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Màté A. Demény,
Elisabeth Scheer,
Giulia Fienga,
Paolo Sassone-Corsi, and
Làszlò Tora*
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104, Department of Transcriptional and Post-Transcriptional Control of Gene Regulation, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France
Received 7 December 2004/ Returned for modification 19 January 2005/ Accepted 23 February 2005
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TAF10 is not only an integral component of TFIID but is also found in the SAGA and TFTC-type coactivator complexes (28). Analysis of a variety of conditional Saccharomyces cerevisiae TAF10 mutants indicated that TAF10 regulates different subsets of genes, some of which are important for cell cycle progression and cell morphology (22, 23, 43). Drosophila melanogaster has two paralogue genes encoding TAF10 homologues (TAF10 and TAF10b), which are differentially expressed during Drosophila embryogenesis (14). D. melanogaster TAF10 was found to be present in both TFIID and TFTC-like complexes, but TAF10b was identified only in TFIID (14, 35). Similarly, in mammalian cells, TAF10 is shared between TFIID and the three closely related GCN5-containing multiprotein complexes: TFTC, PCAF/GCN5, and the STAGA complexes (28). Lack of TAF10 leads to cell cycle arrest and cell death by apoptosis in mouse F9 embryonic carcinoma cells (31). Moreover, in mouse F9 cells lacking TAF10, the integrity of most TFIID is compromised (32). Interestingly, in human HeLa cells, different TFIID complexes containing or lacking TAF10 exist and exhibit functionally distinct properties (6, 21). By generating a functionally null mutation of the TAF10 gene, we showed that TAF10 is required for early mouse development and survival of the pluripotent inner cell mass but not for survival of mouse trophoblast cells. Together, these data suggest that TAF10 is required for transcription of a subset of genes essential for specific cell types and differentiation pathways.
Electron microscopy, yeast genetics, X-ray crystallography, and biochemical experiments have shown that histone fold (HF) motifs mediate many of the subunit interactions within the TFIID and SAGA complexes (11). It was shown that S. cerevisiae TAF3 and TAF8 contain putative HF motifs and that both of these domains selectively heterodimerize with S. cerevisiae TAF10, which also contains a putative HF (13). The mapping of histone-like TAFs in yeast TFIID revealed that these TAF pairs are also formed in three distinct lobes within endogenous TFIID (25). Furthermore, yeast Spt7p, a component of the yeast SAGA complex (17, 49), has an HF motif similar to that of S. cerevisiae TAF3, which selectively heterodimerizes with the HF domain of S. cerevisiae TAF10, defining an additional histone-like pair in the SAGA complex (13). Thus, yeast TAF10 has three HF-containing interaction partners: S. cerevisiae TAF3 and TAF8 and Spt7. The mammalian and Drosophila homologues of TAF3 contain an N-terminal HF domain, which selectively heterodimerizes with members of the TAF10 family (12). The isolated HF of human TAF3 heterodimerizes with that of human TAF10, whereas the HF of D. melanogaster TAF3 is more selective since it interacts only with D. melanogaster TAF10 and not with D. melanogaster TAF10b (12). Drosophila Prodos, also called D. melanogaster TAF8 (45), contains an HF motif, which, in contrast to that of D. melanogaster TAF3, selectively heterodimerizes with D. melanogaster TAF10b but not with D. melanogaster TAF10 (20). Consequently, it has been proposed that Drosophila Prodos is a Drosophila TFIID component (20). Recently, the human homologue of TAF8 (TAFII43) was described as an integral component of TFIID (18). Human TAF8 is an orthologue of mouse Taube Nuss, which is essential for early embryonic mouse developmental events (46). Interestingly, Taube Nuss–/– and TAF10–/– mice show the same very early embryonic lethality, indicating that the lack of either TAF8 or TAF10 leads to selective inner cell mass death (32, 46). These knockout-mouse results further underline the possibility that these two mammalian proteins interact in vivo and have similar roles in the respective TAF-containing complexes. A human factor with significant similarity to yeast Spt7, called STAGA-associated factor 65
or human SPT7Like (hereafter SPT7L), has also been described as a STAGA or TFTC subunit (30).
