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1 Graduate School of Frontier Biosciences, and
2 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
3 Laboratory of Gene Regulation, Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194, Japan
4 Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021, USA
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Abstract |
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 Top Abstract IntroductionResultsExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsExperimental proceduresReferences |
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Mediator orthologs are conserved among metazoans. The human Mediator complexes, thyroid hormone receptor-associated proteins (TRAP)/SRB-MED-containing cofactor (SMCC) (Ito et al. 1999), vitamin D receptor-interacting proteins (DRIP) (Rachez et al. 1999), activator-recruited cofactor (ARC) (Näär et al. 1999), cofactor required for Sp1 activation (CRSP) (Ryu et al. 1999), negative regulator of activated transcription (NAT) (Sun et al. 1998), positive cofactor 2 (PC2) (Malik et al. 2000), and the mouse Mediator complex (Jiang et al. 1998), were purified using various methods and turned out to possess essentially similar subunit compositions (Bourbon et al. 2004). These complexes fall into two broad classes based mainly on whether or not they contain the MED12–MED13–CDK8–Cyclin C module (reviewed in Malik & Roeder 2005). Biochemical studies indicated further that some complexes (TRAP, DRIP, PC2 and CRSP) positively regulate transcription (Rachez et al. 1999; Ryu et al. 1999; Malik et al. 2000), whereas others (NAT and ARC-L) negatively regulate it (Sun et al. 1998; Taatjes et al. 2002); SMCC was reported to have both positive and negative effects (Gu et al. 1999). Structural studies have demonstrated that there are several structural and conformational subtypes, and the largest Mediator complex in S. cerevisiae and in humans, consists of three modules: head, middle, and tail (Taatjes et al. 2002). Although this module composition is well-conserved from yeast to human (Malik & Roeder 2005), the Mediator undergoes conformational changes upon binding to different activators and Pol II (Näär et al. 2002; Taatjes et al. 2002), perhaps to respond to varying environmental situations.
In higher eukaryotes, there are three Cdk/cyclin-containing transcription factors: the general transcription factor TFIIH (containing CDK7/Cyclin H), the TRAP/Mediator complex (containing CDK8/Cyclin C), and the transcription elongation factor P-TEFb (containing CDK9/Cyclin T1), all of which are known to phosphorylate the CTD of the largest subunit of Pol II, thereby regulating Pol II function at important stages in transcription as well as at the later steps of gene expression (reviewed in Orphanides & Reinberg 2002). Although the Mediator complex is known to function at transcription initiation and possibly at the transition to elongation by phosphorylating the CTD of Pol II at the same step in transcription as does TFIIH, because of its compositional complexity it remains the least characterized of these three transcription factors.
In this paper, we established three HeLa cell lines, each expressing an N-terminal HA/FLAG-tagged (HF:) human Mediator subunit, hMED6, hMED7, or hCDK8 (the human homolog of SRB10). We examined complexes derived from these cell lines using biochemical approaches to assess if any related novel and/or uncharacterized Mediator subcomplexes that share some Mediator subunits additionally exist in the cell as well as to further elucidate the functional mechanisms of the previously identified 1.5-MDa human complex and CDK8. We especially focused on CDK8, which regulates transcription both positively and negatively in S. cerevisiae (Holstege et al. 1998; Liu et al. 2004). In humans, on the other hand, only a negative role has been reported for hCDK8 in the Mediator complex NAT, which represses transcription in vitro by phosphorylation of Cyclin C (Akoulitchev et al. 2000). Here, we observed that HF: hCDK8 was present in three complexes with molecular weights of 1.5 MDa, 1 MDa, and 600 kDa that had separate functional activities. To elucidate the mechanisms of these functions, we examined the effects of three Mediator subcomplexes on transcription, CTD phosphorylation of Pol II, and their physical interactions with glutathione S-transferase (GST)-tagged components of the general transcription machinery. In a final study, we examined the effect of knocking down hCDK8 expression with RNA interference on transcriptional activation by Gal4-VP16.
