF1 itself appears to be one of the most labile However, it is also possible that TAF12 is required primarily to subunits, becoming significantly depleted when nearly any of the maintain the stability of TAF4 and that the depletion of the other other TAFs is targeted for depletion. Similarly, a significant subunits is an indirect result of depleting TAF4. amount of TBP becomes destabilized and degraded when any Another potential core component is TAF5 (formerly TAF is targeted, with the exception of TAF2. The fraction of dTAFII80) because reduction of this subunit results in the TBP that remains is likely a combination of ‘‘free’’ TBP released degradation of most other subunits except TAF2, with TAF4 from the destabilized TAFs and TBP present in other complexes affected only moderately. This finding suggests that TAF5 may such as the RNA polymerase I complex SL1, but not TFIIIB, also play a key role in TFIID assembly or stability, which was because TRF1 functionally replaces TBP in the formation of an unexpected because a partial complex containing recombinant RNA polymerase III preinitiation complex in Drosophila (9, 10). Drosophila TAF4, TAF6, and TBP can be assembled on an Taken together, these results suggest that TBP, TAF1, TAF2, immobilized, truncated TAF1 (formerly dTAFII230) in vitro (3). and TAF11 are likely peripheral subunits of TFIID, whereas Thus, the in vitro-assembled complexes may not always accu- TAF4, TAF5, TAF6, TAF9, and TAF12 comprise a core sub- rately reflect the stability and͞or assembly of endogenous com- complex that can retain its integrity in the absence of the plexes in vivo. On the other hand, these data are consistent with peripheral components. the model proposing that TAF5 can dimerize and act as a scaffold to coordinate the two separate lobes of the TFIID TFIID Comprises a Decorated TAF4-Containing Core Complex. To structure (see the Introduction). establish further the possibility of a stable core subcomplex in the Targeting TAF6 by RNAi results in the depletion of all but absence of TAF1 and TBP, we prepared nuclear extracts from TAF2 and TAF4, indicating that it too may play a role in complex either untreated cells or cells treated with TAF1 dsRNA. Using stability. By contrast, RNAi knockdown directed against TAF9 a monoclonal antibody against TAF4, we immunoprecipitated (formerly dTAFII40), a proposed histone-fold partner to TAF6, this core subunit from each nuclear extract and probed the results mainly in the loss of TBP, TAF1, and TAF5. Interestingly, coprecipitating proteins by Western blot. As shown in Fig. 1B,a the levels of its putative partner, TAF6, remain unchanged, full TFIID complex is precipitated by the TAF4 antibody in suggesting that TAF6 is either not an obligate partner of TAF9 untreated control extracts, whereas a subcomplex containing just or it can make contacts with subunits other than TAF9 and that the core subunits, devoid of TBP and TAF1, is efficiently these interactions may be sufficient to hold TAF6 as a stable precipitated from nuclear extracts derived from S2 cells treated component of TFIID even in the absence of TAF9. However, as with TAF1 dsRNA. These data, taken with our findings pre- described above, the reciprocal relationship between TAF6 and sented above, provide evidence that Drosophila holo-TFIID

12348 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605499103 Wright et al. Downloaded by guest on October 2, 2021 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 Fig. 3. The TAF6 NTR is sufﬁcient for TFIID stability. (A) Schematic of the stable cell line expressing the TAF4 CTR under the control of the copper-inducible Drosophila TAF6 protein showing the histone fold, the NTR and CTR con- MtnA promoter was either left untreated or treated with TAF4 dsRNA. These cells structs, and the regions of TAF6 targeted by RNAi. (B) Copper-inducible stable were then either left untreated or treated with copper, as indicated. Whole-cell S2 cell lines were generated expressing either 3ϫ FLAG-tagged TAF6 NTR or lysates were then immunoblotted for the TFIID subunits indicated on the left. 3ϫ FLAG-tagged TAF6 CTR. These cells were treated with dsRNA against TAF6, ␤ -Tubulin was included as a loading control. (C) The M2 anti-FLAG monoclonal and copper and nuclear extracts were prepared. The M2 anti-FLAG antibody antibody was used to immunoprecipitate (IP) 3ϫ FLAG-tagged TAF4 was used to precipitate truncated TAF6-containing complexes, and Western BIOCHEMISTRY CTR-containing complexes from nuclear extracts prepared from either WT con- blotting with antibodies against the proteins indicated at the left of the panel trol S2 cells or the TAF4 CTR cell line treated with TAF4 dsRNA and copper. The was used to detect the