With the use of in vitro interaction assays, human TAF10 has been shown to associate with its three HF-containing interaction partners: TAF8, TAF3, and SPT7L. However, nothing was known about the associations of these factors in living cells. Here we investigate the roles of the mammalian TAF10-interacting partners in the nuclear localization of TAF10. We show that human TAF10 does not have a nuclear localization signal and its nuclear transport is dependent entirely on interaction(s) with TAF3, TAF8, or SPT7L. The potential implications of the regulation of the nuclear localization of TAF10 by its partners are discussed.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunization and antibody production. The QSPDDSDSSYGSHSTDSLMG(C) peptide corresponding to SPT7L amino acids 377 to 396 was synthesized, coupled to ovalbumin, and used for generation of mouse monoclonal antibodies as described in reference 6. All the other antibodies used were previously described (2, 12, 26, 32, 47).
Immunoprecipitation and Western blot analysis. Sf9 cell lysates were prepared as described in reference 26. Proteins of Sf9 cell lysates (from a 75-cm2 Falcon flask) were subjected to immunoprecipitation (IP) with 100 µl protein G-Sepharose (Pharmacia) and approximately 5 µg of the different antibodies (as indicated in the figures). Antibody-protein G-Sepharose-bound protein complexes were washed three times with IP buffer (25 mM Tris-HCl [pH 7.9], 10% [vol/vol] glycerol, 0.1% NP-40, 0.5 mM dithiothreitol, 5 mM MgCl2) containing 0.5 M KCl and twice with IP buffer containing 100 mM KCl. After washing, protein G-antibody-bound proteins were either eluted by an excess of the corresponding epitope peptide or directly boiled in sodium dodecyl sulfate (SDS) sample buffer and separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto nitrocellulose membrane, and probed with the primary antibodies as indicated in the figures. Chemiluminescence detection was performed according to the manufacturer's instructions (Amersham).
Immunofluorescence. Indirect immunofluorescence tests were performed on cells growing on coverslips. Cells were fixed in 2% paraformaldehyde for 10 min and permeabilized in 0.5% Triton X-100 for 10 min. After blocking in 1% bovine serum albumin-phosphate-buffered saline (PBS), they were incubated with anti-TAF10 monoclonal antibody (mAb; 2B11; diluted 1:1,000), antihemagglutinin (anti-HA) mAb (12CA5; diluted 1:1,000), or anti-Flag mAb (M5; diluted 1:1,000) followed by Cy3-labeled anti-mouse secondary antibody (diluted 1:1,000; Jackson Laboratories). When applicable, nuclei were stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride; 50 ng/ml). Images were analyzed by using either a wide-field fluorescence Leica (DMIRBE) microscope with a Cool Snap Ropers camera or a Leica laser-based confocal microscope.
Pull downs.
Six-His-tagged glutathione S-transferase (GST), six-His-tagged importin 
, and six-His-tagged importin ß were expressed, purified, and immobilized on nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagene) as described previously (7, 15). HeLa cells were transfected with 0.5 µg of the expression vectors described above, and whole-cell extracts were prepared in buffer B (20 mM HEPES [pH 7.9], 300 mM KCl, 10% glycerol, 15 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin). Nickel-based pull-down assays were performed by incubating the importin-containing beads with whole-cell extracts for 5 h at 4°C in buffer B. The beads were washed five times with the same buffer. The presence of the CFP, YFP, or Flag fusion proteins in the input or bound to the His-tagged importin and His-tagged importin ß was detected by Western blot analysis using the anti-GFP antibody (NEB) or anti-Flag antibody.
Imaging and fluorescence recovery after photobleaching (FRAP). We used the Leica TCS SP2 (AOBS) confocal microscope operating with a 40-mW argon laser. The laser was tuned to lines of 458 nm and 514 nm. Cells were examined with a 63x/1.3-numerical-aperture oil immersion objective and 2.5x zoom. The gain of photomultiplier tubes and the emission intervals were adjusted to eliminate cross talk between the CFP (470 to 500 nm) and YFP (530 to 600 nm) channels.
