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Molecular and Cellular Biology, January 2000, p. 340-351, Vol. 20, No. 1
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Human TFIID Components TAF_II135 and TAF_II20 and the Yeast SAGA Components ADA1 and TAF_II68 Heterodimerize to Form Histone-Like Pairs

Yann-Gaël Gangloff, Sebastiaan Werten, Christophe Romier, Lucie Carré, Olivier Poch, Dino Moras, and Irwin Davidson^*

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch Cédex, C.U. de Strasbourg, France

Received 22 July 1999/Returned for modification 17 September 1999/Accepted 28 September 1999

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

It has been previously proposed that the transcription complexes TFIID and SAGA comprise a histone octamer-like substructure formed from a heterotetramer of H4-like human hTAF_II80 (or its Drosophila melanogaster dTAF_II60 and yeast [Saccharomyces cerevisiae] yTAF_II60 homologues) and H3-like hTAF_II31 (dTAF_II40 and yTAF_II17) along with two homodimers of H2B-like hTAF_II20 (dTAF_II30 and yTAF_II61/68). However, it has not been formally shown that hTAF_II20 heterodimerizes via its histone fold. By two-hybrid analysis with yeast and biochemical characterization of complexes formed by coexpression in Escherichia coli, we showed that hTAF_II20 does not homodimerize but heterodimerizes with hTAF_II135. Heterodimerization requires the 2 and 3 helices of the hTAF_II20 histone fold and is abolished by mutations in the hydrophobic face of the hTAF_II20 2 helix. Interaction with hTAF_II20 requires a domain of hTAF_II135 which shows sequence homology to H2A. This domain also shows homology to the yeast SAGA component ADA1, and we show that yADA1 heterodimerizes with the histone fold region of yTAF_II61/68, the yeast hTAF_II20 homologue. These results are indicative of a histone fold type of interaction between hTAF_II20-hTAF_II135 and yTAF_II68-yADA1, which therefore constitute novel histone-like pairs in the TFIID and SAGA complexes.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Transcription factor TFIID is one of the general factors required for accurate and regulated initiation by RNA polymerase II. TFIID comprises the TATA-binding protein (TBP) and TBP-associated factors (TAF_IIs) (4, 7). The cDNAs encoding many human TAF_IIs (hTAF_IIs) have been isolated, revealing a striking sequence conservation with yeast and Drosophila TAF_IIs (yTAF_IIs and dTAF_IIs, respectively) (25, 27, 35, 36, 40, and references therein). A subset of TAF_IIs are present not only in TFIID but also in the SAGA, PCAF, STAGA, and TFTC complexes (6, 16, 32, 43, 52; reviewed in references 18, 19, and 53).

TAF_II function in living cells has been studied genetically in yeast and by transfection experiments with mammalian cells. In yeast, a variable requirement for TAF_IIs has been found. Temperature-sensitive mutations in yTAF_II145 result in cell cycle arrest and lethality, but the expression of only a small number of genes is affected (23, 51). In contrast, tightly temperature-sensitive mutations in yTAF_II17, yTAF_II25, yTAF_II60, or yTAF_II61/68 which are also present in the SAGA complex have a more dramatic effect, in which the transcription of the majority of yeast genes is affected (1, 38, 39, 41, 46). An increasing body of results also shows that hTAF_II28, hTAF_II135, and hTAF_II105 can act as specific transcriptional coactivators for nuclear receptors and other activators in mammalian cells (10, 26, 33-35, 45, 55).

It has been proposed that the TFIID, PCAF, and SAGA complexes contain a histone octamer-like substructure (8, 20-22, 54). This is suggested by the fact that three TAF_IIs show obvious sequence homology to histones H4, H3, and H2B: hTAF_II80 (dTAF_II60 and yTAF_II60), hTAF_II31 (dTAF_II40 and yTAF_II17), and hTAF_II20 (dTAF_II30 and yTAF_II61/68), respectively. Structural studies show that dTAF_II60 and dTAF_II40 interact via a histone fold and form an H3-H4-like heterotetramer, although dTAF_II40 does not contain an N helix characteristic of H3 (2, 30, 54). In the mammalian PCAF complex, hTAF_II80 is replaced by another histone fold-containing protein, PAF65, which forms a histone pair with hTAF_II31 (43). hTAF_II20 shows homology to H2B and may contain an C helix characteristic of H2B (30). In the absence of an obvious H2A-like TAF_II, the histone octamer-like structure is postulated to comprise an hTAF_II80 (PAF65)/hTAF_II31 heterotetramer and two hTAF_II20 homodimers.

Although it was not originally noted in sequence comparisons, hTAF_II28 and hTAF_II18 are also histone-like TAF_IIs since they interact via a histone fold motif to form a heterodimer (5, 11). hTAF_II28 is atypical, since it shows equivalent sequence homology to H3, H4, and H2B, but structurally resembles H3 due to the presence of an N helix. hTAF_II18 shows sequence homology to H4 but is likely to contain an C helix typical of H2B. Furthermore, the SAGA, PCAF, TFTC, and STAGA component SPT3 shows extensive sequence homology to the histone fold motifs of both hTAF_II18 and hTAF_II28 in its N- and C-terminal regions, respectively (5, 36). These two regions are separated by a long linker domain which would allow SPT3 to form a histone-like pair by intramolecular interactions. Therefore, while the existence of these additional histone-like pairs in the TFIID, PCAF, TFTC, and SAGA complexes does not rule out the existence of a histone octamer-like structure, this simple model cannot account for all of the histone pairs seen in these complexes.

One essential postulate of the original octamer-like model was the presence of two hTAF_II20 homodimers in TFIID, yet hTAF_II20 has not been shown to homodimerize via its histone fold motif. In this report, we show that hTAF_II20 does not form homodimers in either yeast two-hybrid assays or Escherichia coli. Instead, coexpressed hTAF_II20 and hTAF_II135 interact in two-hybrid assays and this interaction requires the histone fold region of hTAF_II20 and a conserved region in hTAF_II135. Interaction with hTAF_II135 is abolished by mutation of the hydrophobic amino acids in the 2 helix of the hTAF_II20 histone fold, which in histone pairs typically form the hydrophobic interface with the heterodimeric partner. Coexpression of hTAF_II135 in E. coli solubilizes the hTAF_II20 histone fold and leads to the formation of a heterodimeric complex. Therefore, our results show that, rather than forming homodimers, hTAF_II20 heterodimerizes with hTAF_II135.

In contrast, yTAF_II68, the hTAF_II20 homologue, does not interact with hTAF_II135. Analysis of hTAF_II135 interaction with hTAF_II20/yTAF_II68 chimeras locates determinants for partner specificity in the 2-L2-3 segment of the histone fold. Finally, we demonstrate that the SAGA component yADA1 contains a domain with homology to hTAF_II135 which mediates its heterodimerization with the histone fold of yTAF_II68 in two-hybrid assays and in E. coli coexpression. This shared hTAF_II135/ADA1 domain shows homology with the histone fold region of H2A. These results show the existence of additional histone-like pairs in the both the TFIID and SAGA complexes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Construction of recombinant plasmids. Two-hybrid expression vectors and bacterial expression vectors were constructed by PCR using primers with the appropriate restriction sites, and constructs were verified by automated DNA sequencing. Details of constructions are available on request. LexA fusions were constructed in the multicopy vector pBTM116 containing the TRP1 marker, and the VP16 fusions were constructed in the multicopy vector pASV3 containing the LEU2 marker (28, 29).