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Results |
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Biochemical studies have classified human SRB/MED-type Mediator complexes with molecular masses of 1–1.5 MDa into two types: one that augments activator-dependent transcription and one that counteracts it (reviewed in Malik & Roeder 2005). As a first step to further characterize the human Mediator complexes, we established three distinct HeLa cell lines constitutively expressing HF: hMED6, hMED7, or hCDK8 (Fig. 1). We chose these three TRAP/Mediator subunits because (i) hMED6 and hMED7 were reported as core subunits in all the reported SRB/MED-type Mediator complexes, (ii) genetic and biochemical studies of the S. cerevisiae Mediator complex indicate that hMED6 and hMED7 might be located in the head and middle modules, respectively, and (iii) hCDK8 is a kinase subunit that can regulate transcription by phosphorylating the CTD of the largest subunit of Pol II and might also be located in the middle module, but may be a conditional component of the Mediators (Malik & Roeder 2005). hCDK8 has also been reported to negatively regulate transcription in the Mediator complex (Sun et al. 1998; Taatjes et al. 2002). Each HF: subunit was expressed constitutively in the established cell lines, as identified by Western blotting with an anti-HA antibody (12CA5; Fig. 1A). [Note that although its calculated molecular weight is lower (27 243 Da versus 28 423), hMED7 migrates slower than hMED6 in SDS-PAGE because its estimated pI of hMED6 is much lower than that of hMED7 (5.4 versus 8.8).]
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We next tested the effects of the human TMLCs on in vitro transcriptional activation. We examined the 1-MDa fractions in addition to the 1.5-MDa and 600-kDa complex fractions (Fig. 3) because the band profiles were slightly different between the 1.5-MDa and 1-MDa complexes, as described above, and because several laboratories reported that there are transcriptionally positive and negative types of human Mediator complexes (reviewed in Malik & Roeder 2005). Although no complexes affected basal transcription in the absence of the potent transcriptional activator protein Gal4-VP16, the 1.5-MDa and 600-kDa complexes augmented Gal4-VP16-dependent transcriptional activation by about 1.4- to 2.1-fold in its presence (Fig. 3A, lanes 9, 10, 13, and 14 versus lane 2; Fig. 3B, lanes 7, 8, 15, and 16 versus lane 2). Strikingly, however, the 1-MDa complex from the hCDK8-cell line clearly reduced both basal (from the template that gives 290 nt transcripts) and Gal4-VP16-activated transcription (from the template that gives 390 nt transcripts) to an almost undetectable level in the presence of Gal4-VP16. However, the corresponding fractions from the hMED6 and hMED7-cell lines still augmented transcriptional activation, albeit to a reduced extent (about 1.5-fold) (Fig. 3B, lanes 9 and 10 versus Fig. 3A, lanes 11 and 12 and Fig. 3B, lanes 17 and 18). In contrast, this 1-MDa complex did not affect transcription when no Gal4-VP16 was added, regardless of the presence and the absence of the Gal4 binding sites (Fig. 3B, lane 1 versus lanes 5 and 6). Since we have observed three distinct functional complexes, we designated the 1.5-MDa, 1-MDa, and 600-kDa complexes TMLC 1, 2, and 3, respectively.
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CDK8-containing TRAP/Mediator complexes phosphorylate the CTD of the largest subunit of Pol II, particularly at Ser2 and Ser5 of the heptapeptide repeat sequence (YSPTSPS) (Sun et al. 1998; Park et al. 2001). We tested whether the three complexes we isolated (TMLC1, TMLC2, and TMLC3) also had this activity in vitro (Fig. 4). Among the three complexes, TMLC1 showed the CTD kinase activity on the largest subunit of Pol II (Fig. 4, lanes 5, 6, 11, 12, 17, and 18). TMLC2 also showed similar kinase activity (Fig. 4, lanes 8 and 9). The 1-MDa fractions from the hMED6 and hMED7-cell lines, which would correspond to TMLC2, phosphorylated Pol II despite much more weakly (Fig. 4, lanes 14, 15, 20, and 21 compared to lanes 8 and 9). In clear contrast to TMLC1 and TMLC2, the TMLC3 fractions did not show any kinase activity (Fig. 4, lanes 24 and 25).
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Although Pol II is reported to associate with the human Mediator complex (reviewed in Malik & Roeder 2005), no systematic study of the binding of the Mediator complexes to the other components of the general transcription machinery has been carried out. We therefore performed a GST-pull down analysis of TMLC1 from the HF: hMED6 and hMED7-cell lines using GST-fused general transcription factor subunits (Fig. 5, upper and lower panels). Bound TMLC1 was detected by Western blotting. TMLC1 predominantly bound to human TFIIB, TFIIEß, TFIIFß (Rap30), and the XPB, p62 and CDK7 subunits of TFIIH (Fig. 5, lanes 2, 4, 6, 11, 13, and 17). Faint binding to TBP, and the XPD and p52 subunits of TFIIH was also observed (Fig. 5, lanes 7, 12, and 14). Previous results by Y. J. Kim and his colleagues on the binding of the Drosophila Mediator complex to the GST-tagged general transcription components (except for TFIIH subunits) correlate with our result except for the binding to TFIIF subunits (Park et al. 2001). We observed that TMLC1 bound the smaller subunit TFIIFß, but the Drosophila Mediator complex bound to the larger TFIIF
. Considering this discrepancy, we will compare the binding specificities of the Mediator subcomplexes (TMLC1, 2, and 3) to the components of the general transcription machinery in future experiments.
