RUNX3 Directly Interacts with Intracellular Domain of Notch1 and Suppresses Notch Signaling in Hepatocellular Carcinoma Cells

ARTICLE IN PRESS YEXCR-08268; No. of pages: 9; 4C:

EXPERIMENTAL CELL RESEARCH XX (2009) XXX– XXX

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Research Article RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells

Juan Gaoa,1, Yu Chenb,1, Kai-Chun Wub, Jie Liub, Yan-Qiu Zhaob, Yang-Lin Panb, Rui Dub, Guo-Rong Zhenga, Yi-Min Xionga, Hua-Lin Xua,⁎, Dai-Ming Fanb,⁎

a Department of Digestive Diseases, Wuhan General Hospital of Guangzhou Command PLA, Wuhan 430070, Hubei Province, P.R. China b Institute of Digestive Diseases, and State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, Shaanxi Province, P.R. China

ARTICLE INFORMATION ABSTRACT

Article Chronology: RUNX3 takes a strong suppressive effect in many tumors including hepatocellular carcinoma Received 19 June 2009 (HCC). HES-1, a downstream target of Notch signaling, is shown to be decreased in human HCC cell Revised version received line SMMC7721 with RUNX3 gene transfection. Since Notch signaling is oncogenic in HCC, RUNX3 22 September 2009 might exert its inhibitory effect in HCC partly through the suppression on Notch signaling. To Accepted 25 September 2009 investigate the possible mechanism of the down-regulation of HES-1 by RUNX3, we performed Western blot and reporter assay and found that RUNX3 suppressed intracellular domain of Notch1 (ICN1)-mediated transactivation of Notch signaling while it did not alter the expression of ICN1 Keywords: and recombination signal binding protein-Jκ (RBP-J) in SMMC7721 cells. Besides, confocal RUNX3 microscopy, co-immunoprecipitation and GST pull-down assays showed that RUNX3 could co- Notch1 localize with ICN1 and RBP-J, forming a complex with these two molecules in nucleus of RBP-J SMMC7721 cells by its direct interaction with ICN1. Furthermore, RUNX3 was recruited to RBP-J HES-1 recognition motif of HES-1 promoter, which was identified by chromatin immunoprecipitation HCC (ChIP) and electrophoretic mobility shift assay (EMSA). Taken together, these findings indicate that RUNX3 suppresses Notch signaling in HCC SMMC7721 cells by its interaction with ICN1 and thus recruitment to the RBP-J recognition motif of downstream genes of Notch signaling. © 2009 Elsevier Inc. All rights reserved.

Introduction carcinogenesis [2]. RUNX1, 2 and 3 have essential roles in haemopoiesis, bone formation and neurogenesis, respectively RUNX3 belongs to the Runt family of transcriptional factors that [3–5]. By chromosomal translocation events, the formation of consist of three members, RUNX1, RUNX2 and RUNX3 [1].Asa fusion proteins such as RUNX1-ETO or TEL-RUNX1 impairs the transcription factor, RUNX3 can activate or repress target gene normal function of RUNX1 thus leads to myeloid leukemia and transcription by cooperating with some cofactors at the promo- childhood B-cell leukemia [6]. RUNX2 could promote cancer ters and enhancers of target genes. Besides, it can also function as metastasis to bone by regulating downstream genes such as a cofactor itself to assist other transcription factors. RUNX family matrix metalloproteinases (MMPs), vascular endothelial growth members play vital roles in balancing cell differentiation and factor (VEGF), receptor activator of nuclear factor-kappaB ligand proliferation and have important functions in development and (RANKL), etc. [7]. Accumulative evidences have suggested that

⁎ Corresponding authors. H.-L. Xu is to be contacted at Fax: +86 27 87164886; D.-M. Fan, Fax: +86 29 82539041. E-mail addresses: [email protected] (H.-L. Xu), [email protected] (D.-M. Fan). 1 Juan Gao and Yu Chen contributed equally to this work.

Please cite this article as: J. Gao, et al., RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells, Exp. Cell. Res. (2009), doi:10.1016/j.yexcr.2009.09.025 ARTICLE IN PRESS

