GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2009) 14, 17-28. doi:10.1111/j.1365-2443.2008.01246.x
© 2009 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tabata, T.
Right arrow Articles by Ishii, S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Tabata, T.
Right arrow Articles by Ishii, S.

Ski co-repressor complexes maintain the basal repressed state of the TGF-β target gene, SMAD7, via HDAC3 and PRMT5

 Takanori Tabata1,2, Kenji Kokura1, Peter ten Dijke3 and Shunsuke Ishii1,2,*

1 Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
2 Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8577, Japan
3 Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-β-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-β target gene, in TGF-β-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-β-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The proteins encoded by ski and its related gene sno (Nomura et al. 1989) (Ski and Sno) act as co-repressors, and interact with two other co-repressors, N-CoR/SMRT and mSin3A (Nomura et al. 1999). Ski and Sno are required for transcriptional repression by multiple repressors, including Mad, Rb, MeCP2 and Gli3 (Nomura et al. 1999; Tokitou et al. 1999; Kokura et al. 2001; Dai et al. 2002). As Mad and Rb are tumor suppressors, Ski and Sno function as tumor suppressors in certain cell types (Shinagawa et al. 2000, 2001). Ski and Sno also bind to other various transcriptional regulators, including Smads (Akiyoshi et al. 1999; Luo et al. 1999; Stroschein et al. 1999; Sun et al. 1999a; Xu et al. 2000b) and c-Myb (Nomura et al. 2004), to inhibit their activities. Negative regulation of Smad-dependent trans-activation by Ski/Sno is consistent with their transforming activity. In the absence of TGF-β, Smad2 and Smad3 are distributed mainly in the cytoplasm (Xu et al. 2000a). Upon TGF-β stimulation, the type I TGF-β receptor phosphorylates Smad2/3, allowing them to translocate into the nucleus (Xu et al. 2000a) and to form complexes with Smad4 (Chacko et al. 2001), which bind to the promoters of TGF-β-responsive genes (Massagué & Wotton 2000). Smads interact with transcriptional co-activators or co-repressors to regulate transcription through the C-terminal Mad homology-2 (MH2) domains (Massagué & Wotton 2000). TGF-β inhibits cellular proliferation, at least in part, by inducing the expression of p15INK4B, and the transforming activity of Ski/Sno is dependent on their ability to repress Smad activity (He et al. 2003).

Ski/Sno interaction with Smad2/3 is enhanced by TGF-β, whereas Ski/Sno constitutively interact with Smad4 (Akiyoshi et al. 1999; Luo et al. 1999; Stroschein et al. 1999; Sun et al. 1999a; Xu et al. 2000b). The interaction of Ski/Sno with Smads results in the disruption of an active heteromeric Smad complex, and induces the replacement of the transcriptional co-activator p300/CBP by the co-repressor–histone deacetylase (HDAC) complex on Smads (Akiyoshi et al. 1999; Luo et al. 1999; Stroschein et al. 1999; Sun et al. 1999a; Xu et al. 2000b; Wu et al. 2002). Degradation of Ski/Sno is induced upon TGF-β stimulation or constitutively (Stroschein et al. 1999, 2001; Sun et al. 1999b; Bonni et al. 2001; Wan et al. 2001; Nagano et al. 2007). Ski has been proposed as nuclear co-repressors for Smad4 involved in maintaining TGF-β responsive genes, such as Smad7, in a basal repressed state (Denissova & Liu 2004). However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown.

Multiple evidences support the functional importance of HDAC activity in repression mediated by various co-repressors. The large complexes of N-CoR or SMRT contain HDAC3 (Guenther et al. 2000; Li et al. 2000), a member of the class I HDACs; although in the small subfractions, N-CoR interacts with class II HDACs, including HDAC4 and HDAC5 (Huang et al. 2000). However, the mSin3A complexes contain the class I HDACs, HDAC1 and HDAC2 (Zhang et al. 1997). Because repression by various transcription factors, such as Mad and nuclear receptors, requires both N-CoR/SMRT and mSin3 (Alland et al. 1997; Heinzel et al. 1997; Nagy et al. 1997), these results suggest that multiple types of HDACs are required for efficient repression.

In addition to HDACs, histone methylation is also important in mediating repression. Various histone methyltransferases (HMTases), including the lysine and arginine methyltransferases, are known to be critical for repression. For example, human SUV39H1, mammalian homologues of Drosophila Su(var)3–9 and of Schizosaccharomyces pombe clr4, encode histone H3-K9 specific methyltransferases and repress transcription (Rea et al. 2000). The complexes of the co-repressor C-terminal binding protein (CtBP) contain the histone H3-K9 methyltransferases G9a and GLP (Shi et al. 2003). Furthermore, the hSWI/SNF chromatin-remodeling protein complexes (which repress transcription) contain the histone arginine methyltransferase, PRMT5, which methylates the histone H3-R8 (Pal et al. 2003, 2004).

Here, we report the purification and characterization of Ski complexes from TGF-β-untreated HeLa cells. The complexes contained HDAC3 and PRMT5 in addition to Smad2/3/4, suggesting that these complexes maintain the basal repressed state of the TGF-β target genes.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Purification of Ski complexes under high salt conditions

We used the immunoaffinity purification method (Nakatani & Ogryzko 2003), to purify Ski complexes. Ski was stably expressed as fusion proteins with N-terminal FLAG- and HA-epitope tags in HeLa S3 cells by retroviral transduction. The Ski complexes were purified from nuclear extracts, which were prepared using the standard Dignam's buffer (420 mM NaCl), by sequential immunoprecipitation with anti-FLAG and anti-HA antibodies. As a control, we carried out a mock purification from control HeLa cells, which were transduced with the control empty virus. The purified Ski complexes contained four bands which were specific to the Ski complex and were not detected in the fraction from control HeLa cells (Fig. 1A). Mass spectrometric and immunoblotting analyses indicated that these four polypeptides were Ski, Smad4, Smad2 and Smad3 (Fig. 1A,B). The relative ratios of Ski, Smad4, Smad2 and Smad3 ( + IgG), calculated from the density of each band, were 1.00, 1.70, 0.47 and 0.56. To further verify the formation of complexes between Ski and Smads, the Ski complexes were purified using anti-FLAG antibody and then subjected to glycerol gradient centrifugation (Fig. 1C). Western blotting of each fraction showed that fractions 6–12, which corresponded to molecular weights of 158–580 kDa, contained Ski and Smad4/3/2, confirming that Ski and Smad4/3/2 form the complexes. The relative ratios of Smad4 to Smad2/3 in the fractions 6–8 were higher than that in the fractions 10–12. As the calculated molecular weight of the complexes, each containing one molecule of Ski and Smad4/3/2, is approximately 250 kDa, the Ski complexes in fractions 6–12 may contain varying ratios of Ski and Smad2/3/4. One possibility is that these heterogeneous complexes were generated during the purification steps, for example, by releasing Smad proteins. Smad proteins are known to form hetero-oligomers which might be partly disrupted during purification.


