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Genes to Cells (2008) 13, 817-826. doi:10.1111/j.1365-2443.2008.01208.x
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Human mediator kinase subunit CDK11 plays a negative role in viral activator VP16-dependent transcriptional regulation

 Taiki Tsutsui1, Hiroyasu Umemura1, Aki Tanaka1,2, Fumitaka Mizuki1, Yutaka Hirose1,2 and Yoshiaki Ohkuma1,2,*

1 Laboratory of Gene Regulation, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
2 Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Saitama, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Mediator is an essential transcriptional cofactor of RNA polymerase II (Pol II) in eukaryotes. This cofactor is a large complex containing up to 30 subunits and consisting of four modules: head, middle, tail, and CDK/Cyclin. Generally, Mediator connects transcriptional regulators, cofactors, chromatin regulators, and chromatin remodellers, with the pre-initiation complex to provide a platform for the assembly of these factors. Many previous studies have revealed that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits mediating the pivotal roles of Mediator in transcriptional regulation. In addition to CDK8, CDK11 is conserved among vertebrates as a Mediator subunit and closely resembles CDK8. While the role of CDK8 has been studied extensively, little is known of the role of CDK11 in Mediator. We purified human CDK11 (hCDK11)-containing protein complexes from an epitope-tagged hCDK11-expressing HeLa cell line and found that hCDK11 could independently form Mediator complexes devoid of human CDK8 (hCDK8). To investigate the in vivo transcriptional activity of the complex, we employed a luciferase assay. Although hCDK11 has nearly 80% amino acid sequence identity to hCDK8, siRNA-knockdown study revealed that hCDK8 and hCDK11 possess opposing functions in viral activator VP16-dependent transcriptional regulation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Precise temporal and spatial transcriptional regulation of protein-encoding genes by RNA polymerase II (Pol II) is vital to the execution of complex gene expression programs in vertebrates in response to growth, developmental and homeostatic signals (reviewed in Roeder 2005). In response to such signals, Pol II is regulated at several steps: (i) recruitment of Pol II and additional general transcription factors to the transcribing gene promoters and formation of the pre-initiation complex; (ii) initiation of RNA synthesis (promoter clearance); (iii) transcriptional elongation from the promoter proximal region to the terminal region; and (iv) transcriptional termination and re-initiation. The Mediator complex (Mediator) is one of the large protein complexes which is thought to regulate the Pol II recruitment step by interacting with Pol II and general transcription factors, and the subsequent transcription initiation step by regulation of C-terminal domain (CTD) phosphorylation of the largest subunit of Pol II in collaboration with TFIIH (Thompson et al. 1993; Kim et al. 1994; Sun et al. 1998; Baek et al. 2006). In addition, Mediator may regulate the elongation step as well by interacting with TFIIS (Wery et al. 2004; Guglielmi et al. 2007).

Mediator was first discovered by genetic studies in the budding yeast Saccharomyces cerevisiae, in which defects caused by deletion of the CTD were suppressed by a family of proteins (SRB; supressor of RNA polymerase B/Pol II) (Nonet & Young 1989). Several components in Mediator, which were found to regulate environmental responses, had previously been identified by yeast genetic screening (reviewed in Myers & Kornberg 2000). Shortly thereafter, Mediator was discovered to relieve squelching in vitro using a crude nuclear extract (Kelleher et al. 1990). Yeast Mediator was then purified to elucidate its function (Kim et al. 1994). The mammalian Mediator complexes were discovered as activator binding complexes in several independent investigations (Fondell et al. 1996; Rachez et al. 1998; Gu et al. 1999; Näär et al. 1999). Mediators are conserved from yeast to humans and contain more than 20 conserved subunits (reviewed in Malik & Roeder 2005). Several conserved subunits are essential for cell survival, and many others are required in regulation of cell differentiation or proliferation in higher eukaryotes (Gim et al. 2001; Wang et al. 2006; Beyer et al. 2007; Lin et al. 2007; Bosveld et al. 2008).

Among these subunits, CDK8 functions as a serine/threonine kinase and is required for several developmental events in metazoa, but is not required for cell survival (Loncle et al. 2007; Westerling et al. 2007). CDK8 generally functions as a negative regulatory component in Mediator and various repressive functions have been demonstrated to date (Hengartner et al. 1998; Akoulitchev et al. 2000). Recently, however, CDK8 was shown to possess a pivotal role in transcriptional regulation by Mediator (Akoulitchev et al. 2000; Liu et al. 2004; Furumoto et al. 2007). We found at least two Mediator subcomplexes that contain human CDK8 (hCDK8), but exhibit opposite effects on transcriptional activation (Furumoto et al. 2007). Thus, CDK8 plays crucial roles in gene expression (Andrau et al. 2006; Donner et al. 2007).

However, another kinase subunit CDK11/CDK8-L (also called CDC2L6) was recently identified in the human Mediator complex using multidimensional protein identification technology (MudPIT) (Sato et al. 2004). Human CDK11 (hCDK11) is very similar to hCDK8. Mammals and other higher eukaryotes possess several sets of proteins similar to Mediator subunits (MED12L and MED12, and MED13L and MED13). MED13L (also called PROSIT240) is mutated in patients with the congenital heart defect characterized by transposition of the great arteries (Muncke et al. 2003). Thus we hypothesized that hCDK11 possesses a physiologically distinct role from hCDK8. In this study, we purified hCDK11 from HeLa cells and conducted luciferase assays in siRNA-treated cells to examine the regulation of gene expression. First we established N-terminal epitope-tagged hCDK11 expressing HeLa cell lines to affinity purify hCDK11-containing complexes from HeLa nuclear extracts. The molecular weights of the purified protein complexes were estimated by gel filtration, and the cellular distribution of the protein was examined by immunofluorescence using a recombinant hCDK11 expression cell line. Finally, to study the in vivo function of hCDK11, we employed siRNA knockdown analysis in VP16-activated transcription. As a result, we found the following: (i) hCDK11 forms Mediator complexes which possess common components with hCDK8-containing Mediator although contain hCDK11 in place of hCDK8; (ii) half of hCDK11 does not co-localize with hCDK8 in human cells; and (iii) hCDK11-containing Mediator actually plays a negative role in VP16-dependent transcriptional regulation in vivo in clear contrast to hCDK8-containing one.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
CDK11 is conserved among vertebrates

In human Mediators, two similar CDKs (hCDK8 and hCDK11) have been identified using MudPIT (Sato et al. 2004). As shown in Fig. 1A, human CDK11 (hCDK11) is a hCDK8 paralogue and possesses a serine/threonine kinase domain with 97% identity to CDK8. In the full sequence, hCDK11 has approximately 80% identity to hCDK8. The amino acid sequences of hCDK11 and hCDK8 are aligned in Fig. 1B. Intriguingly, searches of available genomes indicate that CDK11 is only present among vertebrates. hCDK11 possesses 38 additional amino acid residues in the C-terminus, compared to hCDK8, and, including a glutamine rich stretch that is characteristic for hCDK11.


Figure 1
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  		Figure 1  Comparison of CDK8 and CDK11. (A) Schematic representation of human CDK8 and CDK11. Numbers indicate amino acid positions. Both hCDK8 and hCDK11 possess a serine/threonine kinase domain (S/T kinase domain). This domain was approximately 97% identical between hCDK8 and hCDK11. (B) Alignment of two CDKs. CDK11 and CDK8 from Homo sapiens (h), Mus musculus (m) and Xenopus tropicalis (xt) are shown. CDK11 is conserved among vertebrates.

 
Affinity purification of epitope tagged hCDK11-containing complexes

As a first step in characterizing hCDK11, we established HeLa cell lines expressing N-terminally HA and FLAG tandem-tagged (HF:) hCDK11 (Fig 2A). hCDK11-containing complexes were affinity purified using anti-FLAG M2-agarose. The eluted fractions were separated by SDS-PAGE and the separated proteins were detected by both silver staining and Western blotting (Fig. 2B and C). In parallel, the same studies were performed for hCDK8-containing complex (Fig. 2B and C, lanes 2). The silver-stained gel indicates that hCDK11 interacts with a large number of nuclear proteins similarly to hCDK8, many of which may be Mediator components (Fig. 2B, lane 3 versus lane 2). Western blotting demonstrated that both hCDK8-containing and hCDK11-containing complexes possessed the same subunits (Fig. 2C lanes 2 and 3). These results suggest that hCDK11 forms Mediator complexes that sharing same components of hCDK8-containing Mediator but does contain hCDK11 in place of hCDK8. In Fig. 2B, two of the major bands present in the mock eluted fraction are the heavy and light chains of anti-FLAG M2 antibody which had dissociated from the M2-agarose beads.


Figure 2
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  		Figure 2  Purification of HA/FLAG-tagged (HF:) hCDK11 and HF:hCDK8. (A) Establishment of HF:hCDK11-expressing HeLa cell lines. Top: HF:hCDK11 was subcloned into the pIRESneo2 expression vector, and the constructed plasmid was transfected into HeLa S3 cells. The pIRESneo2 vector alone was also transfected as a mock control. Bottom: whole lysates from the established clones were subjected to Western blot analysis using an anti-HA antibody (12CA5). Arrows indicate the position of HF:hCDK11. The asterisk denotes a non-specific band. (B) SDS-PAGE of the HF:hCDK8-containing and HF:hCDK11-containing complexes. Nuclear extract (NE) was prepared from either HF:hCDK11 clone #3 cell line or previously reported HF:hCDK8 cell line, loaded onto an anti-FLAG M2 agarose beads, and bound proteins were eluted using the FLAG peptide. Eluates were subjected to SDS-PAGE and the gel was silver stained. 1: Eluate from vector trasfected NE, 2: Eluate from HF:hCDK8 expression cell line NE, 3: Eluate from HF:hCDK11 expression cell line NE. Asterisks indicate the non-specifically leaked mouse IgG heavy chain and light chain. Arrows on the right side of picture indicates each tagged CDK. (C) Western blot analysis of purified hCDK8-containing and hCDK11-containing complexes. Eluates were subjected to SDS-PAGE and Mediator subunits were detected using specific antibodies to these subunits.

 
Cellular localization of hCDK11

To elucidate hCDK11 function in human cells, we examined the localization of HF:hCDK11 expressed in HeLa cells by double staining the cells with anti-HA (12CA5, Roche) and anti-hCDK8 (Santa Cruz Biotechnology) (Fig. 3A,B, and C). Both hCDK11 and hCDK8 exist predominantly in the nucleus (Fig. 3A) and the localization of hCDK8 is consistent with a previous report (Fryer et al. 2004). To determine whether hCDK11 co-localizes with hCDK8, we examined the distribution of both CDKs in the nucleus at higher magnification. The distribution of HF:hCDK11 resembles that of hCDK8, but there are many foci which do not overlap each other when the images are merged (Fig. 3B and C, right panels). As similar patterns were observed in all analyzed cells, it appears that at least a portion of HF:hCDK11 localizes to sites distinct from hCDK8 in the nucleus. The fact that hCDK11 and hCDK8 are concentrated in nuclear foci probably reflects Mediator occupancy of particular promoters. Therefore, the distinct foci suggest that some promoters are predominantly occupied by hCDK11, but not by hCDK8.


Figure 3
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  		Figure 3  Localization of hCDK11 and hCDK8. (A) Immunofluorescence using anti-HA (left) and anti-hCDK8 (middle). DAPI (right) was used to stain nuclei. Both HF:hCDK11 and hCDK8 predominantly localized in the nucleus. The scale bar is 25 µm. (B) Magnified images of immuno-stained nuclei. Left, anti-HA treated; middle, anti-hCDK8 treated; right: merge. The scale bar indicate 5 µm. (C) Further magnified images of cell nucleus. Left, anti-HA; middle, anti-hCDK8; right, merge. HF:hCDK11 displayed a localization pattern similar to hCDK8, but some nuclear foci were predominantly hCDK11 in the merged image. The scale bar is 1 µm.

 
hCDK11 forms Mediator complexes

This difference in localization of the two CDKs raises fundamental questions about hCDK11: (i) Can hCDK11 form Mediator complexes? and (ii) Do they lack hCDK8? To address these issues, affinity purified HF:hCDK11-containing proteins were loaded onto an HPLC-G4000SWXL gel filtration column and fractionated according to molecular size. Each fraction was then examined by SDS-PAGE and protein bands were detected by both silver staining and Western blotting (Fig. 4A and B). HF:hCDK11 was detected at approximately 2 MDa and 100–300 kDa (Fig. 4B, lane 5, and lanes 11 and 12). hCyclin C was co-purified with HF:hCDK11 at both molecular size ranges but, in addition, was detected in two smaller size fractions (Fig. 4B, lanes 6 and 7). hMED6 was detected only at approximately 1.5 MDa (Fig. 4B, lane 5). In contrast, hCDK8 was not detected in these fractions. These results strongly indicate that hCDK11 forms a 2-MDa Mediator complex without hCDK8.


Figure 4
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  		Figure 4  Gel filtration of anti-FLAG M2 agarose eluted proteins. (A) SDS-PAGE of gel filtration fractions. The anti-FLAG M2 agarose eluate was separated using an HPLC TSKgel G4000SWXL gel filtration column (7.5 mm I.D. x 30 cm, Tosoh). Each fraction was loaded onto a PAG mini 4%–20% acrylamide gradient gel (Daiichi Pure Chemicals). Samples of the eluate fractions were separated by SDS-PAGE and the gel was silver stained. (B) Western blotting of gel filtration fractions. Arrows indicate the positions of molecular weight markers. hCyclin C and hMED6 were co-purified with HF:hCDK11 in the around 2 MDa molecular weight fraction.

 
Cellular functions of hCDK11

To investigate the in vivo functions of hCDK11, cellular expression was knockeddown using siRNAs against hCDK11. The effect on Gal4–VP16 activated transcription was then measured using a luciferase assay. The designed siRNAs were transfected into HeLa S3 cells and the amount of expressed mRNA was measured by reverse transcription (RT)-PCR (Fig. 5A). As shown previously (Furumoto et al. 2007), two short inhibitory RNAs (siRNAs), CDK8-119 and CDK8-480, specifically reduced hCDK8 mRNA expression, but did not affect hCDK11 mRNA expression (Fig. 5A, lanes 2 and 3, middle and top panels). Conversely, two siRNAs against hCDK11, CDK11-903, and CDK11-955, specifically reduced hCDK11 mRNA expression, but did not affect hCDK8 expression (Fig. 5A, lanes 4 and 5, top and middle panels). As siRNAs against hCDK11 or hCDK8 specifically reduced the target mRNA, we conducted luciferase assays to explore the function of each hCDK in Gal4–VP16 dependent transcriptional activation (Fig. 5B). Each siRNA was transfected into HeLa S3 cells and, after 2.5 days, cells were transfected with the Gal4 binding domain-containing luciferase reporter plasmid, with or without the Gal4–VP16 expression plasmid. Luciferase activity was calculated relative to luciferase activity in the presence of Gal4–VP16 and a non-target control siRNA. When cells were treated with siRNAs against hCDK8, luciferase activity was reduced to 40% of control (Fig. 5B, hCDK8 119 and 480). In contrast, siRNA against hCDK11 significantly increased luciferase activity by 2.5-folds over the control (Fig. 5B, hCDK11 903 and 955).


Figure 5
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  		Figure 5  Effects of siRNA knockdown of hCDK8 and hCDK11 on Gal4–VP16 activated transcription. (A) Knockdown efficiency of each siRNA. siRNAs, non-target control (NC), CDK8-119, CDK8-480, CDK11-903 and CDK11-955, were transfected into HeLa S3 cells, and RT-PCR analysis was carried out to measure mRNA abundance. Mock indicates no template control. (B) The effects of the knockdown on transcriptional activation were measured by reporter assay in the presence or absence of the Gal4–VP16 activator. All luciferase activities are presented as values relative to Gal4–VP16 transcription in the presence of the NC siRNA normalized to 1.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study we characterized a novel Mediator subunit, hCDK11, which exists predominantly in the nucleus, similar to hCDK8 (Fig. 3), but forms distinct Mediator complexes lacking hCDK8 (Fig. 4B). hCDK11 partially co-localized with hCDK8 but more than half of hCDK11 and hCDK8 were uniquely localized in nuclei (Fig. 3B). The siRNA analyses of VP16-dependent transcriptional activation indicated that hCDK8 and hCDK11 have opposite roles in transcriptional activation (Fig. 5B). As previously reported, hCDK8 acted as a positive regulator in vivo in VP16-dependent transcriptional activation (Furumoto et al. 2007), but, unexpectedly, hCDK11 acted as a negative regulator under the same conditions. This result is striking when we considered the high degree of homology between the amino acid sequences of these hCDKs (more than 80% identity throughout the entire sequence, Fig. 1A). These data suggest that hCDK11 is not just a redundant paralogue of hCDK8, but rather may have roles distinct from hCDK8 in transcriptional regulation.

Half of hCDK11 and hCDK8 uniquely localize in the nucleus

Through immuno-staining studies we demonstrated that both HF:hCDK11 and hCDK8 predominantly localized in the nucleus (Fig. 3A). Both hCDK11 and hCDK8 formed foci, but hCDK11 partially co-localized with hCDK8. Half of the protein did not overlap and uniquely localized at distinct sites (Fig. 3B and C). These results were confirmed by gel filtration following affinity chromatography with anti-FLAG M2 agarose (Fig. 4). The gel filtration chromatography revealed that hCDK11 forms Mediator complexes lacking hCDK8 (Fig. 4B, top and bottom panels). From these results, we hypothesize that hCDK11 plays a unique role by localizing in foci containing gene promoters which hCDK11 alone specifically regulates. We think there are, at present, two possibilities to interpret the opposing roles of the two hCDKs in transcription. One possibility is that hCDK8 binds to the gene essential for transcriptional activation pathway and hCDK11, on the other hand, binds to the gene important for transcriptional repression. The other possibility is that hCDK8 forms complexes with transcriptional co-activators and hCDK11 forms complexes with transcriptional co-repressors. In HeLa cells, the relative amount of hCDK11 expressed was lower than that of hCDK8 (Tsutsui, data not shown). Therefore, in future studies we will use a cell line in which hCDK11 is highly expressed to investigate the roles of endogenous hCDK11 more precisely. In a previous report, hCDK8 was shown to accumulate in nuclear foci (Fryer et al. 2004), but in our study, the distribution of hCDK8 in the nucleus was almost entirely uniform. The differences in localization from these two analyses may arise from differences in antibody specificities or cell culture conditions.

hCDK11 and hCDK8 are mutually exclusive components of Mediator complexes

As shown in Figs 2 and 4, hCDK11 formed a large 2 MDa Mediator complex lacking hCDK8 but containing hCyclin C. hCyclin C co-precipitated with hCDK11 similarly to that with hCDK8 (Fig. 2C, lanes 2 and 3). The hCDK11-containing Mediator was similar in molecular weight to the previously reported large form of Mediator complexes and, therefore, contained several reported Mediator components (Figs 2C and 4B). hCyclin C was also observed in lower molecular weight fractions together with hCDK11. This complex is probably an hCDK11/hCyclin C pair distinct from the 2-MDa Mediator complex. To elucidate the functional differences between hCDK8 and hCDK11, further examination of the in vitro transcription mechanisms will be required.

hCDK11 negatively regulates VP16-dependent transcription, in contrast to hCDK8

In the siRNA knockdowns, VP16-dependent transcription was clearly down-regulated in vivo by hCDK11 (Fig. 5B). However, the precise mechanism of the negative regulation of VP16-dependent transcription is still unclear. In mammalian cells, there are a few reports indicating a negative regulatory role for the Mediator complex in vivo (Fryer et al. 2004; Mo et al. 2004). But, in yeast, several distinct roles have been reported (Hengartner et al. 1998; Chi et al. 2001; Nelson et al. 2003). Since we observed a negative effect of hCDK11 on VP16-dependent transcriptional activation, the most attractive possibility is that hCDK11 forms Mediator complexes that physically interact with negative cofactors and directs transcription of target genes to be negatively regulated. However, there are many other possibilities as well: (i) hCDK11 phosphorylates VP16, which results in its degradation or export from the nucleus to the cytoplasm; (ii) hCDK11 phosphorylates the Pol II CTD and prevents its entry into the pre-initiation complex; or (iii) hCDK11 phosphorylates Cyclin H of TFIIH and inactivates its transcriptional activity. In addition, our siRNA studies could not exclude the possibility of indirect cellular side-effects of siRNAs against hCDK11. At present, however, it is clear that hCDK11 plays a role distinct from hCDK8. Recently, a CDK8-inactive mutation was shown to be lethal to mice in pre-implantation stage even though CDK11 was present (Westerling et al. 2007). This result confirms the distinct roles of CDK11 and CDK8. Studies are currently underway to elucidate these distinct roles in the near future.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Construction of a eukaryotic expression plasmid carrying CDK11 and establishment of epitope-tagged HeLa cell lines

The HA/FLAG-epitope tagged (HF:) CDK11 vector was constructed by two step subcloning. First, the CDK11 open reading frame was amplified by PCR from the full length cDNA clone (Open Biosystems, MHS1010-9203618) using a combination of PCR primers. The oligonucleotide CDK11-1T (5'-CCGTTC ATATGGATTATGATTTCAAGG-3') was designed to create an NdeI site (underlined) at the first methionine codon of hCDK11 cDNA and the oligonucleotide CDK11-1B (5'-GAGTTCTC GAGCTGGTCAGTACCGGTGG-3') was designed to create a XhoI site (underlined) after the stop codon. The amplified fragment was subcloned into the HA/FLAG (AS)-pGEM7 vector (Chiang & Roeder 1993). The fragment with the HA/FLAG-tag at the N-terminus of the hCDK11 open reading frame was then cut out with NcoI and BamHI from the subcloned vector, and was subcloned into the pIRESneo2 vector (Clontech). All PCR products were verified by sequencing using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Human HeLa S3 cells were transfected the HF:CDK11 plasmid using the PolyFect mammalian cell transfection reagent (Qiagen). Five stable cell lines were selected in the presence of 800 µg/mL G418, and clone #3 was established in suspension culture and used for the following studies.

Purification of hCDK11-containing complexes

The HA/FLAG-tagged hCDK11-expressing HeLa cell line (clone #3) or HA/FLAG-tagged hCDK8-expressing HeLa cell line were grown in approximately 50 L of RPMI 1640 medium containing 5% calf serum, and approximately 40 mL of nuclear extract was prepared as described (Dignam et al. 1983). The nuclear extract was adjusted to the composition of 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 1 mL of this nuclear extract was incubated with 50 µL of anti-FLAG M2 agarose (Sigma-Aldrich) for 4 h at 4 °C. After five washes with 1 mL BC300, bound proteins were eluted from beads by incubation for 30 min at 4 °C with 150 µL of BC300 containing 300 µg/mL FLAG peptide.

The eluted fraction was analyzed by Western blotting with anti-HA (Roche, 12CA5), anti-MED1(Santa Cruz, sc-9959), anti-MED6 (Santa Cruz, sc-9434), anti-MED13 (Santa Cruz, sc-12013), anti-MED17 (kindly gifted from Professor Robert G. Roeder), anti-MED18 (Our laboratory stock), anti-MED20 (Santa Cruz, sc-5382), anti-MED23 (Santa Cruz, sc-9431), anti-MED24 (kindly gifted from Professor Robert G. Roeder), and anti-Cyclin C (Santa Cruz, sc-1061). The eluted fraction was also subjected to SDS-PAGE and silver stained. The remaining extract was purified by gel filtration chromatography using a TSK gel G4000SWXL 7.8 mm x 30 cm column (Tosoh) equilibrated with BC300. The purified complex was separated using a PAG Mini 4%–20% acrylamide gradient gel (Daiichi Pure Chemicals), and the separated proteins were transferred to an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore) as described previously (Ohkuma et al. 1995). And incubate with anti-HA or anti-MED6 or anti-Cyclin C or anti-CDK8 (BD, F9–1075). Chemiluminescent signals were detected using the ECL Western blotting signal detection kit (GE Healthcare) and RX-U film (Fuji Film).

Immunofluorescence

HF:CDK11 expressing cells were incubated on a slide glass with DMEM containing 5% calf serum for 24 h at 37 °C and fixed for 15 min by addition of 3.7% (vol/vol) paraformaldehyde in PBS at room temperature. The fixed cells were permeabilized using 0.5% (vol/vol) Triton-X100 in PBS for 5 min on ice. The fixed cells were incubated overnight at 4 °C with an anti-HA mouse monoclonal antibody (1/10 dilution) (Roche, 12CA5) and an anti-CDK8 (Santa Cruz, sc-1521) goat polyclonal antibody (1/500 dilution) in PBS-BAG (1% (vol/vol) BSA, 0.1% sodium azide, 0.5% (vol/vol) gelatin). Alexa 488 or 594-conjugated secondary antibodies (Invitrogen) were diluted in PBS-BAG (1/500 dilution) and samples were incubated in the antibodies for 4 h at room temperature. Finally, after nuclear staining with DAPI, the slides were examined using a Leica TCS-SP5 confocal laser scanning microscope with a 63x oil-immersion objective.

siRNA transfection

Five double-stranded RNA oligonucleotides were designed by Dharmacon. The sequences of each strand of the five siRNAs are as follows:


CDK8-119: sense strand: 5'-CUACAAAGCCAAGAGGAAAdTdT-3'
antisense strand: 5'-UCAUCCUUCCCAUCUUUCCdTdT-3'
Cdk8-480: sense strand: 5'-GGGUGAAGGUCCUGAGCGAdTdT-3'
antisense strand: 5'-UCGCUCAGGACCUUCACCCdTdT-3'
CDK11-903: sense strand: 5'-GAUCCAACCAAGAGAAUUAdTdT-3'
antisense strand: 5'-UAAUUCUCUUGGUUGGAUCdTdT-3'
CDK11-955: sense strand: 5'-GGUCAAGCCUGACAGCAAAdTdT-3'
antisense strand: 5'-UUUGCUGUCAGGCUUGACCdTdT-3'
Non-silencing siRNA: sense strand: 5'-AUUCUAUCACUAGCGUGACUU-3'
antisense strand: 5'-GUCACGCUAGUGAUAGAAUUU-3'

For transfection of siRNAs into a HeLa S3 cell line, cells (2 x 104 cells) were seeded in each well of a 24-well-plate and, after culturing for 1 day, siRNA oligos (final concentration 33 nM) were transfected into the cells using Lipofectoamine 2000 (Invitrogen). After culturing for additional 2.5 days, cells were lysed and total RNA was prepared using an RNeasy mini isolation kit (Qiagen) or luciferase assays were conducted.

Reverse transcription-polymerase chain reaction (RT-PCR)

RNA was reverse transcribed using the Primescript RT reagent Kit (TaKaRa) and the synthesized first strand cDNAs were amplified by the following gene specific primers:


CDK11: forward 5'-GTCAGTCTACCTTAGAGAAAGCCAG-3'
reverse 5'-AACTCCTTGAGAGCAAGAAC-3'
CDK8: forward 5'-CCCTGAACTACTTCTTGGAGC-3'
reverse 5'-CAGCTGGTCATGGTGATAAGG-3'

The β-actin gene specific primers were purchased from Qiagen (QuantiTect, cat no. QT00095431). CDK11 and CDK8 RNAs were amplified by 31 cycles of PCR, and β-actin RNA was amplified by 25 cycles. Amplified fragments were then separated using a 2% agarose gel and detected by SYBR GREEN I (Invitrogen) staining.

Luciferase assays

Luciferase assays were performed based on the method of Osada et al. (1999). HeLa S3 cells cultured for 2.5 days following transfection with siRNAs (described above) were co-transfected with 100 ng of the 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 (the Gal4–VP16 expression plasmid). After 1 day, the cells were lysed and transcriptional activity was measured using a Dual-Luciferase Reporter Assay System (Promega).


   Acknowledgements
 
Authors thank Jun Yanagisawa for critical reading of this manuscript and discussion, and Tadashi Furumoto for advices on establishing the epitope-tagged HeLa cell lines and the siRNA experiments. Authors also thank our colleagues at the University of Toyama for helpful discussions. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y.O.), the Solution Oriented Research for Science and Technology (SORST) (Y.O.), and a Research Grant in the Natural Sciences from the Mitsubishi Foundation (Y.O.).


   Footnotes
 
Communicated by: Hiroshi Handa

* Correspondence: ohkumay{at}pha.u-toyama.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 1 April 2008
Accepted: 7 May 2008





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