Live-cell microscopy was performed on a Zeiss Meta confocal microscope using the 405-nm diode and 514-nm laser line of an Ar laser (nominal output, 40 mW; beam width at specimen, 0.2 µm). All experiments were done at 37°C, and imaging was done with a 63x objective and numerical aperture of 1.3. Scanning was bidirectional at the highest possible rate with a 4x zoom and a pinhole of 1 Airy unit. Laser power for bleaching was maximal. For imaging, the laser power was attenuated to 0.1% of the bleach intensity. FRAP experiments were performed as described in reference 38 with few modifications. Briefly, six single-prebleach images were acquired, followed by two iterative bleach pulses of 223 ms each. Single-section images were then collected at 250-ms intervals for 60 s. Recovery of signal in the bleached region and loss of signal in the unbleached region were measured as average intensity signals in a region comprising at least 50% of the bleached or unbleached area. The sizes of this measurement region were identical in all experiments. All recovery curves were generated from background-subtracted images. The fluorescence signal measured in a region of interest was individually normalized to the prebleach signal in the region of interest according to the following equation: R = (It – Ibg)/(Io – Ibg), where Io is the average intensity in the region of interest during prebleaching, It is the average intensity in the region of interest at time point t, and Ibg is the background signal determined in a region outside of the cell nucleus.
Bleaching of the whole nuclear fluorescent pools was performed using the same settings as before except that two single-prebleach images were acquired, followed by two iterative bleach pulses, and pictures were collected at 2-min intervals for 30 min.
Immunohistochemistry on squash preparations from microdissected seminiferous tubules. Immunofluorescence tests were performed on squash preparations of microdissected tubules from adult mice (37). The slides were fixed with 4% paraformaldehyde in 1x PBS and permeabilized with 0.2% Triton-1x PBS. After blocking for 1 h in 5% bovine serum albumin in 1x PBS-0.05% Tween 20, the slides were incubated overnight at 4°C with the antibody indicated in the figures (anti-TAF10 [4G2] mAb, diluted 1:300; anti-TAF8 [1FR 1A2] mAb, diluted 1:300; or anti-TBP [3G3] mAb, diluted 1:1,000) (6, 21, 32). The slides were then washed with 1x PBS and incubated with a secondary antibody (Cy3-conjugated rabbit anti-mouse, diluted 1:500). Immunostained samples were counterstained with DAPI (Boehringer) before being subjected to microscopy.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When the cellular localization of these proteins was analyzed by fluorescence microscopy, as expected the TAF3-, TAF8-, and SPT7L-containing fusion proteins all localized exclusively to the nucleus (Fig. 3A and data not shown). Surprisingly, and in contrast to endogenous TAF10 protein (Fig. 3B), neither the CFP-TAF10 nor the YFP-TAF10 fusion protein localized exclusively to the nucleus (Fig. 3A). The expression of these proteins was cytoplasmic rather than nuclear (Fig. 3A).
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Next, we examined whether the deletion of the nonessential N-terminal domain of TAF10 would allow its nuclear localization. When a series of truncated TAF10 mutants were used to transfect cells (Fig. 3D), they all remained mostly cytoplasmic, indicating that the N-terminal 100 amino acids of TAF10 do not influence its cellular localization (Fig. 3D). These results suggest that TAF10, in contrast with TAF3, TAF8, and SPT7L, is not able to efficiently enter the nucleus or is actively exported from there.