coprecipitating proteins. (Left) Input. (Right) Immu- input and immunoprecipitated proteins were probed with antibodies directed noprecipitated (IP) proteins. against the subunits indicated on the left. (Left) Input. (Right) Immunoprecipi- tated eluates. Next, we asked whether it is possible to actually isolate a holo-TFIID complex lacking full-length TAF4. To test this consists of a TAF4-containing core complex, which is decorated possibility, we immunoprecipitated the TAF4 CTR from nuclear on the ‘‘outside’’ with TBP, TAF1, TAF2, and TAF11. extracts prepared from untreated S2 cells and from the TAF4 CTR cell line under conditions in which the TAF4 CTR is TAF4 C-Terminal Region (CTR) Rescues TFIID Stability. Given the importance of TAF4 in forming and maintaining a stable TFIID expressed and the endogenous TAF4 is depleted. The antibody complex, we decided to investigate the functional domains of this against the TAF4 CTR efficiently precipitated TFIID lacking critical subunit. TAF4 consists of a C-terminal histone fold, a full-length TAF4, whereas TFIID failed to be precipitated from middle region called the ETO domain found in metazoans, and the control S2 nuclear extracts (Fig. 2C). These data indicate that an N-terminal metazoan-specific glutamine-rich coactivation the TAF4 CTR is necessary and sufficient for nucleating a domain (Fig. 2A) (11, 12). The C-terminal region of human holo-TFIID complex, suggesting that the N-terminal two-thirds TAF4 has previously been shown to be sufficient to incorporate of full-length TAF4, which is present in metazoan organisms, into TFIID (13). We sought to address whether the correspond- very likely evolved to fulfill a nonstructural function within ing portion of Drosophila TAF4 could not only incorporate into TFIID, possibly serving as a coactivation domain suitable for TFIID but also be sufficient to nucleate the assembly of the core contacting activators or other components of the basal machin- subcomplex and rescue the stability of the ensemble. To test this ery, as has been postulated (11). hypothesis, we generated a copper-inducible stable S2 cell line expressing an epitope-tagged C-terminal region of TAF4. These TAF6 N-Terminal Region (NTR) Is Sufficient for TFIID Stability. Unlike cells were either left untreated or treated with TAF4 dsRNA TAF4, TAF6 is conserved from yeast to man over its entire against the 5Ј region of TAF4, which is not present in the length and bears its histone fold in the N terminus. The domain transgenic TAF4 CTR construct (Fig. 2A). The untreated and of TAF6 required for integration into TFIID has not yet been TAF4 RNAi cells were then either left untreated or treated with determined. To map which region of TAF6 is required for TFIID copper to induce expression of the TAF4 CTR. This strategy stability, this subunit was divided into a histone-fold-containing allows us to largely deplete TAF4 and replace it with the TAF4 NTR and a CTR. These constructs were epitope-tagged, placed CTR. As shown in Fig. 2B, when expressed in S2 cells the TAF4 under the control of a copper-inducible promoter, and trans- CTR efficiently rescues the TAF4 RNAi-induced degradation of fected into S2 cells to generate stable cell lines expressing these TAF1, TAF5, TAF6, TAF9, and TBP, suggesting that this truncated versions. These cells were each induced with copper C-terminal region is sufficient to assemble and stabilize most, if and treated with TAF6 dsRNA directed against a region of not all, holo-TFIID. The TAF4 CTR also appears to have a TAF6 that allows the truncated form to be expressed (see Fig. dominant-negative effect, in that full-length TAF4 becomes 3A) while depleting the endogenous protein; after 3 days, the depleted when the CTR is overexpressed, most likely because of cells were harvested and nuclear extracts were prepared. Anti- competition for recognition sites in stable TFIID complexes. bodies against the epitope tag were subsequently used to immu- This finding suggests that the core TAFs may be limiting for the noprecipitate the TAF6 truncations, and the coprecipitating assembly of the complex because overexpression of the TAF4 proteins were detected by Western blotting. As shown in Fig. 3B, CTR does not result in an increase in the amount of holo-TFIID. the TAF6 NTR is able to coprecipitate the other subunits of

Wright et al. PNAS ͉ August 15, 2006 ͉ vol. 103 ͉ no. 33 ͉ 12349 Downloaded by guest on October 2, 2021 TFIID, whereas the CTR failed to do so. No full-length TAF6 was detectable in these immunoprecipitations, indicating that the TAF6 NTR is necessary and sufficient to stabilize TFIID, leaving the CTR to perform other functions, such as binding to core promoter elements and possibly serving as a coactivator target.

