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The highly conserved region of the co-repressor Sin3A functionally int
http://www.100md.com 《核酸研究医学期刊》
     Genetic Institute, Justus-Liebig-University, Heinrich-Buff-Ring 58–62, D-35392 Giessen, Germany and 1 Department of Biochemistry, University of Kuopio, PO Box 1627, Fin-70211 Kuopio, Finland

    *To whom correspondence should be addressed. Tel: +49 641 99 35468; Fax: +49 641 99 35469; Email: aria.baniahmad@gen.bio.uni-giessen.de

    Present address:

    Uwe Dressel, Institute for Molecular Bioscience, University of Queensland, Queensland Bioscience Precinct, Services Road, Brisbane, QLD 4072, Australia

    ABSTRACT

    The Sin3 proteins are evolutionarily conserved co-repressors (CoR) that function as mediators of gene repression for a variety of transcriptional silencers. The paired amphipathic helices of Sin3A were identified and studied as protein–protein interacting domains. Previously we have shown the interaction of Sin3A with the CoR Alien in vivo and in vitro. Here, we show that Alien and Sin3A reside together in vivo with the vitamin D3 receptor on the human 24-hydroxylase (CYP24) promoter containing vitamin D3 response elements by chromatin immunoprecipitation. We delineated and characterized the interaction domains of Sin3A with Alien. Interestingly, the highly conserved region (HCR) of Sin3A, which has not yet been functionally characterized, interacts with Alien. The HCR encompasses only 134 amino acids, shares more than 80% identity with Sin3B and binds to the N-terminus of Alien, which harbours a transferable silencing function. Functionally, co-expression of Sin3A enhances Alien-mediated gene repression and overexpression of the HCR alone leads to the inhibition of Alien-mediated repression and to the induction of the endogenous CYP24 promoter. Our results therefore indicate a novel functional role of the Sin3 HCR and give novel insights into Alien-mediated gene repression.

    INTRODUCTION

    Gene silencing is mediated in part through the recruitment of conserved co-repressors (CoRs) to DNA-bound transcriptional silencers (reviewed in 1–3). CoRs mediate gene repression by binding to basal transcription factors and/or by recruitment of an enzymatic activity that alters chromatin structure. Sin3 was shown to recruit histone deacetylase (HDAC) activity and histone methyltransferase activity, which is thought to lead to a more compact chromatin structure. This in turn leads to lower accessibility of transcriptional activators and basal promoter factors to their target sites and, ultimately, to a reduced transcription rate at that particular gene (4–6).

    Sin3A and Sin3B exhibit in part strong sequence similarities and both proteins are evolutionarily conserved, having homologues in Drosophila and yeast. Sin3 proteins harbour four paired amphipathic helices (PAH) motifs and were shown to inhibit expression of the yeast HO gene (7). Yeast Sin3 was originally also found to repress the human progesterone receptor heterologously expressed in yeast cells (8). In mammalian cells Sin3A and Sin3B were originally found to be involved in Mad/Max-mediated repression and cell cycle control (9–12). Subsequently, an increasing number of transcription factors were identified that interact with Sin3A or Sin3B. Mapping the interaction domains (IDs) of Sin3 with various transcription factors, the PAH2 of Sin3A has been predominantly identified as mediating binding, while other regions of Sin3A have only been sporadically characterized (13–15).

    Mammalian Sin3A is a large protein capable of multiple protein–protein interactions. It is thought that the Sin3 proteins serve as a scaffold on which CoR complexes assemble and recruit HDAC activity. In line with that, several Sin3A CoR protein complexes have been described (14,16–24).

    Another highly conserved protein, Alien, has been shown to have characteristics of a CoR for selected members of the nuclear receptor superfamily. Alien was found to be ubiquitously expressed in vivo and in all cell lines tested. Interestingly, expression of Alien is induced by thyroid hormone in vivo and in neuronal cell culture (25), suggesting that the receptor controls expression of its CoR and thus regulates its own transcriptional silencing. Alien mediates transcriptional silencing in part by recruiting HDAC activity that is sensitive to trichostatin A (TSA), an inhibitor of HDAC activity (26). In addition, by using co-immunoprecipitation and the yeast two-hybrid system, Alien was shown to interact with Sin3A (26).