2 EXPERIMENTAL CELL RESEARCH XX (2009) XXX– XXX

RUNX3 can act as a novel tumor suppressor. CpG hypermethyla- fectant. Since HES-1 is a downstream target gene of Notch tion silencing of RUNX3 gene is found to be associated with lung signaling, we explored the possibility that RUNX3 might play a cancer, uterine cervical cancer, gall bladder carcinoma, and role in Notch signaling pathway in SMMC7721 cells. In this granulosa cell tumor [8–11].Besides,RUNX3expressionis study, we presented evidences that RUNX3 inhibited Notch decreased in 40–60% human gastric cancer specimens due to signaling in SMMC7721 cells, as demonstrated by luciferase promoter hypermethylation and loss of heterozygosity (LOH) at assay. RUNX3 formed a complex with ICN1 and RBP-J in nucleus 1p36 (the site of RUNX3 gene in genome) [12]. RUNX3 leads to of SMMC7721 cells by its direct interaction with ICN1, as growth arrest and apoptosis in gastric cancer, which is found to be demonstrated by confocal microscopy, co-immunoprecipitation related to its up-regulation of p21 and p27 expression, Bim/Bcl-2 and GST pull-down assays. Furthermore, we found that RUNX3 ratio and down-regulation of cyclinD1 expression [13–16]. was recruited to the RBP-J recognition sequence on target gene RUNX3 also shows a suppressive effect on HCC. It was reported promoters of Notch signaling by EMSA and ChIP assays. Our data that the expression and copy number of RUNX3 are reduced in indicate that RUNX3 might inhibit Notch signaling in SMMC7721 HCC [17]. Promoter hypermethylation of RUNX3 gene is detected cells by its recruitment to the RBP-J recognition sequence in 2.7% of normal liver, whereas in 40–45% of HCC tissues and cell through interaction with ICN1. The suppression of Notch lines [18]. Another investigation reported that promoter hyper- signaling might be one of the molecular mechanisms for the methylation is detected in 75–80% of HCC tissues and cell lines negative effect of RUNX3 on the biology of HCC. and LOH is detected in 37.8% of HCC [19]. Therefore, RUNX3 is an important tumor suppressor gene and its deficiency might be involved in the process of HCC. Materials and methods HES-1, a member of Hairy and enhancer of split (HES) family, is a downstream target of Notch signaling pathway. Notch Construction of vectors signaling pathway is composed of Notch receptors, ligands and downstream effectors. Binding of Notch ligands to receptors pBK-RUNX3, which included a full-length RUNX3 cDNA, was a gift leads to a release of the intracellular region of Notch (ICN), from professor Paul J Farrell of Imperial College Faculty of Medicine which translocates to the nucleus and forms a ternary complex [32]. pcDNA3.1/ICN1-myc-His()C was a gift from professor Tom with a transcription factor Suppressor of Hairless or Lag-1 (CSL, Kadesch of University of Pennsylvania School of Medicine [33]. ICN1 also known as CBF1/RBP-J) and transcriptional coactivators of included 1760–2556 amino acid positions of full-length human the mastermind-like (MAML) to activate transcription of a group Notch1 and was cloned into pcDNA3.1/myc-His(-)C at the XhoI and of downstream genes [20]. As the common adaptor of activated KpnI sites. pCMV-RBP-J-myc was constructed as described [34]. Notch receptors, RBP-J recognizes a consensus sequence C/ pGa981-6, a reporter gene plasmid which contained a hexamerized TGTGGGAA (RBP-J recognition sequence, RBP-J binding site) that 50 bp Epstein-Barr virus nuclear antigen 2 response element exists in multiple differentiation-regulating genes such as (EBNA2RE) of the TP-1 promoter in front of the luciferase gene, members of HES family [21]. was strictly dependent on RBP-J [35]. When ICN was co-transfected Notch signaling pathway is an evolutionarily highly conserved with pGa981-6, a strong transactivation of the reporter gene mechanism for cell–cell communication, which is not only mediated by the endogenous RBP-J was detected [34,36].Anegative important in embryonic development and cell fate determination, control plasmid (neg-pGa981-6) was constructed by replacing but also plays vital roles in cancer. Besides its classical effects on EBNA2RE with an irrelevant DNA segment. pcDNA3.1(-) and pRL-TK hematologic malignancies [22,23], researches also showed that were purchased from Invitrogen Life Technologies (Carlsbad, CA, activation of the Notch1 pathway can enhance primary melanoma USA) and Promega Corporation (Madison, WI, USA). Prokaryotic cell growth and enable primary melanoma cells to gain metastatic vector expressing His-Trx tagged ICN1 (His-Trx-ICN1) fusion capability [24]. Down-regulation of Notch1 inhibits cell growth, protein was constructed by amplifying the coding region of ICN1 induces apoptosis, and suppresses cell invasion, metastasis and mentioned above (corresponding to 5278–7668 site of full-length neurovascular development in pancreatic cancer [25,26]. In the human Notch1 cDNA) using primers with the plasmid pcDNA3.1/ research of liver, it was found that Notch signaling promotes the ICN1-myc-His(-)C as template. The primers were as follow: sense growth and proliferation of hepatocytes. For example, the primer 5′-GCGGATCCAAGCGCCGGCGGCAGCATGGCC-3′ and anti- expression of Notch1, Notch2, Notch3, Delta1, Jagged1 mRNA sense primer 5′-GCAAGCTTTTACTTGAACGCCTCCGGGATGC-3′.The exist in hepatocytes of rat models of liver injury and regeneration amplied fragment was inserted into pET-32a(+) (Novagen, [27]. After partial rat liver hepatectomy, both Notch1 and Jagged1 Germany) by digestion with endonuclease BamHI and HindIII, and proteins are up-regulated, ICN1 increases, and Notch1/Jagged1 confirmed by DNA sequencing. Vector expressing His-Trx tagged signaling pathway is activated during rat liver regeneration [28].In RBP-J (His-Trx-RBP-J) fusion protein was constructed by amplifying the research of HCC, it was reported that the levels of Notch1, the full-length RBP-J cDNA using primers with the plasmid pCMV- Jagged1, and HES-1 gene expression are increased in most HCC RBP-J-myc as template. The primers are as follow: sense 5′- samples relative to the adjacent HCC-free liver tissue [29]. Our GCGGATCCATGGCGTGGATTAAAAGGAAAT-3′, and antisense 5′- previous researches also showed that Notch1, Notch4 and Jagged1 GCAAGCTTTTAGGATACCACTGTGGCTGTA-3′. The amplied fragment are up-regulated in HCC [30,31], and Notch1 signaling promotes was inserted into pET-32a(+) by digestion with BamHI and HindIII, the progress of HCC (our unpublished data). These reports indicate and confirmed by DNA sequencing. Vector expressing GST-tagged that Notch signaling is activated in HCC. RUNX3 (GST-RUNX3) fusion protein was constructed by amplifying In our previous study, a gene microarray analysis of RUNX3 human RUNX3 cDNA using primers as: sense 5′-GCGGATCCATGG- stable transfected HCC SMMC7721 cell line identified HES-1 as CATCGAACAGCATCTTCG-3′ and antisense 5′-GCCTCGAGTCAG- one of the down-regulated genes compared with mock trans- TAGGGCCGCCACACGGCC-3′, with the plasmid pBK-RUNX3 as

