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IDENTIFICATION OF NOVEL DIRECT STAT3 TARGET GENES FOR CONTROL OF GROWTH AND DIFFERENTIATION

Marylynn Snyder, Xin-Yun Huang and J. Jillian Zhang*

Department of Physiology and Biophysics, Weill Medical College of Cornell University 1300 York Avenue, New York, NY 10021 Running title: novel direct Stat3 target genes

Address correspondence to: J. Jillian Zhang, Department of Physiology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, Tel: (212) 746-4614, Fax: (212) 746-6226, Email: jjz2002@med.cornell.edu ABSTRACT Stat3 (Signal Transducer and Activator of Transcription 3) is a key regulator of gene expression in response to signaling of the gp130 family cytokines including interleukin 6 (IL-6), oncostatin M (OSM) and leukemia inhibitory factor (LIF). Many efforts have been made to identify Stat3 target genes and to understand the mechanism of how Stat3 regulates gene expression. Using the microarray technique, hundreds of genes have been documented to be potential Stat3 target genes in different cell types. However, only a small fraction of these genes have been proven to be true direct Stat3 target genes. Here, we report the identification of novel direct Stat3 target genes using a genome-wide screening procedure based on the Chromatin Immunoprecipitation (ChIP) method. These novel Stat3 target genes are involved in a diverse array of biological processes such as oncogenesis, cell growth and differentiation. We show that Stat3 can act as both a repressor and activator on its direct target genes. We further show that most of the novel Stat3 direct target genes are dependent on Stat3 for their transcriptional regulation. In addition, using a physiological cell system, we demonstrate that Stat3 is required for the transcriptional regulation of two of the newly identified direct Stat3 target genes important for muscle differentiation. INTRODUCTION The STAT (Signal Transducer and Activator of Transcription) family of transcriptional regulators is activated in response to extracellular signaling proteins including

cytokines and growth factors (1,2). When cytokines bind to their cell surface receptors, the receptor-associated JAK tyrosine kinases become activated and in turn phosphorylate a single tyrosine residue in the STAT molecule. The phosphorylated STATs then enter the nucleus as dimers and bind to specific DNA sequences in the promoters of their target genes to regulate transcription. Although the seven members of the STAT family have similarity in their molecular structure and function, they play diverse physiological roles in a wide variety of biological processes (3).

One member of the STAT family, Stat3, mediates the signaling of cytokines that share the gp130 receptor chain, which include interleukin-6 (IL-6), oncostatin M (OSM) and leukemia inhibitory factor (LIF) (4,5). In response to gp130 ligand stimulation, Stat3 is phosphorylated on Tyr 705 and forms dimers through phosphotyrosine-SH2 domain interactions (6). The dimerized Stat3 molecules enter the nucleus and bind to a consensus DNA sequence in the promoters of its target genes to regulate transcription (7). The transcriptional activity of Stat3 is mediated by its transcription activation domain (TAD) located in the carboxyl-terminal end of the molecule (8). In addition to the tyrosine phosphorylation, the Stat3 TAD contains a serine residue (Ser724) which is also phosphorylated to achieve maximum transcription activity (9,10). Analyses of Stat3-dependent enhancersomes demonstrate that Stat3 interacts and recruits other transcription factors and co-activators to the promoters of its target genes (10-14). Furthermore, non-tyrosine-phosphorylated Stat3 has been shown to be able to activate

http://www.jbc.org/cgi/doi/10.1074/jbc.M706976200The latest version is at JBC Papers in Press. Published on December 7, 2007 as Manuscript M706976200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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transcription in cooperation with other transcription factors such as NFκB (15,16).

Stat3 plays essential roles in a diverse array of cellular processes. For example, Stat3 activation is associated with oncogenesis and tumor metastasis (17,18). Stat3 is also necessary for the normal development of multiple cellular systems, including early embryogenesis, lymphocyte growth, wound healing and postnatal survival, as demonstrated by the various Stat3 knock-out mouse models (19,20). LIF-mediated self-renewal of murine embryonic stem cells also requires Stat3 activation (21-23). Because of these wide-ranging physiological functions of Stat3, it is critical to identify Stat3 target genes to fully understand how Stat3 mediates its effect on these various cellular processes. Several microarray analyses have been carried out and numerous potential downstream targets of Stat3 have been identified (24-28). Particularly among them are genes that regulate cell cycle progression, cell survival/growth and migration, correlating with the concept that Stat3 is a critical factor in oncogenesis and making it a suitable drug target for cancer treatment (29,30). Therefore, it is essential to identify direct Stat3 target genes to develop specific therapeutic treatments as well as to understand how Stat3 regulates gene transcription to achieve diverse physiological impacts. However, the large numbers of potential Stat3 target genes from the microarray studies make it difficult to systemically distinguish the direct Stat3 targets for further analyses. In this report, we used a genome-wide screening method utilizing the chromatin immunoprecipitation (ChIP) assay to identify Stat3 target genes. Because this method is based on the binding of Stat3 to DNA, the target genes identified are direct Stat3 targets. We show here that we have identified direct Stat3 target genes in NIH3T3 cells that are regulated by OSM. These direct Stat3 target genes include some of the genes that have been identified by previous microarray screening. However, a significant number of novel Stat3 target genes were also identified by this method. These novel genes are diverse in their function, including involvement in oncogenesis, neuronal development and muscle differentiation. We further demonstrate

that Stat3 functions as both a transcriptional activator and a repressor for its direct target genes. Using the Stat3-deficient MEF cells, we show that most of the novel Stat3 direct targets are dependent on Stat3 for their transcriptional regulation. In addition, to test the physiological relevance of these novel direct Stat3 target genes, we showed that Stat3 represses two of its direct target genes in a myoblast cell line for muscle differentiation and knock-down of Stat3 in this myoblast cell line prevents their differentiation. Therefore, we have identified a set of novel direct Stat3 target genes important for a wide range of biological processes including the control of cellular growth and differentiation.