    Alien interacts in a hormone-sensitive manner with the thyroid hormone receptor (TR) and the vitamin D3 receptor (VDR) (26,27). The VDR is a transcription factor that regulates target gene expression via recruitment of co-activators and CoRs to specific DNA response elements. 1,25-Dihydroxyvitamin D3 is a natural agonist of the VDR, but in the absence of ligand the receptor can also act as a transcriptional silencer. Both the TR (28) and VDR (29) also influence the cell cycle. The VDR is known to inhibit cell proliferation of commonly occurring hormone-regulated cancers and is therefore considered to be a potentially important drug target for breast and prostate cancer therapy (30–32).

    The VDR binds to a variety of vitamin D3 response elements (VDREs) with DNA sequence half-sites of specific geometry, such as direct hexameric repeats spaced by three nt (DR3) and everted repeats spaced by 9 nt (ER9). We have shown previously that Alien is a CoR for the VDR (27). Alien was shown to enhance VDR-mediated gene repression in the absence of ligand. Interestingly, in contrast to other CoRs, such as NCoR and SMRT, Alien displays VDRE subtype selectivity, by enhancing VDR-mediated gene repression on DR3-type but not on ER9-type VDREs (27).

    The aim of this study was to investigate the mechanisms of Alien-mediated silencing and the role of the Alien–Sin3A interaction in this phenomenon. Therefore, we analysed whether VDR, Alien and Sin3A are co-recruited in a chromatin context and mapped the IDs of Alien and Sin3A. Interestingly, we found that the highly conserved region (HCR) of Sin3A binds to Alien. This region is located outside the four PAHs. Notably, the HCR in isolation acts as a dominant negative inhibitor of Alien-mediated repression, while a longer Sin3A fragment that includes the HCR and two silencing domains enhances Alien-mediated repression. By mapping the silencing domain of Alien we found that the N-terminal region of Alien harbours a transferable silencing domain, which interacts with the HCR, indicating a correlation between silencing and interaction with Sin3A. Furthermore, expression of the HCR alone increases expression of the endogenous, VDR-regulated CYP24 gene. These results provide novel insights into the role of the HCR and the trimeric complex of VDR, Alien and Sin3A in VDR-mediated silencing.

    MATERIALS AND METHODS

    Chromatin immunoprecipitation (ChIP) assays

    Human breast cancer MCF-7 cells were used for ChIP assays of endogenous factors recruited to the human CYP24 gene promoter, which is known to be a primary VDR target (33). ChIP assays and PCR analyses were performed as described by V?is?nen et al. (34). Antibodies for VDR, the retinoid X receptor (RXR) and Sin3A were purchased from Santa Cruz Biotechnology (Heidelberg, Germany) (sc9164, sc553 and sc994, respectively). The anti-Alien antibody was described earlier (26). For hormonal studies the cells were kept in charcoal-depleted serum prior to treatment with a final concentration of 10 nM 1,25(OH)2D3 (dissolved in ethanol) for the indicated time prior to formaldehyde treatment. PCR primers for the distal region of the human CYP24 promoter (–812 to –425, negative control) were 5'-CGTCTGCCAG GGCCCCGGGG-3' and 5'-CATCGTTGGTGCAAGCC ACG-3' and those for the proximal region (–488 to –64, containing the VDRE cluster) were 5'-GTCCAGGCT GGGGGTATCTG-3' and 5'-CGCAGAGGAGGGCGGAG TGG-3'.

    Real-time PCR

    Total RNA and mRNA were extracted using Tri-reagent (Sigma-Aldrich, St Louis, MO) and an Oligotex mini mRNA kit (Qiagen, Hilden, Germany), respectively. An aliquot of 100 ng mRNA was used as a template in a cDNA synthesis reaction using 100 pmol oligo(dT18) primer in the presence of reverse transcriptase (Fermentas, Vilnius, Lithuania). The reaction was performed at 37°C for 1 h. Real-time quantitative PCR was performed in an IQ cycler (Bio-Rad, Hercules, CA) using the dye SybrGreen (Molecular Probes, Leiden, The Netherlands). In PCR reactions, 3 mM MgCl2 was used. The PCR cycling conditions used were: 40 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s. Fold inductions were calculated using the formula: 2– (Ct), where Ct is Ct – Ct(EtOH), Ct is Ct(test gene) – Ct(control gene) and Ct is the cycle at which the threshold is crossed. Gene-specific primer pairs for acidic riboprotein P0 (ARP0, the control gene) and CYP24 were previously described elsewhere (34,35). The primers for human ribosomal protein L13a were 5'-CCTGGAGGAGAAGAGGAAAGAGA-3' (forward) and 5'-TTGAGGACCTCTGTGTATTTGTCAA-3' (reverse). The PCR conditions were the same as for the other genes except that an annealing temperature of 62°C was used instead of 58°C. The control gene ARPO was run at the same time at this higher temperature for correct data normalization. PCR product quality was monitored using post-PCR melt curve analysis at the end of the amplification cycles.