EXPERIMENTAL CELL RESEARCH XX (2009) XXX– XXX 3 template. The amplied fragment was inserted into pGEX4T-1 were co-transfected into SMMC7721 cells cultured in 24-well plate (Amersham Pharmacia, Piscataway, NJ, USA) by digestion with with Lipofectamine™ 2000. pRL-TK was co-transfected as an BamHI and XhoI and confirmed by DNA sequencing. internal control for transfection efficiency. pcDNA3.1(-) and neg- pGa981-6 of the same quantity replacing pcDNA3.1/ICN1-myc-His Cell culture and generation of stable cell line (-)C and pGa981-6, respectively, were used as a negative control. 48 h later, co-transfected cells were processed with Luciferase Human HCC SMMC7721 cells were cultured at 37 °C in Dulbecco's Assay System (Promega) and luciferase assay was performed in modified Eagle's medium (DMEM) (Gibco Industries, Tulsa, OK, Luminometer TD-20/20 (Turner Designs, Fresno, CA, USA).

USA) supplemented with 10% fetal calf serum in 5% CO2-humidified Experiment was repeated at least three times, and data were air. Generation of SMMC7721 cell line that stably expressed RUNX3 analyzed with the Student's t-test. was accomplished through transfection with pBK-RUNX3 using Lipofectamine™ 2000 reagent (Invitrogen), following the manufac- Double immunofluorescence staining turer's protocol. Transgene expressed cells were selected and maintained with 600 μg/ml G418 (Gibco) and named SMMC7721- Double immunostaining for studies on antigen co-localization was RUNX3. Mock transfection was done by transfecting a pBK vector performed with tetraethyl rhodamine isothiocyanate-labeled goat into SMMC7721 cell line and selected. The identification of positive anti-rabbit IgG (Chemicon, Temecula, CA, USA; 1:100) and mixed clones was performed by Western blot analysis with anti- fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse RUNX3 rabbit polyclonal antibody (pAb) (Active Motif, Carlsbad, CA, IgG (Chemicon, 1:80). The primary antibody combinations con- USA, diluted 1:100). For Notch signaling inhibition, SMMC7721 and sisted of the anti-RUNX3 pAb (1:50) and anti-myc mouse SMMC7721-RUNX3 cells were treated with N-[N-(3,5-difluorophe- monoclonal antibody (mAb) (Cell Signaling Technology, Boston, nacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Merck MA, USA; 1:1500). For cell staining, SMMC7721-RUNX3 cells were KGaA, Darmstadt, Germany) at a final concentration of 10 μM plated on glass coverslips overnight and were transiently respectively for the following experiments. transfected with pcDNA3.1/ICN1-myc-His(-)C or pCMV-RBP-J- myc. 48 h after transfection, cells were fixed with 4% paraformal- RT-PCR and Western blot analysis dehyde in PBS. Fixed cells were stained as above and examined using a Fluoview FV1000 laser scanning confocal microscope For RT-PCR, cDNA from the above SMMC7721-RUNX3 cells and (Olympus, Japan). mock transfectant was extracted using the one-step RT-PCR kit (MBI Fermentas, Lithuania) according to the manufacturer's instructions. Co-immunoprecipitation assay PCR was performed using human primer sequences for HES-1 [29]: sense 5′-CCAAAGACAGCATCTGAGCA-3′; antisense 5′- The nuclear extract of SMMC7721-RUNX3 cells was prepared as TCAGCTGGCTCAGACTTTCA-3′. The length of product was 91 bp. mentioned above. The extract was pretreated with Protein G β-Actin was used as a housekeeping gene. Gene expression was Plus/Protein A-Agarose (Calbiochem, Germany) to remove presented by the relative yield of the PCR product from target nonspecifically bound proteins. Immunoprecipitation was car- sequences to that from β-actin gene. Mean values from three ried out with the anti-RUNX3 pAb, anti-Notch1 pAb or anti- independent experiments were taken as results. For Western blot, RBP-J pAb and the agarose beads. After the pretreated nuclear the nuclear protein extract of SMMC7721-RUNX3, mock transfec- extract, antibody and the agarose beads were incubated at 4 °C tant and DAPT-treated SMMC7721 cells were prepared with NE-PER for 4 h, the beads were washed and subjected to SDS-PAGE and Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotech- Western blot with anti-Notch1 pAb (1:300), anti-RBP-J pAb nology, Rockford, IL, USA). Nuclear proteins from the above cells (1:300) and anti-RUNX3 pAb (1:100). In negative control, were separated by SDS-PAGE. After transferring, the nitrocellulose preimmune serum was used to substitute for the above membrane was incubated with anti-Notch1 goat pAb (Santa Cruz antibodies of immunoprecipitation. Biotechnologies, Santa Cruz, CA, USA, 1:300), anti-RBP-J rabbit pAb (Santa Cruz, 1:300) and anti-HES-1 rabbit pAb (Santa Cruz, 1:300) GST pull-down assay and then detected with peroxidase coupled anti-goat IgG or anti- rabbit IgG. Proteins were visualized by enhanced chemilumines- His-Trx-ICN1, His-Trx-RBP-J and GST-RUNX3 fusion proteins were cence. Western blot for Histone H3 (BioLegend, San Diego, CA, USA; produced in Escherichia coli using pET-ICN1, pET-RBP-J and pGEX- 1:200) was used as an internal sample. The bidimensional optical RUNX3, respectively. Fusion proteins as well as control proteins densities of ICN1, RBP-J, HES-1 and Histone H3 proteins on the films (His-Trx and GST) were purified by the Ni2+-chelating resin were quantified and analyzed with Molecular Analyst software (Invitrogen) or glutathione–Sepharose 4B beads (Amersham (Bio-Rad Laboratories, Hercules, CA, USA). Every experiment was Biosciences), respectively, following the supplier's protocols. GST repeated three times. The ratios of ICN1, RBP-J, HES-1 against pull-down assay was then performed. His-Trx-ICN1, His-Trx-RBP-J Histone H3 were calculated. Statistical analysis was done with or His-Trx was incubated with GST or GST-RUNX3 immobilized on Student's t-test using SPSS software (version 10.0, SPSS Inc, Chicago, glutathione–Sepharose 4B beads. Then the beads were incubated IL, USA). Statistical significance was assumed if P <0.05. at 4 °C for 2 h and were washed with a buffer (142.5 mM KCl, 5 mM

