CDX2, a Homeobox Transcription Factor, Upregulates Transcription of the P21/WAF1/CIP1 Gene

CDX2, a homeobox transcription factor, upregulates transcription of the p21/WAF1/CIP1 gene

Yun-Qing Bai1,2, Satoshi Miyake1, Takehisa Iwai2 and Yasuhito Yuasa*,1

1Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo 113-8519, Japan; 2Department of Surgery, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo 113-8519, Japan

The CDX2 homeobox transcription factor plays key roles epithelial phenotype. Previous studies suggested that in intestinal development and homeostasis. CDX2 is CDX2 is a tumor suppressor. Firstly, heterozygous downregulated during colorectal carcinogenesis, whereas Cdx2 knockout mice develop multiple colonic polyps overexpression of CDX2 results in growth inhibition and (Chawengsaksophak et al., 1997). Secondly, the expres- differentiation of colon carcinoma and intestinal cells. sion of CDX2 is downregulated during colorectal However, the means by which CDX2 functions remain carcinogenesis (Ee et al., 1995; Mallo et al., 1997). poorly understood. p21/WAF1/CIP1 is one of the cyclin- Thirdly, CDX2 overexpression seems to inhibit growth dependent kinase inhibitors. In addition to its role in cell and/or promote differentiation of colorectal cancer cells cycle control, p21plays critical roles in differentiation and (Mallo et al., 1998) or undifferentiated intestinal tumor suppression. The overlapping in both the expression epithelial cells (Suh and Traber, 1996). Moreover, the and function of CDX2 and p21in the small intestine and ectopic expression of CDX2/Cdx2 has been reported to colon strongly suggests a link between these two genes. By be associated with intestinal metaplasia formation of the means of luciferase reporter and electrophoretic mobility stomach (Bai et al., 2002; Silberg et al., 2002; Mutoh shift assays, we show here that CDX2 transactivated and et al., 2002). Nevertheless, the molecular mechanisms physically interacted with the promoter of p21 in a p53- underlying these roles of CDX2 remain poorly under- independent manner. Moreover, overexpression of CDX2 stood. increased the mRNA expression of p21 in HT-29 colon The homeobox is a highly conserved 180-bp DNA carcinoma cells, as demonstrated by reverse transcription– sequence encoding a 60-amino-acid motif termed the polymerase chain reaction. These data suggest that p21is homeodomain. The homeodomain, with a helix–turn– a transcriptional target of CDX2. Our results may thus helix structural conformation, is the sequence-specific provide a new mechanism underlying the functions of DNA-binding domain of a family of transcriptional CDX2. regulatory proteins (McGinnis and Krumlauf, 1992). Oncogene (2003) 22, 7942–7949. doi:10.1038/sj.onc.1206634 Homeoproteins of the caudal family bind DNA via an AT-rich sequence whose consensus is A/CTTTATA/G Keywords: CDX2; transcription; p21/WAF1/CIP1 (reviewed by Freund et al., 1998). Previous studies showed that CDX2 binds cis-elements present in the gene promoters of enterocytic markers such as sucrase- isomaltase, lactase-phlorizin hydrolase, phospholipase- A, apolipoprotein B, carbonic anhydrase 1, and Introduction calbindin D9K (Freund et al., 1998). Homeobox genes such as Hox-C8 and HNF-1 have also been demon- Human CDX2 is a member of the caudal-related strated to be transcriptional targets of CDX2 (Freund homeobox gene family (McGinnis and Krumlauf, et al., 1998). In addition, expression of the gut-enriched 1992; Mallo et al., 1997). During early development in Krupel-like factor (GKLF) gene, an epithelial-specific Drosophila, caudal is involved in anterior–posterior transcription factor that functions as a suppressor of cell patterning (Macdonald and Struhl, 1986). The expres- proliferation, is dependent on CDX2 in human colon sion of the rodent Cdx2 homeobox gene is tissue specific cancer cell line RKO (Dang et al., 2001). Importantly, and present from the early embryo to the adult (James CDX2 can regulate the expression of the liver intestine- et al., 1994), and thus it is likely that Cdx2 plays roles in cadherin (LI- cadherin) gene, indicating its key role in both the establishment and maintenance of the intestinal mediating CDX2 function in intestinal cell fate determination (Hinoi et al., 2002). Clearly, further investiga- tions are needed to clarify the complex network involved *Correspondence: Y Yuasa, Department of Molecular Oncology, in CDX2. Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, p21/WAF1/CIP1 (hereafter referred to as p21) was Japan; E-mail: [email protected] one of the first cyclin-dependent kinase (cdk) inhibitors Received 2 January 2003; revised 31 March 2003; accepted 7 April 2003 identified, and it can bind and inhibit G1 cyclin/Cdk CDX2 up-regulates transcription of p21 Y-Q Bai et al 7943 complexes (Harper et al., 1993; Xiong et al., 1993). p21 can induce growth suppression in vivo through cyclin D1-Cdk4 and cyclin E-Cdk2 complexes (Lin et al., 1996), and overexpression of p21 results in cell cycle arrest in G1 (Xiong et al., 1993; Luo et al., 1995) and tumor cell growth suppression (El-Deiry et al., 1993). In addition to its role in cell cycle control, p21 is also believed to inhibit DNA replication through its ability to bind proliferating cell nuclear antigen (PCNA), which is required for both replicative DNA synthesis and DNA repair. However, p21 has no inhibitory effect on the DNA repair function of PCNA (Li et al., 1994; Waga et al., 1994). Unexpectedly, p21 plays critical roles in differentiation, such as the role in control of epithelial self-renewal and commitment to differentiation (Di Cunto et al., 1998). Finally, p21 has been proved to be an unsuspected tumor suppressor, which can mediate at least some of the tumor-suppressing effects of p53 Figure 1 Effect of CDX2 on the p21 promoter. (a) Reporter (reviewed by Dotto, 2000). construct p21-Luc, in which the p21 promoter drives expression of Since firstly CDX2 and p21 are both associated with the firefly luciferase gene, was cotransfected with the expression differentiation induction and tumor growth suppression, vector pcDNA3.1CDX2 or the empty vector in different cells, as indicated. The renilla reporter plasmid (1 : 10 ratio with target and secondly their expression overlaps in the small reporter) was also included in all experiments to normalize intestine and colon (Gartel et al., 1996; Polyak et al., variation in transfection efficiency. Cells were harvested after 48 h 1996; Mallo et al., 1997; Silberg et al., 2000), a possible and assayed for luciferase activity. All values were equalized on the link between these two genes is suggested. In the present basis of the activity observed upon cotransfection with a control study, we first identified nine CDX-core binding renilla expression vector, and expressed as ratio of CDX2- expressing vector to empty vector. These results represent the sequences, TTTAT, in the sense or, ATAAA, the means and standard deviation of three independent experiments. reverse orientation (Suh et al., 1994) within the human (b) p21-Luc, p21-Luc/D1orp21-Luc/D2 were cotransfected with the p21 promoter (À2699 to þ 45 bp). By means of expression vector pcDNA3.1CDX2 or the empty vector in HT-29 luciferase reporter assay, electrophoretic mobility shift cells. p21-Luc/D1 and p21-Luc/D2 were constructed by deleting À1900 to À658 bp or À2504 to À212 bp of the p21-Luc construct, assay (EMSA) and reverse transcription–polymerase respectively, as described under ‘Materials and methods’. Numbers chain reaction (RT–PCR), we then demonstrated that on the promoter refer to positions relative to the transcription start CDX2 activated transcription of the p21 gene through site of the human p21 gene ( þ 1). Note that no CDX binding site direct physical interaction with the p21 promoter. remains upstream of position À1900 bp. The position of p21-WT, which was used as probe for subsequent EMSA, containing three clustered CDX binding sites is also indicated (À471 to À434 bp). The activity of p21-Luc was arbitrarily set at 100%. Variations in Results transfection efficiency were corrected by normalization for expression of renilla luciferase. All transfections were repeated three times in duplicate Transactivation of the p21 promoter by CDX2 To determine whether or not the p21 gene is a transcriptional target of CDX2 and, if so, whether or not p53 four cell lines described above (data not shown), is involved, SAOS-2 (p53À/À) and U-2OS (p53 þ / þ ) indicating specific transactivation by CDX2 of the p21 osteosarcoma cells were cotransfected with the p21- promoter. Since p53 is null in SAOS-2 and mutated in Luc reporter construct and expression vector pcDNA3. HT-29cells, CDX2 transactivated the p21 promoter in a 1CDX2. These two cell lines were selected because they p53-independent manner. are widely used for p53-related studies and lack To determine whether or not the activation of the p21 endogenous CDX2 expression (data not shown). Ac- promoter by CDX2 is dependent on CDX binding sites, cording to the dual-luciferase reporter assay, cotransfec- deletion mutants were made as described in ‘Materials tion of pcDNA3.1CDX2 resulted in 11.1- and 3.3-fold and methods’. As a result, deletion of putative CDX transcriptional activation of the 2744-bp p21 promoter binding sites on the 50 side from À1900 to À658 bp (p21- construct in SAOS-2 and U-2OS, respectively (Figure 1). Luc/D1) or all the binding sites (p21-Luc/D2) in the p21 Then the ability of CDX2 to activate p21 transcription promoter resulted in 35 and 79% reduction in the p21 was further studied in gastrointestinal cancer cell lines promoter activity, respectively (Figure 1b), suggesting GT3TKB and HT-29. The endogenous CDX2 expres- that these binding sites are crucial for CDX2 to sion was below the detectable level in GT3TKB (Bai transactivate the p21 promoter. et al., 2000) and was also very low in HT-29cells. As a result, overexpression of CDX2 induced p21 activity by CDX2 binds to the p21 promoter 6.4- and 2.6-fold in GT3TKB and HT-29(Figure 1), respectively. In contrast, no transcriptional activation of To examine the physical interaction between the p21 p21-Luc was observed after CDX1 transfection in the promoter and the CDX2 homeodomain protein, we