    Plasmids

    The in vitro transcription vectors for mouse Sin3A and Sin3B were kindly provided by Dr R.N.Eisenman (11). Sin3A deletions were generated by insertion of the mouse Sin3A coding region in-frame into the vectors pT7?Sal (36), pcDNA3-ATG, a pcDNA3 vector (Invitrogen, Karlsruhe, Germany) derivative having a Kozak sequence with the ATG translational start codon, or pCITE (Novagen, Schwalbach, Germany) using standard cloning techniques. pGST-linker and GST–Alien were described earlier (26). pGST-Sin3A57–215, pGST-Sin3A215–404 and pGST-Sin3A404–545 were kindly provided by Dr M.Privalsky (37), whereas pGST-Sin3A248–678 and pGST-Sin3A1001–1214 were described earlier (38). GST–Sin3A deletions were generated using pGEX-2T (Amersham Biosciences, Freiburg, Germany) or pGST-linker by in-frame insertions of Sin3A cDNA into the polylinker. Vectors for mammalian expression systems were pUAS4x-TATA-Luc and pUAS4x-TK-Luc as reporters (kindly provided by Dr S.Tenbaum), pUAS6x-tkCAT (39) and pcDNA3-Sin3 deletions and pABGal94-Alien (26) as expression vectors. pAB-HA-Sin3A56–724 was generated by insertion of the ScaI fragment of pVZ-Sin3A (Dr R.N.Eisenman) in frame into pAB?gal (36,40).

    GST pull-down assay

    GST pull-down assays using a TNT in vitro translation kit (Promega, Mannheim, Germany) were performed as follows. Bacterial expression of GST, GST–mSin3A deletions or GST–hAlien was performed by induction of gene expression with 0.2 mM IPTG for 3 h at 37°C in Escherichia coli HB101 cells. The recombinant proteins were purified after lysis of the cells in lysis buffer (60 mM KCl, 20 mM HEPES pH 7.8, 2 mM DTT, 1 mM EDTA, 4 mg/ml lysozyme) by three freeze–thaw cycles. After centrifugation at 4°C at 33 000 r.p.m. for 20 min, the supernatant was incubated with glutathione beads (Amersham Biosciences) for 30 min at room temperature. Bound GST fusion proteins were washed three times with NETNM buffer . GST fusion proteins bound to beads were blocked with 20% denatured milk powder in NETN (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40) for 15 min. The beads were washed twice with NETNM buffer and once with TWB buffer (20 mM HEPES pH 7.9, 60 mM NaCl, 6 mM MgCl2, 8.2% glycerol, 1 mM DTT, 0.1 mM EDTA). In vitro translated, methionine-labelled proteins were generated using the TNT kit (Promega) according to the manufacturer’s protocol and added to the beads in a final volume of 50 μl (in TWB buffer) for 1 h at room temperature. After extensive washing with 6 x 1 ml of NETNM buffer and twice with NETN buffer, SDS loading buffer was added and the bound proteins were separated by SDS–PAGE. In each experiment, the input lane shows 10% of the amount incubated with the GST fusion beads. The GST fusion proteins were stained with Coomassie Brilliant Blue to ensure equal loading, and the bound proteins were visualized by autoradiography.

    Yeast two-hybrid assays

    Yeast two-hybrid assays were described earlier (26,41) using the LexA fusion system. DNA-bound factors are fused to the lexA DNA-binding domain and the activator to the B42 activation domain. ?-galactosidase assays of the lacZ reporter from transformed yeast cells were used for quantification.

    Cell culture and transient transfections

    HeLa, CV1 and HEK293 cells were grown in DMEM with 10% fetal bovine serum (FBS) at 37°C/5% CO2, HD3 cells in DMEM with 8% FCS and 2% chicken serum at 37°C/5% CO2 and MCF-7 cells with 5% FBS at 37°C/5% CO2. HD3 cells were transfected by the DEAE-dextran method (26), HeLa, CV1 and HEK293 cells by the CaPO4 method and MCF-7 cells with N--N,N,N-trimethylammonium methylsulfate (DOTAP; Roth, Karlsruhe, Germany). DOTAP transfection was carried out for 4 h according to the manufacturer’s protocol. For hormonal studies the cells were kept in charcoal-depleted FBS. The hormone 1,25(OH)2D3, a gift from Dr Lise Binderup (LEO Pharma, Ballerup, Denmark), was added 24 h after transfection at a final concentration of 10 nM. Standard deviations represent the variation of the mean. Independent duplicate, triplicate or quadruplicate experiments were performed each time. Each experiment was performed at least three times. pCMV-LacZ was included as an internal control and was used for normalization.