MATERIALS AND METHODS Cell culture and reagents NIH3T3 cells were maintained in DMEM supplemented with 10% fetal bovine serum (Hyclone Laboratories Incorporated). Wild-type and Stat3-deficient mouse embryonic fibroblasts (MEFs, from David Levy, New York University) were grown in DMEM supplemented with 10% fetal bovine serum. C2C12 cells (from Chisa Hidaka, Hospital for Special Surgery) were maintained in DMEM supplemented with 10% fetal bovine serum for growth conditions. C2C12 cells were maintained in DMEM supplemented with 2% horse serum (Sigma) for differentiation conditions. Antibodies: anti-phosphotyrosine-Stat3 (Cell Signaling Technology); anti-Stat3 (Transduction Laboratories). Recombinant mouse OSM was from R&D Systems. Recombinant mouse LIF was from Chemicon International. Genome-wide screening of direct target genes of Stat3 by Chromatin Immunoprecipitation (ChIP)

NIH3T3 cells were grown in 15-cm dishes to 80-90% confluency. Cells were then treated with 25 ng/ml mouse OSM for 30 minutes. ChIP assays were performed as described previously (10) with 2.5 µg of Stat3 antibody. The precipitated genomic DNA was further purified three different ways to determine if different techniques used for the DNA isolation yielded the same target genes. DNA was ethanol precipitated with yeast tRNA

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twice, isolated with Qiaquik PCR Purification Kit once, or agarose gel purified once respectively. For agarose gel purification, DNA fragments ranging in size from 200 bp to 2 kb were excised. DNA was resuspended in 30 µl sterile deionized water and blunted with the DNA Terminator End Kit (Lucigen) according to the manufacturer’s instructions. There was no significant difference in the final numbers of clones obtained with these various DNA purification methods and some genes were repetitaively pulled out with the different methods. The blunted genomic DNA was amplyfied by ligation-mediated PCR with linkers containing EcoRI sites (LMPCR.1: 5′-GCGGTGACCCGGGAGATCTGAATTC-3′ and LMPCR.2: 5’-GAATTCAGATC-3′) as follows. Kinase reaction of LMPCR.2: 9 µl linker (350 ng/µl), 6 µl sterile deionized water, 2 µl kinase buffer, 2 µl 10 mM ATP, and 1 µl T4 polymucleotide kinase (NEB) were incubated at 37°C for 30 min. The kinase was then heat inactivated at 65°C for 10 min. The entire 20 µl LMPCR.2 reaction was annealed to 9 µl (350 ng/µl) LMPCR.1. The reaction was heated to 95°C for 2 min., 65°C for 10 min., 37°C for 10 min, and 25°C for 20 min. A total of 3 µl of the annealed linkers were ligated to 10 µl of blunted ChIP DNA with 1 µl (2000 U) T4 DNA ligase (NEB) at 16°C for 16 hours. Ligation mixtures were purified with Qiaquik PCR Purification Kit to obtain the ChIP DNA/linker mix. The ChIP DNA/linker complex was then PCR amplified in a reaction containing 5 µl of the ChIP DNA-linker complex, 1 µl Platinum Taq DNA Polymerase (Invitrogen), 200 pmol LMPCR.1, 2 mM MgCl2, and 0.75 mM dNTPs in a final volume of 50 µl. PCR was performed with the following conditions: 1 cycle 95°C/50sec, 25 cycles 94°C/15sec, 55°C/30sec, 68°C/2min, followed by 1 cycle at 68°C for 7 min. The PCR-amplified DNA was purified with Qiaquik PCR Purification Kit and then digested with 60 U EcoRI in a 200 µl reaction at 37°C for 2 hr. The EcoRI-digetsed DNA was purified with the Qiaquik PCR Purification Kit again and ligated into the EcoRI site of pBluescript followed by transformation into

DH5α (Invitrogen) and plated onto LB agar plates containing ampicillin and X-gal. Plasmid DNAs were purified from white colonies and sequenced with T7 and T3 primers. Most of the plasmids contained inserts of concatemers of 10-20 bp in length from different genes. Sequences were identified with NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome) against the mouse genome using a combination of the databases nr, refseq_genomic, and refseq_rna. All sequences were considered potential targets regardless of the sequence’s location within the gene, even if the sequence was not located in the promoter region. GeneSpring GX 7.3.1 software (Agilent Technologies) was used to find genes that are common to a microarray list and our ChIP list. Three publications of microarray studies of mouse cell lines were utilized (24-26) to generate three initial lists, which were then combined into one final list by manual editing (removing duplicates) to generate Table 1. Gene-specific Chromatin Immunoprecipitation (ChIP) Analysis

NIH3T3 cells were grown in DMEM media supplemented with 10% FBS in 15-cm dishes to 80-90% confluency. Cells were then either treated with 25 ng/ml mouse OSM or left untreated. ChIP analysis was performed as described previously (10) with 2.5 µg Stat3 antibody (Santa Cruz). DNA from one 15-cm dish was used for a total of six separate PCR reactions.

If promoter sequences for a potential Stat3 target gene could be obtained from NCBI, putative Stat3 sites were identified within these sequences. Some genes do not have promoter sequences available from NCBI and putative Stat3 sites for these genes were identified by obtaining complete genomic sequences from NCBI followed by searching for potential Stat3 sites 1-3 kb upstream of the translational start site. All subsequent gene-specific ChIP primers were designed to flank the putative Stat3 sites and could amplify PCR fragments that were about 200 bp in size. Primers for the ChIP are as follows: Boc: 5′-GTCTCGCTGGTGTCAGCTC-3′ and 5′-ACACACACCACGGCAGAGT-3′; Cln6: 5′-