Yeast strains and two-hybrid assays. The vectors expressing the LexA-TAF_II and VP16-TAF_II fusion proteins were sequentially transformed into Saccharomyces cerevisiae L40 (49) [MATa trp1-901 leu2-3,112 his 3-200 ade2 LYS2::(LexAop)₄-HIS3, URA3::(LexAop)₈-LacZ] by the lithium acetate technique (14). Transformants were selected on Trp Leu plates. For qualitative detection of -galactosidase activity, yeast colonies were replica plated on a nitrocellulose filter and lysed by freezing in liquid nitrogen and the filter was then placed on filter paper presoaked with X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) to turn positive colonies blue. Quantitative -galactosidase assays of individual L40 transformants were determined as previously described (50). Reproducible results were obtained in several independent experiments, and the results of typical experiments are shown in the figures.

Coexpression in E. coli. PCR was used to clone the hTAF_II20 histone fold region (and derivatives) between the NdeI and BamHI sites of the pET15b vector to generate a six-histidine-tagged fusion protein. Deletion mutant forms of the TAF_II135 CR-II domain and yADA1(259-359) were cloned in a modified version of the vector pACYC184 (New England BioLabs) (unpublished data). Plasmid pairs were introduced into E. coli BL21 (DE3), and double transformants were selected on plates containing ampicillin and chloramphenicol. Bacteria were amplified to an optical density at 600 nm of 0.45 and induced for 4 h at 25°C with 1 mM isopropyl--D-thiogalactopyranoside (IPTG). Cell were lysed by sonication in buffer (25 mM Tris HCl [pH 6.0], 0.4 M NaCl), and the soluble fraction was collected after centrifugation at 14,000 rpm for 20 min at 4°C in an Eppendorf centrifuge. Aliquots of the soluble fraction from a 10-ml bacterial culture were then incubated with 50 µl of Co2⁺ beads (TALON metal affinity resin; Clontech) for 30 min at 4°C. The beads were washed three times with 1 ml of lysis buffer and then resuspended in Laemmli buffer. One-fifth of the bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining with Coomassie brilliant blue as shown in the figures. Interactions with the glutathione S-transferase (GST) derivatives were performed by using glutathione-Sepharose (Pharmacia). Binding and washing were done essentially as described above. For gel filtration, the hTAF_II20(57-128)-hTAF_II135(870-952) complex was purified by chromatography on Co2⁺ beads from 1.5 liters of culture. The eluted material was loaded on a Superdex 75 column (Pharmacia), and molecular mass was determined by comparing its elution time with that of known standards in the gel filtration standard kit from Bio-Rad.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The histone fold region of hTAF_II20 interacts with hTAF_II135. To test its ability to form homodimers, full-length hTAF_II20(1-161) was fused to either the LexA DNA-binding domain (DBD) or the VP16 acidic activation domain (AAD) (see Materials and Methods and Fig. 1 and 2B). Both expression vectors were transformed into yeast strain L40, which harbors a LexA-responsive -galactosidase gene. Interaction of the two proteins was evaluated from expression of the LexA-dependent -galactosidase reporter (see Materials and Methods). In such experiments, coexpression of LexA-hTAF_II20 and VP16-hTAF_II20 did not lead to significant activation of the -galactosidase reporter gene (summarized in Fig. 1), showing that hTAF_II20 did not homodimerize in two-hybrid assays.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Summary of the results obtained with LexA- and VP16 AAD-TAFII fusions in yeast two-hybrid assays. The combinations used are indicated along with the amino acid coordinates of the TAFII segments included in each fusion. A plus sign indicates -galactosidase activity significantly higher than that seen with the appropriate LexA or VP16 control, and a minus sign indicates background levels (see Fig. 2C and 4B for examples).

Previous results have shown in vitro interactions between hTAF_II20 and hTAF_II135 and between their Drosophila homologues dTAF_II30 and dTAF_II110 (22, 48, 56). We tested the ability of hTAF_II135 and hTAF_II20 to interact in the yeast two-hybrid assay. Amino acids 372 to 1083 of hTAF_II135 were fused to the LexA DBD or the VP16 AAD (see Materials and Methods and Fig. 4A), and the expression vectors were sequentially introduced into yeast with the corresponding hTAF_II20 expression vectors. Strong -galactosidase activity was observed in yeast expressing complementary hTAF_II20/hTAF_II135 LexA-VP16 AAD pairs (summarized in Fig. 1). These results show that while neither hTAF_II20 nor hTAF_II135 homodimerizes, they do interact with each other.

The C-terminal domains of hTAF_II20, dTAF_II30, and yTAF_II68 contain a putative histone fold motif which shows sequence homology to that of H2B (Fig. 2A). The minimal histone fold motif comprises three  helices, (1, 2, and 3) separated by two loops, L1 and L2. In addition, they may possess an C helix, as observed in H2B. Fusion proteins containing the full hTAF_II20 histone fold or subdomains fused to the VP16 AAD (Fig. 2B) were tested for the ability to interact with LexA-hTAF_II135(870-951) (see below). TAF_II20(57-161), containing the entire histone fold region, interacted with hTAF_II135 as efficiently as full-length hTAF_II20 (Fig. 2C, lanes 1 and 2). Interaction was not affected by deletion of the C helix [hTAF_II20(57-128); Fig. 2C, lane 3]. Interaction was mildly increased when the 1 helix was deleted [hTAF_II20(73-128); Fig. 2C, lane 4], while wild-type levels were seen when the 1 helix and the N-terminal end of the 2-helix were deleted [TAF_II20(87-128); Fig. 2C, lane 5]. In contrast, interaction was abolished when the 3 helix was deleted [hTAF_II20(57-116) and hTAF_II20(73-116); Fig. 2C, lanes 6 and 7]. Therefore, a minimal region including amino acids 87 to 128 of hTAF_II20 containing the C-terminal region of the 2 helix along with L2 and the 3 helix suffices to mediate interaction with hTAF_II135.

View larger version (31K): [in this window] [in a new window]   FIG. 2.   (A) Alignment of the sequences of the histone fold regions of hTAFII20, dTAFII30, and yTAFII68 with those of H2B and NC2. The prefixes h, d, and y are for human, Drosophila melanogaster, and yeast (S. cerevisiae). The positions of the predicted  helices and loops are indicated above the sequence based on homology with H2B (30). In the upper panel, invariant amino acids are in white on a black background and similar amino acids are indicated by gray shading. In the lower panel, conserved hydrophobic residues are in white on a black background and conserved charged or small residues are indicated by gray shading. Amino acids were classified as follows: small residues, P, A, G, S, and T; hydrophobic residues, L, I, V, A, F, M, C, Y, and W; polar-acidic residues, D, E, Q, and N; basic residues, R, K, and H. (B) Schematic structure of VP16-hTAFII20 fusions. The hTAFII20 amino acids at the boundaries of the fusions are indicated. The ability of each fusion to interact with hTAFII135 is shown to the right by a plus or minus sign. (C) Quantification of -galactosidase (-gal) activity in two-hybrid assays. The VP16 AAD-hTAFII20 fusions shown below each lane were assayed in a LexA-hTAFII135(870-951) background as indicated above the graph.