Figure 1
View larger version (23K):
[in this window]
[in a new window]

  		Figure 1  Purification of Ski complexes under high salt conditions. (A) Purification and mass spectrometric analysis of polypeptides associated with Ski. Nuclear extracts were prepared from HeLa S3 cells expressing human Ski tagged with both FLAG and HA epitopes under 420 mM NaCl conditions, and sequentially immunopurified with FLAG and HA antibody affinity resins. Mock-transduced HeLa cells were used as a control. The Ski-associated polypeptides were detected by Coomassie Blue staining. The identities of the associated polypeptides by mass spectrometric analyses are indicated on the right. The asterisks denote the degradation products of Ski. (B) Co-immunoprecipitation of the Ski complexes. Immunopurified Ski complexes were analyzed by Western blotting using the antibodies shown on the left. (C) Glycerol gradient analysis. Ski complexes were separated on a 10–40% glycerol gradient by ultracentrifugation. Fractions were detected by Western blotting. The numbers over the lanes represent the eluted fraction numbers, and arrows indicate protein molecular markers. (D) Complex formation of Ski with Smad4 in the absence of TGF-β. Cell lysates were prepared from HeLa cells cultivated for 24 h under serum-starved conditions, and immunoprecipitated with the antibody shown above each lane. The immunocomplexes were subjected to SDS-PAGE, followed by Western blotting to detect the proteins indicated at the right.

 
It is believed that Smad2 and Smad3 are distributed mainly in the cytoplasm in the absence of TGF-β (Xu et al. 2000a). The presence of Smad2/3 in the Ski complexes suggests that, in the absence of exogenous TGF-β, some Smad2/3 proteins are present in the nucleus, where they form complexes with Ski. However, it is still possible that the nuclear localization of Smad2/3 is because of the presence of a small amount of TGF-β in the serum which was used to cultivate HeLa cells. To examine this, we carried out a co-immunoprecipitation (ChIP) assay using HeLa cells cultivated under serum-deprivation conditions for 24 h. Under these conditions, the anti-Ski antibody co-precipitated Smad2/3/4, whereas the anti-Smad4 antibody co-precipitated Ski (Fig. 1D). These results indicate that minimal quantities of Smad2/3 are capable of forming complexes with nuclear Ski in the absence of TGF-β. Furthermore, these results strongly suggest that non-phosphorylated Smad2/3 are involved in the Ski complex.

Purification of Ski complexes under relatively low salt conditions

As the Ski complexes purified under 420 mM salt concentrations did not contain any component, such as HDAC or HMTase, capable of explaining the molecular mechanism of Ski-mediated transcriptional repression, we then tried to purify Ski complexes using lower salt conditions. When we used a 300-mM salt-containing buffer to prepare extracts, the Ski complexes, purified as described above, contained additional bands compared to the complexes purified under 420 mM salt conditions. Mass spectrometric analyses indicated that these additional polypeptides were the N-CoR/SMRT co-repressors, PRMT5 (type II arginine methyltransferase), transducin (β)-like 1 (TBL1: a WD40 repeat-containing protein) and its related protein TBLR1, HDAC3 and casein kinase 2 (CK2) (Fig. 2A). Western blotting analyses of the Ski complexes, which were purified by anti-FLAG antibody, using specific antibodies indicated that Smad4/3/2, TBLR1, HDAC3 and CK2 were involved in the Ski complexes (Fig. 2B, left panel). When the Ski complexes were purified using anti-FLAG and anti-HA antibodies, both N-CoR and PRMT5 were detected by Western blotting (Fig. 2B, right panel).


Figure 2
View larger version (39K):
[in this window]
[in a new window]

  		Figure 2  Purification of Ski complexes under relatively low salt conditions. (A) Purification and mass spectrometric analysis of proteins associated with Ski. Nuclear extracts from HeLa S3 cells expressing human FLAG/HA-Ski were prepared under 300 mM NaCl salt conditions, and sequentially immunopurified with the FLAG and HA antibody affinity resins. Mock-transduced HeLa S3 cells were used as a control. The Ski-associated proteins were detected by Coomassie Blue staining. The identities of the associated proteins by mass spectrometric analysis are indicated on the right. The asterisks denote the degradation products of Ski. The arrowhead denotes the degradation products of antibodies that may be degraded by nonspecifically immunoprecipitated proteases. (B) Co-immunoprecipitation of the Ski complexes. Immunopurified Ski complexes were analyzed by SDS-PAGE, followed by Western blotting using the antibodies indicated on the left. (C) Glycerol gradient analysis. Immunopurified Ski complexes were separated on a 10–40% glycerol gradient by ultracentrifugation, and fractions were analyzed by SDS-PAGE, followed by Western blotting using the antibodies indicated on the left. The numbers over the lanes represent the eluted fraction numbers, and arrows indicate protein molecular masses.

 
To further confirm the complex formation between Ski and these proteins, the Ski complexes were first purified using anti-HA antibody, and then subjected to glycerol gradient centrifugation, followed by Western blotting. As it is known that anti-FLAG M2-agarose non-specifically enriches for PRMT5 activity (Nishioka & Reinberg 2003), we used anti-HA antibody before glycerol gradient analysis. Western blotting of each fraction showed that fractions 5–11, which corresponded to molecular weights of 232–580 kDa, contained Ski, PRMT5 and HDAC3 in addition to Smad4/3/2 (Fig. 2C). N-CoR/SMRT, TBL1/TBLR1 and CK2 were not detected by Western blotting, possibly because of their low concentrations in the purified complexes or the low reactivity of each antibody. Although the Ski complexes, purified under 300 mM salt conditions, contained additional components compared to those purified under 420 mM salt conditions, their apparent molecular weights are similar. This suggests that these complexes are heterogeneous with regard to their components, for instance, they may contain a varying number of Smad proteins. These results suggest that Smad4/3/2 tightly associate with Ski in the complexes, whereas other components, N-CoR/SMRT, PRMT5, TBL1/TBLR1, HDAC3 and CK2, interact with Ski with relatively weak affinity. Estimation of number of each component in the Ski complexes based on their abundance in the purified complexes is difficult, because the purified complexes may be a mixture of complexes which contain different number of each component. In the case of other co-repressor complexes, the precise estimation of number of each component was also difficult.