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GAAATGCAGAGAACCCAGGA-3′ and 5′-GGAGGGAGGAGGAATGAGAG-3′; Angpt1: 5′-TTCCTGTCAAGTCATCTTGTGAA-3′ and 5′-GCGTCAGCTGCGAGTACATA-3′; Smad9: 5′-CTGGCTCCACTTTCCAAGAG-3′ and 5′-CGGGGAAAGAGGATGAGAC-3′; Perq1: 5′-CAGGGGAAAAGCTGGTGTAG-3′ and 5′-TCCCAAACCTCACCTTGTTC-3′; TNF-R: 5′-ACCTTCTCTCTCCCCTCAGC-3′ and 5′-ATTGACAACGCTCGTGAATG-3′; Pcnt: 5′-GCGCTGGATTCAAGATGG-3′ and 5′-CGGTGGGGAAGAAATCCTA-3′; Pax4: 5′-TTGATGCATGGGAAACTTTG-3′ and 5′-CCTTGTGGCTCTACCCTGAA-3′; Bcl3: 5′-GGCACAATGAGCAGAGTGG-3′ and 5′-CCGACTGAACTGAAGGGACT-3′; Fgl2: 5′-TCCATTTAAAGAGCGGACCTT-3′ and 5′-TTCTTCCCACTAAATGTCACCA-3′; Gbp1: 5′-TCCCAGCCTTAGCTCAGAGA-3′ and 5′-AAAGGGGTGGAGTTTCCAGT-3′; Cdo: 5′-GCCTCCGCTTACTGAAAAAC-3′ and 5′-TCCTAGTCCCCAGAAGAAGGA-3′; CBP: 5′-CTGTTTCCGCGAGCAGGT-3′ and 5′-TGTCATTCGCGGAGAAGC-3′; Ect2: 5′-GAAGGATAAGCGAGGACTGC-3′ and 5′-GTGCTCAGTTCCGGGTTC-3′; FasL: 5′-GCCTGGTTTACCAGCCTTCT-3′ and 5′-TGAGACACCCACTCACTTGC-3′; Peg10: 5′-GTCCGGACTCCCGATACAC-3′ and 5′-AGGCTCGGTGGACCTTCT-3′ . Quantitative Real-Time RT-PCR (qRT-PCR)

NIH3T3 cells were grown in 6-well dishes in DMEM supplemented with 10% FBS until cells were 80% confluent. Cells were then serum-starved for 24 hrs. The cells were then either left untreated or treated with mouse OSM at 25 ng/ml for lengths of time as indicated. C2C12 cells were grown to about 80% confluence in growth medium and then cultured in differentiation medium for 24 hours followed by treatment with mouse OSM at 25 ng/ml or LIF 100ng/ml for lengths of time as indicated. WT MEFs and Stat3-deficient MEFs were grown in DMEM supplemented with 10% FBS to about 80% confluence. The medium was then changed to DMEM without serum for 24 hours followed by treatment with OSM at 25 ng/ml. RNA from these various cells was extracted

using the Trizol method (Invitrogen) and reverse transcribed with Superscript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative Real-Time RT-PCR analysis was performed using the SYBR Green Core PCR Reagent Kit (Applied Biosystems). RT-PCR primers are as follows: TNF-R: 5′-CAGTCTGCAGGGAGTGTGAA-3′ and 5′-CACGCACTGGAAGTGTGTCT-3′; Pcnt: 5′-CCAGATTTCCCCACTCAAGA-3′ and 5′-GTCCTTCCGGACAACTTCAG-3′; Fgl2: 5′-CGTGCTAGGAAGGAGAAGCA-3′ and 5’-CCGGCTTTGTAGTCTTTCCA-3′; Smad9: 5’-GCCTAGCAAGTGTGTCACCA-3′ and 5′AACGGGAACTCACAGCACTC-3’; Boc: 5′-CATGGATGAACGTGACTTGG-3′ and 5′-GGAGATTGGCTAGCGTCACT-3′; Cln6: 5′-TCTCAACAAGCCAAGTGTCG-3′ and 5-CTGGCTCCCATGATGAAAGT-3′; Angpt1: 5’-TCAGTGGCTGCAAAAACTTG-3’ and 5’-TTTCAAGTCGGGATGTTTGA-3′; Bcl3: 5′-GCCAGACTGCAATTCACCT-3′ and 5′-CTCCAGGAGCAGCAGAACA-3′; Perq1: 5′-TGGAGGATGAGGATGAGGAG-3′ and 5′-GGTGAGCTGGAGTTCTCTGG-3′; Gbp1: 5′-TGGAGACTTCACTGGCTCTG-3′ and 5′-CAGCTGGTCCTCCTGTATCC-3′; FasL: 5′-TCCATCTTGTGGGCCTAGAG-3′ and 5′-TCCTAATCCCATTCCAACCA-3′; Cdo: 5′-CAGGAAGCAACTGGAGAAGG-3′ and 5′-CAGGGACACCTTCTGATCGT-3′; mMyog: 5’-GGCATGCAAGGTGTGTAAGA-3’ and 5’-GCGCAGGATCTCCACTTTAG-3’. Stat3 siRNA Stat3 siRNAs and control siRNAs were obtained from Qiagen (cat # SI01435294, SI01435287 for Stat3 and 1022563 for control siRNA). C2C12 cells were grown in DMEM supplemented with 10% FBS (Growth Medium, GM) to a density of 1 x 105 cells. Cells were then transfected with Stat3 siRNAs or control siRNAs according to the manufacturer’s instructions. Transfected cells were grown in GM for 3 days and then cultured in DMEM supplemented with 2% horse serum (Differentiation Medium, DM) for 24 hours. A portion of the transfected cells were analyzed by Western blotting assay to determine the knock-down of Stat3. The transfected cells were further treated with OSM for 4 hours or left

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untreated followed by Real-Time qRT-PCR analyses. Western Blot Analyses Whole cell lysates were separated by SDS-PAGE and blotted with the indicated antibodies followed by chemiluminescence (DuPont).

RESULTS Identification of direct Stat3 target genes by genome-wide ChIP Screening In order to identify direct Stat3 target genes, we used a genome-wide screening method based on ChIP followed by ligation mediated PCR and subcloning. A similar approach has been utilized to identify direct target genes for other transcription factors such as CREB (31). For our screening, we used NIH3T3 cells which have been extensively used as a cell line for studying Stat3 function (17). Oncostatin M (OSM) activates Stat3 robustly in NIH3T3 cells (10) and was chosen as a ligand to activate Stat3 at maximum level for genome wide screening of Stat3 target genes. Since ligand-activated STATs become de-phosphorylated and inactivated rapidly (32,33), a 30 minute OSM-treatment time point was chosen in order to capture as many as possible the direct Stat3 target genes as well as to make it feasible for a scaled-up ChIP assay. NIH 3T3 cells were treated with OSM for 30 min and ChIP assays were performed with a Stat3-specific antibody. The precipitated genomic DNA fragments from the ChIP were then amplified by ligation-mediated PCR and further subcloned into pBluescript followed by sequencing of the cloned inserts (see methods for details). The DNA sequences obtained were used to BLAST search against the mouse genome database. Four separate independent ChIP-subcloning experiments were performed and ~400 genes were identified. These potential Stat3 target genes function in a wide range of biological processes including immune response, oncogenesis, cell cycle control, development, cell adhesion and differentiation. Out of these potential direct Stat3 target genes, about one fourth have been identified previously by microarray analyses of mouse cell lines (24-26) (Table 1). Most of these microarray-identified genes have not been confirmed by ChIP to demonstrate that they are direct Stat3 target

genes. Therefore using the genome-wide screen method based on ChIP, we have identified the direct Stat3 target genes from these microarray studies. Identification of novel direct Stat3 target genes