MgCl2, 10 mM HEPES, pH 7.6, 1 mM EDTA, 0.2% NP-40) for five Reporter assay times to collect the associated protein. The associated proteins were separated by SDS-PAGE, followed by Western blot with an pcDNA3.1/ICN1-myc-His(-)C (0.25 μg), pGa981-6 (0.1 μg), pRL-TK anti-His mAb (Sigma, St. Louis, MO, USA; 1:1000). GST and His-Trx (0.05 μg) and increasing amount of pBK-RUNX3 (0.1, 0.2, 0.4 μg) were used as negative controls for this experiment.

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EMSA Results Then the nuclear extract of SMMC7721-RUNX3 cells was prepared as above. The probe containing RBP-J binding site was obtained by RUNX3 down-regulates the expression of HES-1 and annealing complementary oligomers and biotin was labeled at 3′ suppresses ICN1-mediated transactivation in SMMC7721 cells terminal using terminal deoxynucleotidyl transferase. The sequence of probe was O54 [37]:5′-TCGCGACTCGTGG- We had detected previously the decreased expression of HES-1 in GAAAATGGGCGGAAGGGCACCGTGGGAAAATAGTTCCAGGT-3′ and a gene microarray of SMMC7721-RUNX3 cell line. Now we the mutant probe was 5′-TCGCGACTCCTTGGAAAATGGGCG- confirmed this down-regulation in mRNA and protein level. GAAGGGCACCCTTGGAAAATAGTTCCAGGT-3′. Nuclear proteins Firstly, Western blot showed that the level of RUNX3 protein in (3 μg) were incubated with 20 fM of the labeled probe for SMMC7721-RUNX3 cells was much higher than that in mock 20 min at room temperature and DNA–protein complexes were transfectant (Fig. 1A). Then, a down-regulation of HES-1 mRNA separated on 4% nondenaturing polyacrylamide gel in 0.5×TBE was found in SMMC7721-RUNX3 cells compared with mock transfectant by RT-PCR analysis (Fig. 1B). Next, we detected the (45 mM Tris base, 45 mM boric acid, 1 mM disodium EDTA·2H2- O) with 2.5% glycerol. The complexes were then transferred to protein expression of ICN1, RBP-J and HES-1 in SMMC7721-RUNX3 Nylon Membrane (Pierce), cross-linked for 10–15 min with the cells, mock transfectant, and DAPT-treated SMMC7721 cells. It was membrane face down on a transilluminator equipped with found that DAPT reduced the expression of ICN1 and HES-1 but not 312 nm bulbs and developed using Lightshift Chemiluminescent RBP-J, which indicated that HES-1 expression in SMMC7721 cells EMSA Kit (Pierce). For competition experiment, increasing depends on activation of Notch signaling. And, consistent with the amount of unlabeled wild probe or mutated probe (100, 200, array result, the expression of HES-1 was much lower in 1000 fM) or mutated probe (1000 fM) was added to the reaction SMMC7721-RUNX3 cells than in mock transfectant. Moreover, mixture for 1 h at room temperature before addition of the ICN1 and RBP-J did not show significant change in SMMC7721- labeled probe. For antibody supershift, 2 μg of anti-RUNX3 pAb, RUNX3 cells compared with mock transfectant, which demon- anti-Notch1 pAb or anti-RBP-J pAb was added to the reaction strated that the down-regulation of HES-1 by RUNX3 in this cell mixture for 1 h at room temperature before addition of the line does not result from its effect on the expression of ICN1 or labeled probe. Preimmune serum substituting for the specific RBP-J (Fig. 1C). Since RUNX3 could inhibit the expression of HES-1, antibody was used as a negative control. The supershift analysis we assumed that RUNX3 might suppress ICN1-mediated transac- was also performed with the nuclear proteins from those DAPT- tivation. To address this, pBK-RUNX3 and pcDNA3.1/ICN1-myc-His treated SMMC7721-RUNX3 cells. (-)C were co-transfected into SMMC7721 cells, together with the luciferase reporter plasmid pGa981-6. The luciferase data showed ChIP that expression of ICN1 led to a strong transcriptional activation of the RBP-J-dependent promoter in pGa981-6 and RUNX3 could SMMC7721-RUNX3 cells and DAPT-treated SMMC7721-RUNX3 significantly inhibit the transactivation in a dose-dependent cells were fixed by addition of formaldehyde to 1% and were manner (Fig. 1D). This result showed that RUNX3 suppresses experimented as follows. Cells were incubated at 37 °C for 10 min, Notch signaling in SMMC7721 cells. washed twice with ice-cold phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ RUNX3 co-localizes and interacts with both ICN1 and RBP-J ml pepstatin A, and then collected by centrifugation after scraping. in SMMC7721 cells in vivo Cells were then resuspended in an SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1), and disrupted by sonication. Cell We next examined the possibility of co-localization and physical lysates were centrifuged at 13,000 g for 10 min at 4 °C. The interaction of RUNX3 with ICN1 and RBP-J in vivo. For this, supernatant was collected and diluted in a ChIP dilution buffer pcDNA3.1/ICN1-myc-His(-)C or pCMV-RBP-J-myc was trans- (0.01% SDS, 1.1% Triton X-100, 2 mM EDTA, 167 mM NaCl, 16.7 mM fected into SMMC7721-RUNX3 cells, respectively, and confocal Tris–HCl, pH 8.1) containing protease inhibitors as mentioned staining was conducted. In SMMC7721-RUNX3 cells transiently above. Then the supernatant was precleared with the salmon transfected with pcDNA3.1/ICN1-myc-His(-)C, RUNX3 was sperm DNA-coated protein A agarose (Sigma), followed by stained red in both nucleus and cytoplasm, ICN1-myc was immunoprecipitation using 5 μg of anti-RBP-J pAb, anti-Notch1 stained green in nucleus, and the yellow staining in nucleus in pAb or anti-RUNX3 goat pAb (Santa Cruz) or overnight at 4 °C with dual-labeling experiments indicated overlapping areas of red and rotation. Antibody complexes were collected with the salmon green fluorescent labels and suggested co-localization of ICN1- sperm DNA-coated protein Agarose, followed by extensive myc with RUNX3. In SMMC7721-RUNX3 cells transiently washing with the ChIP dilution buffer. Precipitated antibody transfected with pCMV-RBP-J-myc, RUNX3 was also stained complexes were reverse cross-linked by incubation at 65 °C for red, RBP-J-myc was stained green in nucleus, and the yellow 4 h. The samples were then treated with proteinase K for 2 h at staining in nucleus showed co-localization of RBP-J-myc with 42 °C and then DNA was recovered by phenol/chloroform RUNX3 (Fig. 2A). Confocal analysis showed that both ICN1-myc extraction and ethanol precipitation. The precipitated DNA was and RBP-J-myc could co-localize with RUNX3 in nucleus of amplified by PCR using primers: sense 5′-CAGACCTTGTG- SMMC7721 cells. Since ICN1 and RBP-J could co-localize with CCTGGCG-3′ and antisense 5′-TGTGATCCCTAGGCCCTG-3′, with RUNX3, we further investigated whether these three molecules product length of 173 bp corresponding to the -167 to +6 site of might form a complex in SMMC7721-RUNX3 cells. Immunopre- HES-1 promoter. Preimmune serum substituting for the above cipitation of nuclear proteins from SMMC7721-RUNX3 was antibodies was used as a negative control. carried out using the anti-RUNX3 pAb, anti-Notch1 pAb or