HDAC and HMTase activities in the Ski complexes

Various HDACs are involved in other multiple co-repressor complexes (Guenther et al. 2000; Huang et al. 2000; Li et al. 2000), where they function as key molecules involved in repressing transcription. Furthermore, the arginine methyltransferase, PRMT5 (part of the chromatin remodeling factor hSWI/SNF complex), is involved in the transcriptional repression of target genes by methylating H3-R8 (Pal et al. 2003, 2004). Together with these data, the presence of HDAC3 and PRMT5 in the Ski complexes suggests that these two factors play a key role in Ski-mediated transcriptional repression. We therefore investigated whether, or not, Ski complexes contain HDAC and HMTase activities. We found that the complexes exhibited sufficient activity to remove the acetyl group from histones (Fig. 3A). In addition, the Ski complexes catalyzed the transfer of a methyl group to histone H3 (Fig. 3B). These results show that Ski complexes contain both HDAC and HMTase activities.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Figure 3  Analysis of the enzymatic activity of the Ski complexes. (A) Histone deacetylase activity of the Ski complexes. Immunopurified Ski complexes were incubated with [3H] histones. The reactions were allowed to proceed for 5 h before extraction and quantification of the released [3H] acetate. The data shown are the average results ± standard deviations from four separate experiments. (B) Histone methylation activity of the Ski complexes. Immunopurified Ski complexes were incubated with histones and [14C]SAM. The reactions were allowed to proceed for 90 min, and the reaction products were analyzed by SDS-PAGE. The gel was fixed, dried and analyzed by fluorography. Histones were separated on the same gel as a size marker and stained with Coomassie Blue.

 
Localization of Ski complexes on the promoter of the TGF-β target gene, SMAD7

The presence of Smad proteins in Ski complexes in the absence of TGF-β (Fig. 1D) suggests that the Ski complexes are located at the transcriptional control regions (which contain the Smad-binding sites) of the TGF-β target genes, in order to maintain their basal repressed state. One such TGF-β target gene is SMAD7, which contains the Smad-binding element (nucleotides –203 to –210; +1 is the transcriptional start site) (Fig. 4A). It has been shown that transcription of the SMAD7 gene is induced upon TGF-β stimulation in HepG2 cells, and that Smad2/3/4 directly bind to its promoter region (Nagarajan et al. 1999). Additionally, it was previously shown that SMAD7 is silenced by Ski (Denissova & Liu 2004). Furthermore, Ski was shown to bind to the Smad-binding element in the Smad7 gene promoter together with Smad4 (Denissova & Liu 2004). Using ChIP assays, we investigated whether, or not, the Ski complexes bind to the SMAD7 promoter region. The amounts of Smad site-containing promoter DNA fragment precipitated with antibodies against Ski, Smad4, Smad2/3, HDAC3, PRMT5 or TBLR1 were significantly higher than that precipitated by the control IgG (Fig. 4B). Variation in the amounts of DNA fragment precipitated by each antibody could be because of different immunoprecipitation capacities for each antibody. Thus, our results show that Ski complexes containing Smad2/3/4, HDAC3 and PRMT5 bind to the SMAD7 promoter.


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		Figure 4  Chromatin-immunoprecipitation assays. (A) Schematic representation of the primer set used in the ChIP analysis. A primer pair is indicated with arrows. (B) HepG2 cells were cross-linked with formaldehyde, lysed and sonicated. Soluble chromatin was immunoprecipitated with the antibodies indicated below. The final DNA extraction was amplified by real-time PCR using the primers that cover the SMAD7 promoter region. The data shown are mean ± standard deviations of a representative of three independent experiments.

 
Role of HDAC3 and PRMT5 in maintaining a basal level of SMAD7 expression

To investigate if the Ski complex members, HDAC3 and PRMT5, are required for the repression of SMAD7 transcription, we looked at the effects of the siRNA-mediated knockdown of HDAC3 or PRMT5 on the level of SMAD7 mRNA. Treatment of HepG2 cells with siRNA specific for Ski, PRMT5 or HDAC3 decreased the levels of these proteins to approximately 1/4 to 1/10 (Fig. 5A). The knockdown of Ski, using siRNA, resulted in the up-regulation of SMAD7 mRNA in HepG2 cells (Fig. 5B), as was reported previously (Denissova & Liu 2004). A decrease in HDAC3 or PRMR5 also significantly increased the SMAD7 mRNA level. These results indicate that Ski complexes maintain the basal repressed state of the SMAD7 gene in the absence of TGF-β.


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		Figure 5  Ski complexes repress SMAD7 expression. (A) Ski, HDAC3 and PRMT5 levels were decreased by siRNA treatment. HepG2 cells were transfected with either Ski, HDAC3, PRMT5 or control siRNA. At 72 h after transfection, the cells were lysed in Laemmli buffer, and analyzed by SDS-PAGE followed by Western blotting using anti-Ski, anti-HDAC3 and anti-PRMT5. The amounts of lysate proteins used are shown above each lane. (B) Knockdown of the Ski complex components up-regulates SMAD7 expression. HepG2 cells were transfected with siRNAs for Ski, HDAC3, PRMT5 or GFP. SMAD7 mRNA levels were analyzed by real-time RT-PCR. Each value was normalized to the level of human GAPDH mRNA. The data shown are mean ± standard deviations of a representative of three independent experiments.

 
Role of Smad proteins in Ski-mediated transcriptional repression by Mad

The binding of Ski complexes to Smad sites in the SMAD7 promoter suggests that Smad4/3/2 in Ski complexes are used to recruit the complexes to the Smad site-containing promoters. However, Ski mediates transcriptional repression not only via Smad proteins but also by other multiple DNA-binding transcription factors, such as basic helix-loop-helix Mad protein (Nomura et al. 1999). To examine whether Smad4 in the Ski complexes plays any role in transcriptional repression mediated by factors other than Smad proteins, we generated HepG2 cells in which the Smad4 level was significantly decreased, and used them in luciferase reporter assays. HepG2 cells were infected with a retrovirus expressing the short hairpin-type RNA (shRNA) for SMAD4, or a control empty virus, and the cell pools containing these expression vectors were isolated. In the cells containing the expression vector for SMAD4 shRNA (S4kd), the Smad4 level was approximately 1/5 that of the control cells (Fig. 6A). These cells were transfected with the luciferase reporter containing the Gal4 site and with increasing amounts of the Gal4-Ski expression vector, and the Ski-dependent repression was analyzed. Gal4-Ski repressed the luciferase expression to a similar degree in both in the Smad4 knockdown cells and the control cells (Fig. 6B). A similar level of repression by Gal4-Mad was also observed in both types of cells (Fig. 6C). These results suggest that Smad4 is not required for repression by transcription factors other than Smad proteins.