The majority of the Stat3 target genes isolated by ChIP using NIH 3T3 cells with 30-min OSM stimulation have not been identified previously by microarray analyses. To confirm that we have identified novel direct Stat3 target genes, gene-specific ChIP analyses were further performed on these potential Stat3 target genes. Some of the novel Stat3 target genes (34 genes) were cloned out multiple times and therefore more likely to be true Stat3 target genes (Table 2). We focused on this group of genes to perform the gene-specific ChIP analyses, as well as including several genes from Table 1 and novel genes that have only been identified once by ChIP screening method.

Putative Stat3 binding sites (TTC/GN2-4GAA) (7,34) were searched for in either known promoter sequences of target genes or genomic sequences upstream of transcription or translation start sites (see Supplemental Table 1). ChIP PCR primers were designed to flank the potential Stat3 sites to generate ~200 bp PCR fragments. We performed gene-specific ChIP on 22 genes from Table 2, 9 genes from Table 1 and 1 novel gene that had been isolated once (FasL). 7 of the genes from Table 1 (TNF-R, Pcnt, Bcl3, Gbp1, Pax4, Fgl2, and Cdo) showed an increase in the amount of Stat3 bound to their promoters in response to OSM treatment compared to untreated cells (Fig. 1A) while two genes, Tek and Ltbp3, had constitutive binding of Stat3 (data not shown and Supplemental Table 2). The c-fos promoter is shown as a positive control (Fig. 1A). Of the 22 novel Stat3 target genes tested with gene-specific ChIP assay, 11 of them showed an increase in Stat3-binding to the promoter in response to OSM treatment. Results for Cln6, Perq1, Smad9, Boc, CBP, Ect2, FasL, Angpt1, and Peg10 are shown in Figure 1B. Il28ra and Dzip1 also showed increase in Stat3 bound to their promoters in response to OSM treatment (Data not shown, see Supplemental Table 2). 7 of the novel Stat3 target genes (Notch4, Bag4, V2r4, Sema3g, Itga11, Paxip1

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and Pax1) showed constitutive Stat3 binding to their promoters that were not affected by OSM treatment (data not shown and Supplemental Table 2). Only 4 of the potential direct Stat3 targets (B3bp, Nav1, Ighmbp2, and Ddef2) had no detectable Stat3 binding (data not shown) in either treated or untreated conditions. Since the ChIP primers usually flank only one of several putative Stat3 sites in these genes, it is possible that we did not choose the right Stat3 sites for these four genes. However, these results demonstrate that with preliminary gene-specific ChIP analyses most of the potential novel Stat3 target genes (~82%) identified by the ChIP assay are true direct Stat3 target genes. Stat3 can both activate and repress its direct target genes in response to OSM To further understand how Stat3 regulates its direct target genes, RNA expression of the target genes were analyzed by quantitative Real-Time RT-PCR. NIH3T3 cells were either untreated or treated with OSM for 2 hrs and 4 hrs. cDNA-specific RT-PCR primers were designed for all the target genes that showed inducible Stat3 binding to their promoters. The expression of these direct Stat3 target genes could be either induced or repressed by the activation of Stat3 in NIH3T3 cells in response to OSM treatment (Fig. 2 and Supplemental Table 2). Of the 18 direct target genes tested, 6 (Bcl3, TNF-R, Peg10, Smad9, Gbp1 and FasL) were induced to varying degrees by OSM treatment (Fig. 2A) and 10 (Perq1, Boc, Cdo, Pcnt, Fgl2, Angpt1, Cln6, Pax4, Dzip1 and Ect2) were reduced by two to three folds (7 of them shown in Fig. 2B, Supplemental Table 2). The expression of CBP was not affected by OSM treatment (data not shown). These results are consistent with previously published data of Stat3 acting both as an activator and a repressor on its target genes. Requirement of Stat3 for transcription regulation of novel direct Stat3 target genes

To test whether Stat3 is essential for the transcriptional control of its direct target genes, we utilized the Stat3-deficient mouse embryonic fibroblasts (MEFs) to analyze the expression of these genes. Wild type and Stat3-/- MEFs were serum-starved for 24 hours and then either

treated with OSM for 4 hours or left untreated. Expression of 10 representative direct Stat3 target genes (five from Table 1 and five from Table 2 and including both induced and repressed genes) were analyzed by Real-Time qRT-PCR. For the five genes from Table 1, the OSM-induced repression of one gene (Cdo) was dependent on Stat3 (Fig. 3E); for TNF-R and Bcl3, there was an increase in their basal level of expression and the OSM induced fold increase was lower in Stat3-/- cells (Fig. 3A, B and supplemental Table 2). Two genes (Pcnt and Gbp1) were not dependent on Stat3. For the five novel direct Stat3 target genes from Table 2, four of them (Smad9, Peg10, Perq1 and Boc) are dependent on Stat3 for either the increase or decrease in their expression in response to OSM (Fig. 3F-J). The basal level expression of Perq1 is also lower in Stat3 cells (Fig. 3J). A summary of these results is presented in Supplemental Table 2. These results demonstrate that Stat3 plays a critical role for the transcriptional regulation for most of its direct target genes, particularly the novel target genes identified by the ChIP method in this report. Stat3 represses the expression of Cdo and Boc in myocytes

Two of the Stat3 direct target genes, Cdo and Boc, function together as a receptor complex and are critical for muscle differentiation and axonal guidance (35-38, 40). Specifically for muscle differentiation, the expression of these two genes is required. It has been suggested that Stat3 activation could inhibit muscle cell differentiation through direct binding and inhibition of MyoD activity (39). The direct repression of Cdo and Boc expression by Stat3 observed in NIH3T3 cells provides a novel mechanistic explanation for the above observations.