    Sequence comparison and databank search

    Protein homology search and sequence analyses and sequence alignments were performed at www.ncbi.nlm.nih.gov/blast (blastn/tblastn/tblastx) and www.searchlaunch.bcm.tmc.edu/multialign/multialign.html with the program CLUSTAL_W 1.8.

    RESULTS

    Alien and Sin3A reside simultaneously on the human CYP24 promoter in vivo

    To investigate whether Alien and Sin3A are present on a native promoter at the same time, we performed ChIP experiments on the 1,25(OH)2D3-inducible human CYP24 gene. The VDRE cluster of the human CYP24 promoter consists of two DR3-type response elements, which mediate both VDR-dependent repression and hormone-induced activation (42) (Fig. 1A). Using antibodies against VDR and RXR for ChIP and subsequent PCR analyses, the proximal CYP24 promoter region was co-precipitated and detected (Fig. 1B). A distal control region of the CYP24 promoter without VDREs was not co-precipitated with these antibodies. This suggests a recruitment of both VDR and RXR to the VDREs in vivo in unstimulated cells. This agrees with the view that VDR–RXR heterodimers are involved in regulation of the CYP24 gene promoter when 1,25(OH)2D3 is absent. For an assessment of Alien and Sin3A binding to these regions the respective antibodies were used. In the absence of 1,25(OH)2D3 both Alien and Sin3A co-precipitated with the proximal CYP24 promoter region in the absence of ligand, suggesting that both CoR proteins associate with this region of the CYP24 promoter. No PCR amplification was observed using the antibodies for either Alien or Sin3A when the cells were treated with 1,25(OH)2D3 for 60 min prior to ChIP. This indicates that both Sin3A and Alien dissociate from this promoter region in the presence of a ligand. This implies that Sin3A and Alien may be involved in VDR-mediated gene repression. The distally located region of the CYP24 promoter used as a control in PCR did not amplify in ChIP assays performed with VDR, RXR, Alien or Sin3A antibodies. Taken together, these data indicate that both Alien and Sin3A associate in vivo with a promoter region that harbours functional VDREs and this interaction is ligand-dependent.

    Figure 1. Alien and Sin3A are co-recruited to the CYP24 promoter in vivo. ChIP assays were performed on the 1,25(OH)2D3-inducible human CYP24 promoter in MCF-7 cells. Antibodies against VDR, RXR, Alien and Sin3A were used for ChIP in the absence of hormone or treatment of the cells with 1,25(OH)2D3 for 60 min prior to ChIP. Subsequent PCR was used for detection of precipitated genomic CYP24 promoter. (Top) Schematic view of the human CYP24 promoter and the primer pairs used for PCR. The DR3-type VDRE cluster is highlighted (barrels). (Bottom) Visualized PCR bands from ChIPs, indicative of chromatin recruitment using the indicated antibodies.

    The HCR of Sin3A interacts with the N-terminus of Alien

    To characterize the Alien–Sin3 interaction the binding domains of Sin3A with Alien were first mapped using a GST pull-down assay with bacterially expressed and purified GST or GST–Alien (26) and in vitro translated, 35S-labelled Sin3 proteins. As a controlled starting point we observed that in vitro translated Sin3A and Sin3B proteins bound to GST–Alien but not to GST alone (Fig. 2). We usually obtained a band doublet of in vitro translated Sin3A, which may derive from either an internal translation start site or protein degradation. As an additional control we used in vitro translated luciferase and did not observe an interaction with either GST or GST–Alien. As a loading control, the Coomassie stained gel with the bacterially expressed GST or GST–Alien is shown. These data show that Alien binds to Sin3A in vitro, an interaction previously characterized by ourselves in co-immunoprecipitation and yeast two-hybrid interaction assay experiments.

    Figure 2. Alien binds the CoRs Sin3A and Sin3B. Bacterially expressed GST or GST–Alien was incubated with 35S-labelled, in vitro translated Sin3A or Sin3B in GST pull-down assays. Bound proteins were separated by SDS–PAGE and visualized by autoradiography. In addition, in vitro translated luciferase was used as a negative control. The Coomassie stained gel shows the amount of bacterially expressed proteins used. Arrows indicate migration of the proteins.