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		Figure 6  The effect of Smad4 knockdown on the repression activity of Ski. (A) The generation of Smad4-knockdown cells. HepG2 cells were infected with shRNA-expressing constructs for human SMAD4, or empty vector, using retroviral infection. After selection with puromycin, cells were lysed in Laemmli buffer and analyzed by SDS-PAGE, followed by Western blotting using anti-Smad4 and {alpha}-tubulin as a loading control. The amounts of lysate proteins used are shown above each lane. (B, C) HepG2 cells expressing shRNA for human SMAD4 (S4kd), or empty vector (control), were transiently transfected with a mixture of Gal4 site-containing luciferase reporter, the Gal4-Ski (B), Mad-Gal4 (C) or the Gal4 expression plasmid, and the internal control plasmid. The luciferase activity was measured and the relative level of luciferase activity is indicated. The data shown are mean ± standard deviations of a representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The purification and characterization of Ski complexes indicate that Ski uses HDAC3 and the type II arginine-specific methyltransferase, PRMT5, to repress transcription. There is much biochemical evidence to demonstrate that HDACs are associated with multiple repression complexes, indicating the involvement of HDACs in transcriptional repression. In particular, HDAC3 is a major core-component in the large complexes formed by the co-repressors SMRT and N-CoR, with estimated sizes of 1.5–2 MDa (Guenther et al. 2000; Li et al. 2000). In addition, nuclear PRMT5 forms complexes with the hSWI/SNF chromatin-remodeling proteins BRG and BRM, and is responsible for transcriptional repression via the methylating histone H3-K8 (Pal et al. 2004). These results suggest that both HDAC3 and PRMT5 are required for Ski-mediated transcriptional repression. Moreover, knockdown of either HDAC3 or PRMT5 led to up-regulation of SMAD7 mRNA (Fig. 5B). Interestingly, subunits of the mSin3A/HDAC2 co-repressor complex are known to repress transcription of the c-Myc target genes, carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase and nucleolin, together with hSWI/SNF complexes containing PRMT5 (Pal et al. 2004). Thus, in order to repress certain groups of target genes, synchronized action by both HDAC and PRMT might be necessary. Another example of a co-repressor complex containing different types of histone modification enzymes is the CtBP complex which contains HDAC1/2 and the histone H3-K9 methyltransferase G9a and GLP (Shi et al. 2003).

Recently, it was reported that protein arginine methyltransferase, PRMT1, interacts with the N-terminal domain of Smad6 and methylates it directly (Inamitsu et al. 2006). Therefore, the possibility that PRMT5 in Ski complexes methylates Smad4/3/2 in the same complex might be worth testing.

Previously, we demonstrated that Ski is required for Mad- or thyroid hormone receptor (TR)-mediated repression (Nomura et al. 1999). Both Mad- and TR-mediated repression also require the co-repressors N-CoR/SMRT and mSin3 (Alland et al. 1997; Heinzel et al. 1997; Nagy et al. 1997). The N-CoR and SMRT complexes contain HDAC3 (Guenther et al. 2000; Li et al. 2000), whereas the mSin3A complexes contain HDAC1 and HDAC2 (Zhang et al. 1997). As mSin3A was shown to interact with N-CoR/SMRT in yeast two hybrid assays and co-immunoprecipitation (Alland et al. 1997; Heinzel et al. 1997; Nagy et al. 1997), both complexes may weakly interact. Furthermore, our data indicate that the Ski complexes contain N-CoR/SMRT and HDAC3. These results suggest that three types of HDACs (HDAC1/2/3) are required for Mad- and TR-mediated repression. The reason for the requirement of multiple HDACs could be because of their different specificities. In fact, HDAC3 was reported to preferentially deacetylate histone H4-K5 (Hartman et al. 2005). Alternatively, the activity of these various HDACs could be used differently, depending on the repressors involved and the promoter context, to ensure an effective repression. Several groups have also shown that the third repressor domain of N-CoR and SMRT can directly interact in vitro with class II HDACs, including HDAC4, HDAC5 and HDAC7 (Huang et al. 2000), suggesting that the use of more divergent HDACs is required for the efficient repression of more complete sets of target genes. Furthermore, requirement of Ski for the Mad and TR-dependent repression may indicate that PRMT5 of the Ski complexes in addition to HDACs of the N-CoR and mSin3A complexes is required for efficient repression.

It has been reported that Ski interacts with Smad4 on the SMAD7 promoter to maintain its basal repressed state in the absence of TGF-β (Denissova & Liu 2004). However, there is no evidence to date showing the involvement of Smad2/3 in maintaining the repressed state of the TGF-β target genes. The present study indicates that Ski unexpectedly forms stable complexes with Smad2/3/4, suggesting that the Smad2/3/4 hetero-oligomers in the Ski complexes are responsible for the recruitment of these complexes to the target Smad-binding sites. Knockdown of Smad4 in HepG2 cells did not affect the Gal4–Ski or Gal4–Mad-dependent repression (Fig. 6). These results suggest that Smad4/3/2 in the Ski complexes are required only for repression at the Smad-binding sites, and that they are not required for repression by transcription factors other than Smad proteins.

The Ski complexes purified under 420 mM salt conditions contained Ski, Smad4, Smad2 and Smad3 at molar ratios of 1.00, 1.70, 0.47 and 0.56, calculated from the density of each band. This suggests that the molecular ratio of Ski, Smad4, Smad3 and Smad2 is approximately 2 : 4 : 1 : 1. However, the results from the glycerol density gradient analysis suggested that they appear to contain varying ratios of Ski and Smad2/3/4. It is known that Ski interacts with Smad2/3 via its amino acids 17–212, and with Smad4 via its amino acids 219–312. The Ski-binding surfaces of Smad3 and Smad4 were also precisely analyzed (Wu et al. 2002; Mizuide et al. 2003). It will be interesting, therefore, to see which domains of the Ski and Smad4/3/2 proteins are used to form the Ski complexes.

The Ski complexes also contained CK2 and TBL1/TBLR1, although these components were not detected in the glycerol gradient analysis, possibly because of low concentrations and/or the low efficiency of specific antibodies. Interestingly, CK2 was shown to phosphorylate HDAC3 and stimulate its activity (Zhang et al. 2005). Therefore, CK2 may act as a positive regulator of Ski complexes by phosphorylating HDAC3. Human TBL1 and TBLR1 are highly related WD-40 repeat proteins, sharing 89% sequence identity and are involved in N-CoR/SMRT complexes together with HDAC3. TBL1/TBLR1 can bind to histones H2B and H4 in vitro (Yoon et al. 2005), suggesting that they can stimulate HDAC activity by stabilizing HDAC3–histone interactions. TBL1 and TBLR1 appear to be multifunctional proteins, and to be involved in protein degradation. The Drosophila TBL1 homologue, Ebi, is a putative F-box protein involved in the ubiquitin-dependent degradation of Tramtrack88 (Dong et al. 1999), whereas mammalian TBL1 induces degradation of β-catenin together with Siah1 (Matsuzawa & Reed 2001). Furthermore, TBL1 and TBLR1 are required for transcriptional activation, mediated by nuclear receptors, by inducing the exchange of co-repressors/co-activators through ubiquitin-dependent protein degradation (Perissi et al. 2004). These results raise the possibility that TBL1/TBLR1 may participate in the degradation of the Ski complexes bound to the TGF-β target genes in response to TGF-β stimulation.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Purification and characterization of the Ski complexes