To see if Stat3 activation could repress Cdo/Boc expression in muscle cells to inhibit differentiation, we further analyzed expression of Stat3 target genes in the myoblast C2C12 cell line, which has been used extensively for muscle differentiation (38-40). C2C12 cells were treated with OSM for 2 and 4 hrs followed by Real-Time RT-PCR analyses of Cdo/Boc expression. In response to OSM, Stat3 was activated in these cells through tyrosine

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phosphorylation after 30 minutes of treatment (Figure 4A). When these cells were grown in growth medium, low levels of Boc and Cdo expression are detected while their levels increased significantly when the cells are in differentiation media (Fig. 4B). After treatment with OSM for 2 and 4 hours in differentiation media, expression of Cdo and Boc were repressed (Figure 4B). As a marker for muscle differentiation, the expression of myogenin was increased in differentiation media and OSM treatment significantly inhibited myogenin expression (Fig. 4B). In growth media, OSM treatment decreased the basal level of Cdo and Boc slightly and had no effect on myogenin expression (data not shown). To further confirm that it is Stat3, not other factors activated by OSM, that represses the expression of Cdo and Boc, the C2C12 cells were treated with another gp130 ligand, LIF, which plays important physiological roles for muscle regeneration after injury (41,42). LIF induced activation of Stat3 in C2C12 cells and inhibited the expression of Cdo, Boc and myogenin (Fig. 4C and D). All together, these results suggest that Stat3 activation causes a repression of Boc, Cdo and myogenin transcription in C2C12 myoblasts, which are necessary for myocyte differentiation. Stat3 is necessary for inhibition of muscle differentiation

To further demonstrate that Stat3 plays a direct physiological role in controlling muscle growth and differentiation, we used the siRNA technique to knock down the level of Stat3 in C2C12 myocytes. C2C12 cells were grown in growth media and transfected with Stat3 siRNA or control siRNA. Three days after transfection, the cells were changed to differentiation media for 24 hours. A portion of the transfected cells were analyzed by Western blotting assay to determine the knock-down of Stat3. Stat3 level was significantly reduced in cells transfected with Stat3 siRNA compared with that in cells transfected with control siRNA (Fig. 5A). The transfected cells were further treated with OSM for 4 hours or left untreated followed by Real-Time qRT-PCR analyses for the expression of Cdo, Boc and myogenin. For all three genes, their expressions were no longer repressed in response to OSM in cells transfected with

Stat3siRNA while the control siRNA had no effect (Fig. 5B, C and D). In addition, the basal level of Cdo and myogenin is also decreased in cells with Stat3 knock-down (Fig. 5C and D). These results demonstrate that Stat3 plays a critical physiological role in controlling muscle differentiation by repressing the expression of genes necessary for differentiation.

DISCUSSION Using the ChIP method with an antibody specific for Stat3 followed by ligation-mediated PCR and subcloning, we have identified a whole new set of Stat3 target genes involved in a wide ranging array of cellular processes. Furthermore, these Stat3 target genes are directly regulated by Stat3 through the binding of Stat3 to their promoters. About 25% of these direct target genes have been previously identified by microarray analyses of mouse cell lines. There are several reasons for the relative small overlap of genes identified by these two methods. Firstly, since the microarray experiments often use much longer ligand treatment (hours versus the 30 min time point in this report), many of the Stat3 target genes identified in the microarray experiments are not direct Stat3 target genes, while the ChIP cloning method used here searches for the direct target genes that bind Stat3. Secondly, it has been shown that the kinetics of STAT binding to their target genes vary. Some targets have STAT binding quickly after ligand stimulation but only for a short time period while others may have slower but sustained STAT binding (43,44). Therefore, the 30 min time point of treatment we chose may render it difficult to clone the immediate-early genes such as c-fos (even though by radioisotope labeling we are able to detect it at the 30min time point) and the genes that have slower Stat3-binding kinetics. The gene-specific ChIP analyses of a subset of these direct target genes showed that some of them have inducible binding of Stat3 on their promoters after OSM treatment while others had constitutive Stat3 binding. For these constitutive Stat3-bound genes, while there is no detectable phosphorylation of Y705 on Stat3 in NIH3T3 cells without OSM treatment, we can not rule out the possibility of low level Stat3 activation in NIH3T3 cells grown in serum-

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containing medium. Another explanation for the constitutive binding of Stat3 on these promoter DNAs could be that Stat3 is present on these promoters through interaction with other DNA-bound transcription factors such as NFkB as reported recently (15). About 18% of the target genes analyzed by gene-specific ChIP showed no detectable Stat3 binding. Because most of the promoters are not defined and there are often multiple putative Stat3 sites in the promoter, it is possible that we did not choose the correct Stat3 binding sites when we designed the gene-specific ChIP primers. It is also possible that these are false positive sequences due to contamination of sticky genomic fragments in the immunoprecipitates during ChIP.

The regulation of these target genes by Stat3 was further analyzed by Real-Time RT-PCR in response to OSM treatment. The results indicate that Stat3 can both activate and repress its direct target genes when activated by OSM in NIH3T3 cells. These results are consistent with findings from the microarray analyses, further demonstrating the complex nature of Stat3 activity not only in its diverse involvement in many different biological processes, but also in the ways it controls transcription of its target genes. Analyses of the expression of these direct Stat3 target genes in Stat3-deficient MEFs also showed that a majority of them are dependent on Stat3 for their transcriptional regulation (both induction and repression). Therefore, for mammalian gene promoters which require multiple transcription factors and co-activators to assemble an enhancersome for

precise transcriptional regulation, Stat3, as one component of the enhancersome, often plays a critical role for its direct target genes, although in a few cases its function maybe dispensable (at least in MEFs in response to OSM), perhaps replaced by other STATs. Furthermore, although we identified these direct Stat3 target genes in NIH3T3 cells, some of these target genes have tissue-specific functions. This could be the reason that the expression of some genes are only moderately affected by OSM treatment in NIH3T3 cells because not all the components of a tissue-specific enhancersome are assembled on the promoters in NIH3T3 cells in response to OSM even if the activated Stat3 can bind there. Therefore, to demonstrate the physiological relevance of these Stat3 target genes, we further analyzed the expression of two of the Stat3 target genes in a tissue-specific cell line. The two related Stat3 target genes, Cdo and Boc, have specific functions in myoblast differentiation and axonal guidance (35-38, 40). Using a myoblastic cell line, we showed that Stat3 represses their transcription that is necessary for muscle differentiation. Furthermore, the expression of these genes can no longer be suppressed in myocytes with Stat3 knock-down. The identification of these genes as direct targets for Stat3 uncovers a link in the mechanism of muscle differentiation regulated by Stat3. All together these results demonstrate that Stat3 plays a critical role in controlling muscle growth and differentiation.