In histone folds, the 2 helix is an amphipathic  helix in which many of the hydrophobic residues interact with the hydrophobic residues of the 2 helix of the heterodimeric partner, while the solvent-exposed face comprises mainly polar and charged residues. Alignment of the hTAF_II20 histone fold with that of H2B, whose structure has been determined, shows that a similar arrangement may exist in hTAF_II20. To determine which residues of the hTAF_II20 2 helix were involved in interactions with hTAF_II135, hydrophobic or charged residues were mutated (Fig. 3A). Mutation of I87 and F91 completely abolished interaction with hTAF_II135 [hTAF_II20(57-128) m1; Fig. 3B, lanes 1 and 2). Similarly, mutation of V95 and V96 [hTAF_II20(57-128) m4] and A99 and A103 [hTAF_II20(57-128) m5] also abolished interaction with hTAF_II135 (Fig. 3B, lanes 5 and 6). In contrast, mutation of charged residues D89 and E93 [hTAF_II20(57-128) m2] or E82 [hTAF_II20(57-128) m3] had no effect on interaction with hTAF_II135 (Fig. 3B, lanes 3 and 4). Therefore, the residues which form the hydrophobic interface of the hTAF_II20 histone fold are critical for interaction with hTAF_II135.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   (A) Locations of mutations in the hTAFII20 histone fold. The sequence of the hTAFII20 histone fold is shown along with the mutated amino acids below the wild-type (WT) sequence. (B) Mutations in hTAFII20 which abolish interaction with hTAFII135. The hTAFII20 mutants assayed in each lane are shown below the graph in the background of the vectors schematized above the graph.

A region of the CR-II domain of hTAF_II135 required for interaction with hTAF_II20. TAF_II135 consists of 1,083 amino acids which can be divided into several regions: an N-terminal proline- and alanine-rich region, four glutamine-rich regions which interact with the SP1 and CREB activators, and two conserved regions, CR-I, present in hTAF_II105, dTAF_II110, and Nervy/ETO, as well as other transcription factors, and the long CR-II region, conserved in hTAF_II105 and dTAF_II110 (35, 45). We determined the region of hTAF_II135 required for interaction with hTAF_II20 by using a series of LexA-TAF_II135 fusions (Fig. 4A). The N-terminal region of hTAF_II135(372-805) lacking CR-II did not interact with hTAF_II20 (data not shown), whereas interaction was observed with the hTAF_II135(805-1083) and hTAF_II135(825-1019) fusions containing only the CR-II region (summarized in Fig. 4A; see also Fig. 4B, lane 1).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   (A) Schematic structures of hTAFII135 and LexA two-hybrid fusions. P&A indicates the proline- and alanine-rich region at the N terminus of hTAFII135. Glutamine-rich regions Q1 to Q4 are also indicated, along with conserved domains CR-I and CR-II. The abilities of the LexA-hTAFII135 hybrids to interact with VP16-hTAFII20 are summarized to the right. (B) Determination of the minimal region of hTAFII135 required for interaction with hTAFII20. The LexA-hTAFII135 fusions shown below each lane were assayed in the VP16-hTAFII20 background indicated above the graph. -gal, -galactosidase.

Further CR-II region deletion mutants were made to more precisely define the amino acids required for interaction with hTAF_II20. Progressive deletions from both the N and C termini indicated that amino acids 870 to 911 suffice to mediate interaction with hTAF_II20 (Fig. 4A and B, lanes 2 to 4). However, deletion of a further 10 amino acids from the N terminus [hTAF_II135(880-911)] or deletion of 8 amino acids from the C terminus [hTAF_II135(825-903)] completely abolished interaction with hTAF_II20 (Fig. 4A and B, lanes 5 and 6). Therefore, amino acids 870 to 911 constitute the minimal domain required for interaction with hTAF_II20.

Chimeras between the histone fold domains of hTAF_II20 and its yeast homologue yTAF_II68 interact with hTAF_II135. Yeast yTAF_II68 contains a histone fold sequence which is highly homologous to that of hTAF_II20 and is located at the C terminus of the protein between amino acids 414 and 539 (Fig. 2A and 5A and B). To test the abilities of the yTAF_II68 histone fold to homodimerize and to interact with hTAF_II135, two-hybrid expression vectors were constructed in which the histone fold region was fused to the LexA DBD or the VP16 AAD (Fig. 5A). Upon coexpression in yeast, no interaction was seen between LexA-yTAF_II68(414-539) and VP16-yTAF_II68(414-539) (summarized in Fig. 1), showing that, as observed for hTAF_II20, yTAF_II68 does not homodimerize. However, no interaction between LexA-hTAF_II135 and VP16-yTAF_II68 was observed irrespective of whether the entire histone fold region, including the C or only the 1 to 3 region, was used (summarized in Fig. 1; see also Fig. 5C, lane 2). Thus, despite the high homology between the hTAF_II20 and yTAF_II68 histone fold regions, they differ in the ability to interact with hTAF_II135.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   (A) Schematic structures of yTAFII68 two-hybrid vectors. The location of the histone fold in yTAFII68 is shown schematically. (B) Structure of chimeric hTAFII20-yTAFII68 histone folds. A sequence comparison of the histone folds of hTAFII20 and yTAFII68 is shown in which the conserved residues are highlighted against a black background and the similar residues are shaded in blue. The criteria for identity take into account differences seen in the dTAFII30 sequence as shown in Fig. 2A. In chimeras c1 to c4, the hTAFII20 residues are in red and the yTAFII68 residues are in blue. Their abilities to interact with hTAFII135 are summarized to the right. (C) Interaction of chimeras c1 to c4 with hTAFII135. The chimeras used are shown below each lane, and their activity, as measured in the hTAFII135 background, is indicated above the graph. -gal, -galactosidase.

Comparison of the yTAF_II68 and hTAF_II20 histone fold sequences shows two regions of variability which may contribute to specificity. The L1 loop of yTAF_II68 is longer than that of hTAF_II20, and the sequences are divergent. Similarly, the region of the 2 helix between amino acids I92 and Q101 of hTAF_II20 and L2 show several differences. To determine which regions of the histone fold were responsible for the specificity of interaction, chimeras between the hTAF_II20 and yTAF_II68 histone folds were made in fusions with the VP16 AAD (Fig. 5B).

We first replaced the divergent sequence between V454 and R463 of the yTAF_II68 2 helix with I92 to Q101 of hTAF_II20 (68/20/68 c1, Fig. 5B). This chimera interacted weakly with hTAF_II135 (Fig. 5C, lane 3), and replacement of amino acids V454 to I490 of yTAF_II68 with those of hTAF_II20 allowed full interaction with hTAF_II135 (68/20 c2, lane 4). On the other hand, the converse chimera did not interact with hTAF_II135 (20/68 c3, lane 5) whereas the replacement of I92 to Q101 of hTAF_II20 with the equivalent yTAF_II68 region did not affect interaction with hTAF_II135 (20/68/20 c4). Therefore, interaction with TAF_II135 requires hTAF_II20 amino acids C terminal of I92 which cannot be replaced with their yTAF_II68 counterparts whereas the differences in the L1 loop do not affect interaction with TAF_II135. Within the hTAF_II20 2-L2-3 segment, one determinant of specificity is located between I92 and Q101; however, full interaction requires the entire region.

hTAF_II20 and hTAF_II135 coexpressed in E. coli heterodimerize. To show that hTAF_II20 and hTAF_II135 interact directly and form heterodimers, as suggested by the yeast two-hybrid data, each protein was expressed in E. coli (see Materials and Methods). Expression of the his₆-tagged hTAF_II20(57-128) histone fold region alone resulted in the accumulation of almost totally insoluble protein, little of which was retained on Co2⁺ beads (Fig. 6A, lanes 2 and 3). When expressed alone, untagged hTAF_II135(825-1019) was not significantly retained on the Co2⁺ beads (lanes 4 and 5) while hTAF_II135(870-952) was unstable and failed to accumulate (Fig. 6A, lane 8).