Leptomycin B does not change the localization of TAF10. The above-described results suggested two possibilities concerning the lack of nuclear localization of exogenously expressed TAF10. It is possible that (i) TAF10 is able to enter by itself into the nucleus and that it is then retained there through interaction with one of its partners but that the excess of "free" TAF10 is exported from the nucleus to the cytoplasm through a nuclear export signal (NES) or that (ii) TAF10 is not able to enter efficiently by itself into the nucleus. The nuclear pore complex protein CRM1 is able to recognize specific leucine-rich NESs to mediate protein export from the nucleus (33). CRM1 is a target of the cytotoxin leptomycin B (LMB). To test the above-listed first possibility and to check whether blocking of CRM1-dependent nuclear export changes the cytoplasmic localization of TAF10, we incubated the pYFP-TAF10-transfected cells for 4 h with 20 ng/ml of LMB. There was no influence of LMB on the cellular localization of TAF10 (Fig. 4A), but a GFP-NES sequence fusion protein was retained in the nucleus under the same conditions (data not shown). These data suggest that if TAF10 shuttles between the nucleus and the cytoplasm, it is not exported into the cytoplasm through a CRM1-dependent pathway.
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To determine whether the highly conserved domain of TAF10, containing only the putative HF domain (14, 23), would be sufficient for the nuclear localization of the protein, we tested the subcellular localization of the TAF10 deletion mutant comprising amino acids 100 to 218 [TAF10(100-218)] in the presence of the three HF-containing interaction partners. TAF10(100-218) fused either to CFP or to YFP alone was found mainly in the cytoplasm (Fig. 3D), but in the presence of TAF3, TAF8, or SPT7L fusion proteins, the TAF10(100-218) deletion mutant localized to the nucleus (Fig. 4D). These results further suggest that TAF10 interacts in the cells with its three partners and that the domain of interaction with these three partners is within the evolutionarily conserved putative HF motif of TAF10.
Next, we tested whether the HF domains of the partners were necessary for the nuclear localization of TAF10. To this end, we cotransfected cells with full-length YFP-TAF10 and a TAF8 deletion mutant that lacks the putative HF of TAF8 [TAF8(105-308)] (Fig. 2 and Fig. 4E). When the HF was deleted from TAF8, the truncated protein still localized to the nucleus, but it could not participate in the nuclear translocation of YFP-TAF10. Similar results were obtained when the HF of SPT7L was deleted (data not shown). The above-described results suggest that the interactions of TAF10 with its partners through its HF domain are crucial for its nuclear translocation and/or retention process.
The nuclear localization of TAF10 is dependent on the NLS present in its interaction partners. The hypothesis that the nuclear localization of TAF8, TAF3, and SPT7L is involved in the nuclear translocation of TAF10 prompted us to search for potential short peptides within the amino acid sequences of these proteins that are sufficient and necessary for their nuclear localization. Most frequently, an NLS comprises a short stretch of basic amino acids (16). Such putative NLS sequences in both TAF8 and SPT7L were predicted to be at the very C-terminal ends of the two proteins (297-KKPKIRRKKSLS-308 for TAF8 and 405-KKRMRKI-412 for SPT7L) by the PSORT II program. Thus, we constructed short C-terminal deletion mutants of TAF8 [TAF8(1-294)] and SPT7L [SPT7L(1-403)] fused with YFP or CFP (Fig. 2). First, we observed the localization of the mutants after transfection into HeLa cells. The deletion mutants CFP-TAF8(1-294) and SPT7L(1-403) were completely cytoplasmic (Fig. 5A, right panels; data not shown), suggesting that their NLS sequences are located within the deleted amino acid regions. In contrast, for TAF3 no single C-terminal NLS sequence has been identified; instead, several NLS sequences were predicted by the computer analysis. However, deleting most of them did not change the nuclear localization of TAF3 (data not shown).
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A heterologous NLS sequence fused to TAF10 is sufficient to translocate TAF10 to the nucleus. To test whether TAF10 alone can be efficiently imported into and/or retained in the nucleus in the absence of a coexpressed interaction partner, we fused either the NLS sequence derived from the SV40 T antigen (NLSSV40) or the above-characterized NLS sequence of TAF8 (NLSTAF8) to the N-terminal end of the full-length TAF10 (Fig. 2). As controls we used vectors expressing either CFP-NLSSV40, which contains the NLS from the SV40 T antigen alone, or CFP-NLSTAF8, which contains only the above-characterized NLS of TAF8 (Fig. 5B). When the CFP-TAF10-NLSSV40 and the CFP-TAF10-NLSTAF8 fusion proteins containing heterologous NLSs were expressed in HeLa cells, they were exclusively nuclear (Fig. 5C). The above-described in vivo results together indicate that TAF10 itself does not have an NLS but that once the protein is imported into the nucleus, either by a heterologous NLS or by one of its three partners, it remains nuclear. Thus, the in vivo nuclear localization of TAF10 seems to be regulated by its complex formation with one its HF motif-containing partners, TAF3, TAF8, or SPT7L.