To generate the retroviral vector expressing FLAG- and HA-tagged Ski, the human ski cDNA was cloned into the pOZ-FH-N vector (Nakatani & Ogryzko 2003). The resulting construct was transfected into the amphotropic packaging Phoenix A cells and medium containing the amphotropic virus was prepared. HeLa S3 cells were transduced with a recombinant retrovirus expressing a bicistronic mRNA encoding FLAG-HA-Ski linked to the IL-2 receptor subunit, and the transduced subpopulation was purified by repeated cycles of affinity cell sorting. Cells from a 30-L culture were disrupted in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, protease inhibitors), and the nuclear pellet was collected by centrifugation at 25 000 g for 30 min. The pellet was extracted with buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, protease inhibitors) containing 420 mM or 300 mM NaCl for 30 min at 4 °C. Lysates were collected by centrifugation at 25 000 g for 30 min and diluted with NaCl-free buffer C to decrease the NaCl concentration to 270 mM or 150 mM. The Ski complexes were immunopurified from these nuclear extracts by incubating with anti-FLAG M2 agarose (Sigma) for 5 h with rotation. After extensive rinsing with wash buffer (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, protease inhibitors), the bound proteins were eluted twice from the M2 agarose by incubation for 30 min with 0.15 mg/mL FLAG peptide (Sigma) in the same buffer. The eluates were further purified by immunoprecipitation with anti-HA 12CA5-conjugated protein G-Sepharose (GE Healthcare) overnight with rotation. The bound proteins were eluted twice from the matrix by incubating for 60 min with 0.5 mg/mL HA peptide (Roche) in wash buffer. The purified proteins were separated by 5–20% gradient SDS polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue. The protein bands were excised and analyzed by mass spectrometry at the RIKEN Brain Science Institute Mass Spectrometry Facility.

Glycerol gradient sedimentation

The Ski complexes were immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-Ski under 420 mM NaCl conditions using anti-FLAG antibody or under 300 mM NaCl conditions using anti-HA antibody as described above. The glycerol concentration of the samples was decreased to 6% by adding glycerol-free buffer, and they were then loaded onto a 4.4-mL 10–40% glycerol gradient in wash buffer. After centrifugation at 55 000 r.p.m. for 9 h or 3 h (Beckman, SW55Ti), 200 µL fractions were collected from the top of the gradient. Each fraction was TCA-precipitated and analyzed by SDS-PAGE, followed by Western blotting using anti-FLAG (M2, Sigma), anti-PRMT5 (07-405, Upstate), anti-Smad4 (H-552, Santa Cruz), anti-Smad2/3 (07-408, Upstate), anti-HDAC3 (H-99, Santa Cruz) or anti-Ski (H329, Santa Cruz).

Co-immunoprecipitation

For co-immunoprecipitation of FLAG-HA-Ski, Smad2/3 and CK2, nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-Ski were immunoprecipitated with anti-FLAG agarose (M2, Sigma) for 5 h with rotation. After washing five times with wash buffer (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, protease inhibitors), immunocomplexes were analyzed by SDS-PAGE, followed by Western blotting using anti-Smad2/3 (07–408, Upstate) or anti-CK2 (06–873, Upstate). For co-immunoprecipitation of FLAG-HA-Ski, Smad4, HDAC3 and TBLR1, NP-40 was added to nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-Ski to a final concentration of 0.5% and they were immunoprecipitated with anti-FLAG agarose (M2, Sigma) for 5 h with rotation. After washing five times with wash buffer (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.5% NP-40, protease inhibitors), immunocomplexes were analyzed by SDS-PAGE, followed by Western blotting using anti-FLAG (M2, Sigma), anti-Smad4 (H-552, Santa Cruz Biotechnology), anti-HDAC3 (H-99, Santa Cruz Biotechnology) or anti-TBLR1 (A300–408A, Bethyl Laboratories). For co-immunoprecipitation of FLAG-HA-Ski, PRMT5 and N-CoR, nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-Ski were immunoprecipitated with anti-FLAG agarose (M2, Sigma) for 5 h with rotation. After an extensive wash with wash buffer (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, protease inhibitors), the bound proteins were eluted twice from the M2 agarose by incubation for 30 min with 0.3 mg/mL FLAG peptide (Sigma) in the same buffer. The eluates were further purified by immunoprecipitation with anti-HA 12CA5-conjugated protein G-Sepharose (GE Healthcare) overnight with rotation. After washing five times with wash buffer, the bound proteins were eluted twice from the matrix by incubating for 60 min with 0.5 mg/mL HA peptide (Roche) in wash buffer. The eluates were TCA-precipitated and analyzed by SDS-PAGE, followed by Western blotting using anti-FLAG (M2, Sigma), anti-PRMT5 (07–405, Upstate) or anti-N-CoR (06–892, Upstate).

HDAC assay

Assays for HDAC activity were carried out essentially as described (Nomura et al. 1999). Nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-Ski, were immunoprecipitated with anti-FLAG agarose (M2, Sigma) for 5 h with rotation under 300 mM condition as described for the Ski complex purification, followed by extensive rinsing with wash buffer (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, protease inhibitors). The bound proteins were then eluted twice from the M2 agarose by incubation for 30 min with 0.3 mg/mL FLAG peptide (Sigma) in the same buffer. The eluates were further purified using anti-HA 12CA5-conjugated protein G-Sepharose (GE Healthcare) overnight with rotation. The beads were rinsed five times with wash buffer (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, protease inhibitors) and then resuspended in 100 µL of HDA buffer (15 mM potassium phosphate buffer, pH 7.4, 0.2 mM EDTA, 5% glycerol, protease inhibitors) containing [3H] histones (5000 cpm/reaction). The reactions were carried out at 37 °C for 5 h and stopped by the addition of 10 µL of 12 N HCl. Released [3H] acetate was extracted with 1 mL of ethyl acetate. After centrifugation, 0.9 mL of the upper organic phase was used for liquid scintillation counting.

HMTase assay

Nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-Ski were immunoprecipitated with anti-HA 12CA5-conjugated protein G-Sepharose (GE Healthcare) overnight with rotation less than 300 mM salt condition as described for the Ski complex purification. The beads were then rinsed five times with wash buffer (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, protease inhibitors) and then twice with methylation buffer (50 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 13.3% glycerol, 1 mM EDTA, 0.25 mM DTT, protease inhibitors), and resuspended in methylation buffer containing 10 µg of histones (Roche) and 0.25 µCi of S-Adenosyl-L-[methyl-14C]methionine (GE Healthcare) in a total volume of 50 µL. The reactions were carried out at 30 °C for 90 min and stopped by the addition of 10 µL of 6 x Laemmli buffer. The samples were separated by SDS-PAGE and the gel was fixed in 45% methanol/10% acetic acid for 30 min treated with Amplify Fluorographic Reagent (GE Healthcare) for 2 h, dried and analyzed by fluorography. To identify the methylated histones, the histones (Roche) were separated in the same gel and stained with Coomassie Blue.