View larger version (24K): [in this window] [in a new window]   FIG. 6.   Coexpression of hTAFII20 and hTAFII135 in E. coli. (A) Bacteria were transformed to express the proteins shown above the lanes, and total extracts (E) were analyzed by SDS-PAGE and staining with Coomassie brilliant blue. The proteins from each extract bound to Co2+ beads (B) are shown in the adjacent lanes. The migration of the molecular mass standards in lane M (in kilodaltons) are shown on the left. (B) Elution profile of the purified hTAFII20(57-128)-hTAFII135(870-952) complex from a Superdex 75 gel filtration column. The lower panel shows the Coomassie brilliant blue staining of the relevant eluted fractions after SDS-PAGE. (C) yADA1 interacts with the histone fold of yTAFII68 and hTAFII20. Bacterial extracts expressing the proteins shown above each lane were chromatographed on glutathione-Sepharose, and the bound proteins were analyzed by SDS-PAGE and staining with Coomassie brilliant blue. Lane 11 shows the proteins bound to Co2+ beads following coexpression of his6-hTAFII20(57-128) and yADA1(259-359).

In contrast, coexpression of hTAF_II135(825-1019) resulted in solubilization of his₆-hTAF_II20(57-128) and the formation of an hTAF_II20/hTAF_II135 complex, since the untagged hTAF_II135(825-1019) protein was now retained on the Co2⁺ beads in stoichiometric amounts with his₆-hTAF_II20(57-128) (Fig. 6A, lanes 6 and 7 compared with lanes 4 and 5). Likewise, coexpression of his₆-hTAF_II20(57-128) stabilized hTAF_II135(870-952) and resulted in the formation of a soluble complex which could be retained on Co2⁺ beads (Fig. 6A, lanes 10 and 11 compared with lanes 8 and 9). These results show that coexpression of hTAF_II20 and hTAF_II135 promotes the formation of a stable, soluble complex while each protein alone is insoluble or unstable.

To determine whether the hTAF_II20/hTAF_II135 complex is a heterodimer or a heterotetramer, the purified complex formed between his₆-hTAF_II20(57-128) and hTAF_II135(870-952) was analyzed by gel filtration. The complex eluted from a Superdex 75 column as a single species with an apparent native molecular mass of approximately 20 kDa indicative of a heterodimer (Fig. 6B). Therefore, the histone fold region of hTAF_II20 forms a heterodimer with hTAF_II135.

Similar coexpression experiments were performed with the hTAF_II135 derivatives and a GST fusion of the histone fold region of yTAF_II68. In agreement with the yeast two-hybrid data, no hTAF_II135 was retained on glutathione beads along with GST-yTAF_II68 (data not shown). There is thus no interaction between E. coli-expressed histone fold regions of yTAF_II68 and hTAF_II135.

Yeast ADA1 interacts with the histone fold regions of hTAF_II20 and yTAF_II68. It has previously been reported that there is no yeast homologue for TAF_II135 (35, 40). Nevertheless, our finding that yTAF_II68 does not homodimerize implies that there is a heterodimeric partner(s) for the yTAF_II68 histone fold in TFIID and SAGA. To identify potential partners, we used the sequence of the minimal region of hTAF_II135 required for interaction with hTAF_II20 to blast search the yeast genome. One of the highest-scoring proteins found in this way was the SAGA component ADA1 (24, 47).

The sequences of both yeast ADA1 and a putative Schizosaccharomyces pombe ADA1 homologue could be aligned with those of hTAF_II135, hTAF_II105, and dTAF_II110, and this region is predicted to adopt a helix-strand-helix conformation (Fig. 7A). This alignment shows the presence of many conserved positions occupied by hydrophobic residues. At several other well-conserved positions, a hydrophobic residue is sometimes replaced by a threonine. Two conserved basic residues are also observed in the 1 helix and in L2 (Fig. 7A). Interestingly, most of these residues are also conserved in the 1 and 2 helices of H2A and in the  subunit of the transcriptional repressor NC2, which has been classified as an H2A-like protein (15, 37).

View larger version (45K): [in this window] [in a new window]   FIG. 7.   (A) Sequence comparison of the putative histone fold regions of hTAFII135, hTAFII105, dTAFII110, yADA1, and a putative S. pombe ADA1 (accession no. CAA21903) with H2A and NC2. In the 1-L1-2 segment, residues which are conserved in the ADA1, TAFII, and H2A/NC2 families are in white against a black background. Residues which are conserved only between the TAFII and ADA1 families are shaded in grey. In the L2-3 segment, additional alignments with the 3 helices of dTAFII40, dTAFII60, hTAFII18, hH3, hH4, and hTAFII20 are shown. Highly conserved residues are in white against a black background. The location of a potential buried intramolecular hydrogen bond between the R(K) and D residues is indicated. (B) Schematic structures of yADA1 two-hybrid vectors. (C) Two-hybrid interactions with yADA1. In the left graph, the proteins tested for interaction with yADA1(259-488) are shown below the lanes. In the right graph, the yADA1 deletion mutants tested for interaction with yTAFII68(414-490) are shown. -gal, -galactosidase.

To test the ability of this region of ADA1 to interact with yTAF_II68 and hTAF_II20, amino acids 259 to 488 or 259 to 359 were fused to the LexA DBD and transformed into yeast (Fig. 7B). A strong interaction was seen between both of the yADA1 constructs and yTAF_II68(414-490) or hTAF_II20(57-128) (Fig. 7C, lanes 1, 3, 7, and 8, and data not shown). Strong interactions were also seen with the 68/20/68 c3 and c4 chimeras (Fig. 7C, lane 2, and data not shown). Importantly, yADA1 interaction with hTAF_II20 was abolished by mutations m1, m4, and m5 in the hydrophobic core of the hTAF_II20 2 helix (lane 4 and data not shown). As expected, however, no interaction was observed with hTAF_II135(372-1083) (lane 5) or with the VP16 AAD alone (lane 6). These results demonstrate that the region of yADA1 with homology to hTAF_II135 and H2A interacts specifically with the histone fold regions of yTAF_II68 and hTAF_II20 but not with hTAF_II135.

We also tested the ability of yADA1 to form complexes with yTAF_II68 and hTAF_II20 in E. coli. GST fusions of the histone fold regions of hTAF_II20(57-128) or hTAF_II20(57-161) or yTAF_II68(414-490) or yTAF_II68(414-539) were coexpressed in E. coli with an untagged version of yADA1(259-359). When expressed alone, yADA1 was globally insoluble and did not bind to glutathione or Co2⁺ beads (Fig. 6C, lane 1, and data not shown). In contrast, when coexpressed with the GST-histone fold fusion of yTAF_II68 or hTAF_II20, yADA1(259-359) was retained on glutathione beads (Fig. 6C, lanes 7 to 10). Note also that coexpression with yADA1(259-359) significantly increased the solubility of the GST-TAF_II fusions (compare lanes 3 to 6 and 7 to 10). Similarly, coexpression of yADA1(259-359) solubilized his₆-hTAF_II20(57-128) and resulted in the formation of a heterodimeric complex which could be retained on Co2⁺ beads. (Fig. 6C, lane 11). These results show that yADA1 interacts directly with the histone fold domains of yTAF_II68 or hTAF_II20 to form stable complexes.