    For fine mapping, several Sin3A deletions were created and tested for interaction with Alien (Fig. 3A). The N-terminal 678 amino acids of Sin3A interacts, while the N-terminal 479 amino acids of the CoR lack interaction with Alien. As a specificity control we co-translated both full-length Sin3A and Sin3A1–479 and incubated the mixture with either GST or GST–Alien. Only the full-length Sin3A protein was retained, but not the first 479 amino acids (Fig. 3A, second panel). This suggested that the ID for Alien is located between amino acids 479 and 678. This was confirmed using this portion of Sin3A in GST pull-down experiments. Further fine mapping of this region revealed that Alien specifically interacts with Sin3A545–678, termed ID1, but not with Sin3A404–545. An additional Alien ID of Sin3A was mapped to amino acids 1001–1137 (Fig. 3A) and was termed ID2. To confirm the Sin3A Alien IDs, we reversed the interaction partners by using bacterially expressed GST–Sin3A deletions and in vitro translated Alien (Fig. 3B). Both Sin3A545–678 (ID1) and Sin3A1001–1137 (ID2) exhibited strong binding to Alien, whereas Sin3A1–545 harbouring PAH1–PAH3 did not show binding to Alien.

    Figure 3. Two domains of Sin3A, the HCR and ID2, interact with Alien. (A) GST pull-down assays using deletions of 35S-labelled, in vitro translated Sin3A with GST or GST–Alien. The deletion mutant Sin3A1–479 and full-length Sin3A were co-incubated with GST or GST–Alien as specificity controls. (B) GST pull-down assays with bacterially expressed Sin3A deletions incubated with 35S-labelled, in vitro translated human Alien. (C) Schematic view and homologies of Sin3A and Sin3B. The positions of PAHs 1–4 are indicated. The two identified IDs of Sin3A with Alien are shown in black. The homologies between Sin3A and Sin3B are graphically presented as percentage identical amino acids. The HCRs of Sin3A and Sin3B share 81% identity at the amino acid level and maps to amino acids 545–678 of Sin3A. Note that Sin3B is smaller in size, with only 954 amino acids.

    Sequence comparisons between mouse Sin3A and Sin3B revealed that, in addition to the PAH domains, an additional HCR exists. This is located C-terminal to PAH3 and has 81% amino acid sequence identity between Sin3A and Sin3B. This HCR exhibits the highest regional homology between Sin3A and Sin3B (Fig. 3C) and overlaps the region identified as ID1. Therefore, henceforth ID1 will be termed the HCR.

    Although the HCR is part of the previously identified domain HDAC interaction domain (HID) (43) and of the sites on Sin3A that mediate the interactions with SAP45 (mSDS3), SAP130 and SAP180 (RBP1), the isolated HCR, even when combined with PAH3, does not interact with these factors (16,43). This indicates that the interaction of Alien with the HCR is distinct from that of these factors.

    Based on our findings, the HCR of Sin3A was selected for further interaction analysis with Alien. To verify the identified IDs the yeast two-hybrid assay was used (Fig. 3). Full-length Alien (Alien1–305) as the activator (B42 fusion) exhibited strong interaction with the Sin3A56–724 harbouring the HCR used as bait (lexA fusion). The deletion fragment, Alien1–128, also interacted with Sin3A, although more weakly than Alien1–305. Interestingly, this N-terminal portion of Alien does not interact with TR or another member of the nuclear receptor superfamily, DAX1, in the same assay system (40), indicating a specific interaction with Sin3A. Other deletions, including Alien1–32 and those that interact with TR, such as Alien128–305, did not show significant interaction with Sin3A56–724 (Fig. 4). The units yielded were similar to those obtained with both the empty bait vector and the empty activator vector. These findings suggest that the first 128 amino acids of Alien participate in the interaction of this protein with Sin3A. To verify that the isolated HCR is sufficient to mediate the Sin3A–Alien interaction, we used Sin3A545–678 as bait. Alien1–305 and Alien1–128 showed an interaction of similar strength with Sin3A545–678, which was in accordance with our GST pull-down experiments. As negative controls we used the empty bait vector or the empty activator vector. Since, within the range of standard deviation, comparable units were obtained with Alien128–305 with or without Sin3A as bait it may indicate that the C-terminal part of Alien does not bind significantly to the HCR of Sin3A (Fig. 4). Based on these data, we conclude that the HCR of Sin3 interacts with Alien in yeast. More specifically, the HCR of Sin3A interacts with the N-terminus of Alien (Alien1–128).