Chromatin-immunoprecipitation (ChIP) assay

HepG2 cells were cross-linked with 1.5% formaldehyde at 37 °C for 20 min. Glycine was added to a final concentration of 187.5 mM, and the cells were incubated for 5 min. Cells were washed three times with ice-cold phosphate-buffered saline (PBS) containing protease inhibitors. Cells were scraped, collected by centrifugation, and resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.0 and protease inhibitors). After incubation at 4 °C for 10 min, cells were sonicated and centrifuged at 17 400 g for 20 min at 8 °C. The supernatant was diluted 10 times with dilution buffer (50 mM Tris–HCl, pH 8.0, 167 mM NaCl, 0.275% TritonX-100, 0.0275% sodium deoxycholate, protease inhibitors) for immunoprecipitation using the antibody against Smad4 and TBLR1, or diluted 20 times with the dilution buffer for Ski, HDAC3, PRMT5 and Smad2/3. Immunoprecipitation was carried out overnight at 4 °C with 4 µg of anti-Ski (H-329, Santa Cruz), anti-Smad4 (H-552, Santa Cruz), anti-HDAC3 (H-99, Santa Cruz), anti-TBLR1 (A300–408A, Bethyl Laboratory), anti-PRMT5 (07–405, Upstate) or anti-Smad3 (FL425, Santa Cruz). Protein-G Sepharose (GE Healthcare) was added for approximately 24 h. The beads were washed once with 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.25% Triton-X100, 0.025% SDS, 0.025% sodium deoxycholate; once with 50 mM Tris–HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.25% Triton-X100, 0.025% SDS, 0.025% sodium deoxycholate; once with 10 mM Tris–HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.125% NP-40, 0.125% sodium deoxycholate; and twice with 10 mM Tris–HCl, pH8.1, 1 mM EDTA. The beads were then incubated for 6 h at 65 °C in 1% SDS, 100 mM NaHCO3, 250 mM NaCl, 10 mM DTT and proteins were digested for 1 h at 45 °C by adding EDTA to a final concentration of 10 mM, Tris–HCl (pH 6.8) to a concentration of 40 mM, and proteinase K to a concentration of 0.2 mg/mL. The DNA was purified by using QIAquick PCR Purification Kit (QIAGEN) and analyzed by real-time PCR using the ABI 7500 Real-Time PCR System and QuantiTect Probe PCR Kit (QIAGEN) according to the manufacturer's instructions. The primers for the amplification of the human SMAD7 promoter region were as follows: forward 5'-CGAAACACAATCGCTTTTTTTTT-3', reverse 5'-CCCTC TGCTCGGCTGGTT-3' and the TaqMan probe was 5'-6-carboxyfluorescein [FAM]-CTAGACGGCCACGTGACGAGGCC-6-carboxytetramethylrhodamine [TAMRA]-3'.

Generation of HepG2 cells expressing the shRNA against SMAD4

To generate the Smad4-knockdown cells, the shRNA-expressing retroviral vector [pRetroSuper empty (pRS) or pRetroSuper-Smad4 (S4kd)] (Deckers et al. 2006) was transfected into the amphotropic packaging Phoenix A cell line and medium containing the amphotropic virus was prepared. HepG2 cells were transduced with the recombinant retrovirus and selected with puromycin (1.5 µg/mL). To confirm the knockdown of Smad4, cells were lysed in Laemmli buffer and analyzed by SDS-PAGE, followed by Western blotting using anti-Smad4 (H-552, Santa Cruz); anti-{alpha}-tubulin (Sigma).

Luciferase reporter assay

Using Lipofectamine 2000 (Invitrogen), HepG2 cells expressing the shRNA against SMAD4 were transfected, in 6-well plates, with a mixture containing 0.5 µg of the luciferase reporter (in which six tandem repeats of the Gal4-binding site were linked to the TK promoter), various amounts (0.1, 0.5, 1.0, 1.5 or 2.0 µg) of Gal4–Ski or Mad-Gal4, which consist of the Gal4 DNA-binding domain and wild-type Ski or the mSin3-interacting domain of Mad (Nomura et al. 1999), respectively, and 0.1 µg of the internal control plasmid pRL-TK (Promega), in which the sea-pansy luciferase gene is linked to the TK promoter. The total amount of DNA was adjusted to 2.6 µg by adding the Gal4 expression plasmid. The luciferase assays were carried out 24 h after transfection using the dual-luciferase assay system (Promega).

siRNA treatment and real-time quantitative RT-PCR

Ski siRNA was purchased from Dharmacon Inc. Other siRNA target sequences were as follows: for HDAC3, 5'-GATGCTG AACCATGCACCT-3'; for PRMT5, 5'-CCGCTATTGCACC TTGGAA-3'; for GFP, 5'-CTACAACAGCCACAACGTC-3'. To confirm the gene-silencing effect of siRNAs, HepG2 cells were transfected with siRNAs (final 200 nM) using Oligofectamine (Invitrogen) according to the manufacturer's instructions. At 72 h after transfection, cells were lysed in Laemmli buffer and analyzed by SDS-PAGE, followed by Western blotting using anti-Ski (H-329, Santa Cruz), anti-PRMT5 (07–405, Upstate) or anti-HDAC3 (H-99, Santa Cruz). To investigate the effect of the knockdown on the expression of SMAD7 mRNA, HepG2 cells were transfected with siRNAs (final 60 nM) targeting HDAC3 or PRMT5, using Oligofectamine (Invitrogen), and at 72 h after transfection, total RNA was isolated using ISOGEN (Nippon Gene). To knockdown Ski, HepG2 cells were transfected with Ski siRNA (final 60 nM) using Oligofectamine (Invitrogen), and at 48 h after transfection, cells were divided into three plates. After an additional 24 h cultivation, the siRNA transfection was repeated. At 72 h after the second transfection, total RNA was isolated using ISOGEN (Nippon Gene). Real-time quantitative RT-PCR was carried out using the ABI 7500 Real-Time PCR System and the QuantiTect Probe RT-PCR Kit (QIAGEN) according to the manufacturer's instructions. The thermal cycling parameters were 50 °C for 30 min, 95 °C for 15 min and 45 cycles of 94 °C for 15 s, 60 °C for 1 min. The primers used were as follows: smad7, 5'-TCCAG ATACCCGATGGATTTTC-3' and 5'-CCCTGTTTCAGCGG AGGAA-3', with the TaqMan probe 5'-FAM-CAAACCAACTG CAGACTGTCCAGATGCT-TAMRA-3'; GAPDH, 5'-CAAC GGATTTGGTCGTATTGG-3' and 5'-GGCAACAATATCC ACTTTACCAGAGT-3', with the TaqMan probe 5'-FAM-CCT GGTCACCAGGGCTGCTT-TAMRA-3'.