FRAP measurements indicate that cytoplasmic TAF10 does not bind to any partner or structure until it is transported into the nucleus by TAF8 or an NLS. FRAP is a technique that can be used to quantitatively measure the kinetics of binding of proteins to unperturbed structures in living cells. The rationale for this approach is based on the property that FRAP kinetics reflect the overall mobility of a protein. For proteins that do not interact with any cellular structures, FRAP kinetics are a direct reflection of their translational motion properties. In contrast, for proteins that bind to relatively immobile structures such as chromatin, binding events slow down the protein's overall mobility (reference 39 and references therein). We used FRAP measurements to compare the binding properties of TAF10 in the cytoplasmic and nuclear compartments. To this end, after irreversible bleaching of a circular region of interest (ROI) by using a 250-ms pulse with a 514-nm laser, we measured the recovery of fluorescence intensity in the bleached spot. When YFP-TAF10 was overexpressed alone in HeLa cells, the ROI was selected in the cytoplasm and the time to reach half of the maximal recovery (t50) was short (t50 was less than 250 ms) and comparable to the time for recovery of the YFP protein (Fig. 6A), suggesting that TAF10 diffuses rather freely and does not bind to any cytoplasmic partner or compartment. When TAF8 was coexpressed along with YFP-TAF10, an ROI within the nucleoplasm was bleached and the fluorescence recovery of YFP-TAF10 was assessed. In this case, 50% recovery of YFP-TAF10 was obtained in 3 s and complete recovery in 40 s (Fig. 6A). The fact that this recovery rate is significantly slower than that measured for YFP or TAF10 alone (3 s versus 250 ms) (Fig. 6A) is consistent with the notion that the slower mobility of TAF10 in the nucleus is a result of the binding of TAF10 to a nuclear structure, such as chromatin, possibly after an interaction with TAF8, and possible subsequent incorporation into complexes such as TFIID/TFTC. Note that comparable t50s (between 3 and 8 s) were observed for other chromatin-associated proteins, such as BRG1, PCAF, and XPB (39). Importantly, a similar slow recovery kinetic was observed in the nucleus for YFP-TAF10-NLSTAF8, which contains a heterologous NLS sequence (Fig. 6A). These data together support the idea that when TAF10 is in the nucleus it can bind to similar nuclear proteins and structures, as YFP-TAF10 cotranslocated with TAF8, further indicating that the interaction partners of TAF10 play a role primarily in the nuclear accumulation of the factor but that once it is in the nucleus it can dynamically incorporate into TAF-containing complexes and/or bind to nuclear structures.
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In contrast to TAF10 alone, TAF10/TAF8 and the TAF10/TAF3 HF-containing pairs bind to importin ß in vitro.