The histone fold region of hTAF_II135 is required for coactivator activity in mammalian cells. We have previously shown that coexpression of hTAF_II135 or hTAF_II28 potentiates transcriptional activation by ligand-dependent activation function 2 of various nuclear receptors (33, 35). In hTAF_II28, the histone fold region plays a critical role in this function (26). To test the requirement for the histone fold region of hTAF_II135 in this process, we generated an hTAF_II135 deletion mutant in which the minimal region required for interaction with hTAF_II20 (amino acids 870 to 910) had been removed.

As previously described, coexpression of hTAF_II135(372-1083) in Cos cells strongly increased activation by a chimeric fusion protein comprising the DBD of the yeast activator GAL4 (G4) fused to activation function 2 of the nuclear receptor for all-trans retinoic acid (RAR) from a minimal G4-responsive promoter (Fig. 8, lanes 2, 4, and 5). In contrast to our previous findings, the CR-II region alone (amino acids 805 to 1083) was also able to elicit such an effect, albeit less strongly than amino acids 372 to 1083 (Fig. 8, lanes 7 and 8). Deletion of amino acids 870 to 910 from the CR-II domain completely abrogated the transcriptional coactivator effect (lanes 10 and 11). Therefore, the histone fold within the CR-II domain is essential for the coactivator activity of hTAF_II135.

View larger version (34K): [in this window] [in a new window]   FIG. 8.   The hTAFII135 histone fold is required for coactivator activity. Quantitative analysis of chloramphenicol acetyltransferase (CAT) activity. The structures of the activator and reporter plasmids are schematized above the graph. The plasmids transfected in each lane are shown below the graph. Transfections contained 50 nM RAR, 2 µg of the chloramphenicol acetyltransferase reporter plasmid, and 2 µg of a luciferase internal control (35), along with 0.25 µg of the G4-RAR chimera and 3 or 5 µg of the hTAFII135 expression vectors, as indicated. Quantitation was done on a Fujix Bas 2000.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

hTAF_II20-hTAF_II135, a novel histone-like pair in the TFIID complex. In this report, we show that hTAF_II20 interacts with hTAF_II135 in yeast two-hybrid assays. In these assays, interaction with hTAF_II135 requires a minimal region of the hTAF_II20 histone fold comprising the C-terminal region of the 2 helix, L2, and the 3 helix. This is in good agreement with the region previously observed in in vitro interactions (22). Interaction with hTAF_II135 is abolished by mutation of hydrophobic amino acids in the hTAF_II20 2 helix. Many of the equivalent residues in H2B participate in interactions with H2A in the nucleosome (30). These observations indicate that hTAF_II20-hTAF_II135 interaction also involves the hydrophobic interface formed by residues of the hTAF_II20 2 helix.

Interestingly, despite the fact that the histone fold region of yTAF_II68 is highly homologous to that of hTAF_II20, no interaction between yTAF_II68 and hTAF_II135 was observed. However, interaction with hTAF_II135 was partially restored when amino acids V454 to R463 of the yTAF_II68 2 helix were replaced with I92 to Q101 of hTAF_II20 and fully restored when the whole 2-L2-3 segment was exchanged. The region from I92 to Q101 therefore contains specific determinants for interaction with hTAF_II135. However, the remainder of the L2-3 region, which appears highly conserved, must contain further important determinants and only the combination of these two regions allows full interaction with hTAF_II135. Thus, even apparently minor differences in the primary amino acid sequence can have important consequences in terms of partner specificity.

The histone fold region of hTAF_II20 is insoluble when expressed alone in E. coli. This is what is observed when many other histone fold proteins are expressed in the absence of their heterodimeric partners and is not what would be expected if the hTAF_II20 histone fold were to homodimerize. Similarly, the hTAF_II135 CR-II region required for interaction with hTAF_II20 is unstable and does not accumulate. In contrast, coexpression of these two proteins results in the formation of a soluble, stable complex which has the native molecular mass of a heterodimer. Indeed, in analogous coexpression experiments, hTAF_II28 solubilized hTAF_II18 and the resulting complex eluted as a heterodimer while the histone fold region of hTAF_II31 solubilized that of hTAF_II80 and the resulting complex eluted as a heterotetramer. However, in mixing and matching experiments, no complexes other than hTAF_II28-hTAF_II18, hTAF_II31-hTAF_II80, and hTAF_II20-hTAF_II135 were formed (unpublished data). Therefore, there are determinants within the histone folds of these TAF_IIs which impose strict partner specificity rules.

Taken together, the interaction mapping data obtained with yeast and the biochemical characterization of the bacterially expressed proteins strongly suggest that hTAF_II20 and hTAF_II135 interact directly via a histone fold and form a novel histone-like pair in the TFIID complex (Fig. 9).

View larger version (22K): [in this window] [in a new window]   FIG. 9.   Summary of the structures and some of the molecular interactions within the TFIID and SAGA complexes. In hTFIID, ribbon representations of the structures of the TBP (42), dTAFII60/TAFII40 (54), and hTAFII28/hTAFII18 are shown (5). Other TAFIIs are depicted as colored ovals. Some of the molecular interactions are shown either by overlapping of the ovals or by arrows. In SAGA, the predicted structure of SPT3 is indicated along with the TBP and dTAFII60/TAFII40, whose histone fold domain is assumed to adopt the same structure as that of yTAFII60/TAFII17. Potential interactions, as suggested in references 9, 13, 16, 17, 31, 44, and 47, are depicted. aa, amino acids.

Previous models have proposed that hTAF_II20 homodimers contribute to the formation of a histone octamer-like structure within TFIID. In two-hybrid assays, no interaction between lexA-hTAF_II20 and VP16-hTAF_II20 was observed although each of these proteins interacted with hTAF_II135. Furthermore, when radiolabelled full-length recombinant hTAF_II20 was used to probe immunopurified hTFIID in far-Western blot experiments, interaction with hTAF_II135 was clearly seen but no interaction with hTAF_II20 was observed (our unpublished data).

It has been shown that dTAF_II30 and hTAF_II20 can oligomerize (22, 56). For hTAF_II20, oligomerization was observed with a fragment comprising the C-terminal half of the 2 helix, L2, and the 3 helix. In contrast, as no oligomerization was seen with the full 1-3 region, it is unlikely to correspond to homodimerization of the histone fold. It is possible that in such experiments the short subdomains of hTAF_II20 used were misfolded and formed aggregates due to the presence of many hydrophobic residues. Indeed, our coexpression experiments show that accumulation of the soluble hTAF_II20 histone fold domain requires the presence of hTAF_II135. Taken together, all of our data argue against the existence of hTAF_II20 homodimers and for the formation of heterodimers with hTAF_II135.