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One of the Mediator subunits, CDK8, is known to be a component of the labile Mediator submodule consisting of CDK8, Cyclin C, MED12, and MED13. S. cerevisiae SRB10 (the yeast homolog of hCDK8) regulates transcription both positively and negatively (Holstege et al. 1998; Liu et al. 2004). In humans, however, the only functions that have been reported for hCDK8 have been negative; hCDK8 phosphorylates the Cyclin H subunit of TFIIH (Akoulitchev et al. 2000) and inactivates it and phosphorylates Hes-1 after recruitment by Mastermind and causes its degradation (Fryer et al. 2004). To further elucidate the mechanisms of hCDK8 function, we knocked it down using RNA interference. Five siRNA oligos were designed on hCDK8 mRNA (Fig. 6A and Experimental procedures), and their effects on hCDK8 expression were examined (Fig. 6B). Four of the five efficiently reduced hCDK8 expression, (Fig. 6B lanes 3 and 4 versus lanes 5–12) but CDK8-105 was less effective than the others.
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We selected two siRNAs (CDK8-119 and CDK8-480) to further elucidate the hCDK8 function. To judge the effectiveness and specificity of each siRNA, siRNA-treated HeLa cells were co-transfected with either of two hCDK8 cDNA plasmids, one of which expresses hCDK8 that is wild type except for a point mutation in the sequence corresponding to the siRNA that makes it resistant to the siRNA-mediated degradation (CDK8sir) and the other of which expresses hCDK8 with a mutation in the kinase domain, D151A, in addition to the siRNA-resistance mutation (CDK8sir D151A). The level of transcription in Gal4-VP16-stimulated control cells (treated with non-silencing siRNA or the control vector) was defined as "1." When the cells were treated with siRNA CDK8-119, the transcription activity was reduced to 40% of the control and siRNA CDK8-480 further reduced the transcription activity to 20% of the control (Fig. 7A). Transcription was not repressed when the mutant plasmid CDK8sir D151A was co-transfected; instead, transcription was repressed to as low as 40% of control. Treatment with siRNA CDK8-480 gave similar results, but transcription was reduced to a greater extent, to 20% of the level in the cells co-transfected with control vector. Treatment with CDK8-480 and the siRNA-resistant plasmids (CDK8sir and CDK8sir D151A) also gave results similar to those obtained with siRNA CDK8-119 and the siRNA-resistant plasmids.
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Discussion
In this study, we identified and characterized three distinct TMLC1-3 using HeLa cell lines expressing epitope-tagged human Mediator subunits (hMED6, hMED7, and hCDK8). All three complexes shared a subset of Mediator-specific subunits, yet the complexes functioned differently in transcription: TMLC1 augmented both transcriptional activation and CTD phosphorylation of Pol II, TMLC2 counteracted transcriptional activation but augumented CTD phosphorylation, and TMLC3 augmented transcription but failed to stimulate phosphorylation. A GST-pull down assay to evaluate the interaction with general transcription factors revealed that TMLC1 predominantly bound to TFIIB, TFIIEß, TFIIFß, and the XPB, p62, and Cdk7 subunits of TFIIH. Our binding profiles strengthen the previous interpretations that Mediator function in transcription initiation and the transition from initiation to elongation. We are convinced that our approach makes it possible to better define the functions of the human Mediator complexes (Sun et al. 1998; Näär et al. 1999; Rachez et al. 1999; Ryu et al. 1999; Malik et al. 2000, 2005; Taatjes et al. 2002) and to further elucidate the structures and functions of these complexes.