    Figure 4. The N-terminus of Alien interacts with the HCR of Sin3A. Yeast two-hybrid assays using either Sin3A56–724 or Sin3A545–678 (the isolated HCR) as bait and full-length Alien or deletions thereof as the activator. The ?-galactosidase yield values are presented as Miller units. Negative controls included the use of empty vectors. The standard deviations are indicated.

    Alien1–128 harbours a transferable silencing function

    Since Alien and Sin3A have been previously shown to be involved in repression of gene expression, we wondered whether the interaction of Alien with Sin3A correlates with Alien-mediated silencing. Using Gal–Alien deletions in transient reporter assays a strong repression capability of full-length Alien was observed (Fig. 5). Several cell lines were tested, the erythroid cell line HD3 (Fig. 5) and HEK 293 cells (not shown) with strong Alien-mediated gene repression and the CV1 cell line with weak Alien-mediated silencing (Fig. 5). Alien1–32 showed no significant repression function, while Alien1–128 efficiently repressed reporter gene activity in CV1 cells. This suggests that the ID of Alien in the presence of Sin3A exhibits silencing function. In addition, Alien128–305 (the C-terminus) repressed promoter activity, suggesting that the C-terminus also harbours a transferable silencing function. Since this domain does not overlap with the Alien N-terminus, it suggests that Alien possesses two silencing domains. Further delineation using additional Alien deletions indicated that the second silencing domain resides in the amino acids 266–305 region of Alien. Similar results were obtained using the HD3 cell line. Thus, Alien harbours two transferable silencing domains: one located in the N-terminus (amino acids 1–128), which coincides with the Sin3A ID, and a second in the C-terminus (amino acids 266–305).

    Figure 5. Alien harbours two silencing domains. Fusions of Alien to the first 94 amino acids of the Gal DNA binding domain were used in reporter assays. Expression vectors for Gal94 or Gal–Alien deletions (3 pmol) were co-transfected with the reporter pUAS6x-tkCAT (1.5 pmol) into HD3 or CV1 cells. The values obtained with the gal DBD were set arbitrarily as 1 and the values obtained with the Gal–Alien fusions are shown as fold repression. The two identified silencing domains of Alien are highlighted as grey boxes.

    Sin3A enhances Alien-mediated silencing

    To investigate the functional role of Sin3A on Alien-mediated repression, Sin3A56–724, which harbours the HCR and two silencing domains (37), was overexpressed together with Gal–Alien fusions in reporter assays. Conditions were chosen from preceding titration experiments (not shown) under which Alien exhibits weak transcriptional repression (Fig. 6A) by using low amounts of Gal–Alien expression vector and CV1 cells. Expression of Sin3A amino acids 56–724 together with Gal–Alien resulted in a significant repression of reporter gene activity. Using the expression vector encoding the Gal DBD as a control only a slight activation, if at all, was observed by overexpression of Sin3A56–724. The transcriptional activity of the Gal–Alien1–32 deletion, which lacks both interaction with Sin3A and silencing function, was not changed by Sin3A56–724, while Gal–Alien1–128, which interacts with Sin3A, exhibited increased silencing (Fig. 6A). Similar results were obtained using HeLa cells (not shown). This indicates that Sin3A acts as a functional enhancing partner for Alien. Thus, in line with the interaction data the co-expression of Sin3A increases Alien-mediated silencing.

    Figure 6. The Sin3A N-terminus enhances while the HCR represses Alien-mediated silencing. (A) Sin3A increases the silencing capability of Alien. The Sin3A N-terminus (0.8 pmol) was overexpressed together with either the Gal DBD or Gal–Alien or gal–Alien deletions (0.15 pmol) in reporter assays with UAS4x-TATA-Luc as the reporter. In dishes without Sin3A the empty vector was used. The values obtained with Gal DBD alone in the absence of Sin3A were arbitrarily set as 1 and the values obtained with the indicated expression vectors are calculated relative to Gal DBD and are indicated as fold repression or fold activation. The values obtained were normalized to those of the co-transfected ?-galactosidase expression vector (0.05 μg). (B) Expression of the HCR inhibits Alien-mediated silencing. Transient transfection assays with the Gal DBD or Gal–Alien (1 μg) in the presence of Sin3A545–678 (HCR) or Sin3A1001–1137 expression vectors or the empty vector (10 μg) in HEK 293 cells using the reporter UAS4xTk-Luc. The values obtained were normalized to those of the co-transfected ?-galactosidase expression vector (0.1 μg) and calculated relative to those obtained with the Gal DBD alone and are shown as fold repression.