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Fig. 1 – RUNX3 down-regulates the expression of HES-1 and suppresses ICN1-mediated transactivation in SMMC7721 cells. Identification of SMMC7721-RUNX3 transfectant. SMMC7721 cells were transfected with pBK-RUNX3 and were selected with G418. Proteins from SMMC7721-RUNX3 and mock transfected SMMC7721 were analyzed with anti-RUNX3 pAb. β-Actin was taken as an internal control. Data were normalized by setting the ratio of RUNX3 of mock transfectant against β-actin at 100% (A). Down-regulation of HES-1 mRNA by RUNX3. PCR was conducted with cDNAs from SMMC7721-RUNX3 and mock transfectant as templates using a human HES-1 primer. β-Actin was taken as an internal control (B). Down-regulation of HES-1 protein by RUNX3. Nuclear proteins from SMMC7721-RUNX3, mock transfectant and DAPT-treated SMMC7721 cells were analyzed with anti-Notch1 pAb, anti-RBP-J pAb and anti-HES-1 pAb. Histone H3 was taken as an internal control. Data were normalized by setting the ratios of ICN1, RBP-J or HES-1 of mock transfectant against Histone H3 at 100%. Results are mean±SD of three independent experiments. ⁎P<0.05, Student's t-test (C). Suppression of ICN1-mediated transactivation by RUNX3. pGa981-6, the reporter gene plasmid for Notch signaling, was co-transfected with plasmid expressing ICN1 and increasing amount of plasmid expressing RUNX3. pcDNA3.1() or neg-pGa981-6 of the same quantity as above was used as a negative control. Luciferase assay was performed in a luminometer. Representative of three independent experiments (D).