Three human TMLCs function distinctly in transcription
Recently, extensive studies have identified several important structural and functional features of Mediator and its related complexes. First, the structures and functions of the Mediator complexes are more conserved from yeast to human than we expected (Bourbon et al. 2004; Sato et al. 2004; Malik & Roeder 2005); for example, recent progress in sequencing of the genomes of various species led to the identification of orthologs of Sin4 and Gal11 in higher eukaryotes, hMED23 (hSur2) and hMED16 (TRAP95), respectively (Boyer et al. 1999; Ito et al. 1999). Second, two closely related Mediator complexes consisting of more than 20 SRB/MED-related subunits are conserved among eukaryotes; one positively regulates and the other negatively regulates transcriptional activation (reviewed in Malik & Roeder 2005). Third, another Mediator-related complex with a molecular weight around 600 kDa was reported as Mediator core (Medc) in S. cerevisiae (Liu et al. 2001), and as Mediator complex C1 in Drosophila (Gu et al. 2002). Indeed, Kim and coworkers identified three Drosophila Mediator complexes altogether (Gu et al. 2002), but their systematic structural and functional studies of these complexes have just begun.
In this study, we identified three distinct human Mediator-like complexes, TMLC1, TMLC2, and TMLC3, after purification of the nuclear extracts of three HeLa cell lines expressing epitope-tagged hMED6, hMED7, and hCDK8. The 1.5-MDa TMLC1 complex was isolated from all three cell lines, the 1-MDa TMLC2 complex was isolated solely from the hCDK8-cell line, and the 600-kDa TMLC3 complex was isolated solely from the hMED6-cell line. Functional studies of the transcriptional activation and CTD phosphorylation of Pol II demonstrated that the three complexes were functionally distinct. Previous models suggested that Mediator complexes containing hCDK8 negatively regulate transcription (Sun et al. 1998; Taatjes et al. 2002), but TMLC1 and TMLC2, which both contain hCDK8, behave differently (Fig. 2); TMLC1 augments transcriptional activation despite being isolated from the hCDK8-cell line, whereas TMCL2 represses transcription, consistent with the previous models. We observed that the TMLC1 fractions from the hCDK8-cell line contained higher amount of hMED12 than those from the other two cell lines (Fig. 1C, lane 2 versus lanes 3 and 4). This is likely to be the case because the TMLC1 fractions from the hCDK8 cell-line might consist of nearly 100% of the hCDK8 submodule-containing Mediator complexes whereas the TMLC1 fractions from the other cell line may mostly consist of the hCDK8 submodule-lacking Mediator complexes and less amount of the submodule-containing Mediator complexes. The important observation is that they both confer positive effects on Gal4-VP16-mediated transcriptional activation (Fig. 3A,B). Therefore, it is highly possible that as we purified it further, TMLC1 will be found to be consisting of two subtypes. The activity of human TMLC1 was similar to that of the major form of the yeast Mediator complex (Pol II-Med) identified by Liu et al. (2001), which also augments transcriptional activation despite containing SRB10 (yeast CDK8). The human ARC-L and PC2/CRSP complexes possess molecular masses of 2- and 1.25-MDa, respectively, which are similar to those of TMLC1 and TMLC2 (Taatjes et al. 2002), but their activities were opposite to those of TMLC1 and TMLC2; ARC-L, which contains hCDK8, negatively regulates transcription, and PC2/CRSP, which does not contain hCDK8 but does contain hMED26 (CRSP70), positively regulates transcriptional activation.
Our isolated TMLC3 does not resemble any of the previously identified Mediator complexes. Although like PC2, which has been described as an active complex of TRAP/Mediator (Malik et al. 2000, 2005), TMLC3 lacks CDK-Cyclin submodules (CDK8-Cyclin C-MED12-MED13; Fig. 2A), and it displays a distinct subunit structure based on the SDS-PAGE band pattern. The inability of TMLC3 to phosphorylate the Pol II CTD correlates well with the absence of this module (Fig. 4). Thus, TMLC3 effects on Pol II are likely to be indirect; that is, it stimulates transcriptional activation without modulating Pol II. Therefore, overall, we conclude that TMLC1 and TMLC3 affect transcriptional activation through distinct mechanisms.
The human Mediator subunit hCDK8 plays a positive role in transcriptional activation
Human Mediator complexes containing hCDK8 are reported to repress transcription by phosphorylating the CTD of Pol II and the Cyclin H subunit of TFIIH (Sun et al. 1998; Akoulitchev et al. 2000). When the Pol II CTD is phosphorylated, it cannot form a transcription preinitiation complex (Lu et al. 1991). To gain further insight into the functions of CDK8, we knocked it down using siRNA and studied its activity with a luciferase assay. Transcriptional activation by Gal4-VP16 was suppressed by both CDK8-119 and CDK8-480 siRNAs (Fig. 7A), indicating that hCDK8 functions positively in transcription in vivo. Co-transfection studies with the siRNA-resistant hCDK8 expression plasmid (either wild type or the kinase mutant D151A) confirmed this positive role because restoration of normal levels of wild-type hCDK8 restored the transcription activity, but introduction of the kinase mutant did not. This positive role perfectly corresponds to the CTD phosphorylation of both Ser2 and Ser5 (Fig. 7B, lanes 1, 3, and 6 versus lanes 2, 4, 5, and 7). Considering these results, we can imagine that it is the CTD kinase activity of hCDK8 of the Mediator complex that positively regulates transcription. It is still not known why Ser2 phosphorylation was suppressed by hCDK8 siRNA as much as Ser5 phosphorylation, because yeast CDK8 (SRB10) has been reported to predominantly phosphorylates Ser5 (Hengartner et al. 1998).