    Interestingly, comparing Sin3A levels of CV1 cells with weak Alien-mediated silencing with that of HEK 293 cells with strong Alien-mediated silencing revealed that CV1 cells express much lower levels of mSin3 (Supplementary Material). This difference in the Sin3 protein levels of the two cell lines might indicate that mSin3 is a limiting factor for Alien-mediated silencing.

    The HCR of Sin3A inhibits Alien-mediated repression in vivo

    Previous results suggest that the combination of the silencing domains of Sin3A plus the Alien interaction domain HCR functionally enhances Alien-mediated silencing by recruiting silencing function to Alien. Conversely, we wondered whether the HCR alone, lacking the N-terminal silencing domain of Sin3A, might inhibit Alien-mediated repression. For that purpose a cell line in which Alien is a potent repressor (and for which low amounts of Alien expression plasmid can be used) is required as a prerequisite and as a suitable basis for the inhibition of Alien-mediated silencing by overexpression of the HCR, which might act as a competitor for endogenous Sin3A. We used HEK 293 cells in which Alien exhibits strong silencing function as compared to CV1 cells. Interestingly, this correlates with the endogenous levels of Sin3A protein as shown by western analysis, revealing low levels of endogenous mSin3 in CV1 cells and much higher levels in HEK 293 cells (Supplementary Material). This indicates that CV1 cells are useful to introduce Sin3A as a gain of function and HEK 293 cells are useful for competition for endogenous Sin3A. Thus, to analyse the potential role of the isolated HCR domain we overexpressed Sin3A545–678 together with Gal–Alien or Gal alone as control. Interestingly, the HCR domain repressed Alien-mediated silencing, which was virtually abolished under the conditions used (Fig 6B). Using Sin3A1001–1137 (ID2), a similar but less pronounced inhibition of Alien-mediated gene silencing was obtained. Based on the strong inhibitory effect on Alien-mediated silencing this indicates that the HCR acts in a dominant negative manner.

    As a further proof of this phenomenon we overexpressed the HCR (Sin3A545–678) in MCF-7 cells and observed the effects on endogenous CYP24 basal expression by real-time PCR (Fig. 7). ARP0 gene expression, which is unaffected by VDR, was used as an internal control. As a further negative control expression of ribosomal protein L13, known to be a reliable standard for gene expression (44), was monitored. Expression of the HCR did not significantly change expression of L13A, neither in the absence nor in the presence of 1,25(OH)2D3. In agreement with the previous data (Fig. 6B), we observed an increase in basal mRNA expression of the endogenous CYP24 gene when Sin3A545–678 was expressed in the absence of hormone. The level of expression of the CYP24 gene was unaffected (3-fold) (Fig. 7) after 60 min of 1,25(OH)2D3 treatment. This indicates that the Alien–Sin3A HCR interaction is involved in repression of this gene. Thus, it suggests that the Alien–Sin3A interaction via the HCR is involved in VDR-mediated gene regulation.

    Figure 7. The Sin3A HCR increases endogenous CYP24 gene expression. The HCR (Sin3A545–678) was overexpressed in MCF-7 cells and human CYP24 mRNA or human ribosomal protein L13a were monitored using real-time PCR. ARPO mRNA was used as an internal control. The mRNA was isolated from MCF-7 cells grown in hormone-depleted serum and cells treated additionally with 10 nM 1,25(OH)2D3 for 60 min transfected with Sin3A545–678 or empty vector (pCDNA3-ATG). Columns represent fold inductions and bars indicate standard deviations. Values obtained in the absence of hormone transfected with the control vector were set arbitrarily as 1. The results are derived from three independent treatments.