Oncogene CDX2 up-regulates transcription of p21 Y-Q Bai et al 7944 performed EMSA. The oligonucleotides, p21-WT and nuclear extract was added. The DNA/protein complexes p21-Mut, used in this study are shown in Figures 1b were competed for by 50–500-fold excess unlabeled wild- and 2a. The reasons for choosing these oligonucleotides type oligonucleotide p21-WT (lanes 3–5), but were not were as follows. (1) Deletion mutant analysis indicated competed for by even 500-fold unlabeled excess mutant- that the portion from À658 to À212 bp contains type oligonucleotide p21-Mut (lanes 6–8) or nonspeciﬁc important CDX2 binding sites as described above oligonucleotide (data not shown), suggesting that the (Figure 1b). (2) Three CDX-core binding sequences, CDX-core binding sites in p21-WT are critical for their TTTAT, are located within the DNA fragment of less interaction with CDX2. Gel shifts with nuclear extract than 30 bp in the p21 promoter, suggesting that these prepared from CDX1-transfected or parental COS-7 oligonucleotides may represent the most possible region cells yielded no complex formation (data not shown). for the interaction with CDX2. (3) Another home- To further determine the speciﬁcity of the interaction odomain protein, HOXA10, has been demonstrated to between p21-WT and CDX2, we incubated the radi- bind to this region in the p21 promoter (Bromleigh and olabeled p21-WT probe and nuclear extract from Freedman, 2000). CDX2-transfected COS-7 cells in the absence or p21-WT was labeled, incubated in the presence of presence of a polyclonal CDX2 antibody. The CDX2 nuclear extract from COS-7 cells transfected with antibody recognized the nuclear protein bound to the CDX2, and then migrated through a 5.5% nondenatur- p21-WT probe, resulting in reduced gel mobility of C1 ing polyacrylamide gel. The autoradiograph in Figure 2b and C2, and a supershift (lane 9). In contrast, no reveals the position of the rapidly migrating unbound supershift was observed for the binding reaction carried probe at the base of the gel and several DNA/protein out in the presence of nonimmune serum (lane 10). As complexes of slower mobility that were formed after the upper two DNA/protein complexes mostly super- incubation with the COS-7 nuclear extract (lane 2), shifted upwardly, we reasoned that these two bands whereas no band shift occurred in lane 1, in which no represent the p21-WT/CDX2 complexes (indicated by C1 and C2). The C1 (lower band) and C2 (upper band) complexes may be formed from binding of CDX2 monomer and dimer on the p21-WT DNA element, respectively, similar to those of the previous report (Suh et al., 1994). These results suggest that homeodomain protein CDX2 interacts physically with the p21-WT cis- element.