In hTAF_II135, interaction with hTAF_II20 minimally requires amino acids 870 to 911 of the hTAF_II135 CR-II region. This region is conserved in hTAF_II105, which, in contrast to previous reports (12), also heterodimerizes with hTAF_II20 in yeast two-hybrid assays (our unpublished data). This minimal region shows homology to a region of yADA1 which can also mediate specific interactions with yTAF_II68 and hTAF_II20. Both the hTAF_II135 and yADA1 regions can be further aligned with the 1-L1-2 region of H2A and the H2A-like protein NC2 (and the C subunit of transcription factor NFY [our unpublished data]). This homology with H2A and H2A-like proteins is consistent with the observation that their partners hTAF_II20 and yTAF_II68 are H2B-like proteins. Most of the conserved residues are hydrophobic, with the exceptions of a conserved arginine in the 1 helix, a polar-acidic residue in the 2 helix, and a basic residue at the beginning of L2. The homology between the TAF_II135/ADA1 and H2A/NC2 families is comparable to that seen between the hTAF_II20 and H2B/NC2 families in that the majority of the conserved residues are the hydrophobic amino acids which make up the heterodimerization interface (30).

The alignment predicts that the minimal domain of hTAF_II135 required for interaction with hTAF_II20 would contain the 1-L1-2 region of the histone fold. This is in good agreement with the observation that the minimal domain of hTAF_II20 required for interaction is the 2-L2-3 region. Due to the head-to-tail arrangement of the histone fold partners, the 1-L1-2 region of hTAF_II135 would be juxtaposed with the 2-L2-3 region of hTAF_II20. The histone fold is postulated to arise from gene duplication of two helix-strand-helix segments, HSH1 and HSH2, corresponding to the N- and C-terminal halves of the histone fold (3). The minimal TAF_II20 and TAF_II135 domains necessary for their mutual interactions correspond well to these predicted HSH2 and HSH1 segments, respectively.

By using only the homology with H2A/NC2, it was not possible to locate an 3 helix in the TAF_II135 and ADA1 families. However, by taking into account other known histone fold 3 sequences, homology between the TAF_II135/ADA1 families and dTAF_II40, dTAF_II60, and hTAF_II20 could be found. In this alignment, a D(V/I/L) pair is found to be conserved. In H3, H4, and dTAF_II60, this D residue forms a buried intramolecular hydrogen bond with a conserved R(K) residue in the L2 loop which stabilizes the folding of the 3 helix back over the 2 helix (30, 54). In other histone folds (H2B and dTAF_II40), this pair is conserved but no interaction is observed. This D(V/I/L) pair is therefore a useful signature which allows us to identify a potential hTAF_II135/ADA1 3 helix at this position. It is interesting to notice from this alignment that the hTAF_II135 region required for interaction with hTAF_II20 is well conserved and similar to H2A, whereas the more divergent 3 region is not absolutely required for interaction.

The histone fold region is critically required for coexpressed hTAF_II135 to potentiate transcriptional activation by the RAR. We previously reported that hTAF_II135 coactivator activity requires a domain N terminal to the CR-II region (35). In the subsequent experiments reported here, however, we did see activity of the CR-II region alone, although it was weaker than that seen when the N-terminal region was present. Notwithstanding this discrepancy, deletion of the HSH1 segment of the hTAF_II135 histone fold completely abrogates coactivator activity.

In hTAF_II28, deletion of the histone fold region also abrogates coactivator activity (33). We have further shown that specific residues in the 2 helix of the hTAF_II28 histone fold are critical for this coactivator activity (26). These residues are located on the solvent-exposed face of this helix and in the interface with hTAF_II18. This suggests that hTAF_II28 acts by integrating into the endogenous TFIID via interactions with hTAF_II18 and that the solvent-exposed face of the 2 helix interacts with other factors required for coactivator activity (for a discussion, see reference 26). Further experiments are required to determine whether a similar mechanism operates in the case of hTAF_II135.

yTAF_II68-yADA1, a novel histone-like pair in the SAGA complex. Our present results show that yADA1 is a heterodimeric partner for yTAF_II68, providing a direct physical link between the TAF_II and yADA protein families. yADA1 and yTAF_II68 have been identified as components of the SAGA complex (16, 44). Previous results have shown that the histone fold region of yTAF_II68 is necessary and sufficient for integration in the SAGA complex (40, 41). Shifting of tightly temperature-sensitive mutants of yTAF_II68 with mutations in the histone fold domain to the nonpermissive temperature results in the disappearance of yADA1 and disruption of the SAGA complex (38). Similarly, in yADA1 mutant strains, the integrity of the SAGA complex is severely compromised (47). These observations are consistent with our present data, and together they show that the yTAF_II68-yADA1 pair is a critical structural element in the SAGA complex. However, in another yTAF_II68 temperature-sensitive strain, yTAF_II68 is depleted from SAGA prepared from cells grown at the nonpermissive temperature along with SPT3, suggesting that there is also an intimate relationship between these two histone fold proteins (16).

Our results lead to a better understanding of the molecular interactions within the SAGA complex (Fig. 9). If there is a histone octamer-like core in SAGA, it may comprise a yTAF_II60-yTAF_II17 heterotetramer and at least one yTAF_II68-yADA1 heterodimer. Furthermore, the yTAF_II68-yADA1 interaction provides a mechanism by which the yTAF_II60-yTAF_II17 heterotetramer, which is also present in TFIID, can be recruited into SAGA. This TAF_II substructure is linked to the other SAGA components via heterodimerization with ADA1, which itself interacts with ADA3 (our unpublished data). As the interactions among ADA2, ADA3, and GCN5 have been well characterized (9), this results in the identification of a well-defined substructure within the SAGA complex.

While the genetic data are consistent with an important yADA1-yTAF_II68 interaction in the SAGA complex, both biochemical and genetic results indicate that yADA1 is likely not a component of yTFIID. The genes encoding the yTAF_IIs are all essential, while the yADA1 gene is not. In addition, yTBP does not coprecipitate with yADA1 (24). Mutation of yTAF_II68 results in the disappearance of yADA1, but deletion of yADA1 does not result in the disappearance of yTAF_II68. This indicates that most, if not all, of ADA1 is complexed with yTAF_II68 in SAGA or related complexes while, as previously noted (38), the majority of yTAF_II68 is in TFIID, where it would not depend on the presence of yADA1 for stability but may be complexed with another partner, since it does not homodimerize. No homologue for TAF_II135 has been described in yeast, whose genome does not encode a protein with obvious homology to TAF_II135. The sequence of the yTAF_II68 partner in TFIID must therefore diverge from that of TAF_II135, and we have not yet identified potential partners. Given the high degree of homology between the other human and yeast TAF_IIs, it is quite remarkable that TAF_II135 is an exception to this rule.

Our results show that there are three histone-like pairs in the TFIID complex, hTAF_II31-hTAF_II80, hTAF_II20-hTAF_II135, and hTAF_II28-hTAF_II18, and in the SAGA complex, yTAF_II17-yTAF_II60, yTAF_II68-yADA1, and SPT3. Consequently, in each case, there are more pairs than can be accommodated in a simple octamer-like model. Further experiments will reveal how these three pairs interact together and/or with other proteins to form the TFIID and SAGA complexes.

	ACKNOWLEDGMENTS

We thank S. Hollenberg for the generous gift of yeast strain L40; R. Losson, J. Ortiz, C. Gaudon, and M. Cervino for help with yeast assays and material; M. Abrink and L. Perletti for critical reading of the manuscript; S. Vicaire and D. Stephane for DNA sequencing; the staff of the cell culture and oligonucleotide facilities; and B. Boulay, J. M. Lafontaine, R. Buchert, and C. Werlé for illustrations.