anti-RBP-J pAb. Co-precipitated proteins were detected by RUNX3 is recruited to the RBP-J binding site on target gene Western blot. As shown in Fig. 2B, these three proteins could promoters of Notch signaling by ICN1 be simultaneously co-precipitated, suggesting that ICN1, RBP-J and RUNX3 might form a three-molecule complex in nucleus of Since RUNX3 could form a complex with ICN1 and RBP-J in the SMMC7721 cells. No specific interaction was observed from co- nucleus of SMMC7721 cells, we wondered whether RUNX3 could be precipitated proteins by preimmune serum, indicating the recruited to target gene promoters of Notch signaling. EMSA and specificity of the interaction. Thus, ICN1, RBP-J and RUNX3 ChIP experiments were employed to examine this speculation. For form a complex in the nucleus of SMMC7721 cells. EMSA, biotin-labeled probe including the RBP-J binding site was incubated with the nuclear extract of SMMC7721-RUNX3, and RUNX3 interacts directly with ICN1 but not RBP-J in vitro DNA–protein complex was analyzed with PAGE. As shown in Fig. 4A, shifted band was detected with nuclear extract from To detect the direct physical interaction between RUNX3, SMMC7721-RUNX3 cells. This band weakened when competitive Notch1 and RBP-J in vitro, GST pull-down analysis was wild cold probe was added into the reaction in a dose-dependent performed. His-Trx-ICN1, His-Trx-RBP-J fusion protein and His- manner but showed no change when the mutant probe was Trx tag were expressed in E. coli and purified. The purified included, which demonstrated the specific binding between wild proteins were separately incubated with glutathione–Sepharose probe and nuclear protein. The shifted band was supershifted by the beads coated with purified GST-RUNX3 or GST. Associated addition of the anti-RUNX3 pAb, anti-Notch1 pAb or anti-RBP-J pAb proteins were washed down and detected by Western blot but not preimmune serum. This result showed that the shifted band with the anti-His mAb. The result showed that His-Trx-ICN1 was was a specific complex formed by RUNX3, ICN1, RBP-J and the probe pulled down by GST-RUNX3 and meant that ICN1 could directly including the RBP-J binding site. We also conducted supershift interact with RUNX3 in vitro. No pull-down could be detected analysis in DAPT-treated SMMC7721-RUNX3 cells and found that between His-Trx-ICN1 and GST protein, His-Trx-RBP-J and GST- DAPT ablated the supershift band caused by anti-RUNX3 pAb and RUNX3, or His-Trx and GST-RUNX3 (Fig. 3). This result anti-Notch1 pAb but not anti-RBP-J pAb, which indicated that suggested that RUNX3 directly interacts with ICN1 but not RUNX3 can bind the RBP-J binding site only through its interaction RBP-J. with ICN1. Recruitment of RUNX3 to the RBP-J binding sequence in

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Fig. 2 – Interaction of RUNX3 with ICN1 and RBP-J in vivo. RUNX3 co-localizes with ICN1 and RBP-J in nucleus of SMMC7721 cells by double immunofluorescence staining. SMMC7721-RUNX3 cells transiently transfected with ICN1-myc or RBP-J-myc were stained with the anti-RUNX3 and anti-myc antibodies. The secondary antibody was tetraethyl rhodamine isothiocyanate-labeled goat anti-rabbit IgG (red) and FITC-conjugated goat anti-mouse IgG (green), respectively. Co-localization of antigens resulted in a yellow staining (A). RUNX3 interacts with ICN1 and RBP-J in nucleus of SMMC7721 cells by co-immunoprecipitation assay. The nuclear extract of SMMC7721-RUNX3 cells was incubated with anti-RUNX3 pAb, anti-Notch1 pAb or anti-RBP-J pAb and the agarose beads. Co-precipitated proteins were detected with the above pAbs. Preimmune serum was as a negative control (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

vivo was demonstrated by ChIP. As shown in Fig. 4B, the preci- was amplified when precipitated with preimmune serum. No pitated DNA was amplified using primers targeting the 173-bp positive band appeared with anti-RUNX3 pAb or anti-Notch1 pAb fragment containing the RBP-J binding site from HES-1 promoter. precipitation from DAPT-treated SMMC7721-RUNX3 cells, which The specific positive band was amplified in precipitates with the indicated that the RUNX3 is associated with the specific target anti-RBP-J pAb, anti-RUNX3 pAb or anti-Notch1 pAb while no band sequence by formation of the three-molecule complex. Thus, our results indicated that RUNX3 is recruited to the RBP-J binding site on target gene promoters of Notch signaling by ICN1.

Dicussion

HCC is one of the leading causes of death in the world. Lots of researches have been done to elucidate the genetic background and molecular mechanism underlying the development and progression of this disease. Previous reports have shown that RUNX3 takes a strong suppressive effect on HCC while Notch signaling contributes to the malignant phenotype of HCC. In the Fig. 3 – Interaction between RUNX3 and ICN1 in vitro. His-Trx-NIC, present study, we demonstrated that RUNX3 suppressed Notch His-Trx-RBPJ and His-Trx proteins expressed in E. coli were signaling in HCC cell line SMMC7721 by its interaction with ICN1 purified and GST pull-down assay was performed by incubating and thus recruitment to the RBP-J recognition motif on down- the above fusion proteins with purified GST or GST-RUNX3 stream gene promoters of Notch signaling. Our results suggest that immobilized on glutathione–Sepharose 4B beads. Bound proteins RUNX3 might exert its inhibitory effect on HCC partly through the were analyzed by Western blot using the anti-His antibody. suppression of Notch signaling.

EXPERIMENTAL CELL RESEARCH XX (2009) XXX– XXX 7

Fig. 4 – Recruitment of RUNX3 to the RBP-J binding site by ICN1. EMSA assay (A). Left: Nuclear extract were prepared from SMMC7721-RUNX3 cells and incubated with the biotin-labeled probe containing RBP-J binding site. Protein–probe complex were analyzed using PAGE. For the competition assay, the cold probe of increasing amount was included in the reaction. For the supershift assay, the anti-RUNX3 pAb, anti-Notch1 pAb or anti-RBP-J pAb was added into the incubation. Free probe and different protein–probe complexes were indicated. W, wild-type RBP-J site; M, mutant RBP-J site. Right, supershift analysis was conducted in DAPT-treated SMMC7721-RUNX3 cells with the above three pAbs. Preimmune serum was as a negative control. ChIP assay (B). SMMC7721-RUNX3 cells and DAPT treated SMMC7721 cells were collected as indicated. ChIP was performed using the anti-RBP-J pAb, anti-Notch1 pAb or anti-RUNX3 pAb and co-precipitated DNA was amplified using primers targeting the HES-1 promoter fragment containing the RBP-J binding site. The amplified fragment is 173 bp. Preimmune serum was as a negative control.