P21 expression was increased by CDX2 overexpression in colon carcinoma cell line HT-29 Based on the findings of the reporter assay and EMSA, we further examined whether or not CDX2 can transactivate transcription of the endogenous p21 gene. Preconfluent HT-29cells were transiently transfected with the CDX2 expression vector or the empty vector as a control. Total RNA was isolated 24 h after transfection from either transfected cells or parental cells cultured in parallel. Semiquantitative RT–PCR analysis showed that p21 mRNA expression was increased in HT-29cells transfected with CDX2 compared with those transfected with the empty vector and parental cells (Figure 3, upper panel). Higher expression of CDX2 in Figure 2 EMSA of the physical interaction between the CDX2 the CDX2-transfected cells was also confirmed (Figure 3, homeoprotein and p21 promoter. (a) Synthetic oligonucleotides middle panel), whereas the internal standard GAPDH p21-WT (wild type) and p21-Mut (mutated) are shown. Note that mRNA level remained constant (Figure 3, lower panel). the CDX-core binding sequences TTTAT (in bold) in p21-WT were changed into GCGTG (in bold) in p21-Mut. The complementary We confirmed the nucleotide sequence of the RT–PCR oligonucleotides were annealed and end-labeled with 32P. (b) products by sequencing (data not shown). Nuclear extract (5 mg) from COS-7 cells transfected with CDX2 To confirm the findings of semiquantitative RT–PCR, was incubated with the 32P-labeled p21-WT oligonucleotide probe we performed LightCycler real-time PCR. Specificity of alone (lane 2) or in the presence of 50, 100, or 500 Â unlabeled p21- real-time PCR was documented with agarose gel WT (lanes 3–5) or p21-Mut (lanes 6–8) oligonucleotide. In lane 1, only 32P-labeled p21-WT oligonucleotide was added. Samples were electrophoresis and resulted in a single product with loaded on a 5.5% acrylamide gel. In addition, gel supershifts were desired length (p21, 417 bp; GAPDH, 457 bp). In carried out with nuclear extract from CDX2-transfected COS-7 addition, a LightCycler melting curve analysis was cells and the 32P-labeled p21-WT probe. The reaction mixture was performed, which resulted in single product specific incubated with 2.5 ml of CDX2 antibody (lane 9) or nonimmune 1 serum (lane 10) for 15 min at room temperature. The arrows melting temperatures as follows: p21, 88.8 C (Figure 4b), indicate the free probe (FP), complex 1 (C1), complex 2 (C2), and and GAPDH, 88.61C (Figure 4d). Representative supershift (SS). amplification of p21 and GAPDH is shown in Figure