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FOOTNOTES

We thank Dr. Chisa Hidaka from Hospital of Special Surgery for the C2C12 cells. We thank all lab members for their help and support. This work is supported by American Heart Association Grant 0455896T to J.J.Z and NIH Grant AG023202 to X.-Y. H.

FIGURE LEGENDS

Figure 1. Induction of Stat3 binding to the promoters of target genes

NIH3T3 cells were treated with mouse OSM for 30 minutes or left untreated followed by ChIP

analyses with Stat3 antibodies and PCR primers designed to include potential Stat3-binding sites

in the promoters of the indicated genes. (A) Stat3 target genes identified by both microarray

analyses and the ChIP method as listed in Table 1. (B) Novel Stat3 target genes as listed in

Table 2. c-fos is shown as a positive control. Quantitation was performed with a

Phosphorimager and the ChIP signals were normalized by the input signals. Fold induction is

presented as a ratio of values of OSM-treated samples to untreated samples. For samples with an

*, fold induction could not be obtained due to undetectable signal in the untreated samples.

Figure 2. Stat3 activates and represses its direct target genes

NIH3T3 cells were grown in DMEM containing 10% FBS in 6-well dishes then serum starved

for 24hrs. Cells were then left untreated or treated with OSM for 2 or 4 hrs followed by RNA

extraction and quantitative Real-Time RT-PCR analyses. Stat3 target genes that are induced by

OSM are presented in (A) and the OSM-repressed Stat3 target genes are presented in (B).

Expression of each gene was normalized to GAPDH and the results are presented as fold of

induction with the expression of a specific gene from the untreated sample set as 1. The

mean+SD of three independent experiments are shown.

Figure 3. Requirement of Stat3 for transcriptional control of direct Stat3 target genes

Stat3 WT (+/+) and Stat3-deficient (-/-) MEFs were grown in DMEM supplemented with 10%

FBS to about 80% confluence. The medium was then changed to DMEM without serum for 24

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hours. Cells were either treated with OSM for 4 hours or left untreated. RNA samples from

these cells were analyzed by Real-Time RT-PCR for the expression of five Stat3 target genes

from Table 1 (A-E) and five novel Stat3 target genes from Table 2 (F-J). Relative expression is

standardized to GAPDH with the values from the untreated sample of Stat3 WT set as one. The

mean+SD of three independent experiments are shown.

Figure 4. Activation of Stat3 inhibits the expression of two direct target genes Cdo and Boc

in myocytes

A. and C. C2C12 myoblasts were cultured in DMEM containing 10% FBS and treated with

OSM (A) or LIF (C) for 30 minutes or left untreated. Whole cell extracts were prepared and

Western blot analysis was performed with antibodies against phosphorylated Stat3 (PY-Stat3) or

total Stat3.

B. and D. C2C12 cells were grown either in growth-inducing media (growth) or differentiation-

inducing media (differentiation). The cells were then treated with OSM for 0, 2, or 4 hours (B)

or LIF for 4 hours (D) followed by RNA extraction and Real-Time RT-PCR analyses for the

expression of Boc, Cdo, myogenin and GAPDH. Relative expression is standardized to GAPDH

and the mean+SD of three independent experiments are shown.

Figure 5. Stat3 is required for OSM-induced repression of Cdo and Boc and myocyte

differentiation

C2C12 cells were grown in growth media and transfected with either control siRNA or Stat3

siRNAs for 3 days. The transfected cells were then cultured in differentiation medium for 24

hours. A portion of the transfected cells were analyzed by Western blotting assays to determine

the effect of knock-down of Stat3 (A). The cells were either further treated with OSM for 4

hours or left untreated followed by Real-Time RT-PCR analyses for the expression of Boc, Cdo,

myogenin and GAPDH (B-D). Relative expression is standardized to GAPDH with the values

from the untreated sample of cells transfected with control siRNA set as one. The mean+SD of

three independent experiments are shown.

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Snyder Table 1. List of Stat3 target genes identified by both microarray and ChIP

Gene Accession Number

Function Cd22 antigen (CD22) NM_009845 Co-receptor activity, cell adhesion Cytotoxic and regulatory T cell molecule (Crtam) NM_019465 Protein binding, cell adhesion Insulin-like growth factor 2 receptor (Igf2r) NM_010515 Receptor/Transporter Tumor nerosis factor receptor superfamily member 1a (TNF-R, Tnfrsf1a) NM_011609 Receptor Transforming growth factor, beta 2 (Tgfb2) NM_009367 Development, apoptosis, proliferation Cyclin B1 (Ccnb1) NM_172301 Kinase activity, cell cycle Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (Mtbp) NM_134092 Transcription Retinoblastoma 1 (Rb1) NM_009029 Cell cycle, transcription, differentiation Tumor susceptibility gene 101 (Tsg101) NM_021884 Transcription, cell cycle, transport PRKC, apoptosis WT1 regulator (Pawr) NM_054056 Transcription, apoptosis Cyclin D2 (Ccnd2) NM_009829 Cell cycle Insulinoma-associated-1 (Insm1) NM_016889 Differentiation, development Odd Oz/ten-m homolog 1 (Odz1) NM_011855 Membrane-associated FMS-like tyrosine kinase 3 (Flt3) NM_010229 Kinase activity MAD homolog 5 (Smad5) NM_008541 Transcription, signaling,development Caveolin 3 (Cav3) NM_007617 Endocytosis General transcription factor II H, polypeptide 2 (Gtf2h2) NM_022011 Transcription SRY-box containing gene 11 (Sox11) NM_009234 Transcription CCAAT/enhancer binding protein (C/EBP), gamma (Cebpg) NM_009884 Differentiation, immune response Guanylate nucleotide binding protein 1 (Gbp1) NM_010259 Immune response, GTP binding Bone morphogenetic protein 4 (Bmp4) NM_007554 Cytokine activity, differentiation,