In this study, we found that increased expression of RUNX3 in has been demonstrated to play a positive role in HCC, this suppres- SMMC7721 repressed the transactivation of ICN1 to Notch signaling sion might be a reason for the negative effect of RUNX3 on HCC. reporter gene and thus down-regulated the target gene HES-1 ex- Furthermore, we studied the mechanism of the regulation of pression. Our result shows that RUNX3 and Notch pathway take Notch signaling by RUNX3. Since RUNX3 did not alter the contrary roles in HCC. Both Runt family of transcriptional factors and expression of ICN1 or RBP-J in SMMC7721 cells, we investigated Notch signaling pathway play important roles in cell differentiation the possibility of its physical interaction with these two Notch and development, and reports have demonstrated the interactive molecules. Double immunofluorescence staining and co-immuno- effects of RUNX and Notch pathway in different contexts. In some precipitation analysis showed that RUNX3 formed a complex with cases, they show a cooperative effect. For examples, in mammalian ICN1 and RBP-J in the nucleus of SMMC7721 cells. By GST pull- cells, transducin-like enhancer of split (TLE) can interact with and act down assay, we found that RUNX3 directly interacted with ICN1 as transcriptional corepressors for HES-1, while members of the but not RBP-J. Since ICN1 binds to RBP-J at the RBP-J binding site on RUNX family of transcription factors can negatively regulate the target gene promoters of Notch signaling, this result hints strongly corepressor role of TLE for HES-1 [38]. And morpholino knock-down that the nuclear complex formed by RUNX3, ICN1 and RBP-J may be of RUNX1 function completely abolishes the increase of hematopoi- located at the RBP-J binding site. Thus, we performed EMSA assay etic stem cell number induced by Notch stimulation [39]. They were and confirmed that RUNX3 was associated with the RBP-J binding also reported to act contrarily in other contexts. Researches found site. To detect the association in vivo, we further conducted ChIP that expression of the fushi tarazu (ftz) gene depends on activation assay and found that RUNX3 bound with a 173-bp HES-1 promoter by runt and repression by hairy in Drosophila [40]. Besides, the hairy- fragment containing the RBP-J binding site. These results indicate related family of transcriptional repressors (Hrt), which are acti- that RUNX3 is recruited to the RBP-J recognition sequence by its vated by Notch1 signaling, physically interacts with RUNX2 and direct interaction with ICN1 and thus RBP-J. represses RUNX2 transcriptional activity in the developing aortic As a transcriptional factor, RUNX3 in most cases functions as a valve of mice [41]. Our investigation provides new evidences for the potent repressor of transcription by itself or by recruitment of other contrary effect of Runt family and Notch pathway by demonstrating corepressors, such as mammalian Swi-indepenent 3a (mSin3A), that RUNX3 inhibits Notch signaling in HCC. Since Notch signaling TLE, and histone deacetylases (HDACs) [42]. RUNX3 can also interact