A TATA-less, DPE-Containing Promoter Is Dependent on TAF1 and TAF4. Having analyzed the effects of depleting individual TAFs or TBP on the integrity of holo-TFIID in Drosophila cells, we next investigated the functional consequences of some of these RNAi-mediated alterations in TFIID composition. We previously showed that although TFIID plays a role at the metallothionein A (MtnA) gene to potentiate an appropriately tuned response to heavy-metal stimuli, some of the TAFs actually appeared to be negatively regulating transcription, whereas other TAFs contributed very little to MtnA expression (8). In previous studies, a correlation was found between TFIID dependence and TATA-less core promoters (14). Also, the DPE is thought to be recognized by TFIID (15). The WT Drosophila MtnA promoter contains a strong TATA box and initiator element but no DPE, perhaps partly explaining the observed Fig. 4. Transcription from a TATA-less, DPE-containing promoter requires weak TFIID dependence. We therefore asked whether mutating TAF1 and TAF4. (A) Diagrams of the reporter constructs used to transfect S2 cells. (Upper) WT MtnA promoter driving luciferase expression. It contains a the TATA box and adding a consensus DPE to the MtnA core TATA box (TATAAAA) and an initiator (TCAGTT), but no DPE (AATCATC promoter would reveal a more central role for the TAFs in starting at position ϩ28). (Lower) WT MtnA promoter with a mutated TATA transcription activation of the MtnA gene. To test this possibility, box (GCGCCCC) and a DPE (AGACGTG) inserted at position ϩ28 from the we used either a construct containing the WT MtnA promoter transcription start site. (B) S2 cells were either left untreated (NT) or treated driving the expression of the firefly luciferase gene (MtnA-luc) with dsRNA directed against TAF1, TAF4, or TAF5 and transfected with either or a construct in which the WT MtnA TATA box was mutated the WT MtnA-luc reporter or the mMtnAϩDPE-luc reporter and actin-Renilla and a consensus DPE sequence was inserted downstream from to control for transfection efﬁciency. After 3 days, the transfected cells were the transcription start site (mMtnAϩDPE-luc) to transfect S2 treated with copper to induce transcription. Six hours later, luciferase expres- cells that were treated with dsRNA directed against individual sion was determined, normalized to Renilla expression, and plotted as fraction of untreated luminescence. TAFs. After 3 days, the cells were treated with copper to induce transcription. Luciferase expression was analyzed and normalized to a cotransfected control vector containing the actin 5C functional characterization of the MtnA promoter reveals a promoter driving expression of Renilla luciferase. As expected, specific role for both TAF1 and TAF4 in transcription activation targeted depletion of TAF1, TAF4, or TAF5 had either no effect from a TATA-less, DPE-containing promoter that is not ob- or slightly increased expression of the WT MtnA-luc promoter served with a TATA-containing, DPE-less promoter. These (Fig. 4). By contrast, depletion of TAF1 resulted in a severe results, taken together, suggest highly gene-specific and TAF decrease (five times) in luciferase expression from the ϩ subunit-selective transactions that contribute to the formation of mMtnA DPE-luc construct, indicating that the TATA-less but an ‘‘activated’’ preinitiation complex at endogenous promoters. DPE-containing core promoter architecture reveals a significant role for TFIID at this altered promoter compared with the WT Discussion MtnA promoter. Not surprisingly, targeting TAF4 by RNAi also Model of Holo-TFIID Assembly. The findings described above allow resulted in a substantial decrease in transcription from the us to build a model of TFIID assembly wherein the TAF4 CTR mutant MtnA promoter, although the effect was not as severe as participates in forming