Oncogene CDX2 up-regulates transcription of p21 Y-Q Bai et al 7945 undifferentiated intestine cell line, led to arrest of proliferation for several days followed by a period of growth, resulting in multicellular structures containing a well-formed columnar layer of cells with multiple morphological characteristics of intestinal epithelial cells (Suh and Traber, 1996). On the other hand, exit from the cell cycle is a prerequisite for terminal differentiation, and p21 expression is induced during terminal differentiation in vitro and in vivo (reviewed by Gartel Figure 3 Semiquantitative RT–PCR analysis of p21 expression in and Tyner, 1999), suggesting that p21 plays a critical HT-29cells. HT-29cells were transfected with the CDX2 role in the differentiation process. Thus, it is interesting expression vector or the empty vector as a control. Total RNA to speculate that CDX2 functions as a proliferation was isolated 24 h after transfection from either the transfected cells inhibitor and differentiation promoter through its or parental cells, and then cDNA was synthesized. Standard PCR reactions were performed with 30 cycles for p21 (upper panel) and transactivation of p21 in addition to other previously CDX2 (middle panel), and 21 cycles for GAPDH (lower panel). reported genes, such as GKLF (Dang et al., 2001) and These cycles were observed to be within the logarithmic phase of LI-cadherin (Hinoi et al., 2002). Of note, it remains the amplification. The PCR products (5 ml) were electrophoresed in unknown whether or not other family members of cdk 2% agarose gels containing 0.5 mg/ml ethidium bromide. The gels were illuminated with UV light and then photographed. Lane 1, inhibitor or other factors related to the cell cycle are parental cells; lane 2, empty vector-transfected cells; lane 3, CDX2- involved. transfected cells Our findings are supported by the following previous observations. (1) The expression patterns of Cdx2 and p21 overlap each other along the vertical intestinal axis in mice (Gartel et al., 1996; Silberg et al., 2000). In the 4a and c, respectively, the results of which are listed in normal proximal intestine, both genes are strongly Table 1. LightCycler real-time PCR was performed in expressed at the crypt–villus junction into villus, duplicate in two independent experiments, and the whereas in the normal colon the expression of Cdx2 results are shown in Figure 4e. The p21 mRNA and p21 is limited to the upper crypt and surface expression level was expressed as the ratio of the epithelium. (2) The expression of both CDX2 and p21 concentrations of p21 and GAPDH. As shown in increased as Caco-2 colon carcinoma cells differentiated, Figure 4e, the p21 expression level in CDX2-transfected which differentiate spontaneously after reaching con- cells was 3.7- and 2.4-fold higher than those in the empty fluency (Gartel et al., 1996; Lorentz et al., 1997). (3) vector-transfected and parental HT-29cells, respec- During colorectal tumorigenesis, CDX2 and p21 were tively. In contrast, the p21 mRNA level in CDX1- downregulated and stronger expression of these two transfected cells was similar to those in the empty genes was demonstrated in portions with higher vector-transfected and parental HT-29cells in a parallel differentiation in tumors (Ee et al., 1995; Polyak et al., study (data not shown). These results suggest that 1996; Mallo et al., 1997; Bukholm and Nesland, 2000). CDX2, but not CDX1, can transactivate the transcrip- Since mutations in the p21 gene are extremely rare in tion of the p21 gene. human tumors (Gartel and Tyner, 1999), decreased expression of CDX2 might contribute to the down- regulation of p21 in addition to methylation in its Discussion promoter (Allan et al., 2000) or inactivation of its upstream genes like p53 (Gartel and Tyner, 1999). These The intestinal epithelium is continuously renewed from results strongly suggest that CDX2 and p21 may act in stem cells anchored near the bottom of invaginations concert in the control of epithelial self-renewal and called intestinal crypts or glands. With the exception of commitment to differentiation, and in carcinogenesis as Paneth cells, the epithelial cells migrate upwards toward well. the crypt mouth where proliferation stops, and differ- Expression of p21 is controlled through both p53- entiation is turned on and proceeds along the villi in the dependent and p53-independent mechanisms. After small intestine or the upper part of the glands in the DNA damage, p21 transcription is activated by p53 colon. Homeobox transcription factor CDX2 seems to (Brugarolas et al., 1995). However, a variety of agents have critical functions in the intestinal development, that promote growth arrest and differentiation also differentiation, and maintenance of the intestinal activate p21 transcription through p53-independent phenotype (Silberg et al., 2000). However, the means pathways by means of different transcriptional factors by which it does so remain poorly defined. Here, we (Gartel and Tyner, 1999). Interestingly, overexpression demonstrated that the p21 gene is a transcriptional of homeobox gene gax or HOXA10 also induced cell target of CDX2, and our results thus provided a new proliferation inhibition or differentiation via p53-inde- mechanism whereby CDX2 functions. pendent upregulation of p21 (Smith et al., 1997; Previous studies showed that overexpression of CDX2 Bromleigh and Freedman, 2000). In our study, a p53- in HT-29colon carcinoma cells resulted in a decrease in independent mechanism is suggested because overex- cell growth rate by half (Mallo et al., 1998). Moreover, pression of CDX2 activated transcription of exogenous conditional overexpression of Cdx2 in IEC-6, an and endogenous p21 in p53 null or mutated cells.

Oncogene CDX2 up-regulates transcription of p21 Y-Q Bai et al 7946

Figure 4 LightCyler real-time PCR analysis of p21 expression in HT-29cells. Cell transfection, RNA isolation, and cDNA synthesis were performed in the same way as for semiquantitative RT–PCR. LightCycler PCR was performed as described in Materials and methods. Representative amplification of p21 and GAPDH is shown in (a) and (c), respectively, in which the real-time SYBR Green I fluorescence history versus cycle number of various templates is indicated. The calculated results in (a) and (c) are listed in Table 1. The Fit Point Method was performed for determination of concentration and CP in this study. LightCycler melting curve analyses demonstrated single product specific melting temperatures for p21 (b) and GAPDH (d), as indicated by w. LightCyler real-time PCR was performed in duplicate in two independent experiments, and the results are shown in (e). The p21 mRNA level was expressed as the ratio of the concentrations of p21 and GAPDH

Table 1 p21 and GAPDH mRNA expression after CDX2 transfection in HT-29cells a p21b GAPDHb

Cells Concentration Crossing point Concentration Crossing point

CDX2-transfected 127.2 25.91143 13.5 pcDNA3.1-transfected 48.0 27.2 126913.3 Parental 70.8 26.7 1121 13.5

aThese are the results of the LightCycler reactions shown in Figure 4a and c. bConcentrations and crossing points were calculated as described in Materials and methods.