Different NLSs have been reported to interact with distinct importins (16). The best-studied interactions involve the import receptor for classical NLS-containing proteins, consisting of an importin ß/importin dimer. Importin recognizes the cargo NLS but requires importin ß to cross the nuclear pore complex and to deliver the cargo protein inside the nucleus (16). To further investigate the mechanism of the translocation of the TAF10/TAF3, TAF10/TAF8, and TAF10/SPT7L heterodimers into the cell nucleus, we performed in vitro pull-down experiments. To this end, Ni-NTA resin-bound six-His-tagged GST (as a control), six-His-tagged importin (Rch-1), and six-His-tagged importin ß (15) were incubated with extracts from HeLa cells transfected with either a TAF10 expression vector alone or combinations of expression vectors expressing TAF10 with each of its interacting partners (Fig. 7). When extracts from TAF10-transfected cells, in which TAF10 is mostly cytoplasmic, were analyzed, a very weak interaction with importin or importin ß was observed (Fig. 7A). On the other hand, when extracts containing coexpressed TAF10/TAF8 or TAF10/TAF3 were incubated with the different importin-containing beads, we observed efficient binding of TAF10 to importin ß (Fig. 7B and E, lower panels). Control experiments showed that YFP-TAF8 and Flag-TAF3, when used individually for transfection, interacted with both importins but predominantly with importin . The above-described results suggest that TAF10 alone does not bind to either importin or ß but that it can bind to importin ß through its dimerization with either TAF3 or TAF8. These findings are in good agreement with our in vivo data and further show that the importin /ß-dependent nuclear translocation process of TAF10 involves its dimerization with either TAF3 or TAF8.
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or ß, we repeated the previous experiments with extracts from HeLa cells either cotransfected with TAF10 and the NLS-lacking TAF8(1-294), which both remain in the cytoplasm, or transfected with the TAF10-NLSTAF8 expression vector, which is exclusively nuclear. When TAF8 lacks its NLS, as in the TAF8(1-294) mutant, it cannot bind to either importin or importin ß and consequently TAF10 does not interact with importin ß (Fig. 7C). However, when TAF10 protein contains a heterologous NLS, it can efficiently interact with importin and mainly with importin ß (Fig. 7D). The data given above together indicate that TAF10 needs to have an NLS-containing partner, or a heterologous NLS fused to it, to be able to bind to importin ß.
YFP-SPT7L was not found to bind to either importin or importin ß, and coexpression with TAF10 did not change the affinity of either protein for the importins (Fig. 7F). Thus, our results also suggest that for nuclear translocation the TAF10/SPT7L complex uses a different pathway than importin /ß. These in vitro interaction studies further show that the nuclear import of TAF10 is regulated through its interaction partners.
Endogenous TAF10 and TAF8 are cytoplasmic in primary adult spermatocytes. In order to gain further insight into the nuclear transport of TAF10 in a natural setting, we have investigated its intracellular localization at various stages of the differentiation process of adult male germ cells. Both TAF8 and TAF10 are expressed in these cells and are likely to participate in the complex transcriptional program which takes place postmeiotically (44). Our analysis reveals that the proteins display highly similar distribution patterns, being cytoplasmic in primary adult spermatocytes and then nuclear in round spermatids. Notably, both TAF8 and TAF10 localize in the perinuclear cytoplasmic region in spermatocytes but are excluded from the transcriptionally inactive, highly heterochromatic chromocenter in the round spermatids (Fig. 8). TBP, in contrast, remains nuclear at all times during these differentiation steps (Fig. 8) (27). Thus, the processes of nuclear transport of TAF8 and TAF10 appear to occur in concert, possibly to allow the proper activation of the postmeiotic transcriptional program.