8 EXPERIMENTAL CELL RESEARCH XX (2009) XXX– XXX with other DNA-binding transcription factors to regulate target [2] J.A. Coffman, Runx transcription factors and the developmental genes transcription [43]. Besides regulation of gene transcription via balance between cell proliferation and differentiation, Cell Biol. Int. 27 (2003) 315–324. binding to the promoter directly, RUNX3 can act as a transcription [3] Q. Wang, T. Stacy, M. Binder, M. Marin-Padilla, A.H. Sharpe, N.A. cofactor and incorporate with other transcription factors. For Speck, Disruption of the Cbfa2 gene causes necrosis and instance, in the study of Drosophila hematopoiesis, the RUNX factor hemorrhaging in the central nervous systems and blocks Lozenge directly interacts with the GATA factor Serpent, and assists definitive hematopoiesis, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) the GATA factor to induce transcription from a GATA-responsive 3444–3449. promoter. Several lines of evidences indicate that this functional [4] F. Otto, A.P. Thornell, T. Crompton, A. Denzel, K.C. Gilmour, I.R. relationship between RUNX and GATA factors may be conserved Rosewell, G.W. Stamp, R.S. Beddington, S. Mundlos, B.R. Olsen, P.B. Selby, M.J. Owen, Cbfa1, a candidate gene for cleidocranial from Drosophila to mammals [44]. In our study, we analyzed HES-1 dysplasia syndrome, is essential for osteoblast differentiation and promoter sequence (-2000 to +700, from http://dbtss.hgc.jp/)for bone development, Cell 89 (1997) 765–771. Runt domain binding sequence and found no putative consensus [5] D. Levanon, D. Bettoun, C. Harris-Cerruti, E. Woolf, V. Negreanu, R. Runt domain binding site sequence PyGPyGGT on the HES-1 pro- Eilam, Y. Bernstein, D. Goldenberg, C. Xiao, M. Fliegauf, E. Kremer, moter, which supports RUNX3 as a transcription cofactor here. F. Otto, O. Brenner, A. Lev-Tov, Y. Groner, The Runx3 transcription Moreover, DAPT abolished the association of RUNX3 with the target factor regulates development and survival of TrkC dorsal root – sequence, which actually excludes the possibility of the direct ganglia neurons, EMBO J. 21 (2002) 3454 3463. [6] N.A. Speck, D.G. Gilliland, Core-binding factors in haematopoiesis binding of these two elements. Thus, RUNX3 might function as a and leukaemia, Nat. Rev., Cancer 2 (2002) 502–513. transcription cofactor in its regulation of Notch signaling in our case. [7] J. Pratap, J.B. Lian, A. Javed, G.L. Barnes, A.J. van Wijnen, J.L. Stein, Multiple molecular complexes in cytoplasm and in nucleus regulate G.S. Stein, Regulatory roles of Runx2 in metastatic tumor and Notch signaling pathway. In the absence of transactivators, RBP-J cancer cell interactions with bone, Cancer Metastasis Rev. 25 functions as a transcription suppressor that recruits corepressor (2006) 589–600. complexes containing HDACs, CBF1-interacting corepressor (CIR) to [8] V.S. Dhillon, M. Shahid, S.A. Husain, CpG methylation of the FHIT, FANCF, cyclin-D2, BRCA2 and RUNX3 genes in Granulosa cell target promoters and inhibits their expression [45,46]. Since RUNX3 tumors (GCTs) of ovarian origin, Mol. Cancer 3 (2004) 33. can interact with some corepressors, we suppose that RUNX3 might [9] X.Z. Chi, J.O. Yang, K.Y. Lee, K. Ito, C. Sakakura, Q.L. Li, H.R. Kim, E.J. work as a molecular scaffold to recruit corepressors such as HDACs Cha, Y.H. Lee, A. Kaneda, T. Ushijima, W.J. Kim, Y. Ito, S.C. Bae, to target gene promoters of Notch signaling and therefore inhibit RUNX3 suppresses gastric epithelial cell growth by inducing p21 Notch signaling pathway. This speculation needs further experi- (WAF1/Cip1) expression in cooperation with transforming ments to prove. growth factor {beta}-activated SMAD, Mol. Cell. Biol. 25 (2005) – Taken together, our study shows that RUNX3 is associated with 8097 8107. [10] Q.L. Li, H.R. Kim, W.J. Kim, J.K. Choi, Y.H. Lee, H.M. Kim, L.S. Li, H. the RBP-J recognition sequence by its interaction with ICN1 and Kim, J. Chang, Y. Ito, K.Y. Lee, S.C. Bae, Transcriptional silencing of functions to inhibit the activity of Notch signaling in SMMC7721 cells. the RUNX3 gene by CpG hypermethylation is associated with lung Since Notch signaling pathway has been reported to play a cancer, Biochem. Biophys. Res. Commun. 314 (2004) 223–228. carcinogenic role in HCC, our result suggests that the suppressive [11] T. Takahashi, N. Shivapurkar, E. Riquelme, H. Shigematsu, J. Reddy, effect of RUNX3 on HCC might be partly accomplished by its inhibition M. Suzuki, K. Miyajima, X. Zhou, B.N. Bekele, A.F. Gazdar, I.I. of Notch signaling. Our investigation provides a new molecular Wistuba, Aberrant promoter hypermethylation of multiple genes mechanism for the suppressive role of RUNX3 in HCC and indicates in gall bladder carcinoma and chrocholecystitis, Clin. Cancer Res. 10 (2004) 6126–6133. RUNX3 as a potential therapeutic modality for the treatment of HCC. [12] Q.L. Li, K. Ito, C. Sakakura, H. Fukamachi, K. Inoue, X.Z. Chi, K.Y. Lee, S. Nomura, C.W. Lee, S.B. Han, H.M. Kim, W.J. Kim, H. Yamamoto, N. Yamashita, T. Yano, T. Ikeda, S. Itohara, J. Inazawa, T. Abe, A. Acknowledgments Hagiwara, H. Yamagishi, A. Ooe, A. Kaneda, T. Sugimura, T. Ushijima, S.C. Bae, Y. Ito, Causal relationship between the loss of RUNX3 expression and gastric cancer, Cell 109 (2002) 113–124. This work was supported by National Natural Science Foundation [13] S. Kang, J.W. Kim, G.H. Kang, N.H. Park, Y.S. Song, S.B. Kang, H.P. of China (Key Item, grant 30530780) (Daiming Fan), National Lee, Polymorphism in folate- and methionine-metabolizing Natural Science Foundation of China (grant 30500597) (Yanqiu enzyme and aberrant CpG island hypermethylation in uterine Zhao) and Youth Scientific Foundation of Health Department of cervical cancer, Gynecol. Oncol. 96 (2005) 173–180. Hubei Province (grant QJX2008-11) (Juan Gao). [14] C. Guo, J. Ding, L. Yao, L. Sun, T. Lin, Y. Song, L. Sun, D.M. Fan, We thank Prof. Paul J Farrell (Imperial College Faculty of Medicine, Tumor suppressor gene Runx3 sensitizes gastric cancer cells to UK) and Prof. Tom Kadesch (University of Pennsylvania School of chemotherapeutic drugs by down-regulating Bcl-2, MDR-1 and MRP-1, Int. J. Cancer 116 (2005) 155–160. Medicine, USA) for their generous gifts of pBK-RUNX3 and pcDNA3.1/ [15] D. Wei, W. Gong, S.C. Oh, Q. Li, W.D. Kim, L. Wang, X. Le, J. Yao, T.T. ICN1-myc-His(-)C plasmids, respectively, and Prof. Shoujing Yang Wu, S. 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