Nevertheless, previous studies suggest that other epithelium and synergistically activate the promoters of transcription factors may cooperate with CDX2 in intestinal genes, such as LPH or SI (Krasinski et al., transactivation of p21. GATA-4, -5 and -6 zinc-ﬁnger 2001; van Wering et al., 2002). Moreover, HNF-1a, and hepatocyte nuclear factor-1a (HNF-1a) homeodo- GATA-4 and Cdx2 can interact physically and activate main transcription factors are expressed in the intestinal SI promoter activity in cotransfection experiments,

Oncogene CDX2 up-regulates transcription of p21 Y-Q Bai et al 7947 where GATA-4 requires the presence of HNF-1a and containing 10% fetal bovine serum (GIBCO BRL) and Cdx2 (Boudreau et al., 2002). These data suggest that 50 mg/ml kanamycin. The cells were plated at 5 Â 104 cells per zinc-finger/homeodomain interactions are an important well (24-well plate) 1 day before transfection. After incubation pathway for cooperative activation of gene transcrip- for 24 h, the cells (50–80% confluent) were transfected with tion. Finally, GATA-6 can induce G cell cycle arrest via 350 ng pcDNA3.1CDX2 or pcDNA3.1CDX1, 100 ng p21-Luc 1 reporter plasmid or its deleted constructs and 10 ng pRL-SV40 upregulation of p21 (Perlman et al., 1998), and Sp1 and vector using TransIT-LT1 transfection reagent (Mirus, Madi- Sp3, members of the zinc-finger family, activate p21 son, WI, USA) according to the protocol of the manufacturer. transcription (Gartel et al., 2000; Koutsodontis et al., The pcDNA3.1 empty vector was used as a negative control. 2001). The pRL-SV40 vector containing the SV40 early promoter Unlike CDX2, CDX1 has pro-oncogenic potential. upstream of renilla luciferase was cotransfected as an internal Firstly, its expression is restricted to the proliferative control. Cells were harvested 48 h after transfection, and compartment of the intestinal epithelium (Subramanian luciferase activity was measured with a Dual-Luciferase et al., 1998). Secondly, CDX1 overexpression stimulates Reporter Assay System (Promega) as described by the the proliferation of IEC6 cells (Soubeyran et al., 1999). manufacturer in Lumicounter 700 (Microtech Niti-On, Chiba, Thirdly, Cdx1 is upregulated through oncogenic activa- Japan). Each transfection was performed in triplicate and experiments were repeated three times. The results were tion of the Ras and Wnt/b-catenin pathways (Lorentz expressed as fold activation, that is, the ratio of normalized et al., 1999; Lickert et al., 2000). However, the role of luciferase activity of the CDX2 expression construct to that of CDX1 in carcinogenesis remains controversial. For the empty vector. example, the expression of CDX1 was found to be downregulated during colorectal carcinogenesis (Mallo et al., 1997). Recently, Cdx1 was found to function as a Electrophoretic mobility shift assay negative regulator in the transcription of the p21 gene in To prepare nuclear extracts, COS-7 cells were transfected with colon adenocarcinoma SW480 cells (Moucadel et al., CDX2 expression vector pcDNA3.1CDX2 and the empty 2002). Our data did not show the upregulation of p21 pcDNA3.1 vector using a previously described procedure (Bai expression by CDX1 either. These data indicate the et al., 2002). Cells were harvested 72 h after transfection by difference between CDX2 and CDX1 in the transcrip- scraping and then resuspended in 4 ml precooled phosphate- buffered saline (PBS), followed by centrifugation at 500 g and tional regulation of the p21 gene. 41C for 5 min. The cells were resuspended in five packed cell In summary, we have shown that the CDX2 home- volumes of cold