   Acknowledgements
 
We are grateful to Y. Nakatani for the pOZ-FH-N vector, and to the staff of the Research Resources Center at the RIKEN Brain Science Institute for the mass spectrometric analysis. This work was supported by Grants-in-aid for Scientific Research and by grants from the Genome Network project of the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Tadashi Yamamoto

* Correspondence: sishii{at}rtc.riken.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Akiyoshi, S., Inoue, H., Hanai, J., Kusanagi, K., Nemoto, N., Miyazono, K. & Kawabata, M. (1999) c-Ski acts as a transcriptional co-repressor in transforming growth factor-β signaling through interaction with smads. J. Biol. Chem. 274, 35269–35277.[Abstract/Free Full Text]

Alland, L., Muhle, R., Hou Jr, H., Potes, J., Chin, L., Screiber-Agus, N. & DePinho, R.A. (1997) Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387, 49–55.[CrossRef][Medline]

Bonni, S., Wang, H.R., Causing, C.G., Kavsak, P., Stroschein, S.L., Luo, K. & Wrana, J.L. (2001) TGF-β induces assembly of a Smad2-Smurf2 ubiquitin ligase complex that targets SnoN for degradation. Nat. Cell Biol. 3, 587–595.[CrossRef][Medline]

Chacko, B.M., Qin, B., Correia, J.J. Lam, S.S., de Caestecker, M.P. & Lin, K. (2001) The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization. Nat. Struct. Biol. 8, 248–253.[CrossRef][Medline]

Dai, P., Shinagawa, T., Nomura, T., Harada, I., Kaul, S.C., Wadhwa, R., Khan, M.M., Akimaru, H., Sasaki, H., Colmenares, C. & Ishii, S. (2002) Ski is involved in transcriptional regulation by the repressor and full-length forms of Gli3. Genes Dev. 16, 2843–2848.[Abstract/Free Full Text]

Deckers, M., van Dinther, M., Buijs, J., Que, I., Lowik, C., van der Pluijm, G. & ten Dijke, P. (2006) The tumor suppressor Smad4 is required for transforming growth factor β-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells. Cancer Res. 66, 2202–2209.[Abstract/Free Full Text]

Denissova, N.G. & Liu, F. (2004) Repression of endogenous Smad7 by Ski. J. Biol. Chem. 279, 28143–29148.[Abstract/Free Full Text]

Dong, X., Tsuda, L., Zavitz, K.H., Lin, M., Li, S., Carthew, R.W. & Zipursky, S.L. (1999) ebi regulates epidermal growth factor receptor signaling pathways in Drosophila. Genes Dev. 13, 954–965.[Abstract/Free Full Text]

Guenther, M.G., Lane, W.S., Fischle, W., Verdin, E., Lazar, M.A. & Shiekhattar, R. (2000) A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness. Genes Dev. 14, 1048–1057.[Abstract/Free Full Text]

Hartman, H.B., Yu, J., Alenghat, T., Ishizuka, T. & Lazar, M.A. (2005) The histone-binding code of nuclear receptor co-repressors matches the substrate specificity of histone deacetylase 3. EMBO Rep. 6, 445–451.[CrossRef][Medline]

He, J., Tegen, S.B., Krawitz, A.R., Martin, G.S. & Luo, K. (2003) The transforming activity of Ski and SnoN is dependent on their ability to repress the activity of Smad proteins. J. Biol. Chem. 278, 30540–30547.[Abstract/Free Full Text]

Heinzel, T., Lavinsky, R.M., Mullen, T.M., Söderstrom, M., Laherty, C.D., Torchia, J., Yang, W.M., Brard, G., Ngo, S.D., Davie, J.R., Seto, E., Eisenman, R.N., Rose, D.W., Glass, C.K. & Rosenfeld, M.G. (1997) A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387, 43–48.[CrossRef][Medline]

Huang, E.Y., Zhang, J., Miska, E.A., Guenther, M.G., Kouzarides, T. & Lazar, M.A. (2000) Nuclear receptor corepressors partner with class II histone deacetylases in a Sin3-independent repression pathway. Genes Dev. 14, 45–54.[Abstract/Free Full Text]

Inamitsu, M., Itoh, S., Hellman, U., ten Dijke, P. & Kato, M. (2006) Methylation of Smad6 by protein arginine N-methyltransferase 1. FEBS Lett. 580, 6603–6611.[CrossRef][Medline]

Kokura, K., Kaul, S.C., Wadhwa, R., Nomura, T., Khan, M.M., Shinagawa, T., Yasukawa, T., Colmenares, C. & Ishii, S. (2001) The Ski protein family is required for MeCP2-mediated transcriptional repression. J. Biol. Chem. 276, 34115–34121.[Abstract/Free Full Text]

Li, J., Wang, J., Wang, J., Nawaz, Z., Liu, J.M., Qin, J. & Wong, J. (2000) Both corepressor proteins SMRT and N-CoR exist in large protein complexes containing HDAC3. EMBO J. 19, 4342–4350.[CrossRef][Medline]

Luo, K., Stroschein, S.L., Wang, W., Chen, D., Martens, E., Zhou, S. & Zhou, Q. (1999) The Ski oncoprotein interacts with the Smad proteins to repress TGFβ signaling. Genes Dev. 13, 2196–2206.[Abstract/Free Full Text]

Massagué, J. & Wotton, D. (2000) Transcriptional control by the TGF-β/Smad signaling system. EMBO J. 19, 1745–1754.[CrossRef][Medline]

Matsuzawa, S.I. & Reed, J.C. (2001) Siah-1, SIP, and Ebi collaborate in a novel pathway for β-catenin degradation linked to p53 responses. Mol. Cell 7, 915–926.[CrossRef][Medline]

Mizuide, M., Hara, T., Furuya, T., Takeda, M., Kusanagi, K., Inada, Y., Mori, M., Imamura, T., Miyazawa, K. & Miyazono, K. (2003) Two short segments of Smad3 are important for specific interaction of Smad3 with c-Ski and SnoN. J. Biol. Chem. 278, 531–536.[Abstract/Free Full Text]

Nagano, Y., Mavrakis, K.J., Lee, K.L., Fujii, T., Koinuma, D., Sase, H., Yuki, K., Isogaya, K., Saitoh, M., Imamura, T., Episkopou, V., Miyazono, K. & Miyazawa, K. (2007) Arkadia induces degradation of SnoN and c-Ski to enhance transforming growth factor-β signaling. J. Biol. Chem. 282, 20492–20501.[Abstract/Free Full Text]

Nagarajan, R.P., Zhang, J., Li, W. & Chen, Y. (1999) Regulation of Smad7 promoter by direct association with Smad3 and Smad4. J. Biol. Chem. 274, 33412–33418.[Abstract/Free Full Text]

Nagy, L., Kao, H.Y., Chakravarti, D., Lin, R.J., Hassig, C.A., Ayer, D.E., Schreiber, S.L. & Evans, R.M. (1997) Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89, 373–380.[CrossRef][Medline]