development Tenascin XB (Tnxb) NM_031176 Receptor binding Tenascin C (Tnc) NM_011607 Receptor binding, cell adhesion Protein kinase, cAMP dependent, catalytic, alpha (Prkaca) NM_008854 Kinase activity Fibroblast growth factor 7 (Fgf7) NM_008008 Growth factor activity, proliferation Myelin-associated oligodendrocytic basic protein (Mobp) NM_001039364 Structural constituent myelin sheath MAD homolog 1 (Smad1) NM_008539 Transcription, development,

proliferation Signal transducer and activator of transcription 3 (Stat3) NM_213660 Transcription, signaling X-box binding protein 1 (Xbp1) NM_013842 Transcription Growth factor receptor bound protein 10 (Grb10) NM_010345 Receptor activity, signaling Minichromosome maintenance deficient 6 (Mcm6) NM_008567 DNA replication, helicase Paternally expressed 3 (Peg3) NM_008817 Apoptosis Mad homolog 7 (Smad7) NM_001042660 Transcription Nuclear factor of kappa light polypeptide gene enhancer in B-cells, p105 (Nfkb1) NM_008689 Transcription, apoptosis, development Activating transcription factor 1(Atf1) NM_007497 Transcription Tumor necrosis factor (ligand) superfamily, member 11 (Tnfsf11) NM_011613 Cytokine activity, differentiation,

development Cyclin D3 (Ccnd3) NM_001081636 Cell cycle, kinase Ros1 proto-oncogene (Ros1) NM_011282 Kinase, receptor, cell cycle regulation DNA methyltransferase 3A (Dnmt3a) NM_007872 Methyltransferase, transcription Catenin (cadherin associated protein), delta 2 (Ctnnd2) NM_008729 Transcription, cell adhesion Breast cancer 2 (Brca2) NM_009765 Transcription, DNA repair GATA binding protein 6 (Gata6) NM_010258 Transcription, development Fos-like antigen 2 (Fosl2) NM_008037 Transcription Recombination activating gene 1 (Rag1) NM_009019 B-cell differentiation, recombination Fas-associated via death domain (Fadd) NM_010175 Apoptosis Neural cell adhesion molecule 2 (Ncam2) NM_010954 Cell adhesion Disintegrin and metallopeptidase domain 15 (Adam15) NM_009614 Proteolysis, peptidase activity Calmodulin 3 (Calm3) NM_007590 Cell cycle, signaling Minichromosome maintenance deficient 7 (Mcm7) NM_008568 DNA replication, cell cycle Minichromosome maintenance deficient 2 (Mcm2) NM_008564 DNA replication, cell cycle Cadherin 10 (Cdh10) NM_009865 Cell adhesion Protein kinase C, epsilon (Prkce) NM_011104 Protein kinase, signaling Caspase 7 (Casp7) NM_007611 Apoptosis Nuclear factor of kappa light chain gene enhancer in B-cells 2, p49/p100 (Nfkb2) NM_019408 Transcription, development Cyclin A1 (Ccna2) NM_009828 Cell cycle Disintegrin and metallopeptidase domain 8 (Adam8) NM_007403 Cell adhesion, proteolysis SRY-box containing gene 6 (Sox6) NM_001025560 Transcription, differentiation,

development Interferon regulatory factor 6 (Irf6) NM_016851 Transcription

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General transcription factor II H, polypeptide 4 (Gtf2h4) NM_010364 Transcription RAS p21 protein activator 3 (Rasa3) NM_009025 GTPase activator, signal transduction Suppressor of Ty 5 homolog (Supt5h) NM_013676 Transcription Fibroblast growth factor receptor 1 (Fgfr1) NM_001079908 Growth factor receptor, development N-myc downstream regulated gene 3 (Ndrg3) NM_013865 Differentiation Breast cancer 1 (Brca1) NM_009764 DNA repair, replication, apoptosis Seven in absentia 2 (Siah2) NM_009174 Apoptosis, development DNA methyltransferase 1 (Dnmt1) NM_010066 DNA methylation, transcription Janus kinase (Jak2) NM_008413 Kinase, signal transduction Bone morphogenetic protein 8a (Bmp8a) NM_007558 Development, differentiation Vav2 oncogene (Vav2) NM_009500 Cell migration, signal transduction Neurofibromatosis 1 (Nf1) NM_010897 Ras protein signal transduction Estrogen receptor 1 (alpha) (Esr1) NM_007956 Receptor activity, signal transduction Paired box gene 4 (Pax4) NM_011038 Transcription, differentiation Odd Oz/ten-m homolog 3 (Odz3) NM_011857 Membrane-associated protein Apoptosis inhibitor 5 (Api5) NM_007466 Apoptosis B-cell leukemia/lymphoma 3 (Bcl3) NM_033601 Transcription, immune response,

differentiation Cadherin 2 (Cdh2) NM_007664 Cell adhesion Protein inhibitor of activated STAT 3 (Pias3) NM_018812 Transcription Calcium/calmodulin-dependent protein kinase II, delta (Camk2d) NM_023813 Kinase activity, calmodulin binding GATA binding protein 4 (Gata4) NM_008092 Transcription, development Caspase 9 (Casp9) NM_015733 Apoptosis Fibrinogen-like protein 2 (Fgl2) NM_008013 Receptor binding, signal transduction Interferon-related developmental regulator 1 (Ifrd1) NM_013562 Development, differentiation Transcriptional regulator, SIN3B (Sin3b) NM_009188 Transcription Angiopoietin 2 (Angpt2) NM_007426 Development Platelet/endothelial cell adhesion molecule 1 (Pecam1) NM_001032378 Cell adhesion Suppressor of cytokine signaling 3 (Socs3) NM_007707 Signal transduction Disintegrin and metallopeptidase domain 23 (Adam23) NM_011780 Cell adhesion Rho guanine nucleotide exchange factor (GEF) 1 (Arhgef1) NM_008488 GTPase activiator, signaling Ubiquitin specific peptidase 9, X chromosome (Usp9x) NM_009481 Ubiquitination Pericentrin (Pcnt) NM_008787 Cilium biogenesis, spindle

organization Platelet derived growth factor alpha polypeptide (Pdgfra) NM_011058 Receptor activity, development Latent transforming growth factor beta binding protein (Ltbp3) NM_008520 Growth factor binding, development