low salt buffer (10 mm HEPES, pH 7.9, 10 mm odomain protein transactivated the transcription of p21 KCl, 1.5 mm MgCl2, 0.5% Nonidet P-40, 1 mm dithiothreitol cdk inhibitor via its binding to the promoter of p21. and 0.5 mm phenylmethylsulfonyl fluoride (PMSF)), and These data suggest that p21 is probably one of the incubated on ice for 10 min. The cells were then homogenized mediators for CDX2 to act in both normal and with 10 slow up-and-down strokes in a glass Dounce pathologic processes. Our results thus may shed new homogenizer using a type B pestle, followed by centrifugation insights into the mechanism underlying the functions of at 13 000 g and 41C for 5 min. The supernatant was removed CDX2. and the nuclear pellet was washed once with 80 ml low salt buffer. The nuclear pellet was resuspended in 40 ml high salt buffer (20 mm HEPES, pH 7.9, 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, 1 mm dithiothreitol, and 0.5 mm PMSF) and Materials and methods then incubated for 30 min on ice with gentle agitation. After centrifugation (13 000 g for 10 min at 41C), the supernatant Plasmids was recovered, mixed with an equivalent volume of glycerol m m The human p21/WAF1/CIP1 promoter luciferase reporter buffer (20 m HEPES, pH 7.9, 100 m KCl, 20% glycerol, m (p21-Luc) construct was described previously (Irwin et al., and 0.2 m EDTA). After the protein concentration had been 2000). This reporter construct contains the p21 promoter from determined using the Bio-Rad Protein Assay reagent (Bio- À2699 to þ 45 bp in the pGL2-basic vector that lacks Rad, Richmond, CA, USA) according to the directions of the eucaryotic promoter and enhancer sequences. Using restriction manufacturer, the nuclear extract was stored at À801C. 0 enzyme AflII, putative CDX binding sites on the 50 side from Synthetic wild-type oligonucleotides p21-WT 5 - 0 1900 to 658 bp of the p21-Luc construct were excised, TCGTTTTTATAATTTATGAATTTTTATGTATTAATG-3 À À 0 resulting in p21-Luc/D1 containing three clustered CDX (À435 bp to À471 bp) and 5 -ACATTAATACATAAAAAT 0 binding sites located from 471 to 434 bp. Further, p21- TCATAAATTATAAAAACG-3 (À434 to À470 bp) within the À À 0 Luc/D2 without any CDX binding site was created by PstI, p21 promoter, and mutated oligonucleotides p21-Mut 5 -TC 0 which deleted the region from 2504 to 212 bp (Figure 1b). GTTGCGTGAAGCGTGGAATTGCGTGGTATTAATG-3 À À 0 CDX2 and CDX1 expression constructs have been described and 5 -ACATTAATACCACGCAATTCCACGCTTCACG- 0 previously (Bai et al., 2002), which were created by subcloning CAACG-3 were used for EMSA (Figure 2a). Note that the human CDX2 or CDX1 complementary DNA (cDNA) inserts CDX-core binding sequences TTTAT (in bold) in p21-WT containing the full coding region into a mammalian expression were changed to GCGTG (in bold) in p21-Mut. Annealing was vector, pcDNA3.1 (Invitrogen, Groningen, Netherlands). performed as follows. Synthetic complementary oligonucleotides (10 nmol) were mixed in annealing buffer (20 mm Tris- HCl, pH 7.5, 10 mm MgCl , and 50 mm NaCl), the final Cell culture, transfection and luciferase reporter assay 2 volume being 200 ml, incubated 5 min at 941C, and then cooled Human osteosarcoma cell lines SAOS-2 and U-2OS, human down slowly to room temperature. Annealed oligonucleotide gastric cancer cell line GT3TKB and human colon adenocar- probe (10 pmol) was end-labeled using 10 U of T4 polynucleo- 32 cinoma cell line HT-29were grown at 37 1C in a 5% CO2,95% tide kinase and 5 ml of 7000 Ci/mmol [g- P] adenosine tripho- air atmosphere in Dulbecco’s modified Eagle’s medium sphate (Amersham Pharmacia Biotech). The labeled probe was