a stable core subcomplex along with the the decrease observed when TAF1 is depleted. This result was TAF6 NTR. Because the depletion of TAF5, TAF9, or TAF12 somewhat surprising because TAF4 RNAi results in the degra- by RNAi results in the depletion of many of the other TFIID dation of most of the TAFs, including TAF1, as well as a significant amount of TBP. We suspect that the ‘‘TFIID’’ activity subunits, they are also likely to be part of the stable core detected in the TAF4 RNAi cells is likely due to a residual subcomplex. We postulate that after formation of this stable core amount of TAF1 remaining in these cells (see Fig. 1A). In subcomplex, holo-TFIID is subsequently generated by the ad- contrast to the depletion of TAF4 by RNAi, the targeted dition of TBP, TAF1, TAF2, and TAF11 as peripheral subunits reduction of TAF5 levels had no detectable effect on the (Fig. 5). mMtnAϩDPE-luc promoter, which was certainly unexpected because of the previously observed similarities between the Assembly of Multiple Distinct TAF-Containing Complexes. In addition effects of knocking down TAF4 and TAF5 on overall TFIID to representing key subunits of TFIID, a subset of the TAFs are stability (Fig. 1A). This intriguing result suggests that a reduction also found in other multiprotein complexes, such as Spt-Ada- of TAF1 levels alone during TAF4 depletion is not likely to be Gin5-acetyltransferase (SAGA) and TBP-free TAF-containing solely responsible for the observed reduction in mMtnAϩDPE- complex (TFTC) (16–18). It is interesting to note that the stable luc reporter activity. Instead, these findings suggest that TAF4 core subcomplex described here, although not found in yeast may be specifically required to potentiate activation of the SAGA, is found in the human TFTC complex. Indeed, the altered promoter. Alternatively, it is possible that a TFIID characteristic trilobed architecture of holo-TFIID can also be component that we have not specifically monitored becomes seen in human TFTC (6). Furthermore, a subcomplex of human differentially depleted between the TAF4 and TAF5 knockdown TAF4, TAF5, TAF6, TAF8, TAF9, TAF10, and TAF12, which cells. These data also suggest that the stable core subcomplex contains our stable core TAF subunits, plus TAF8 and TAF10, may not be required for full activation of a TFIID-dependent also can form a trilobed structure when coexpressed in insect promoter. Our analysis of TFIID integrity coupled with the cells (4). This finding suggests that the TFIID-specific peripheral

12350 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605499103 Wright et al. Downloaded by guest on October 2, 2021 the same promoter with the TATA box mutated and a DPE inserted downstream, coupled with RNAi knockdown of specific TAFs, we found that transcription from the MtnA promoter becomes significantly more dependent on TFIID when the TATA box is mutated and a DPE is inserted. Transcription from this altered promoter depended heavily on TAF1 and TAF4, but not on TAF5, TAF6, or TAF9. Previous studies identified a conserved C-terminal region of yeast TAF1 that was required for association of TFIID with endogenous promoters (23). Perhaps this same region of Drosophila TAF1 is required for TFIID to bind efficiently to TATA-less, DPE-containing promoters but is dispensable at promoters containing a TATA box. In strong TATA-containing promoters, the TBP–TATA interaction may be sufficient for stable binding of the complex to promoter DNA. Another possibility is that one or more of the multiple enzymatic activities of TAF1 become necessary for full activation of the MtnA promoter when the TATA box is mutated and a DPE is inserted. Fig. 5. Model of TFIID assembly in vivo. TFIID consists of a stable core Although TAF4 has long been known to have coactivator subcomplex made up of TAF4, TAF5, TAF6, TAF9, and TAF12, which becomes activity, our results suggest that it may also influence, directly or decorated with TBP, TAF1, TAF2, and TAF11. Subunit stoichiometry is adapted indirectly, core promoter recognition because a change in core from Sanders et al. (7). promoter elements at the MtnA promoter led to an increased dependence on TAF4 for transcription activation. An alternative possibility is that the change from a TATA-containing, DPE-less subunits (i.e., TAF1, TAF11, and TBP) may not be necessary to core promoter to a TATA-less, DPE-containing promoter may form a trilobed architecture. The Drosophila SAGA- or TFTC- unmask alternative coactivator usage, causing the activator, like complex has not been thoroughly characterized, so it is not MTF-1, to rely more heavily on TAF4 as a coactivator. Indeed,

known whether all of the TAF components of the core subcom- activators have been known to target multiple coactivators and BIOCHEMISTRY plex are also found in these other complexes. However, TAF5, other components of the transcription machinery (24). Our in TAF9, and TAF10 were indeed found in a SAGA-like complex, vivo functional analysis suggests an intriguing role for both TAF1 together with other proteins associated with the human TFTC and TAF4 in mediating transcription from a TATA-less, DPE- complex, such as GCN5, Spt3, and Tra1 (19). We therefore containing promoter that is not apparent when using a TATA- speculate that the stable core subcomplex we have identified in containing, DPE-less promoter. this study may serve as a common precursor to both TFIID and TFTC-like complexes. For the generation of TFIID, TBP, TAF1, Materials and Methods TAF2, and TAF11 would be representative peripheral subunits Cell Culture and RNAi. S2 cells were grown at 25°C in M3ϩBPYE decorating the stable core subcomplex, as described above. To medium (Drosophila Genomics Resource Center, Bloomington, generate a TFTC-like complex, dGCN5, dSpt3, dTra1, and IN). RNAi was performed as described by using 20 ␮g of dsRNA dAda2b would serve as alternative peripheral subunits associ- per 106 cells (25). Oligonucleotide sequences used to generate ated with the same stable core subcomplex that is nucleated by RNAi constructs are available on request. TAF4 and TAF5. Extract Preparation and Immunoprecipitations. Whole-cell lysates Testis-Specific Partial Core Subcomplex. In addition to the 8–12 were prepared by harvesting cells, washing once with 1ϫ PBS, and ubiquitously expressed TAFs, recent reports have identified lysing cells in 1ϫ SDS sample buffer. Nuclear extracts were tissue-specific TAF homologs in both Drosophila and mammals. prepared as follows. Approximately 108 cells were harvested and Although only one tissue-specific TAF has been characterized in washed once with 1ϫ PBS. All subsequent steps were performed at mammals [TAF4b (20)], five tissue-specific TAF homologs have 4°C or on ice. The cells were resuspended in 2 packed-cell pellet vol been described in Drosophila (21, 22). These tissue-specific TAFs of buffer I (15 mM Hepes, pH 7.6͞10 mM KCl͞2 mM MgCl2͞0.5 are expressed in the developing spermatocytes within the testis mM EDTA͞0.5 mM EGTA͞350 mM sucrose͞1mMDTT͞0.2 mM and are termed no hitter (nht), cannonball (can), meiosis I arrest PMSF). Triton X-100 was added to 0.1%, and cells were processed (mia), spermatocyte arrest (sa), and ryan express (rye), which are with a Dounce homogenizer. Nuclei were then pelleted at 7,650 ϫ cell-type-specific homologs of TAF4, TAF5, TAF6, TAF8, and g and resuspended in 4 packed-cell pellet vol of buffer AB [15 mM TAF12, respectively. Remarkably, with the exception of TAF9, Hepes, pH 7.6͞110 mM KCl͞2 mM MgCl2͞0.5 mM EDTA͞0.5 there is a testis-specific homolog corresponding to each of the mM EGTA͞1mMDTT͞0.2 mM PMSF͞1ϫ Complete protease subunits of the stable core subcomplex described here, which inhibitor (Roche, Indianapolis, IN)]. Nuclei were lysed by adding suggests that a distinct tissue-specific core subcomplex may be 1͞10th vol of 4 M (NH4)2SO4 and then mixing for 30 min. The lysate formed in the testis. Because the testis TAFs have not been was cleared by centrifugation at 37,000 rpm in a TL-100 ultracen- characterized biochemically, it is not known whether these trifuge (Beckman, Mountain View, CA). Next, the extract was tissue-specific TAFs associate with the ubiquitously expressed precipitated with 1 vol of saturated (NH4)2SO4, centrifuged at TAFs or TBP. It will be interesting to discover whether the testis 16,000 ϫ g, and dissolved in 0.2 packed-cell pellet vol of buffer C TAFs form a stable core subcomplex similar to their ubiquitously [25 mM Hepes, pH 7.6͞150 mM KCl͞0.1 mM EDTA͞10% (vol/ expressed counterparts that serves as a platform for the gener- vol) glycerol͞1mMDTT͞0.2 mM PMSF͞1ϫ Complete protease ation of a tissue-specific holo-TFIID or TFTC, or whether they inhibitor]. The nuclear extract was desalted by using a 0.5-ml have evolved to scaffold an entirely different set of proteins. Sephadex G-25 (GE Healthcare, Uppsala, Sweden) spin column equilibrated with buffer C. Immunoprecipitations were performed Core Promoter Architecture-Specific Activity of TAF1 and TAF4. Using by adding 25 ␮l of antibody-saturated protein G–Sepharose beads two reporter constructs, one containing the WT TATA- (GE Healthcare) or M2–agarose (Sigma, St. Louis, MO) to Ϸ150 containing, DPE-less MtnA promoter and the other containing ␮g of nuclear extract diluted to 250 ␮lin1ϫ PBS and mixing

Wright et al. PNAS ͉ August 15, 2006 ͉ vol. 103 ͉ no. 33 ͉ 12351 Downloaded by guest on October 2, 2021 overnight at 4°C. The beads were then washed five times with 1ϫ site by using the same technique. The TATA mutation and DPE PBS plus 0.5 M NaCl and 0.1% Triton X-100 and eluted with 2 bead sequence are described in ref. 26. Cells were transfected in vol of 1ϫ PBS plus 0.5% N-lauroylsarcosine. triplicate by using Effectene reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Transcription from TAF Truncation Constructs and Cell Lines. The TAF4 CTR construct the reporters was induced by adding 0.5 mM copper sulfate for was made by PCR-amplifying a DNA fragment corresponding to 6 h. Luciferase assays were performed by using the Dual- residues 623–921 and ligating the fragment in frame into a vector Luciferase assay system (Promega, Madison, WI) according to containing the MtnA promoter, an N-terminal 3ϫ FLAG tag, the manufacturer’s recommendations. Data presented in Fig. 4B and the blasticidin-resistance gene. This vector was used to represent the average of three independent experiments. transfect S2 cells, and resistant cells were selected with blasticidin. The TAF6 NTR and CTR were made as described above. Antibodies. The anti-TAF1 30H9 (3), anti-TAF2 (27), anti-TAF4 The TAF6 NTR construct encodes residues 1–227, and the TAF6 3E12 (8), anti-TAF5 3D10 (28), and anti-TAF9 4H6 (29) antibodies were described previously. The anti-TAF6 25B4 antibody CTR construct encodes residues 230–592. Expression of the and the anti-TBP 3C3 antibody were screened from previously TAF truncations was induced with 0.5 mM CuSO . Oligonucle- 4 described hybridomas (11). The M2 anti-FLAG antibody otide sequences used are available on request. (Sigma) was used to detect and immunoprecipitate the TAF4 CTR, TAF6 NTR, and TAF6 CTR. Luciferase Assays. The MtnA-luc reporter was described in ref. 8. ϩ The mMtnA DPE reporter was made by mutating the TATA We thank M. Deato and Y. Fong for the critical review of the manuscript, box of the MtnA-luc construct by PCR-based mutagenesis. A M. Haggart for technical assistance, and members of the Tjian laboratory DPE was then inserted downstream from the transcription start for helpful suggestions.

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