Although the positive effect of hCDK8 on transcription was clearly demonstrated in vivo, hCDK8 might have a negative regulatory function as well. For instance, we observed both positive and negative effects on in vitro transcriptional activation using two distinct hCDK8-containing subcomplexes (Fig. 3B). These contradictory functions are supported by various yeast studies. It is widely accepted that the CDK8 subunit in the Mediator complex negatively regulates transcription in vivo by phosphorylating the CTD of Pol II prior to formation of the preinitiation complex, or other transcription factors like Ste12 and the transcriptional co-repressor Tup1 (Hengartner et al. 1998; Nelson et al. 2003; Green & Johnson 2004). In contrast, it was also reported that CDK8 is required for efficient transcriptional activation by Gal4 and Sip4 (Vincent et al. 2001). It is clear that the subunit components of the TMLC1 and TMLC2 complexes are different; in particular, the lower band of the doublet band just above 220 kDa (possibly hMED12) and several subunits smaller than 30 kDa (especially those smaller than 15 kDa) display a lower stoichiometry in TMLC2 relative to TMLC1 (Fig. 3C). Since CDK8 is reported to form the so-called CDK8 submodule with three other Mediator subunits (Cyclin C, MED12, and MED13), our observation that the content of MED12 is not stoichiometric in TMLC2 might be noteworthy. Thus, an interesting possibility is that the Mediator complex possessing an intact CDK8 submodule has a positive role in transcriptional activation, but a partial CDK8 submodule that contains only CDK8 and Cyclin C has a negative role. At present, it is difficult to consider how the lower stoichiometry of the smaller subunits would directly convert the transcriptionally positive TMLC1 to the negative form TMLC2. In humans, at least two Mediator subcomplexes with distinct molecular masses have been reported so far (Ito et al. 1999; Malik et al. 2000, 2005; Taatjes et al. 2002) but extensive studies to elucidate their biological role are still underway. We need to further purify all three TMLCs to identify their constituent subunits by mass spectrometry and to elucidate their different functional mechanisms.
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Experimental procedures |
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For the basal transcription assays, the plasmid pML(C2AT)
-50 containing the adenovirus type 2 major late (AdML) promoter was used as a template (Sawadogo & Roeder 1985). To study transcriptional activation, the plasmid pG5HM(C2AT) was used as the test template (Chiang et al. 1993), with the plasmid pML(C2AT)-53Sh as the baseline control (Ohkuma et al. 1995). pG5HM(C2AT) contains five GAL4-binding sites and the core promoter, as described previously (Ohkuma et al. 1995). The two templates pML(C2AT)-50 and pG5HM(C2AT) give 390-nucleotide (nt) transcripts, and pML(C2AT)-53Sh gives a 290-nt transcript.
Antibodies
We used the following antibodies for Western blotting and immunoprecipitation assays of the human TRAP/Mediator complex subunits. Rabbit polyclonal antibodies against hMED1 (C-19; sc-5334), hMED6 (C-16; sc-9434), hMED12 (A-18; sc-5374), hMED13 (E-20; sc-12013), hMED23 (L-20; sc-9431), 
-tubulin (C-20; sc-7396), and the largest subunit of Pol II (N-20; sc-899) were purchased from Santa Cruz Biotechnology, the rabbit polyclonal antibody against hCDK8 was purchased from BioSource International Inc., and we prepared the rabbit polyclonal antibody against hMED18 was prepared by ourselves (Furumoto & Ohkuma, in preparation). The rabbit anti-hMED7, anti-hMED17 and anti-hMED20 polyclonal antibodies, and the mouse anti-HA monoclonal antibody (12CA5) are as described elsewhere (Malik et al. 2000; Watanabe et al. 2000). The mouse monoclonal antibodies (IgM) against phosphorylated Ser2 (H5) and Ser5 (H14) of the CTD heptapeptide repeat sequence of Pol II were purchased from Berkeley Antibody Company.