S.W. and C.R. were supported by EMBO fellowships. This work was supported by grants from the CNRS, the INSERM, the Hôpital Universitaire de Strasbourg, the Ministère de la Recherche et de la Technologie, the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer, and the Human Frontier Science Programme.

	FOOTNOTES

^* Corresponding author. Mailing address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B.P. 163-67404, Illkirch Cédex, C.U. de Strasbourg, France. Phone: 33 3 88 65 34 40 (45). Fax: 33 3 88 65 32 01. E-mail: irwin{at}titus.u-strasbg.fr.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

1.	Apone, L. M., C. A. Virbasius, F. C. Holstege, J. Wang, R. A. Young, and M. R. Green. 1998. Broad, but not universal, transcriptional requirement for yTAFII17, a histone H3-like TAFII present in TFIID and SAGA. Mol. Cell 2:653-661[CrossRef][Medline].
2.	Arents, G., R. W. Burlingame, B. C. Wang, W. E. Love, and E. N. Moudrianakis. 1991. The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix. Proc. Natl. Acad. Sci. USA 88:10148-10152[Abstract/Free Full Text].
3.	Arents, G., and E. N. Moudrianakis. 1993. Topography of the histone octamer surface: repeating structural motifs utilized in the docking of nucleosomal DNA. Proc. Natl. Acad. Sci. USA 90:10489-10493[Abstract/Free Full Text].
4.	Bell, B., and L. Tora. 1999. Regulation of gene expression by multiple forms of TFIID and other novel TAFII-containing complexes. Exp. Cell Res. 246:11-19[CrossRef][Medline].
5.	Birck, C., O. Poch, C. Romier, M. Ruff, G. Mengus, A. C. Lavigne, I. Davidson, and D. Moras. 1998. Human TAFII28 and TAFII18 interact through a histone fold encoded by atypical evolutionary conserved motifs also found in the SPT3 family. Cell 94:239-249[CrossRef][Medline].
6.	Brand, M., K. Yamamoto, A. Staub, and L. Tora. 1999. Identification of TATA-binding protein-free TAFII-containing complex subunits suggests a role in nucleosome acetylation and signal transduction. J. Biol. Chem. 274:18285-18289[Abstract/Free Full Text].
7.	Burley, S. K., and R. G. Roeder. 1996. Biochemistry and structural biology of transcription factor IID (TFIID). Annu. Rev. Biochem. 65:769-799[CrossRef][Medline].
8.	Burley, S. K., X. Xie, K. L. Clark, and F. Shu. 1997. Histone-like transcription factors in eukaryotes. Curr. Opin. Struct. Biol. 7:94-102[CrossRef][Medline].
9.	Candau, R., and S. L. Berger. 1996. Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo. J. Biol. Chem. 271:5237-5245[Abstract/Free Full Text].
10.	Caron, C., G. Mengus, V. Dubrowskaya, A. Roisin, I. Davidson, and P. Jalinot. 1997. Human TAFII28 interacts with the human T cell leukemia virus type I Tax transactivator and promotes its transcriptional activity. Proc. Natl. Acad. Sci. USA 9:3662-3667.
11.	Davidson, I., C. Romier, A. C. Lavigne, C. Birck, G. Mengus, O. Poch, and D. Moras. 1998. Functional and structural analysis of the subunits of human transcription factor TFIID. Cold Spring Harbor Symp. Quant. Biol. 63:233-241[CrossRef][Medline].
12.	Dikstein, R., S. Zhou, and R. Tjian. 1996. Human TAFII105 is a cell type-specific TFIID subunit related to hTAFII130. Cell 87:137-146[CrossRef][Medline].
13.	Eisenmann, D. M., K. M. Arndt, S. L. Ricupero, J. W. Rooney, and F. Winston. 1992. SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 6:121-131.
14.	Gietz, R. D., R. H. Schiestl, A. R. Willems, and R. A. Woods. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355-360[CrossRef][Medline].
15.	Goppelt, A., G. Stelzer, F. Lottspeich, and M. Meisterernst. 1996. A mechanism for repression of class II gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains. EMBO J. 15:3105-3116[Medline].
16.	Grant, P. A., D. Schieltz, M. G. Pray-Grant, D. J. Steger, J. C. Reese, J. R. Yates, and J. L. Workman. 1998. A subset of TAFIIs are integral components of the SAGA complex required for nucleosome acetylation and transcriptional stimulation. Cell 94:45-53[CrossRef][Medline].
17.	Grant, P. A., D. Schieltz, M. G. Pray-Grant, J. R. Yates, and J. L. Workman. 1998. The ATM-related cofactor Tra1 is a component of the purified SAGA complex. Mol. Cell 2:863-867[CrossRef][Medline].
18.	Grant, P. A., D. E. Sterner, L. J. Duggan, J. L. Workman, and S. L. Berger. 1998. The SAGA unfolds: convergence of transcription regulators in chromatin-modifying complexes. Trends Cell Biol. 8:193-197[CrossRef][Medline].
19.	Grant, P. A., and J. L. Workman. 1998. Transcription. A lesson in sharing? Nature 396:410-411[Medline]. (News.)
20.	Hoffmann, A., C. M. Chiang, T. Oelgeschlager, X. Xie, S. K. Burley, Y. Nakatani, and R. G. Roeder. 1996. A histone octamer-like structure within TFIID. Nature 380:356-359[CrossRef][Medline].
21.	Hoffmann, A., T. Oelgeschlager, and R. G. Roeder. 1997. Considerations of transcriptional control mechanisms: do TFIID-core promoter complexes recapitulate nucleosome-like functions? Proc. Natl. Acad. Sci. USA 94:8928-8935[Abstract/Free Full Text].
22.	Hoffmann, A., and R. G. Roeder. 1996. Cloning and characterization of human TAF20/15. Multiple interactions suggest a central role in TFIID complex formation. J. Biol. Chem. 271:18194-18202[Abstract/Free Full Text].
23.	Holstege, F. C., E. G. Jennings, J. J. Wyrick, T. I. Lee, C. J. Hengartner, M. R. Green, T. R. Golub, E. S. Lander, and R. A. Young. 1998. Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95:717-728[CrossRef][Medline].
24.	Horiuchi, J., N. Silverman, B. Pina, G. A. Marcus, and L. Guarente. 1997. ADA1, a novel component of the ADA/GCN5 complex, has broader effects than GCN5, ADA2, or ADA3. Mol. Cell. Biol. 17:3220-3228[Abstract].
25.	Jacq, X., C. Brou, Y. Lutz, I. Davidson, P. Chambon, and L. Tora. 1994. Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor. Cell 79:107-117[CrossRef][Medline].
26.	Lavigne, A. C., Y. G. Gangloff, L. Carr, G. Mengus, C. Birck, O. Poch, C. Romier, D. Moras, and I. Davidson. 1999. Synergistic transcriptional activation by TATA-binding protein and hTAFII28 requires specific amino acids of the hTAFII28 histone fold. Mol. Cell. Biol. 19:5050-5060[Abstract/Free Full Text].
27.	Lavigne, A. C., G. Mengus, M. May, V. Dubrovskaya, L. Tora, P. Chambon, and I. Davidson. 1996. Multiple interactions between hTAFII55 and other TFIID subunits. Requirements for the formation of stable ternary complexes between hTAFII55 and the TATA-binding protein. J. Biol. Chem. 271:19774-19780[Abstract/Free Full Text].