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DISCUSSION |
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Here we found a new role for the HF-containing TAFs by investigating the regulation of TAF10 nuclear localization by its three HF-containing interaction partners. We observed that overexpressed TAF10, in contrast to the native protein, is more abundant in the cytoplasm of HeLa cells than in the nucleus and can be transferred to the nucleus upon overexpression of one of its partners. In agreement with the in vitro data, this result indicates that TAF10 interacts with its three HF-containing partners, TAF3, TAF8, and SPT7L, also in vivo. Moreover, we observed that if we eliminate the weak fluorescent nuclear pool of TAF10 by photobleaching, the remaining cytoplasmic TAF10 pool does not translocate to the nucleus through a passive diffusion mechanism. In contrast, YFP and YFP-tet proteins freely diffuse through the two subcellular compartments. These data together with previous observations with Drosophila cells (14) suggest that the expression levels of endogenous partners of TAF10 (TAF3, TAF8, and SPT7L) can be limited in the cells and that "free" TAF10, which is not part of any histone fold dimer combination, remains cytoplasmic (see also below). We investigated two different possibilities for the failure of the "free" or overexpressed pool of TAF10 to localize to the nucleus: (i) the overexpressed protein cannot be retained in the nucleus and is rapidly exported to the cytoplasm due to the limited amounts of the endogenous retention partner in the nucleus or (ii) TAF10, due to the lack of a functional NLS or because a cytoplasmic protein masks its NLS, can be imported into the nucleus only as a dimer with one of its NLS-containing partners and thus the "free" TAF10 protein would remain cytoplasmic. Several lines of evidence confirmed the second possibility. LMB treatment did not change the cytoplasmic localization of TAF10, suggesting that it is not exported into the cytoplasm through a CRM1-dependent pathway. Interestingly, the nuclear export of another TAF, TAF4b (formerly TAFII105), is mediated by a composite nuclear export signal and thus the nuclear export of TAF4b was suggested to happen through a CRM1-independent nuclear export pathway (42). Nevertheless, the cytoplasmic localization of all the truncated derivatives of TAF10 excluded the existence of an export signal insensitive to LMB and rather indicated the absence of a domain that can function as a nuclear localization signal. This conclusion was also supported by the observation that a heterologous NLS can drive TAF10 into the nucleus. The observation that the overexpressed CFP-TAF10-NLSSV40 and CFP-TAF10-NLSTAF8 fusion proteins remain nuclear excludes export or cytoplasmic retention as a mechanism for the cytoplasmic accumulation of the exogenously expressed TAF10 protein. The above-mentioned conclusion is in complete agreement with the fluorescent recovery kinetics after photobleaching measurements. The diffusion is measured in FRAP experiments over a micrometer range, and the transient interaction of a protein with subcellular structures is rate limiting and slows down the overall recovery rate of a given protein in a FRAP assay. As the t50 measured for TAF10 in the cytoplasm ranges from 10 to 100 ms, similar to the rate of free diffusion of YFP or CFP itself, our data suggest that TAF10, when excluded from the nucleus, moves like a "free" protein without binding to any partner or cytoplasmic subcellular structures. Although YFP-TAF10 does not seem to bind tightly to any cytoplasmic structure or partner, we cannot exclude the possibility of transient association of TAF10 with a cytoplasmic partner that would somehow counteract the passive diffusion of YFP-TAF10 to the nucleus. Alternatively, it could be possible that TAF10 in the cytoplasm forms dimers or oligomers (as suggested earlier [21]), which then would not be able to diffuse passively through the nuclear pore.
We also investigated the possible mechanism by which TAF10 enters into the nucleus. The lack of nuclear localization of TAF10 by its truncated partners, from which the NLSs have been deleted, suggested that TAF10 does not have an NLS and enters the nucleus only with its NLS-containing partners. Moreover, we showed that TAF10 could not efficiently interact with importin and ß when overexpressed alone. On the other hand, TAF3 and TAF8 interact efficiently with importin , probably through their NLSs, and by dimerizing with TAF10 through their histone fold domains can bring TAF10 to importin ß. Transport receptors cannot always bind their cargoes directly; in some cases they need an adapter molecule, which complicates the transport cycles considerably. The best-studied adapter is importin , which mediates import of proteins that carry mostly classical NLSs. Importin binds both the cargo and importin ß in the cytoplasm. Importin ß accounts for contacts with the nuclear pore complex (8, 16) and mediates translocation of the trimeric NLS-containing factor-importin /ß complex from the cytoplasmic to the nuclear side of the nuclear pore complex. TAF10 lacks a functional NLS; thus, it does not bind to importin and cannot subsequently bind to importin ß. TAF8 or TAF3 alone is able to bind to importin , but in the presence of TAF10, TAF8 or TAF3 binds predominantly to importin ß. It is possible that in the TAF8-TAF10 (or TAF3-TAF10) heterodimer, the conformation of TAF8 (or TAF3) is changed and thus the dimer has a higher affinity for importin ß. The fact that the third interaction partner of TAF10, SPT7L, uses an import pathway other than the importin /ß complex to achieve the nuclear import of the corresponding dimer suggests a different regulatory mechanism for the nuclear import and the consequent incorporation of the SPT7L-TAF10 dimer into the TFTC complex.