Cell adhesion molecule-related/down regulated by oncogenes (Cdo) NM_021339 Muscle differentiation, development,

cell adhesion Txk tyrosine kinase (Txk) NM_013698 Kinase activity CD44 antigen (CD44) NM_009851 Protein binding Lymphotoxin B (Ltb) NM_008518 Immune response Neuropeptide Y receptor Y2 (Npy2r) NM_008731 Signal transduction Receptor (calcitonin) activity modifying protein 3 (Ramp3) NM_019511 Receptor activity Transforming growth factor, beta receptor III (Tgfbr3) NM_011578 Receptor activity Insulin-like growth factor binding protein 4 (Igfbp4) NM_010517 Growth factor, cell growth regulation Insulin-like growth factor binding protein 1 (Igfbp1) NM_008341 Cell growth Lymphoid-restricted membrane protein (Lrmp) NM_008511 Membrane associated Eph receptor B2 (Ephb2) NM_010142 Receptor, axon guidance Cone-rod homeobox containing gene (Crx) NM_007770 Transcription Forkhead box D2 (Foxd2) NM_008593 Transcription Notch gene homolog 2 (Notch2) NM_010928 Signal transduction Toll-like receptor 6 (Tlr6) NM_011604 Receptor, immune response Activating transcription factor 2 (Atf2) NM_001025093 Transcription p300/CBP-associated factor (Pcaf) NM_020005 Histone acetylation, transcritpion Transcription factor 12 (Tcf12) NM_011544 Transcription Cathepsin C (Ctsc) NM_009982 Proteolysis SRY-box containing gene 13 (Sox13) NM_011439 Transcription Myosin Va (Myo5a) NM_010864 Differentiation, development, transport Endothelial-specific receptor tyrosine kinase (Tek) NM_013690 Cell adhesion, migration, kinase

A complete list of genes that were identified both by ChIP-subcloning in this report and by microarray analyses reported previously (24-26).

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Snyder Table 2. List of novel direct Stat3 target genes identified by ChIP

Gene Accession Number

Function

Immunoglobulin lambda chain complex (Ig1) NG_004051 Immune response Histone deacetylase 4 (Hdac4) NM_207225 Transcription, proliferation RNA binding motif protein 9 (Rbm9) NM_175387 Transcription, signal transduction Paternally expressed 10 (Peg10) NM_130877 Development Biregional cell adhesion molecule-related/regulated by oncogenes (Boc) NM_172506 Muscle differentiation, cell adhesion Paired box gene 1 (Pax1) NM_008780 Transcription, differentiation, development Spectrin beta 2 (Spnb2) NM_175836 DNA methylation, Smad nuclear translocation Immunoglobulin mu binding protein 2 (Ighmbp2) NM_009212 Transcription Cell adhesion molecule 4 (Cadm4, Igsf4c) NM_153112 Integral to membrane Integrin, alpha 11(Itga11) NM_176922 Cell adhesion migration Serum response factor binding protein 1 (Srfbp1) NM_026040 Transcription CCAAT/enhancer binding protein (C/EBP), alpha (Cebpa) NM_007678 Transcription, development, differentiation Interleukin 28 receptor alpha (Il28ra) NM_174851 Receptor activity MAD homolog 9 (Smad9) NM_019483 Transcription, development PDLIM1 interacting kinase 1 like (Pdik1l) NM_146156 Kinase, cell division PAX interacting (with transcription-activation domain) protein 1 (Paxip1) NM_018878 Proliferation Bcl3 binding protein (B3bp) NM_001024917 ATP binding, endonuclease Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3G (Sema3g)

NM_001025379 Development Development and differentiation enhancing factor 2 (Ddef2) NM_001004364 GTPase activator activity Ect2 oncogene (Ect2) NM_007900 Rho protein signal transduction Angiopoietin-like 2 (Angptl2) NM_011923 Signal transduction Catenin (cadhein associated protein), alpha 2 (Ctnna2) NM_009819 Cell adhesion Porcupine homolog (Porcn) NM_023638 Wnt receptor signaling pathway Vomeronasal 2, receptor, 4 (V2r4, V2r42) NM_009493 Receptor activity PERQ amino rich, with GYF domain 1 (Perq1) NM_031408 Signal transduction Ceroid-lipofuscinosis, neuronal 6 (Cln6) NM_001033175 Lysosome organization, visual perception BCL2-associated athanogene 4 (Bag4) NM_026121 Apoptosis Angiopoietin 1 (Angpt1) NM_009640 Angiogenesis, development, differentiation DAZ interacting protein 1 (Dzip1) NM_025943 Transcription, development, differentiation Notch gene homolog 4 (Notch4) NM_010929 Signal transduction, differentiation, development Neuron navigator 1 (Nav1) NM_173437 Neuron migration COBW domain containing 1 (Cbwd1) NM_146097 Not determined CREB binding protein (Crebbp, CBP) NM_001025432 Transcription, histone acetylation Gamma-aminobutyric acid (GABA) A receptor, alpha 5 (Gabra5) NM_176942 Signaling

This list contains novel direct Stat3 target genes that were cloned out multiple times from OSM-stimulated 3T3 cells by the ChIP-subcloning method.

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TNF-R Pcnt- + - +- +

c-fos- +Bcl3 Gbp1

- +Fgl2- ++-

Pax4

Boc- +

Cln6- +

Perq1- +

Smad9- +

+Cdo-

3.7

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3.3

FasL- +

Angpt1- +

4.1 *

- + - +CBP Ect2

4 4.2 2 5

7.5 5 3.5 2.5 10 11.5

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Marylynn Snyder, Xin-Yun Huang and J. Jillian Zhangdifferentiation

Identification of novel direct STAT3 target genes for control of growth and

published online December 7, 2007J. Biol. Chem. 

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