    DISCUSSION

    Alien has characteristics of a CoR for selected members of the nuclear receptor superfamily, such as TR and VDR, and mediates their transcriptional repression function. This suppression of gene activity is in part HDAC-dependent. A portion of this HDAC dependency may be mediated by the recruitment of Sin3A by Alien (26). The aim of this study was to obtain mechanistic insights into the role of Sin3a in Alien-mediated silencing. The interaction of endogenous Sin3A with Alien was previously shown by co-immunoprecipitation and confirmed by yeast two-hybrid assays (26). Furthermore, TSA in part relieved Alien-mediated silencing (26), which is in agreement with the findings that Sin3A exists in complexes with HDAC activity (14,16). Here, we show for the first time that Alien is recruited to chromatin in vivo. The recruitment of Alien to the unstimulated endogenous human CYP24 proximal promoter, which harbours functional DR3-type VDREs. The presence of Alien on this promoter is regulated by 1,25(OH)2D3. Accordingly, the acetylation of histone H4 on the human CYP24 promoter is increased within 1 h by addition of 1,25(OH)2D3 (34). These facts taken together strongly indicate that Alien is a bona fide CoR for VDR. This is in line with our previous findings of a hormone-sensitive interaction of Alien with the VDR (27). Similarly, Sin3A is recruited to the same promoter region as Alien and this recruitment is also regulated by 1,25(OH)2D3. This suggests the possible existence of a CoR complex composed of at least the Alien, Sin3A and VDR proteins. In agreement with this, Sin3A and VDR were found to be complexed in vivo (45). Thus, these results are in agreement with our previous findings and strongly suggest that both endogenous Alien and Sin3A are co-recruited in vivo in the context of native chromatin.

    To gain mechanistic insights we identified the IDs of Sin3A with Alien in vitro and analysed the functional significance of this interaction in vivo. Interestingly, a HCR between Sin3A and Sin3B of 134 amino acids in length mediates the interaction with Alien and functionally inhibits Alien-mediated silencing in a dominant negative manner both in transient transfection assays and at the level of the CYP24 endogenous promoter. Protein database searches with the Sin3A HCR sequence revealed that the HCR is highly conserved in the Sin3A orthologues from different species. Xenopus and Drosophila HCRs share 95% and 67% identity to the human, rat and mouse HCRs, respectively. Such a high homology is indicative of important biological functions. Notably, we did not find additional proteins in the database with sequences resembling the Sin3A HCR.

    The N-terminus of Alien interacts with the HCR. In line with that we identified a silencing domain in the N-terminus of Alien, while the Alien C-terminus was shown to interact with nuclear receptors (40). This may suggest that Alien may serve as a bridge between Sin3A and DNA-bound transcriptional silencers. The N-terminus of Sin3A harbours two potent silencing domains (37). It is likely that these silencing domains combined with the HCR are responsible for the enhancement of Alien-mediated silencing. Expressing the HCR of Sin3A alone we observed a dominant negative effect on Alien-mediated silencing at a native promoter and in reporter gene assays. This suggests that the HCR in isolation competes for available Alien protein in living cells. Combined with a silencing domain, the HCR may act as a CoR.

    Most studies analysing the interaction using a wide variety of different transcriptional repressors revealed that the PAH domains of Sin3A participated in it. For example, PAH3 alone is sufficient to bind to SAP30 (46). The IDs of the CoRs SMRT and NCoR with Sin3A have been mapped and were shown to involve PAH1 or PAH2 and PAH3 of Sin3A, respectively (9,37). Interestingly, the PAH domains were indispensable for the interaction with Alien (Fig. 3). Thus, the SMRT and NCoR interactions with Sin3A is distinct from that of Alien. This indicates that interactions of different nuclear receptor CoRs with Sin3A involve distinct domains in this protein. Furthermore, for SAP45, SAP130 and SAP180 the entire sequences between PAH3 and PAH4, which are identical to the HID (43), were required for binding to Sin3A (16). However, Sin3A deletions matching the regions of the HCR and PAH3 were insufficient to bind to these factors. Therefore, the dominant negative effects of the HCR in both inhibition of Alien-mediated silencing and an increase in endogenous CYP24 gene expression may be specifically attributed to the Alien–Sin3A HCR interaction.

    We propose a model of Alien-mediated silencing through recruitment of Sin3 to its N-terminus and interaction with DNA-bound transcriptional silencers at the C-terminus. Taken together, our observations show that Sin3A is able to interact with other factors outside its PAH domains and reveal a novel role for the evolutionarily HCR of Sin3A. In addition, the hormone-regulated recruitment of Alien to an endogenous VDR target gene emphasizes its role in nuclear receptor function.

    SUPPLEMENTARY MATERIAL

    ACKNOWLEDGEMENTS

    We are grateful to Dr R. N. Eisenman for providing the Sin3A and Sin3B cDNAs, Dr M. Privalsky for three pGST–Sin3A fusions, Dr R.Brent for the yeast two-hybrid system and Dr L. Binderup for 1,25(OH)2D3. This work was supported by the Academy of Finland, grant no. 50319 (to C.C.) and SFB397 of the German Research Council.

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