Recent development in in vivo imaging technologies allowed us to start to investigate fundamental mechanistic questions about how transcription complexes assemble and thus to better understand how they regulate transcription in the cells. In this respect, the important questions to answer are whether the different complexes, which have been purified biochemically as intact entities and which have been suggested to be recruited to the preinitiation complex as multisubunit complexes, are (i) assembled as subcomplexes or whole complexes in the cytoplasm, (ii) assembled as subcomplexes or whole complexes in the nucleus, or (iii) assembled within the nucleoplasm before DNA binding or on the promoter of a given gene. The TAF-containing complexes TFIID and TFTC have a unique characteristic because most of their components form dimers through their histone fold domains (11, 25). The present study on the nuclear localization of TAF10 reveals a new regulatory mechanism in the assembly of the TAF-containing complexes. We show that the first step of the assembly of these complexes takes place in the cytoplasm by the formation of histone fold dimers and is followed by coimport into the nucleus. Our biochemical and in vivo data together show that once TAF10 is imported into the nucleus, it presumably incorporates efficiently into the complexes and binds to chromatin, suggesting that in the case of TAF10 this first step of assembly in the cytoplasm is fundamentally important for the completion of the whole process.
Drosophila has two distinct homologues of human TAF10, D. melanogaster TAF10 and TAF10b, which are encoded by two paralogue genes. It has been shown that the subcellular localization of both D. melanogaster TAF10 and TAF10b proteins changes during development and can also depend on the cell type (14). Here we report that the intracellular localization of mouse TAF10 during adult spermatogenesis is regulated in concert with one of its partners, TAF8. Because of the lack of antibodies against TAF3 and SPT7L, which would display a specific signal in male germ cells, we cannot exclude the possibility that these two other partners may be regulated in a similar manner. We have found that TAF10 is cytoplasmic in pachytene spermatocytes and nuclear in round spermatids following the localization of TAF8 in the same cell types. This observation reinforces the notion that the concerted nuclear transport of TAF10 and TAF8 is indeed a physiologically relevant event, as it coincides with the postmeiotic wave of transcriptional activation (44). Importantly, other TAFs, such as TAF7L, show this developmentally regulated intracellular localization as well (40), suggesting a general control mechanism of the composition of TFIID and other TAF-containing complexes during spermatogenesis.
Similar regulatory interactions that specifically affect the subcellular localization of proteins have been described (4). In particular, the nuclear localization of the extradenticle and PBX1 proteins is regionally restricted during Drosophila and mammalian development and depends on the nuclear localization of their interacting proteins HTH and PREP1 (4).
Here we describe a novel mechanism in which HF-containing proteins can regulate the local action of their interacting partners by facilitating their import into the nucleus. This also suggests that in its cytoplasm, depending on with which interaction partner TAF10 heterodimerizes, the cell determines into which TAF-containing complex (TFIID or TFTC) TAF10 will be incorporated once transported into the nucleus. Thus, in this study we have unraveled a novel regulatory role for the three histone fold-containing interacting partners of TAF10. The question of whether this is a general role for most of the known histone fold-containing TAFs or other proteins during cellular differentiation or metazoan development remains to be answered.
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ACKNOWLEDGMENTS |
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E.S. was supported by an EMBO long-term fellowship, M.A.D. by a fellowship from the European Community (grant HPRN-CT-2000-00088), and G.F. by a fellowship of the Fondation de la Recherche Médicale. This work was supported by funds from INSERM, CNRS, Hôpital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, and the Front Nationale de la Science ACI and by European Community RTN (HPRN-CT-2000-00087, HPRN-CT-2000-00088, and HPRN-CT 00504228), STREP (LSHG-CT-2004-502950), and AICR (03-084) grants.
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FOOTNOTES |
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Present address: National Cancer Institute, NIH, 41 Library Drive, Bldg. 41 B507, Bethesda, MD 20892.
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