Nakatani, Y. & Ogryzko, V. (2003) Immunoaffinity purification of mammalian protein complexes. Methods Enzymol. 370, 430–444.[Medline]

Nishioka, K. & Reinberg, D. (2003) Methods and tips for the purification of human histone methyltransferases. Methods 31, 49–58.[CrossRef][Medline]

Nomura, N., Sasamoto, S., Ishii, S., Date, T., Matsui, M. & Ishizaki, R. (1989) Isolation of human cDNA clones of ski and the ski-related gene, sno. Nucleic Acids Res. 17, 5489–5500.[Abstract/Free Full Text]

Nomura, T., Khan, M.M., Kaul, S.C., Dong, H.D., Wadhwa, R., Colmenares, C., Kohno, I. & Ishii, S. (1999) Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor. Genes Dev. 13, 412–423.[Abstract/Free Full Text]

Nomura, T., Tanikawa, I., Akimaru, H., Kanei-Ishii, C., Ichikawa-Iwata, E., Khan, M.M., Ito, H. & Ishii, S. (2004) Oncogenic activation of c-Myb correlates with a loss of negative regulation by TIF1β and Ski. J. Biol. Chem. 279, 16715–16726.[Abstract/Free Full Text]

Pal, S., Vishwanath, S.N., Erdjument-Bromage, H., Tempst, P. & Sif, S. (2004) Human SWI/SNF-associated PRMMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes. Mol. Cell. Biol. 24, 9630–9645.[Abstract/Free Full Text]

Pal, S., Yun, R., Datta, A., Lacomis, L., Erdjument-Bromage, H., Kumar, J., Tempst, P. & Sif, S. (2003) mSin3A/histone deacetylase 2- and PRMT5-containing Brg1 complex is involved in transcriptional repression of the Myc target gene cad. Mol. Cell. Biol. 23, 7475–7487.[Abstract/Free Full Text]

Perissi, V., Aggarwal, A., Glass, C.K., Rose, D.W. & Rosenfeld, M.G. (2004) A corepressor/coactivator exchange complex required for transcriptional activation by nuclear receptors and other regulated transcription factors. Cell 116, 511–526.[CrossRef][Medline]

Rea, S., Eisenhaber, F., O’Carroll, D., Strahl, B.D., Sun, Z.W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C.P., Allis, C.D. & Jenuwein, T. (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593–599.[CrossRef][Medline]

Shi, Y., Sawada, J., Sui, G., Affar E.B., Whetstine, J.R., Lan, F., Ogawa, H., Luke, M.P., Nakatani, Y. & Shi, Y. (2003) Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature 422, 735–738.[CrossRef][Medline]

Shinagawa, T., Dong, H.D., Xu, M., Maekawa, T. & Ishii, S. (2000) The sno gene, which encodes a component of the histone deacetylase complex, acts as a tumor suppressor in mice. EMBO J. 19, 2280–2291.[CrossRef][Medline]

Shinagawa, T., Nomura, T., Colmenares, C., Ohira, M., Nakagawara, A. & Ishii, S. (2001) Increased susceptibility to tumorigenesis of ski-deficient heterozygous mice. Oncogene 20, 8100–8108.[CrossRef][Medline]

Stroschein, S.L., Bonni, S., Wrana, J.L. & Luo, K. (2001) Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN. Genes Dev. 15, 2822–2836.[Abstract/Free Full Text]

Stroschein, S.L., Wang, W., Zhou, S., Zhou, Q. & Luo, K. (1999) Negative feedback regulation of TGF-β signaling by the SnoN oncoprotein. Science 286, 771–774.[Abstract/Free Full Text]

Sun, Y., Liu, X., Eaton, E.N., Lane, W.S., Lodish, H.F. & Weinberg, R.A. (1999a) Interaction of the Ski oncoprotein with Smad3 regulates TGF-β signaling. Mol. Cell 4, 499–509.[CrossRef][Medline]

Sun, Y., Liu, X., Ng-Eaton, E., Lodish, H.F. & Weinberg, R.A. (1999b) SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor β signaling. Proc. Natl. Acad. Sci. USA 96, 12442–12447.[Abstract/Free Full Text]

Tokitou, F., Nomura, T., Khan, M.M., Kaul, S.C., Wadhwa, R., Yasukawa, T., Kohno, I. & Ishii, S. (1999) Viral Ski inhibits retinoblastoma protein (Rb)-mediated transcriptional repression in a dominant negative fashion. J. Biol. Chem. 274, 4485–4488.[Abstract/Free Full Text]

Wan, Y., Liu, X. & Kirschner, M.W. (2001) The anaphase-promoting complex mediates TGF-β signaling by targeting SnoN for destruction. Mol. Cell 8, 1027–1039.[CrossRef][Medline]

Wu, J.W., Krawitz, A.R., Chai, J., Li, W., Zhang, F., Luo, K. & Shi, Y. (2002) Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-β signaling. Cell 111, 357–367.[CrossRef][Medline]

Xu, L., Chen, Y.G. & Massague, J. (2000a) The nuclear import function of Smad2 is masked by SARA and unmasked by TGFβ-dependent phosphorylation. Nat. Cell Biol. 2, 559–562.[CrossRef][Medline]

Xu, W., Angelis, K., Danielpour, D., Haddad, M.M., Bischof, O., Campisi, J., Stavnezer, E. & Medranon, E.E. (2000b) Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type β transforming growth factor. Proc. Natl. Acad. Sci. USA 97, 5924–5929.[Abstract/Free Full Text]

Yoon, H.G., Choi, Y., Cole, P.A. & Wong, J. (2005) Reading and function of a histone code involved in targeting corepressor complexes for repression. Mol. Cell. Biol. 25, 324–335.[Abstract/Free Full Text]

Zhang, X., Ozawa, Y., Lee, H., Wen, Y.D., Tan, T.H., Wadzinski, B.E. & Seto, E. (2005) Histone deacetylase 3 (HDAC3) activity is regulated by interaction with protein serine/threonine phophatase 4. Genes Dev. 19, 827–839.[Abstract/Free Full Text]

Zhang, Y., Iratni, R., Erdjument-Bromage, H., Tempst, P. & Reinberg, D. (1997) Histone deacetylases and SAP18, a novel polypeptide, are components of a human Sin3 complex. Cell 89, 357–364.[CrossRef][Medline]

Accepted: 1 October 2008




This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. M. Band, M. Bjorklund, and M. Laiho
The Phosphatidylinositol 3-Kinase/Akt Pathway Regulates Transforming Growth Factor-{beta} Signaling by Destabilizing Ski and Inducing Smad7
J. Biol. Chem., December 18, 2009; 284(51): 35441 - 35449.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tabata, T.
Right arrow Articles by Ishii, S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Tabata, T.
Right arrow Articles by Ishii, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS