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• W2080396646 abstract "SAFB2 physically interacts with p53 : shown by pull down ( view interaction ) The tumour suppressor p53 is a multifunctional protein, which in response to diverse cellular stresses regulates target genes that induce cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism [1, 2]. Notably, excess p53 activity is accompanied by unwanted effects [3]. Thus, tight regulation of p53 is essential for maintaining normal cell growth and preventing tumorigenesis. Under normal conditions of growth, a small fraction of nuclear p53 was found associated with the nuclear matrix both in primary cultures of normal mammalian cells as well as in transformed cell lines [4, 5]. The structural basis and function of nuclear matrix-bound p53 are poorly understood. Interestingly, nuclear matrix-bound p53 was shown to increase significantly following exposure of cells to genotoxic stress [5]. Furthermore, matrix association was also observed in certain transformed cell lines expressing endogenous mutants of p53, indicating that wild-type conformational structure of p53 is not required for binding to the nuclear matrix [5]. Scaffold attachment factor B1 [SAFB1; also known as HET (Hsp27-ERE-TATA-binding protein) or HAP (hnRNP A1-associated protein)] was originally isolated on the basis of its ability to bind the scaffold or matrix attachment regions (S/MARs) of genomic DNA [6]. These DNA elements, which usually comprise AT-rich sequences, are thought to modulate gene expression through the compartmentalization of chromatin into topologically separated loops [7]. SAFB1 contains several domains, such as a SAF-box (aa 35–67), an RNA recognition motif (RRM, aa 409–482), a nuclear localization signal (NLS, aa 599–614), a Glu/Arg-rich region (aa 619–699) and a Gly-rich region (aa 785–899) that have been shown to interact with transcription and pre-mRNA processing components, thereby mediating the assembly of various transcription and/or splicing regulatory complexes in the vicinity of nuclear matrix [8-10]. Transcriptional repression seems to be the most prominent function of SAFB1. While SAFB1 was initially reported to repress estrogen-dependent transcription [11] recent studies suggest that it may function as a more general transcriptional repressor, mediating repression mainly of immune regulators and apoptotic genes [12, 13]. In the present study we established that a fraction of nuclear p53 binds to and co-localizes with SAFB1 in cells treated with chemotherapeutic drugs. Binding of p53 to SAFB1 was shown to be functionally significant, since SAFB1 suppressed p53-mediated reporter gene expression in p53-null cells (K562). In addition, knock-down of SAFB1 resulted in an increase of p53 activity in HepG2 cells treated with 5-fluorouracil (5-FU). Plasmids expressing wild-type HA (haemagglutinin)-tagged p53 (pcDNA3/HA-p53) and mutants p53R248 W (pcDNA-4TO-p53HUR248 W) and p53R273H (pcDNA-4TO-p53HUR273H) were kindly provided by Dr. D. Kardassis (School of Medicine, University of Crete, Heraklion, Greece) and Dr. W. Deppert (Heinrich-Pette-Institut, Tumor Virology, Hamburg, Germany) respectively. To express p53 as a GST fusion protein, p53 cDNA was digested with EcoRI and BamHI from pcDNA3/HA-p53, repurified, and subcloned into the BamHI and SmaI sites of pGEX-4T1 (Amersham Biosciences). GFP-tagged SAFB1 (pGFP3-SAFB1) was kindly provided by Dr. D. Elliott (Institute of Human Genetics, University of Newcastle, Newcastle, UK). GST-SAFB1(1–240), GST-SAFB1(240–600) and GST-SAFB2(641–953) were kindly provided by Dr S. Oesterreich (Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA, see also [14]). GST-SAFB1(709–915) has been previously described [15]. To construct pGEX-SAFB1(566–706) the cDNA fragment coding for amino acids 566–706 of SAFB1 was amplified by PCR from pGFP3-SAFB1, using as primers: sense 5′-CGCGGATCCATGGATAAATCCAAAGGGGTGCC-3′ and antisense 5′-CGGGAATTCCCGCTCCTGCTCATAGCGCAG-3′, containing the underlined BamHI and EcoRI sites respectively. The PCR fragment was digested with BamHI and EcoRI, repurified and subcloned into the BamHI and EcoRI sites of pGEX-4T1. Plasmids encoding siRNAs targeting SAFB1 were generated using the siSTRIKE U6 Hairpin Cloning System (Promega). Briefly, siSAFB1 encodes a siRNA targeting nucleotides 1739–1757 (5′-GGACTGTAGTAATGGATAA-3′) of the human SAFB1 mRNA (NM_002967.2), while scrSAFB1 encodes a scrambled version of this sequence (5′-GAATGACTGATGAGATAGT-3′) and was used as control. Both the SAFB1 targeted and the control nucleotide were aligned against the Genebank database sequences to ensure specificity. Plasmids were purified using the Qiagen MaxiPrep protocol (Qiagen). HepG2 and 293T cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics, while K562 cells were maintained in RPMI medium plus 10% FBS and antibiotics. HepG2 cells were treated with 50 μg/ml 5-FU or 200 nM mithramycin for 36 h, prior harvesting. 293T cells were transfected using the calcium phosphate method as previously described [16]. K562 cells were co-electroporated with the respective constructs for transcription assays at 155 V and 1050 μF using the Gene Pulser Xcell™ Electroporation System (Bio-Rad), according to the manufacturer's instructions. HepG2 cells were electroporated with 20 μg of siSAFB1 or scrSAFB1 plasmid at 165 V and 1050 μF and then they were allowed to recover for 12 h before addition of 2.3 μg/ml of puromycin. After three weeks of selection under puromycin, cells were analyzed for SAFB1 expression and used in transcription assays. HepG2 cells were transfected with the transcription constructs using the calcium phosphate method. Cell fractionation protocols were employed as described in [17]. Gel loading was adjusted to give equivalent cell numbers in each lane. GST pull-down and co-immunoprecipitation experiments were performed as previously described [16]. Transcription assays were based on the ability of p53 to promote transcription of a (−2325/+8) p21-CAT reporter gene in the presence or absence of SAFB1. In K562 cells (p53 null cells) the transfection mixture contained 2 μg of the (−2325/+8) p21-CAT reporter plasmid, 3 μg of CMV β-gal plasmid, 0.5 μg of pcDNA3/HA-p53 and when indicated increasing concentrations (1, 2 and 3 μg) of pGFP3-SAFB1. In HepG2 cells (p53 positive cells) expressing either scrSAFB1 or siSAFB1, the transfection mixture contained 2 μg of the (−2325/+8) p21-CAT reporter plasmid and 3 μg of CMV β-gal plasmid. In each case vector DNA was added as necessary to achieve a constant amount of transfected DNA (15 μg). In HepG2 cells 50 μg/ml 5-FU were added 12 h after transfection. Forty eight hours post transfection both K562 and HepG2 cells were harvested and CAT activities were determined using 14C-chloramphenicol and acetyl-CoA as previously described [18]. The results represent the mean of at least three independent transfection experiments, each carried out in duplicate. HepG2 cells seeded on coverslips were washed three times with PBS. The samples were then fixed with 1% formaldehyde in PBS, permeabilized with 0.2% Triton X-100 and blocked with 0.5% fish skin gelatin. Probing with the relevant primary [mouse monoclonal anti-SAFB1 (Αbcam Inc.), diluted 1:150; rabbit polyclonal anti-p53 (FL-393, Santa Cruz), diluted 1:50] and secondary (FITC-conjugated goat anti-mouse, diluted 1:400; RRX-conjugated goat anti-rabbit, diluted 1:350) antibodies, was performed according to Maison et al. [19]. A yeast two-hybrid screen using as a bait a 528-bp fragment coding for the NH2-terminal domain of SRPK1a (SRPK1aNt) was performed as previously described [16]. SRPK1a is a much less studied alternatively spliced form of SRPK1 that contains an insertion of 171 amino acids at its NH2-terminal domain [16]. To isolate proteins that interact specifically with SRPK1a a yeast two-hybrid screen was performed using as a bait SRPK1aNt (encoding amino acids 1–176 of SRPK1a). Screening of ∼8 × 106 recombinant clones led to the isolation of 21 clones that remained positive for β-galactosidase activity when co-transformed with the SRPK1aNt fusion protein but not with the DNA binding domain of GAL4 alone. As previously described, three of the positive clones that showed the strongest interaction, were different isolates of SAFB1 [16]. Proceeding with the characterization of the remaining positive clones, we noticed that two clones, showing weaker interaction than SAFB1, encoded p53. On the basis of the p53 sequences isolated by the two-hybrid screen, COOH-terminal residues 254–390 of p53 (accession number Q549C9) appear to be sufficient for the interaction with SRPK1aNt. The observed interaction seems to be specific, because both p53 clones showed a very weak interaction with SRPK1 (Supplementary Fig. 1A). Using pull-down assays we confirmed that FLAG-SRPK1a overexpressed in 293T cells was able to interact with GST-p53 in vitro (Supplementary Fig. 1B). The interaction of SRPK1a with p53 was further demonstrated in vivo by co-immunoprecipitation/Western blotting analysis (Supplementary Fig. 1C). Given that both SAFB1 and p53 were shown to interact with SRPK1a and that a fraction of nuclear p53 was previously found associated with the nuclear matrix [5], we sought to investigate whether there is an interaction between SAFB1 and p53. In this respect it should be noted that neither SAFB1 nor p53 contain an RS domain and consequently they are not phosphorylated by SRPKs (data not shown). As a first step, we studied the subcellular distribution of endogenous p53 and SAFB1 in HepG2 cells by biochemical fractionation. HepG2 cells have been used in the past as a model system to study p53 because they express a wild-type p53 protein that can be activated to elicit normal p53 function [20, 21]. Under normal conditions of growth, p53 protein was almost equally distributed between the cytoplasm and the nucleus, while only a minimal amount of p53 was found associated with the nuclear matrix fraction (Fig. 1 A). SAFB1 was exclusively nuclear. A sub-population of SAFB1 molecules was soluble and detected in the nucleoplasmic fraction, while the majority of SAFB1 protein was found associated with the nuclear matrix (Fig. 1A). To test whether the distribution of SAFB1 and p53 could be modulated by treatment of cells with genotoxic agents, HepG2 cells were treated with 50 μg/ml 5-FU or 200 nM mithramycin for 36 h, prior to fractionation. The rationale for choosing these two drugs was that 5-FU and mithramycin are both known to upregulate p53 but through different mechanisms. More specifically, 5-FU inhibits the cell's ability to synthesize properly DNA and RNA, while mithramycin interferes with the transcription of genes that bear GC-rich motifs in their promoters. As shown in Fig. 1B, the distribution pattern of SAFB1 was unaffected, whereas as expected, both drugs induced an increase in the total amount of cellular p53. The increase was far more pronounced in the nuclear fraction. Roughly equivalent amounts of p53 were detectable in the nucleoplasmic and nuclear matrix fractions (Fig. 1B). Following these observations, we next asked whether native cellular SAFB1 and p53 proteins interact with each other in HepG2 cells treated with 50 μg/ml 5-FU for 36 h. Fig. 2 A shows that an anti-SAFB1 antibody can immunoprecipitate p53 from HepG2 cells (left panel), while the anti-p53 monoclonal antibody DO-1 was also able to immunoprecipitate SAFB1 (right panel), but less efficiently. Having shown that SAFB1 interacts with p53, we next mapped the region in SAFB1 mediating this interaction. To this end we generated a series of GST-SAFB1 deletion constructs (Fig. 3 A), and confirmed the correct protein expression (Fig. 3B). Performing GST-pull down assays with the N-terminal (aa 1–260), central (aa 240–600), Glu/Arg rich (aa 566–706) and C-terminal (aa 709–915) regions of SAFB1 and 293T cell extracts overexpressing HA-p53, we identified the C-terminal region of SAFB1 as the p53-interacting domain (Fig. 3C). To further analyze the localization and interaction between SAFB1 and p53 we performed confocal microscopy co-localization studies (Fig. 4 ). Under normal conditions of growth, p53 dispersed throughout the cytoplasm and the nucleus of HepG2 cells with no apparent co-localization with SAFB1. Treatment of HepG2 cells with 5-FU resulted in a significant increase of the nuclear concentration of p53 and a clearly visible co-localization with SAFB1 in several nuclear speckles. As the mouse monoclonal anti-SAFB1 antibody used in the co-immunoprecipitation and immunofluorescent experiments of this study may also target SAFB2, a highly related family member of SAFB1 [22], we tested whether the interaction of p53 with SAFB1 was specific or a similar interaction may also occur with SAFB2. GST-pull down assays employing the C-terminal regions of SAFB1 and SAFB2 (GST-SAFB1(709–915) and GST-SAFB2(641–953) respectively) and 293T cell extracts overexpressing HA-p53 showed that both SAFB proteins may interact with p53 (Supplementary Fig. 2). Interestingly, there have been some previous reports that certain p53 mutants, containing DNA contact mutations, interact stronger with the nuclear matrix, and especially the S/MAR elements of DNA, than the wild-type protein [23, 24]. We tested two such p53 mutants, p53R273H, that was used in the previous studies [23, 24] and also p53R248W, for their ability to interact with SAFB1. As shown in Supplementary Fig. 3, p53R248W was able to interact with the C-terminal domain of SAFB1 with the same efficiency as wtp53, while the p53R273H/SAFB1 interaction was weaker. Given that p53 functions as a gene-specific transcriptional activator, we investigated whether SAFB1 has any effect on the transcriptional activity of p53. To this end we initially used K562 cells that do not express p53 and a p21 promoter-containing CAT reporter system. p21(WAF1/CIP1) is a major transcriptional target of p53 and it plays a critical role in p53-dependent cell cycle arrest [25]. An advantage of using the p53-null K562 cell line was that exogenously expressed p53 exhibited a subcellular distribution pattern similar to the one observed in 5-FU-treated HepG2 cells (Fig. 5 A, left panel). Compared to cells transfected with p53 alone, cells co-transfected with p53 and SAFB1 exhibited a 0.2–4-fold decrease in CAT activity, depending on the concentration of SAFB1 (Fig. 5B). Since SAFB1 was proposed to function as a more general transcriptional inhibitor [12, 13] and therefore overexpression of SAFB1 could result in smaller amounts of p53 protein synthesized from the expression plasmid which would itself result in decreased CAT activity, we performed Western blotting analysis of p53 from the cell extracts that were used in the transcription assays. As shown in Fig. 5C SAFB1 did not affect the protein levels of p53. The functional significance of the SAFB1-p53 interaction was further demonstrated by knocking-down SAFB1 in HepG2 cells. To this end HepG2 cells were electroporated with 20 μg of siSAFB1 or scrSAFB1 and after three weeks of selection under puromycin they were used in transcription assays, in the absence and presence of 50 μg/ml 5-FU. The reduced protein levels of SAFB1 were confirmed by Western blotting (Fig. 6 A, left panel), while the protein levels of p53 remained unaffected upon expression of siSAFB1 (Fig. 6A, right panel). As shown in Fig. 6B, the activity of the p21 reporter, in the absence of 5-FU, was practically not affected upon treatment with siSAFB1. This is reasonable taking into consideration that in the absence of 5-FU a minimal co-localization of SAFB1 and p53 is observed. In the presence of 5-FU, knock-down of SAFB1 resulted in a ∼3.5-fold increase of CAT activity, further confirming the role of SAFB1 as a transcriptional repressor of p53. The tumor suppressor p53 regulates cell cycle progression and apoptosis in response to various types of stress, whereas excess p53 activity creates unwanted effects. Therefore, tight regulation of p53 is essential for maintaining normal cell growth. In addition, a tight regulation of p53 is required following stress response. As in unstressed cells, the activity of p53 needs to be precisely adjusted once the cellular machinery has counteracted the stress. The principal mechanisms governing p53 activity appear to be exerted at the protein level. These include regulation of p53 stability, post-translational modifications and conformational changes that control the DNA binding activity of p53 [26]. The central component in p53 regulation is the p53-interacting protein MDM2/HDM2 [27]. Binding of MDM2 to p53 inhibits p53's transcriptional activity. In addition MDM2 is a RING-finger protein that functions as a ubiquitin ligase for p53, leading to its proteosomal degradation.p53 degradation occurs mainly on cytoplasmic proteasomes and, hence, has an absolute requirement for nuclear export of p53 via the CRM1 pathway [28]. Yet, cytoplasmic export of ubiquitinated p53 is relatively slow and consumes significant energy in the form of Ran-GTP for the CRM1 pathway [29]. Accumulating evidence suggest that p53 levels and activity may also be regulated inside the nucleus and that the nuclear matrix is actively involved in this regulation, either positively or negatively. More specifically, the interaction of certain p53 mutants with the S/MAR elements of DNA resulted in gain of function that was proposed to result through the reorganization of chromatin and the recruitment of specific transcriptional complexes on these elements [24]. On the other hand, SMAR1, a nuclear matrix-associated protein that, like SAFB proteins, interacts with MARs (Matrix Attachment Regions), was reported to form a ternary complex with MDM2-p53 and negatively regulate p53-mediated transcription [30]. In addition, P2P-R (also known as PACT and Rbbp6), a hnRNP-related protein that interacts with SAFB1, was recently shown to interact with HDM2 and enhance HDM2-mediated ubiquitination and degradation of p53 as a result of the increase of the p53–HDM2 affinity [31-33]. SAFB1 (and potentially SAFB2) further expands the list of nuclear matrix-associated proteins that negatively regulate p53 activity. This function of SAFB1 is consistent with previously published evidence documenting that SAFB1 may function as transcriptional repressor via its C-terminal domain, the same region that was shown to interact with p53 [11-14]. An intriguing speculation based on these data is that there may be various nuclear matrix-binding sites for p53 that fine-tune its activity. For example the R273 mutation may potentiate the binding of p53 to S/MAR elements resulting in gain of function [23, 24], whereas the same mutation reduces the affinity of p53 for SAFB1 (Supplementary Fig. 3, this study). The SAFB1-mediated down-regulation of p53 is most likely part of a feedback mechanism functioning only under stress conditions, since the amount of p53 associated with SAFB1 without the addition of genotoxic agents (see 1, 4) is practically negligible. In this respect the inactivation of p53 by SAFB1 is not in contradiction with previous reports proposing that loss of SAFB1 results in increased proliferation [13, 34]. It is also interesting to note that SAFB proteins have been shown to inhibit proliferation mainly through repression of nuclear receptors and particularly the estrogen receptor [12, 22]. Given the opposing roles of ER and p53 (with ER promoting cell proliferation and p53 slowing it down) but also their functional interactions [35, 36] an additional interesting hypothesis is that SAFB proteins may fine tune the mutual transcriptional silencing of these two molecules, thus regulating cell growth. When the cells need to proliferate SAFB would inhibit the cell growth suppressive functions of p53 and release ER, whereas when the cells need to stop proliferating SAFB would shift towards inhibiting the expression of ER responsive genes and releasing p53. This shift could be mediated through the ability of SAFB1 to participate in different transcription regulatory complexes associated with the nuclear matrix [8, 9]. Such complexes are probably transiently formed inside the nucleus [37]. Depending on the cellular activities, a fraction of nuclear SRPKs may be recruited to specific SAFB-complexes and inactivated by interacting with SAFB molecules [15]. The inhibition of RS domain-dependent phosphorylation may affect the composition of these transient complexes per se, since various SAFB partners contain RS domains, thus influencing gene transcription and/or mRNA splicing [38]. For example, P2P-R was shown to be a substrate or SRPK1a [39], while ZO-2, another SAFB1 interacting protein that was shown to inhibit proliferation through downregulation of cyclin D1 [40, 41], also contains an RS domain and may be targeted by SRPKs. In this respect, elucidating the structure and the molecular mechanisms governing the assembly of nuclear matrix-associated complexes will unravel important information concerning the regulation of the transcriptional machinery. We thank Dimitris Kardassis, David Elliot, Steffi Oesterreich for kindly providing us with pcDNA3/HA-p53, pGFP3-SAFB1 and pGEX-2TK-SAFB1/SAFB2 constructs respectively. We also thank Wolfgang Deppert for the pcDNA-4TO-p53HUR273H and pcDNA-4TO-p53HUR248W constructs. T.G., E.G. and M.H.-C. were supported by grants from the Greek Ministry of Education (HPAKΛEITOΣ grants in the context of the E.U. funded ΕΠΕΑΕΚ II program). P.P. was a recipient of a fellowship from the Greek National Fellowship Foundation, while N.V. was a recipient of a fellowship from Onassis Foundation. R.E.S. is supported by Varigenix, Inc. in his role as company president. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2010.11.054. Supplementary Fig. 1. The NH2-terminal domain of SRPK1a interacts with p53 in yeast. (A) S. cerevisiae strain pJ69-4A was co-transformed with (A) pGBT9-SRPK1aNt and pVP16-p53, (B) pGBT9-SRPK1aNt and pVP16, (C) pGBT9 and pVP16-p53, (D) pGBT9-SRPK1 and pVP16-p53, (E) pGBT9-SRPK1 and pVP16, and (F) pGBT9 and pVP16. The strains were streaked in the same order on plates containing SC minus Trp, Leu, Ade, His, and 50 mM AT (left) or SC minus Trp, Leu, Ade, His, and 5 mM AT (right). The p53 clone used in these experiments encodes amino acids 254-390 of mouse p53, while the full-length coding region of SRPK1 was sunbcloned to pGBT9. (B) GST-p53 or GST alone were incubated with extracts from 293T cells overexpressing FLAG-SRPK1a. The complexes were recovered by pull-down with glutathione-Sepharose beads and analyzed on 10% SDS-polyacrylamide gels. Bound SRPK1a was detected with the M5 anti-FLAG monoclonal antibody. A standard amount of cell extract, one-tenth of which is shown, was used in each binding assay. (C) 293T cells were co-transfected with plasmids expressing FLAG-SRPK1a and HA-p53. Complexes between SRPK1a and p53 were immunoprecipitated with the M5 anti-FLAG monoclonal antibody (left panel) or the anti-p53 (DO-1) monoclonal antibody (right panel) and analyzed on 10% SDS-polyacrylamide gels. Bound p53 and SRPK1a were detected with the DO-1 (left panel) and the M5 anti-FLAG monoclonal antibodies (right panel) respectively. One-tenth of the cell extract used in each immunoprecipitation assay is shown in the first lane. No immunoprecipitation of p53 or SRPK1a was observed when an irrelevant anti-GFP monoclonal antibody was used as control (middle lane in both panels). Supplementary Fig. 2. SAFB2 also interacts with p53. (A) SDS–PAGE analysis and Coomassie Blue staining of GST-SAFB1(709-915) and GST-SAFB2(641-953) (B) GST pull-down assays employing 293T cell extracts overexpressing p53 and GST-SAFB proteins as indicated. Bound p53 was detected with the DO-1 monoclonal antibody. A standard amount of cell extract, one-tenth of which is shown, was used in each binding assay. Supplementary Fig. 3. Interaction of p53 mutants with SAFB1. GST pull-down assays employing GST-SAFB1(709-915) and K562 cell extracts overexpressing p53, p53R273H and p53R248W respectively. Bound p53 was detected with the DO-1 monoclonal antibody. A standard amount of cell extract, one-tenth of which is shown, was used in each binding assay. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W2080396646 title "SAFB1 interacts with and suppresses the transcriptional activity of p53" @default.
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