28.	LeDouarin, B., A. L. Nielsen, J. M. Garnier, H. Ichinose, F. Jeanmougin, R. Losson, and P. Chambon. 1996. A possible involvement of TIF1 alpha and TIF1 beta in the epigenetic control of transcription by nuclear receptors. EMBO J. 15:6701-6715[Medline].
29.	LeDouarin, B., A. L. Nielsen, J. You, P. Chambon, and R. Losson. 1997. TIF1 alpha: a chromatin-specific mediator for the ligand-dependent activation function AF-2 of nuclear receptors? Biochem. Soc. Trans. 25:605-612[Medline].
30.	Luger, K., A. W. Mader, R. K. Richmond, D. F. Sargent, and T. J. Richmond. 1997. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389:251-260[CrossRef][Medline].
31.	Madison, J. M., and F. Winston. 1997. Evidence that Spt3 functionally interacts with Mot1, TFIIA, and TATA-binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae. Mol. Cell. Biol. 17:287-295[Abstract].
32.	Martinez, E., T. K. Kundu, J. Fu, and R. G. Roeder. 1998. A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID. J. Biol. Chem. 273:23781-23785[Abstract/Free Full Text].
33.	May, M., G. Mengus, A. C. Lavigne, P. Chambon, and I. Davidson. 1996. Human TAFII28 promotes transcriptional stimulation by activation function 2 of the retinoid X receptors. EMBO J. 15:3093-3104[Medline].
34.	Mazzarelli, J. M., G. Mengus, I. Davidson, and R. P. Ricciardi. 1997. The transactivation domain of adenovirus E1A interacts with the C terminus of human TAFII135. J. Virol. 71:7978-7983[Abstract].
35.	Mengus, G., M. May, L. Carre, P. Chambon, and I. Davidson. 1997. Human TAFII135 potentiates transcriptional activation by the AF-2s of the retinoic acid, vitamin D3, and thyroid hormone receptors in mammalian cells. Genes Dev. 11:1381-1395[Abstract/Free Full Text].
36.	Mengus, G., M. May, X. Jacq, A. Staub, L. Tora, P. Chambon, and I. Davidson. 1995. Cloning and characterization of hTAFII18, hTAFII20 and hTAFII28: three subunits of the human transcription factor TFIID. EMBO J. 14:1520-1531[Medline].
37.	Mermelstein, F., K. Yeung, J. Cao, J. A. Inostroza, H. Erdjument-Bromage, K. Eagelson, D. Landsman, P. Levitt, P. Tempst, and D. Reinberg. 1996. Requirement of a corepressor for Dr1-mediated repression of transcription. Genes Dev. 10:1033-1048[Abstract/Free Full Text].
38.	Michel, B., P. Komarnitsky, and S. Buratowski. 1998. Histone-like TAFs are essential for transcription in vivo. Mol. Cell 2:663-673[CrossRef][Medline].
39.	Moqtaderi, Z., M. Keaveney, and K. Struhl. 1998. The histone H3-like TAF is broadly required for transcription in yeast. Mol. Cell 2:675-682[CrossRef][Medline].
40.	Moqtaderi, Z., J. D. Yale, K. Struhl, and S. Buratowski. 1996. Yeast homologues of higher eukaryotic TFIID subunits. Proc. Natl. Acad. Sci. USA 93:14654-14658[Abstract/Free Full Text].
41.	Natarajan, K., B. M. Jackson, E. Rhee, and A. G. Hinnebusch. 1998. yTAFII61 has a general role in RNA polymerase II transcription and is required by Gcn4p to recruit the SAGA coactivator complex. Mol. Cell 2:683-692[CrossRef][Medline].
42.	Nikolov, D. B., H. Chen, E. D. Halay, A. Hoffman, R. G. Roeder, and S. K. Burley. 1996. Crystal structure of a human TATA box-binding protein/TATA element complex. Proc. Natl. Acad. Sci. USA 93:4862-4867[Abstract/Free Full Text].
43.	Ogryzko, V. V., T. Kotani, X. Zhang, R. L. Schlitz, T. Howard, X. J. Yang, B. H. Howard, J. Qin, and Y. Nakatani. 1998. Histone-like TAFs within the PCAF histone acetylase complex. Cell 94:35-44[CrossRef][Medline].
44.	Roberts, S. M., and F. Winston. 1997. Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Srb/mediator complexes. Genetics 147:451-465[Abstract].
45.	Saluja, D., M. F. Vassallo, and N. Tanese. 1998. Distinct subdomains of human TAFII130 are required for interactions with glutamine-rich transcriptional activators. Mol. Cell Biol. 18:5734-5743[Abstract/Free Full Text].
46.	Sanders, S. L., E. R. Klebanow, and P. A. Weil. 1999. TAF25p, a non-histone-like subunit of TFIID and SAGA complexes, is essential for total mRNA gene transcription in vivo. J. Biol. Chem. 274:18847-18850[Abstract/Free Full Text].
47.	Sterner, D. E., P. A. Grant, S. M. Roberts, L. J. Duggan, R. Belotserkovskaya, L. A. Pacella, F. Winston, J. L. Workman, and S. L. Berger. 1999. Functional organization of the yeast SAGA complex: distinct components involved in structural integrity, nucleosome acetylation, and TATA-binding protein interaction. Mol. Cell. Biol. 19:86-98[Abstract/Free Full Text].
48.	Tanese, N., D. Saluja, M. F. Vassallo, J. L. Chen, and A. Admon. 1996. Molecular cloning and analysis of two subunits of the human TFIID complex: hTAFII130 and hTAFII100. Proc. Natl. Acad. Sci. USA 93:13611-13616[Abstract/Free Full Text].
49.	Vojtek, A. B., S. M. Hollenberg, and J. A. Cooper. 1993. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74:205-214[CrossRef][Medline].
50.	vom Baur, E., M. Harbers, S. J. Um, A. Benecke, P. Chambon, and R. Losson. 1998. The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors. Genes Dev. 12:1278-1289[Abstract/Free Full Text].
51.	Walker, S. S., W. C. Shen, J. C. Reese, L. M. Apone, and M. R. Green. 1997. Yeast TAFII145 required for transcription of G1/S cyclin genes and regulated by the cellular growth state. Cell 90:607-614[CrossRef][Medline].
52.	Wieczorek, E., M. Brand, X. Jacq, and L. Tora. 1998. Function of TAFII-containing complex without TBP in transcription by RNA polymerase II. Nature 393:187-191[CrossRef][Medline].
53.	Workman, J. L., and R. E. Kingston. 1998. Alteration of nucleosome structure as a mechanism of transcriptional regulation. Annu. Rev. Biochem. 67:545-579[CrossRef][Medline].
54.	Xie, X., T. Kokubo, S. L. Cohen, U. A. Mirza, A. Hoffmann, B. T. Chait, R. G. Roeder, Y. Nakatani, and S. K. Burley. 1996. Structural similarity between TAFs and the heterotetrameric core of the histone octamer. Nature 380:316-322[CrossRef][Medline].
55.	Yamit-Hezi, A., and R. Dikstein. 1998. TAFII105 mediates activation of anti-apoptotic genes by NF-kappaB. EMBO J. 17:5161-5169[CrossRef][Medline].
56.	Yokomori, K., J. L. Chen, A. Admon, S. Zhou, and R. Tjian. 1993. Molecular cloning and characterization of dTAFII30 alpha and dTAFII30 beta: two small subunits of Drosophila TFIID. Genes Dev. 7:2587-2597[Abstract/Free Full Text].

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