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• W2045796723 abstract "Yeast TFIID comprises the TATA binding protein and 14 TBP-associated factors (TAFIIs), nine of which contain histone-fold domains (HFDs). The C-terminal region of the TFIID-specific yTAF4 (yTAFII48) containing the HFD shares strong sequence similarity with Drosophila(d)TAF4 (dTAFII110) and human TAF4 (hTAFII135). A structure/function analysis of yTAF4 demonstrates that the HFD, a short conserved C-terminal domain (CCTD), and the region separating them are all required for yTAF4 function. Temperature-sensitive mutations in the yTAF4 HFD α2 helix or the CCTD can be suppressed upon overexpression of yTAF12 (yTAFII68). Moreover, coexpression in Escherichia coli indicates direct yTAF4-yTAF12 heterodimerization optimally requires both the yTAF4 HFD and CCTD. The x-ray crystal structure of the orthologous hTAF4-hTAF12 histone-like heterodimer indicates that the α3 region within the predicted TAF4 HFD is unstructured and does not correspond to thebona fide α3 helix. Our functional and biochemical analysis of yTAF4, rather provides strong evidence that the HFD α3 helix of the TAF4 family lies within the CCTD. These results reveal an unexpected and novel HFD organization in which the α3 helix is separated from the α2 helix by an extended loop containing a conserved functional domain. Yeast TFIID comprises the TATA binding protein and 14 TBP-associated factors (TAFIIs), nine of which contain histone-fold domains (HFDs). The C-terminal region of the TFIID-specific yTAF4 (yTAFII48) containing the HFD shares strong sequence similarity with Drosophila(d)TAF4 (dTAFII110) and human TAF4 (hTAFII135). A structure/function analysis of yTAF4 demonstrates that the HFD, a short conserved C-terminal domain (CCTD), and the region separating them are all required for yTAF4 function. Temperature-sensitive mutations in the yTAF4 HFD α2 helix or the CCTD can be suppressed upon overexpression of yTAF12 (yTAFII68). Moreover, coexpression in Escherichia coli indicates direct yTAF4-yTAF12 heterodimerization optimally requires both the yTAF4 HFD and CCTD. The x-ray crystal structure of the orthologous hTAF4-hTAF12 histone-like heterodimer indicates that the α3 region within the predicted TAF4 HFD is unstructured and does not correspond to thebona fide α3 helix. Our functional and biochemical analysis of yTAF4, rather provides strong evidence that the HFD α3 helix of the TAF4 family lies within the CCTD. These results reveal an unexpected and novel HFD organization in which the α3 helix is separated from the α2 helix by an extended loop containing a conserved functional domain. Accurate transcription initiation at protein-coding genes by RNA polymerase II requires the assembly of a multiprotein complex around the mRNA start site (1Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Google Scholar). Transcription factor TFIID is one of the general factors involved in this process. TFIID comprises the TATA binding protein (TBP), 1The abbreviations used are: TBP, TATA-binding protein; TAFII, TBP-associated factor; HFD, histone-fold domain; TS, temperature sensitive; CCTD, conserved C-terminal domain; y, yeast; h, human.1The abbreviations used are: TBP, TATA-binding protein; TAFII, TBP-associated factor; HFD, histone-fold domain; TS, temperature sensitive; CCTD, conserved C-terminal domain; y, yeast; h, human.responsible for specific binding to the TATA element found in many RNA polymerase II promoters, and a set of TBP-associated factors (TAFIIs) (2Bell B. Tora L. Exp. Cell Res. 1999; 246: 11-19Google Scholar, 3Gangloff Y. Romier C. Thuault S. Werten S. Davidson I. Trends Biochem. Sci. 2001; 26: 250-257Google Scholar). A subset of TAFIIs are present not only in TFIID but also in the SAGA, PCAF, STAGA, and TFTC complexes that lack TBP but are involved in RNA polymerase II transcription (4Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Google Scholar, 5Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Google Scholar, 6Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates J.R. Workman J.L. Cell. 1998; 94: 45-53Google Scholar, 7Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Google Scholar, 8Martinez E. Kundu T.K. Fu J. Roeder R.G. J. Biol. Chem. 1998; 273: 23781-23785Google Scholar). A subset of TAFIIs are also found in a macromolecular complex containing Drosophilapolycomb group proteins (9Saurin A.J. Shao Z. Erdjument-Bromage H. Tempst P. Kingston R.E. Nature. 2001; 412: 655-660Google Scholar). The function of TAFIIs has been studied in several organisms. In yeast, the genes encoding all of the TFIID components, with the exception of yTAF14, are essential for viability. Genetic studies have shown that TAFIIs play an important role in transcriptional regulation of many genes (10Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Google Scholar). Temperature-sensitive (TS) mutations in yTAF1 and yTAF5 provoke cell cycle arrest where the expression of only a small number of genes is affected (11Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Google Scholar, 12Apone L.M. Virbasius C.M. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Google Scholar). In contrast, TS mutations in yTAF6, yTAF9, yTAF10, and yTAF12 or the TFIID-specific yTAF11 lead to a dramatic decrease in overall transcription levels (13Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Google Scholar, 14Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Google Scholar, 15Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Google Scholar, 16Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Google Scholar, 17Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Google Scholar, 18Sanders S.L. Klebanow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Google Scholar). Studies in Drosophila have shown that TAFIIs are involved in the transcriptional regulation of genes during development (19Soldatov A. Nabirochkina E. Georgieva S. Belenkaja T. Georgiev P. Mol. Cell. Biol. 1999; 19: 3769-3778Google Scholar, 20Zhou J. Zwicker J. Szymanski P. Levine M. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13483-13488Google Scholar, 21Hernandez-Hernandez A. Ferrus A. Mol. Cell. Biol. 2001; 21: 614-623Google Scholar, 22Wassarman D.A. Aoyagi N. Pile L.A. Schlag E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1154-1159Google Scholar). In mammalian cells, TAFIIs again seem to be involved in cell cycle control and are essential for the viability of proliferating cells (23Metzger D. Scheer E. Soldatov A. Tora L. EMBO J. 1999; 18: 4823-4834Google Scholar, 24Chen Z. Manley J.L. Mol. Cell. Biol. 2000; 20: 5064-5076Google Scholar, 25Sekiguchi T. Nohiro Y. Nakamura Y. Hisamoto N. Nishimoto T. Mol. Cell. Biol. 1991; 11: 3317-3325Google Scholar, 26Perletti L. Kopf E. Carre L. Davidson I. BMC Mol. Biol. 2001; 2 (www.biomedcentral.com/1471-2199/2/4)Google Scholar). The histone-fold domain (HFD) plays an important role in the structural organization of TFIID. Sequence comparisons and structural studies indicated that TAF6 and TAF9 contain HFDs similar to those of core histones H4 and H3, which interact to form an H3-H4-like heterotetramer (27Xie X. Kokubo T. Cohen S.L. Mirza U.A. Hoffmann A. Chait B.T. Roeder R.G. Nakatani Y. Burley S.K. Nature. 1996; 380: 316-322Google Scholar, 28Hoffmann A. Chiang C.M. Oelgeschlager T. Xie X. Burley S.K. Nakatani Y. Roeder R.G. Nature. 1996; 380: 356-359Google Scholar). hTAF4 and hTAF12 heterodimerize via HFDs similar to those of H2A and H2B, respectively (29Gangloff Y.G. Werten S. Romier C. Carre L. Poch O. Moras D. Davidson I. Mol. Cell. Biol. 2000; 20: 340-351Google Scholar). It has been suggested that the TAF6-TAF9 heterotetramer may associate with the TAF4-TAF12 heterodimer to form an octameric substructure within TFIID (28Hoffmann A. Chiang C.M. Oelgeschlager T. Xie X. Burley S.K. Nakatani Y. Roeder R.G. Nature. 1996; 380: 356-359Google Scholar, 29Gangloff Y.G. Werten S. Romier C. Carre L. Poch O. Moras D. Davidson I. Mol. Cell. Biol. 2000; 20: 340-351Google Scholar). The equivalent yTAFIIs have been shown to assemble in vitro to form a macromolecular complex with stoichiometry (yTAF6-yTAF9)2-2(yTAF4-yTAF12) consistent with that of a histone-like octamer (30Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Google Scholar). The possible existence of such a structurein vivo is supported by high copy suppressor genetic interactions in yeast (15Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Google Scholar, 30Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Google Scholar) and the finding that these four yTAFIIs colocalize within the same subdomain of native yeast TFIID (31Leurent C. Sanders S. Ruhlman C. Mallouh V. Weil P.A. Kirschner D.B. Tora L. Schultz P. EMBO J. 2002; 21: 3424-3433Google Scholar). X-ray crystallography has shown that hTAF11 and hTAF13 heterodimerize via HFDs, and this interaction is in agreement with a correspondingin vivo genetic interaction between their yeast orthologues (14Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Google Scholar, 32Birck C. Poch O. Romier C. Ruff M. Mengus G. Lavigne A.C. Davidson I. Moras D. Cell. 1998; 94: 239-249Google Scholar). Biochemical studies have also indicated that yTAF3 and yTAF8 contain HFDs that heterodimerize with a HFD in yTAF10 (33Gangloff Y.G. Sanders S.L. Romier C. Kirschner D. Weil P.A. Tora L. Davidson I. Mol. Cell. Biol. 2001; 21: 1841-1853Google Scholar). A similar result was obtained in studies of their metazoan orthologues (21Hernandez-Hernandez A. Ferrus A. Mol. Cell. Biol. 2001; 21: 614-623Google Scholar, 34Gangloff Y.G. Pointud J.C. Thuault S. Carre L. Romier C. Muratoglu S. Brand M. Tora L. Couderc J.L. Davidson I. Mol. Cell. Biol. 2001; 21: 5109-5121Google Scholar). The existence of these heterodimers in native yeast TFIID is supported by immunoelectron microscopy, which shows colocalization of these proteins (31Leurent C. Sanders S. Ruhlman C. Mallouh V. Weil P.A. Kirschner D.B. Tora L. Schultz P. EMBO J. 2002; 21: 3424-3433Google Scholar). Hence, nine yTAFIIs contain HFDs that specifically heterodimerize to form five histone-like pairs (for review, see Ref. 3Gangloff Y. Romier C. Thuault S. Werten S. Davidson I. Trends Biochem. Sci. 2001; 26: 250-257Google Scholar). TSG2/yTAF4 shows significant sequence similarity to the metazoan TAF4 and TAF4b proteins (35Reese J.C. Zhang Z. Kurpad H. J. Biol. Chem. 2000; 275: 17391-17398Google Scholar, 36Sanders S.L. Weil P.A. J. Biol. Chem. 2000; 275: 13895-13900Google Scholar). Overexpression of yTAF4 suppresses the TS phenotype of a mutation in yTAF12, and these TAFIIs interact physically with one another (30Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Google Scholar, 35Reese J.C. Zhang Z. Kurpad H. J. Biol. Chem. 2000; 275: 17391-17398Google Scholar), although the precise domains required for their interaction have not been described. Although the yTAF4 HFD shows sequence similarity to those of its metazoan counterparts, additional shared regions of similarity exist in the C terminus. To better understand the function of each region and their contribution to heterodimerization with yTAF12, we have made a detailed structure-function analysis of yTAF4. We show that the HFD is essential, but not sufficient, for yTAF4 function, which additionally requires the short highly conserved C-terminal domain (CCTD) and the intervening linker region. We demonstrate a strong genetic interaction between the yTAF4 CCTD and yTAF12 in vivo, and coexpression in Escherichia coli shows that the CCTD also contributes to direct yTAF4-yTAF12 heterodimerization in vitro. Together with the structure of the orthologous hTAF4-hTAF12 heterodimer (48Werten S. Mitschler A. Romier C. Gangloff Y.-G. Thuault S. Davidson I. Moras D. J. Biol. Chem. 2002; 277: 45502-45509Google Scholar), our results provide evidence that the α3 helix of the TAF4 HFD is located within the CCTD and reveal a novel HFD organization in which the α2 helix is separated from the α3 helix by an extended L2 loop containing a functional domain. The yeast strains used in this study are: YSLS46 (MATaleu2Δ0 ura3Δ0 his3Δ1 met15Δ0 KANΔtaf4 [pRS416ADH-TAF4]), used for plasmid shuffling of TAF4, was derived from YSLS40 (36Sanders S.L. Weil P.A. J. Biol. Chem. 2000; 275: 13895-13900Google Scholar) by sporulation and tetrad dissection; YSLS46/4 (MATaleu2Δ0 ura3Δ0 his3Δ1 met15Δ0 KANΔtaf4 [pAS3-TAF4]); YSLS46/4m4(MATaleu2Δ0 ura3Δ0 his3Δ1 met15Δ0 KANΔtaf4 [pAS3-TAF4(M219P)]); YSLS46/4 (186–388) (MATaleu2Δ0 ura3Δ0 his3Δ1 met15Δ0 KANΔtaf4 [pAS3-TAF4 (186–388)]); and YSLS46/4m5(MATaleu2Δ0 ura3Δ0 his3Δ1 met15Δ0 KANΔtaf4 [pAS3-TAF4 (186–388)(R362A, D363A)]). All yeast and bacterial expression vectors were constructed by PCR using oligonucleotide primers with the appropriate restriction sites (details available on request). All constructs were verified by restriction enzyme analysis and automated sequencing. For complementation experiments, wild-type or mutated TAFIIs were cloned in pAS3 with a Leu marker as previously described (33Gangloff Y.G. Sanders S.L. Romier C. Kirschner D. Weil P.A. Tora L. Davidson I. Mol. Cell. Biol. 2001; 21: 1841-1853Google Scholar). A low copy expression plasmid containing the native yTAF4 promoter was constructed by introducing a cassette comprising 500 bp of sequence upstream of the yTAF4 ATG into the pRS415 vector (37Mumberg D. Muller R. Funk M. Gene. 1995; 156: 119-122Google Scholar) (pRS415taf4p). Downstream of the ATG, a sequence encoding the FLAG epitope was inserted followed by restriction sites allowing the in-frame cloning of the wild-type or mutated yTAF4 derivatives. All strains were transformed by the LiAc technique. For complementation assays, the indicated plasmids were transformed and the wild-type yTAF4 URA3 plasmid was shuffled out by two passes on media containing 5-fluororotic acid as previously described (33Gangloff Y.G. Sanders S.L. Romier C. Kirschner D. Weil P.A. Tora L. Davidson I. Mol. Cell. Biol. 2001; 21: 1841-1853Google Scholar). For suppression of the yTAF4 mutant strains, cells were transformed with high-copy-number vectors with URA3 or HIS3 markers expressing the indicated yTAFII. Transformed yeast were then grown at the indicated temperatures. In all experiments, cultures were grown in yeast extract-peptone dextrose medium. Auxotrophic selections were performed in the appropriate synthetic dextrose medium. Total RNA was isolated from strains grown at the indicated temperatures for the indicated times as described above. In each case the cultures were immediately cooled, pelleted for 5 min at 4000 rpm, washed twice in cold H2O, repelleted, and frozen at −80 °C. Total RNA was isolated by the hot phenol/chloroform method and subsequently quantified (38Schroeder S.C. Weil P.A. Nucleic Acids Res. 1998; 26: 4186-4195Google Scholar). Analysis of poly(A)+ RNA was performed on slot blots by hybridization with an oligo(dT) probe and quantified by Phosphorimager analysis (Amersham Biosciences) as previously described (18Sanders S.L. Klebanow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Google Scholar). All samples were analyzed in triplicate, and the average values ± standard error are shown. Coexpression inE. coli was performed as previously described (29Gangloff Y.G. Werten S. Romier C. Carre L. Poch O. Moras D. Davidson I. Mol. Cell. Biol. 2000; 20: 340-351Google Scholar). The derivative of yTAF12 was expressed as a histidine-tagged fusion protein in pET-15b. Native untagged derivatives of yTAF4 were expressed from a modified version of the vector pACYC184 (PerkinElmer Life Sciences) (39Fribourg S. Romier C. Werten S. Gangloff Y.G. Poterszman A. Moras D. J. Mol. Biol. 2001; 306: 363-373Google Scholar). Comparison of the amino acid sequence of yTAF4 to those of its metazoan orthologues hTAF4, dTAF4, and hTAF4b indicates strong similarity in the C-terminal domain of the proteins. This region can be divided into three sub-domains, the HFD, a short strongly conserved domain at the extreme C terminus (Conserved C-Terminal Domain, CCTD), and an intervening linker region (Fig.1). To address the requirement of these domains for function in vivo, a set of deletion mutants were constructed (Fig. 2 A) and tested for their ability to rescue the growth of the yeasttaf4Δ null strain.Figure 2Schematic description of yTAF4 deletion mutants. A, yeast TAF4 is schematically depicted. Non-conserved regions are shown as a thin black line, the linker region between the HFD and the CCTD as athick black line, the α-helices of the HFD are indicated as dark blue boxes, and the CCTD by alight blue box. In yTAF4c1–3, the hTAF4 CCTD and/or α3 helices are differentiated from their yTAF4 counterparts bycoloring and in yTAF4c2 and c3 the hTAF4 linker region is shown by an open box. The presence of substituted amino acids is indicated with an asterisk. The amino acid coordinates of the deletion end points, the HFD, and the CCTD are indicated. The ability of each construct to complement the growth of the taf4Δ strain when expressed from a high copy vector or a low copy vector under the control of the TAF4 promoter is indicated to the right. TS indicates temperature sensitive growth.B, the location of amino acid substitutions within the HFD and CCTD is indicated. The wild-type amino acid sequence is indicated on the first line and the mutated residues are shown below.View Large Image Figure ViewerDownload (PPT) In plasmid shuffle experiments, full-length wild-type yTAF4 rescued the growth of the taf4Δ null strain (Fig.3 A, sector 1, summarized in Fig. 2 A), although no rescue was seen with the expression vector alone (Fig. 3 A, sector 2). Growth was also seen when the non-conserved N-terminal region upstream of the HFD was deleted (Fig. 3 A, sector 4 and Fig. 3 B, sector 1; yTAF4 (186–388)). Similarly, deletion of the α1 helix and the α1 helix and loop L1 of the HFD did not abolish yTAF4 function (Fig. 3 B, sectors 2 and 3; yTAF4 (203–388) and yTAF4 (213–388)), which was lost upon additional deletion of the N-terminal half of the α2 helix (Fig. 3 B, sector 4; yTAF4 (231–388), summarized in Fig. 2 A). In agreement with the observation that the α1 helix could be deleted without abolishing function, a mutation within this helix also did not affect growth (Fig. 2 B,yTAF4m3 and Fig. 3 A, sector 8). In contrast, mutations in the α2 helix either completely abolished function (Fig. 2 B, yTAF4m1, m2 and Fig. 3 A, sectors 6 and 7) or led to a TS phenotype (yTAF4m4, Fig. 3 C, sector 1). Hence, the central α2 helix of the HFD is critical for yTAF4 function. Surprisingly however, mutation (yTAF4 (186–388)m6, Fig. 2,A and B) or deletion (yTAF4Δ (251–261), Fig.2 A) of the α3 helix did not affect the ability to efficiently complement (Fig. 3 E, sectors 2 and3, summarized in Fig. 2 A). Although the above results show that yTAF4 HFD is required for function, no complementation is seen with the construct yTAF4 (186–280) containing the HFD alone (Fig. 3 A, sector 3), indicating that the linker region and/or the CCTD are also required. Indeed, deletion of the CCTD completely abolished function (Fig. 3 A, sector 5; yTAF4 (1–356)). Mutation of two highly conserved residues within the yTAF4 CCTD led to a TS phenotype when introduced into the (186–388) deletion mutant (Fig.3 C, sector 2; mutation yTAF4 (186–388)m5). In addition, deletion of the linker region between the presumed α3 helix of the HFD and the CCTD led to a loss of function (yTAF4Δ (288–329), summarized in Fig. 2 A). Similarly, deletion of the entire linker region and the presumed α3 helix (yTAF4Δ (251–358), Fig.2 A), which brings the CCTD into close proximity to the α2 helix (see also below), also led to a loss of function (Fig.3 E, sector 4). Interestingly, neither the hTAF4 CCTD alone, nor the combination of the hTAF4 CCTD and linker regions can substitute for the equivalent yTAF4 regions and support yeast growth (yTAF4c1 and yTAF4c2, Fig. 3 B, sector 5and Fig. 3 E, sector 5). Finally, exchanging the yTAF4 linker region by that of hTAF4 did not permit complementation (yTAF4c3, Fig. 3 E, sector 6). Some of the above complementation results were performed with a high copy expression vector containing the strong alcohol dehydrogenase promoter. To exclude the possibility that some of the derivatives may complement due to increased expression, those bearing deletion and point mutations for which complementation was observed were also expressed under the control of the natural yTAF4 promoter sequence in a low copy vector (as described under “Experimental Procedures”). As observed above, wild-type yTAF4 and the (186–388) derivative containing only the conserved regions both supported growth (Fig.3 D, sector 1 and 7). When expressed from this vector, deletion or mutation of the α1 helix did not affect complementation (summarized in Fig. 2 A and 3 F,sector 3). However, deletion of the α1 helix and L1 loop of the HFD led to impaired growth at 28 °C and a TS phenotype (Fig.3 D, sector 6 and Fig. 3 F, sector 4). Similar to the high copy vector, complementation and a TS phenotype were observed with yTAF4 (186–388)m5 (Fig. 3 D,sector 3 and Fig. 3 F, sector 5). No TS phenotype was, however, observed when the mutation was introduced in the context of the full-length protein (Fig. 3 F, yTAF4 (1–388)m5). The only significant difference with the low copy vector was observed with yTAF4 (1–388)m4 containing a mutation in the α2 helix. When expressed from a high copy expression vector, this derivative complemented at 28 °C and led to a TS phenotype (summarized in Fig. 2 A), whereas when expressed from the low copy vector, no complementation was seen (Fig. 3 D,sectors 4 and 5). Taken together, the above results show that the α2 helix of the HFD is critical for function, but that the α1 helix is not absolutely required. The CCTD and the intervening region are also essential domains, because their deletion results in a loss of function even when expressed from a high copy vector. Therefore, the evolutionarily conserved regions of yTAF4 all contribute to function in vivo. As shown above, we have isolated mutations in the CCTD or in the HFD with a TS phenotype. We overexpressed the other histone-like yTAFIIs in strains harboring each mutation in order to test their ability to suppress this TS phenotype and hence to interact genetically with yTAF4. With the strain harboring the yTAF4 (1–388)m4 mutation in the HFD, overexpression of wild-type yTAF4 efficiently rescued growth as expected (summarized in Fig.4 A). However, of the other yTAFIIs tested, only the expression of yTAF12 was able to rescue the growth at the non-permissive temperature, whereas all strains grew at the permissive temperature (summarized in Fig.4 A). A similar result was obtained using mutation yTAF4 (186–388)m5 in the CCTD (Fig. 4, A and B). These results show strong and selective genetic interactions between yTAF12 and the HFD and CCTD of yTAF4. Direct physical interactions between yTAF4 and yTAF12 have previously been observed (30Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Google Scholar, 35Reese J.C. Zhang Z. Kurpad H. J. Biol. Chem. 2000; 275: 17391-17398Google Scholar), however, the domains of yTAF4 required for this have not been determined. To address this question, yTAF4 deletion mutants were tested for their ability to heterodimerize with a histidine-tagged derivative of the yTAF12 HFD when coexpressed in E. coli, a powerful method for investigating heterodimerization of TAFIIs (33Gangloff Y.G. Sanders S.L. Romier C. Kirschner D. Weil P.A. Tora L. Davidson I. Mol. Cell. Biol. 2001; 21: 1841-1853Google Scholar, 39Fribourg S. Romier C. Werten S. Gangloff Y.G. Poterszman A. Moras D. J. Mol. Biol. 2001; 306: 363-373Google Scholar). After coexpression, the bacterial extracts were separated on cobalt agarose beads, and the retained proteins were analyzed by electrophoresis and staining with Coomassie Brilliant Blue. When coexpressed with the histidine-tagged yTAF12 (409–491), efficient heterodimerization was observed with full-length wild-type yTAF4, which was retained on the beads only in the presence of yTAF12 (Fig.5 B, lanes 2 and 3). As previously observed with other HFDs, coexpression and heterodimerization also led to an increase in solubility of the yTAF12 HFD (lanes 1 and 3). Heterodimerization was also observed with yTAF4 (144–388) in which the N-terminal region has been deleted (lane 5). In contrast, only small amounts of heterodimer were seen with the construct yTAF4 (144–280) in which the conserved C-terminal sequence was deleted (lane 7). We also tested the ability of several other constructs equivalent to those tested in the complementation experiments for their ability to heterodimerize. Deletion of the linker region between the presumed α3 helix and the CCTD resulted in efficient production of the heterodimer (yTAF4 (144–388)Δ1,lane 9), showing that this domain is not required for heterodimerization. Moreover, a deletion of the presumed α3 helix and linker region, leaving only a short loop region and bringing the CCTD in close proximity to the α2 helix also resulted in highly efficient heterodimer production (yTAF4 (144–388)Δ2, lane 11). These results show that deletion of the CCTD, but not the linker region strongly impairs heterodimer production while bringing the CCTD close to the α2 helix results in highly efficient heterodimer production. Heterodimerization with yTAF12 was strongly impaired by mutations m1, m2, and m4 within the α2 helix of the yTAF4 HFD (Fig. 5 C,lanes 1–3). Similarly, mutation m5 in the CCTD and replacement of the yTAF4 CCTD by that of hTAF4 (yTAF4 c1) also impaired heterodimerization compared with that seen with the equivalent wild-type construction (compare lanes 4,5, and 6). This observation shows that the CCTD plays a direct role in heterodimerization with yTAF12. Here we describe two novel TS mutants in yTAF4. To determine the effect of each mutation on overall RNA polymerase II transcription, the levels of poly(A)+ RNA were examined in each strain following the shift to 37 °C. As a control in these experiments, we included the previously characterized yTAF10 mutant strain (G210E) bearing a mutation in the HFD. In this strain, bulk poly(A)+ levels are strongly reduced (18Sanders S.L. Klebanow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Google Scholar). Total RNA was prepared from each yeast strain grown at 28 °C, and after growth at 37 °C for between 15 min and 2 h, poly(A)+ levels were quantified by hybridization with an oligo(dT) probe and PhosphorImaging. As previously described, the level of poly(A)+ mRNA fell off rapidly to less than 25% of the control value after shift of the yTAF10(G210E) mutant strain to 37 °C (Fig.6). Mutation of the CCTD also led to a strong reduction in poly(A)+ mRNA levels, which decreased to around 30% (Fig. 6). In contrast, the mutation in the α2 helix of the HFD had a less dramatic effect (Fig. 6). These results show that the integrity of the CCTD is rather broadly required for transcription. In this report, we show that the conserved region of yTAF4 is required for yeast viability. We demonstrate that the CCTD of yTAF4 interacts genetically with yTAF12, is broadly required for transcription in vivo, and is required for optimal heterodimerization with yTAF12 in vitro. Together with the structure of the human TAF4-TAF12 heterodimer in the accompanying paper (48Werten S. Mitschler A. Romier C. Gangloff Y.-G. Thuault S. Davidson I. Moras D. J. Biol. Chem. 2002; 277: 45502-45509Google Scholar), our results provide evidence that the HFD of the TAF4 family has an unexpected organization where the α3 helix is located within the CCTD. We have previously proposed that TAF4 contains a HFD that mediates heterodimerization with that of TAF12 (29Gangloff Y.G. Werten S. Romier C. Carre L. Poch O. Moras D. Davidson I. Mol. Cell. Biol. 2000; 20: 340-351Google Scholar). Analysis of yTAF4 shows that the HFD is essential for functionin vivo. Mutations in the α2 helix of the HFD abolish function or lead to a TS phenotype. These mutations also impair heterodimerization in in vitro coexpression experiments. In parallel with our study, the x-ray crystal structure of the hTAF4-hTAF12 histone-like pair was determined (48Werten S. Mitschler A. Romier C. Gangloff Y.-G. Thuault S. Davidson I. Moras D. J. Biol. Chem. 2002; 277: 45502-45509Google Scholar), indicating that hTAF12 adopts a canonical histone fold and showing the presence of an α1-L1-α2 in hTAF4. From the structure, it can be seen that the above mutations are indeed located within the heterodimerization interface. The correlation with complementation indicates that yTAF4-yTAF12 heterodimerization is essential for function in vivo. It is, however, surprising that deletion or mutation of the α1 helix that contributes significantly to the heterodimerization interface seen in the structure does not have a more radical effect. In contrast to yTAF12, yTAF10, or yTAF3 (16Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Google Scholar, 17Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Google Scholar, 18Sanders S.L. Klebanow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Google Scholar, 33Gangloff Y.G. Sanders S.L. Romier C. Kirschner D. Weil P.A. Tora L. Davidson I. Mol. Cell. Biol. 2001; 21: 1841-1853Google Scholar, 40Kirschner D. Saunders S.L. Vom Bauer E. Gangloff Y.-G. Davidson I. Weil P.A. Tora L. Mol. Cel. Biol. 2001; 22: 3178-3193Google Scholar), the previously ascribed yTAF4 HFD is not by itself sufficient for growth, which requires the entire conserved C-terminal region. Deletion of the CCTD results in a loss of function. Impaired heterodimerization with yTAF12 is also seen when the CCTD is deleted and with mutant m5 in the CCTD which shows a TS phenotype in vivo. The yTAF4c1 in which the yTAF4 CCTD is replaced by that of hTAF4 does not complementin vivo and displays impaired heterodimerization. The hTAF4 and yTAF4 CCTDs are closely related but not identical. There are several amino acid substitutions and an additional amino acid in the human sequence. Any one or several of these evolutionary changes may hinder heterodimerization and any other function(s) of the CCTD and lead to a loss of the ability to complement. In contrast to the above, deletion of the linker region does not affect heterodimerization but abolishes function in vivo. Hence, this loss of function cannot be ascribed to a loss of heterodimerization with yTAF12 suggesting that the conserved linker region plays a distinct role in vivo perhaps interacting with other TFIID subunits or mediating interactions of TFIID with other cellular proteins. As for the CCTD, species specificity is also observed, as the hTAF4 linker region cannot substitute for the equivalent region of yTAF4. Mutation of the yTAF4 CCTD has a dramatic effect on bulk mRNA levels upon shift to 37 °C, indicating that this domain is rather broadly required for transcription. This mutation is more detrimental than the m4 mutation in the yTAF4 HFD. However, it is important to note that the m5 mutation is present in the context of a deletion of the N-terminal region. In fact, the m5 mutation has an effect only in this context, but not in the context of the full-length protein. The fact that deletion of the N-terminal region alone has no effect suggests that there is a sequence within the N-terminal region, perhaps an additional αN helix as found in histone H3 or hTAF11 (32Birck C. Poch O. Romier C. Ruff M. Mengus G. Lavigne A.C. Davidson I. Moras D. Cell. 1998; 94: 239-249Google Scholar), which plays a partially redundant role with the CCTD and can suppress the effect of mutation, but not deletion of the CCTD. Nevertheless, our results suggest that distinct mutations within yTAF4 can have differential effects on gene expression, a conclusion confirmed by gene-specific effects of these yTAF4 mutations. 2S. T. and I. D., manuscript in preparation. The conclusion that the full spectrum of activity of a given TAF cannot be derived from the study of a single mutated allele has been underscored by analysis of distinct mutations in yTAF10 or yTAF5, which have dramatically different effects both on gene transcription and cell-cycle progression (40Kirschner D. Saunders S.L. Vom Bauer E. Gangloff Y.-G. Davidson I. Weil P.A. Tora L. Mol. Cel. Biol. 2001; 22: 3178-3193Google Scholar, 41Kirchner J. Sanders S.L. Klebanow E. Weil P.A. Mol. Cell. Biol. 2001; 21: 6668-6680Google Scholar, 42Durso R.J. Fisher A.K. Albright-Frey T.J. Reese J.C. Mol. Cell. Biol. 2001; 21: 7331-7344Google Scholar). A mutation in the yTAF4 α2 helix (M219P) leads to a TS phenotype. The introduction of proline residues in the α2 helix of several other yTAFIIs has also previously been shown to generate TS phenotypes and has proven useful for examining genetic interactions in yeast (15Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Google Scholar, 33Gangloff Y.G. Sanders S.L. Romier C. Kirschner D. Weil P.A. Tora L. Davidson I. Mol. Cell. Biol. 2001; 21: 1841-1853Google Scholar). Using the TS mutation in the yTAF4 α2 helix, we find a strong and selective genetic interaction with yTAF12, whose overexpression rescues the growth at restrictive temperature. These results are complementary to those of Reese et al. (35Reese J.C. Zhang Z. Kurpad H. J. Biol. Chem. 2000; 275: 17391-17398Google Scholar), who have shown that overexpression of yTAF4 could rescue the TS phenotype of a yTAF12 TS mutant containing a partial deletion of the α3 helix. Hence, there is a reciprocal genetic interaction in vivo between the HFDs of yTAF4 and yTAF12, suggesting that this pair exists within the native TFIID. The existence of this pair in vivo is also supported by the colocalization of yTAF4 and yTAF12 observed in immunoelectron microscopy within native yeast TFIID (31Leurent C. Sanders S. Ruhlman C. Mallouh V. Weil P.A. Kirschner D.B. Tora L. Schultz P. EMBO J. 2002; 21: 3424-3433Google Scholar). Our results extend those cited above by demonstrating a further selective genetic interaction between yTAF12 and the yTAF4 CCTD. While this work was in progress, Selleck et al. (30Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Google Scholar) reported that a TS mutation in the HFD of yTAF4 could be suppressed by overexpression of yTAF12. A reevaluation of this observation (43Selleck W. Howley R. Fang Q. Podolny V. Fried M. Buratowski S. Tan S. Nat. Struct. Biol. 2002; 9: 231Google Scholar) indicates that the mutation conferring temperature sensitivity to this yTAF4 derivative is L365P, which does not lie within the presumed HFD region, but within the CCTD (see Fig. 1 B). This observation provides independent corroboration of a genetic interaction between yTAF12 and the yTAF4 CCTD. Given this strong genetic interaction, it was not surprising to find that yTAF4 directly heterodimerizes with yTAF12 when coexpressed inE. coli. These results extend those of Reese et al. (35Reese J.C. Zhang Z. Kurpad H. J. Biol. Chem. 2000; 275: 17391-17398Google Scholar),who showed an interaction between yTAF4 and yTAF12 in glutathione S-transferase-pulldown experiments. Sellecket al. (30Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Google Scholar) have reported heterodimerization of full-length yTAF4 and an N-terminally deleted derivative of yTAF12 upon coexpression in E. coli. Here we show that heterodimerization requires only the yTAF12 HFD, but that for yTAF4 the CCTD is additionally required. Full-length yTAF4 or only the conserved region heterodimerize efficiently with the HFD of yTAF12. In contrast, with only the yTAF4 HFD greatly reduced production of the heterodimer was obtained. Mutations in the CCTD impaired heterodimerization, whereas deletion of the presumed α3 helix as well as the linker region did not impair heterodimer production, but rather led to an optimal yield. Hence, when the CCTD is deleted or mutated, but not when the intervening linker region including the putative α3 helix is deleted, a reduction in heterodimer production is observed. This observation is in agreement with genetic suppressor results in vivo and shows that the CCTD directly contributes to efficient heterodimerization with yTAF12. With the determination of the hTAF4-hTAF12 structure, it was noted that the amino acids encoding the presumed hTAF4 α3 helix were present within the crystal, but were disorganized, suggesting that this region probably does not correspond to the α3 helix. Therefore, either hTAF4 belongs to a novel class of HFD which does not contain an α3 helix or an α3 helix is located elsewhere within the C-terminal domain. Several observations made during the analysis of yTAF4 suggest that an α3 helix is located within the CCTD. The CCTD is highly conserved in the TAF4 family. Within the CCTD, a region of strong similarity to other experimentally determined HFD α3 helices can be observed with a conserved D(L/V/M/I) pair (Fig. 1 B). This Asp residue, which is mutated in our m5 derivative plays an important role in several HFDs by forming an intramolecular bond with an arginine residue in the L2 loop, whereas the L365P mutation described by Sellecket al. (30Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Google Scholar) is located immediately following the DL pair (see asterisks in Fig. 1 B). Hence, the mutations conferring a TS phenotype and impairing heterodimerization lie within a region showing high sequence similarity to known α3 helices (Fig.1 B). The suppression of these mutations upon overexpression of yTAF12 and the impaired heterodimerization seen in vitroprovides strong evidence that this is indeed the α3 helix of the TAF4 HFD. In contrast, deletion or mutation of the previously ascribed α3 helix has no effect on yTAF4 function in vivo and does not affect heterodimerization in vitro. Together, these observations point to the possible presence of an α3 helix within the CCTD, showing that the TAF4 HFD has an unexpected organization with an extended linker between the α2 and α3 helices. The presence of an extended L2 loop within the HFD of yTAF10 and yTAF11 has previously been noted (30Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Google Scholar, 33Gangloff Y.G. Sanders S.L. Romier C. Kirschner D. Weil P.A. Tora L. Davidson I. Mol. Cell. Biol. 2001; 21: 1841-1853Google Scholar, 40Kirschner D. Saunders S.L. Vom Bauer E. Gangloff Y.-G. Davidson I. Weil P.A. Tora L. Mol. Cel. Biol. 2001; 22: 3178-3193Google Scholar). However, in contrast to yTAF4, these loops are not present in the metazoan orthologues and can be deleted in the yeast proteins without loss of function. In the TAF4 family, the extended loop region is conserved and cannot be deleted without loss of function. Hence, the yTAF4 HFD has a novel organization in which a functional domain is located within the L2 loop. In addition to mediating heterodimerization with TAF12, the conserved region of TAF4 also mediates interaction with the Q-rich region of the cAMP-response element-binding protein transcriptional activator (44Asahara H. Santoso B. Guzman E. Du K. Cole P.A. Davidson I. Montminy M. Mol. Cell. Biol. 2001; 21: 7892-7900Google Scholar). It has been suggested that proteins harboring poly(Q) expansions interact with the conserved region of TAF4 and provoke the development of neurodegenerative diseases by sequestration of TAF4 and interference with cAMP-response element-binding protein function (45Shimohata T. Nakajima T. Yamada M. Uchida C. Onodera O. Naruse S. Kimura T. Koide R. Nozaki K. Sano Y. Ishiguro H. Sakoe K. Ooshima T. Sato A. Ikeuchi T. Oyake M. Sato T. Aoyagi Y. Hozumi I. Nagatsu T. Takiyama Y. Nishizawa M. Goto J. Kanazawa I. Davidson I. Tanese N. Nat. Genet. 2000; 26: 29-36Google Scholar). Moreover, TAF4 interacts with the adenovirus E1A protein, and this interaction absolutely requires the CCTD (46Mazzarelli J.M. Mengus G. Davidson I. Ricciardi R.P. J. Virol. 1997; 71: 7978-7983Google Scholar). Finally, the TAF4 linker and CCTD regions interact with the general transcription factor TFIIA (47Guermah M. Tao Y. Roeder R.G. Mol. Cell. Biol. 2001; 21: 6882-6894Google Scholar). Given this plethora of interactions, it is not surprising that the conserved region of TAF4 has a complex organization and contains several functional elements. We thank L. Carré for excellent technical assistance, S. Vicaire and D. Stephane for DNA sequencing, the staff of the oligonucleotide facilities, and B. Boulay for help with illustrations." @default.
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• W2045796723 title "Functional Analysis of the TFIID-specific Yeast TAF4 (yTAFII48) Reveals an Unexpected Organization of Its Histone-fold Domain" @default.
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