Construction of bacterial expression plasmids
To construct the plasmids expressing the human Mediator subunits, the cDNA sequences of hMED6, hMED7, and hCDK8 were retrieved from the Entrez DNA sequence database system. The GENBANK accession number of each cDNA was as follows: hMED6, NM_005466 [GenBank] ; hMED7, AF031383; and hCDK8, NM_001260 [GenBank] . The full-length cDNAs were isolated by PCR using the following combinations of PCR primers. For hMED6, the oligonucleotide HMED6-1T (5'-GAGCCTCATATGGCGGCGGTGGATATC-3') was designed to create an NdeI site (underlined) at the first methionine codon of hMed6 cDNA, and the oligonucleotide HMED6-1B (5'-GGACACTCGAGTACTCACTGAAGTCTCATC-3') was designed to create an XhoI site (underlined) after the stop codon. For hMED7, the oligonucleotide HMED7-1T (5'-CTCAGGCATATGGGTGAACCACAGCAAG-3') was used to create an NdeI site (underlined) at the first methionine codon of hMED7 cDNA, and the oligonucleotide HMED7-1B (5'-GTCTTGGATCCTTCATGGTCTTTCATTCATC-3') was used to create a BamHI site (underlined) after the stop codon. For hCDK8, the oligonucleotide HSR10-1T (5'-GACACCATATGGACTATGACTTTAAAGTG-3') was used to create an NdeI site (underlined) at the first methionine codon of hMED6 cDNA, and the oligonucleotide HSR10-1B (5'-CGTTCTCTCGAGTGCAGCTCAGTACCGATG-3') was used to create an XhoI site (underlined) after the stop codon. An NdeI-XhoI fragment of hMED6 cDNA, an NdeI-BamHI fragment of hMED7 cDNA, and an NdeI-XhoI fragment of hCDK8 cDNA were subcloned into the 6His-pET11d vector (Merck Biosciences). All PCR products were checked by sequencing using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).
Expression and purification of recombinant proteins
Recombinant proteins were expressed in Escherichia coli BL21(DE3)pLysS by induction with isopropyl-ß-D-thiogalactopyranoside (IPTG). The E. coli cells were harvested and resuspended with buffer B (BB500; 20 mM Tris–HCl [pH 7.9 at 4 °C], 0.5 mM EGTA, 10% [vol/vol] glycerol, 1 mM phenylethylsulfonyl fluoride [PMSF], 2 µg/mL antipain, 2 µg/mL aprotinin, 1 µg/mL leupeptin, 0.8 µg/mL pepstatin, 10 mM 2-mercaptoethanol, and 500 mM NaCl) (Ohkuma et al. 1995). After sonication, soluble bacterial lysates or the bacterial pellet solubilized with 2 M guanidine–HCl (pH 7.9) were used for purification depending on the protein solubilities (Watanabe et al. 2000). Six histidine (6His)-tagged proteins were purified through Ni-nitrilotriacetic acid (NTA) Sepharose (GE Healthcare Bio-Sciences) by eluting with buffer D (BD500; 20 mM Tris–HCl [pH 7.9 at 4 °C], 20% [vol/vol] glycerol, 1 mM PMSF, 10 mM 2-mercaptoethanol, and 500 mM KCl) containing 100 mM imidazole–HCl (pH 7.9). HA-tagged and GST-fusion proteins were also similarly expressed in E. coli BL21(DE3)pLysS by IPTG induction. E. coli cells from 50 to 100 mL of culture were harvested, resuspended in 1 mL of BB500, and sonicated. Soluble lysates were separated from insoluble debris by ultracentrifugation and stored at –80 °C until use.
Construction of eukaryotic expression plasmids and establishment of HeLa cell lines
We used two steps to construct the HF: hMED6, hMED7, and hCDK8 plasmids for establishing HeLa cell lines constitutively expressing these tagged subunits. First, the coding regions of hMED6, hMED7, and hCDK8 were digested from the above-described bacterial expression plasmids with NdeI and EcoRI and subcloned into the corresponding sites of the HA/FLAG(AS)-pGEM7 vector (Chiang et al. 1993). Second, these subunit coding regions with HA/FLAG-tags at the N-termini were cut out with EcoRV and EcoRI (New England Biolabs), and subcloned into the SmaI/EcoRI fragment of the pIRESneo2 mammalian expression vector (BD Biosciences).
Human HeLa S3 cells were transfected with the HF: hMED6, hMED7, and hCDK8 plasmids using the PolyFect mammalian cell transfection reagent (Qiagen). Stable cell lines were screened in the presence of 800 µg/mL G418. We selected two cell lines of the tagged subunits and established them in suspension culture.
Purification of Mediator and its related complexes
HeLa cells lines were grown in about 100 L of RPMI 1640 medium containing 10% calf serum, and about 100 mL of nuclear extracts (10 mg/mL protein) were prepared as described (Dignam et al. 1983). Each nuclear extract (100 mL) was adjusted to the same buffer composition as buffer C (20 mM Tris–HCl [pH 7.9 at 4 °C], 20% [vol/vol] glycerol, 0.5 mM EDTA [pH 8.0], 0.5 mM PMSF, 2 µg/mL antipain, 2 µg/mL aprotinin, 1 µg/mL leupeptin, 0.8 µg/mL pepstatin, 0.05% [vol/vol] NP40, and 10 mM 2-mercaptoethanol) containing 300 mM KCl (BC300) and loaded onto a 2-mL column of anti-FLAG M2 monoclonal antibody agarose (Sigma-Aldrich) at 4 °C. After washes with 10 mL BC300, the column was incubated overnight with 3 mL of 300 µg/mL FLAG peptide (Sigma-Aldrich) and the HF: TRAP/Mediator complexes were efficiently eluted. To fractionate the various Mediator subtypes and remove the free form of the tagged subunits, the eluted peak fractions were subjected to a TSKgel G4000SW high-performance liquid chromatography (HPLC) gel filtration column (2.15 x 30 cm; Tosoh) equilibrated with BC300. The HF:Mediator complexes were monitored by Western blotting with the anti-HA (12CA5) antibody. After SDS-PAGE in a PAG Mini 4%–20% acrylamide gradient gel (Daiichi Pure Chemicals), the separated proteins were transferred to an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore) as described previously (Ohkuma et al. 1995). Chemiluminescent signals were detected using the SuperSignal detection system (PIERCE) and RX-U film (Fuji Film).
In vitro transcription assays
TFIIH was purified from HeLa nuclear extracts as previously described (Watanabe et al. 2000). Pol II was purified from HeLa nuclear pellets by DE52, A25, P11, and HPLC-DEAE 5PW chromatography as described elsewhere (Watanabe et al. 2000). FLAG-tagged TBP (f-TBP)-containing TFIID (f-TFIID) was purified from nuclear extracts of f-TBP-expressing HeLa cells (cell line #3–10) through phosphocellulose (P11, Whatman) and anti-FLAG M2 monoclonal antibody agarose (Sigma-Aldrich) column chromatographies as described (Chiang et al. 1993). In vitro transcription was carried out as described (Ohkuma et al. 1995). To observe transcriptional activation, 20 ng of f-TFIID was used instead of 20 ng of 6His-TBP. As an activator, we used 40 ng of GAL4-VP16 containing the C-terminal acidic activation domain of VP16 (residues 413–490) fused to GAL4(1–94) (residues 1–94). Autoradiography was performed at –80 °C with Fuji RX-U X-ray film. The incorporation of [-32P] CTP (GE Healthcare Bio-Sciences) into transcripts was quantified using the Fuji BAS2500 Bio-Imaging analyzer.
Kinase assays
Assays were carried out essentially as described (Ohkuma & Roeder 1994) in the presence of Pol II. Phosphorylation reactions were performed at 30 °C for 1 h and stopped by the addition of 75 µL of phosphorylation stop solution (10 mM EDTA, 0.1% NP40, and 0.05% SDS). Phosphorylated proteins were TCA-precipitated, analyzed by SDS-PAGE (5.5% acrylamide), and detected by autoradiography performed at –80 °C with Fuji RX-U X-ray film.
GST pull-down assays
GST-fusion proteins were used for protein interaction assays. Each TRAP/Mediator complex (500 ng) was incubated with lysates containing 300 ng of GST-proteins together with 5 µL (packed volume) of Glutathione-Sepharose 4B (GE Healthcare Bio-Sciences) in a 500-µL reaction volume of buffer C with 100 mM KCl (BC100) containing 200 µg/mL BSA for 4 h at 4 °C with rotation. The glutathione-Sepharose resin was washed twice with 500 µL of buffer C with 200 mM KCl (BC200), and once with 500 µL of BC100, boiled in SDS sample buffer, and analyzed by SDS-PAGE (12% acrylamide). Pulled down proteins were detected by Western blotting with the anti-HA antibody (12CA5).
siRNA oligos
Five double-stranded RNA oligonucleotides were designed by B-Bridge International and generated by Dharmacon. The sequences of each strand of the five siRNA are as follows:
Cdk8-105: sense strand: 5'-CUACAAAGCCAAGAGGAAAdTdT-3'antisense strand: 5'-UUUCCUCUUGGCUUUGUAGdTdT-3'
Cdk8-119: sense strand: 5'-GGAAAGAUGGGAAGGAUGAdTdT-3'
antisense strand: 5'-UCAUCCUUCCCAUCUUUCCdTdT-3'
Cdk8-301: sense strand: 5'-GAACAUGACCUCUGGCAUAdTdT-3'
antisense strand: 5'-UAUGCCAGAGGUCAUGUUCdTdT-3'
Cdk8-480: sense strand: 5'-GGGUGAAGGUCCUGAGCGAdTdT-3'
antisense strand: 5'-UCGCUCAGGACCUUCACCCdTdT-3'
Cdk8-777: sense strand: 5'-GGGAUUUCCUGCAGAUAAAdTdT-3'
antisense strand: 5'-GGGAUUUCCUGCAGAUAAAdTdT-3'
The sequence of the non-silencing siRNA was 5'-AUUCUAUCACUAGCGUGACUU-3' (Dharmacon).
siRNA-resistant hCDK8 cDNAs
Two siRNA-resistant cDNAs were designed. For CDK8sir against Cdk8-119 siRNA, hCDK8 cDNA in the pBluescript cloning vector (Stratagene) was mutated at three sequential codons simultaneously corresponding to the amino acid residues 124D (GAT to GAC), 125G (GGG to GGA), and 126K (AAG to AAA) by using the site-directed mutagenesis kit (Molecular Biology Laboratory, Japan) together with the mutant oligo 1 (5'-AAAGCCAAGAGGAAA GACGGAAAAGATGATAAAGACTAT-3'). In the sequence, three changed nucleotides were underlined. For CDK8sir against Cdk8-480 siRNA, hCDK8 cDNA in the pBluescript cloning vector was similarly mutated at three sequential codons simultaneously corresponding to the amino acid residues 484E (GAA to GAG), 485G (GGT to GGC), and 486P (CCT to CCA) by using the site-directed mutagenesis kit together with the mutant oligo 2 (5'-ATTTTAGTTATGGGTGAGGGCCCAGAGCGAGGAAGAGTA-3'). In the sequence, three changed nucleotides were underlined. Similarly to the construction of the above-described wild-type hCDK8 expression plasmid, those mutated hCDK8 cDNAs were subcloned into the HA/FLAG(AS)-pGEM7 vector (Chiang et al. 1993), and further into the pIRESneo2 mammalian expression vector (BD Biosciences).
CTD kinase mutation in hCDK8
A point mutation was created in the pBluescript cloning vector (Stratagene) at the site essential for the CTD kinase activity of hCDK8 (D151A) by using the site-directed mutagenesis kit (Medical & Biological Laboratories) together with a mutant oligonucleotide (5'-TGGGTGTTGCACAGAGCTTTGAAACCTGCTAAT-3'). In the sequence, one mutated nucleotide is underlined. Similarly to the construction of the above-described hCDK8 expression plasmid, those mutated hCDK8 cDNAs were subcloned into the HA/FLAG(AS)-pGEM7 vector (Chiang et al. 1993), and further into the pIRESneo2 mammalian expression vector (BD Biosciences).
Luciferase assay
Luciferase assays were performed based on the method of Osada et al. (1999). HeLa S3 cells (2 x 104) were seeded into each well of 24-well plates. After 1 day, siRNA oligos (final concentration 33 nM) were transfected into cells using Lipofectamine 2000 (Invitrogen). Cells were cultured for 2.5 days and, after washing with PBS, were cotransfected with 100 ng of pE1b-TATA-luciferase reporter plasmid, 0.5 ng of pRL-TK (Renilla luciferase used as an internal control), and 0.5 ng of pM-VP16 (expression vector for Gal4-VP16). After 1 day, the cells were lysed and their transcription activities were quantitated using a PicaGene Dual SeaPansy Luminescence kit (Toyo Ink).
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Acknowledgements |
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Footnotes |
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aPresent Address: Department of Medical Technology, Faculty of Health Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142, Japan
* Correspondence: E-mail: ohkumay{at}pha.u-toyama.ac.jp |
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