Oncogene CDX2 up-regulates transcription of p21 Y-Q Bai et al 7948 purified by size-exclusion chromatography through a 0.5-ml scripts were amplified from the same cDNA samples with the column of Sephadex G-25 (Amersham Pharmacia Biotech). following primer pair, 50-GACCACAGTCCATGCCATCAC- Nuclear extract (5 mg) was incubated in the presence of 10 fmol 30 (sense) and 50-GTCCACCACCCTGTTGCTGTA-30 (anti- labeled probe with 1 mg poly dI:dC (Amersham Pharmacia sense) for 21 cycles, which were observed to be within the Biotech) with or without 100–500-fold excess unlabeled probe logarithmic phase of amplification. The PCR products (5 ml) in 20 mm HEPES, pH 7.9, 50 mm KCl, 2 mm MgCl2, 10% were electrophoresed in 2% agarose gels containing 0.5 mg/ml glycerol, 300 mg/ml BSA, 0.1 mm EDTA, and 0.5 mm dithio- ethidium bromide. The gels were illuminated with UV light threitol for 15 min at room temperature. DNA–protein and then photographed. complexes were resolved on a 5.5% TBE (90 mm Tris borate m and 2 m EDTA, pH 8.3) polyacrylamide gel and then LightCycler real-time PCR exposed to radiograph film for autoradiograph detection. Gel supershifts were carried out with nuclear extracts and the Based on the findings of semiquantitative PCR, we further gel shift method described above in the presence of 2.5 ml of the performed LightCycler real-time PCR. First, conditions for antibody against CDX2, as described previously (Bai et al., real-time PCRs were optimized with regard to Taq DNA 2002). polymerase, forward and reverse primers, MgCl2 concentrations and various annealing temperatures. RT–PCR amplifica- RNA extraction and cDNA synthesis tion products were separated by 2% agarose gel electrophoresis, and LightCycler melting curve analyses were HT-29cells were seeded at 1.25 Â 106 per 10-mm plate. After also performed. The optimized conditions were transferred on incubation for 24 h, the cells were transfected with 10 mg the following protocol. For LightCycler reaction, a mastermix pcDNA3.1CDX2 or the empty pcDNA3.1 vector using of the following reaction components was prepared to the TransIT-LT1 transfection reagent (PanVera, WI, USA) indicated final concentrations: 8 mlH2O, 2.4 ml MgCl2 (3 mm), according to the protocol of the manufacturer. In addition, 1 ml forward primer (0.5 mm), 1 ml reverse primer (0.5 mm), cells without transfection were cultured in parallel as a control. 2 ml10Â PCR buffer, 3.2 ml dNTP (0.2 mm), 0.4 ml Taq Total RNA was extracted 24 h after transfection using an DNA polymerase (2 U), and 1 ml SYBR Green I (0.5 Â , RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to TAKARA Biomedicals, Kyoto, Japan). LightCycler glass the protocol recommended by the manufacturer. RNA capillaries were filled with the LightCycler mastermix concentration was determined by spectrophotometry, and (19 ml) and 1 ml cDNA was added as a PCR template. The absence of RNA degradation was confirmed by agarose gel capillaries were closed, centrifuged, and placed into the electrophoresis. The isolated RNA (1 mg) was preincubated LightCycler rotor. The following run protocol was used: with 0.4 mg 12–18 mer oligo(dT) at 701C for 10 min, and then denaturation program (951C for 1 min), amplification and with 10 mm dNTP, 0.1 M DTT, and 1 ml Superscript II (RNase quantification program repeated 45 times for p21 and 40 times H[À] reverse transcriptase; Life Technologies Inc., Gaithers- for GAPDH (951C for 0 s, 601C for 5 s for p21 or 671C for 5 s burg, MD, USA) at 421C for 1 h (Bai et al., 2000). for GAPDH,721C for 20 s with a single fluorescence measurement), melting curve program (65–951C with a heating Semiquantitative PCR analysis rate of 0.11C per second and a continuous fluorescence 1 The synthesized cDNA was then amplified by the PCR measurement) and finally a cooling step to 40 C. The crossing method. The PCR reactions for p21 and CDX2 were point (CP) is defined as the point at which the fluorescence performed with both 30 and 35 cycles, in a 25 ml mixture rises appreciably above the background fluorescence. In this comprising 1 ml reverse transcriptase reaction mixture, 5% study, the Fit Point Method was performed for determination of concentration and CP using LightCycler Software version dimethyl sulfoxide, 2.5 ml10Â PCR buffer, 4 ml 1.25 mm dNTP (Pharmacia), 10 pmol each oligonucleotide primer pair, and 3.3 (Roche Molecular Biochemicals). The standard curve was p21 GAPDH 0.5 U Taq DNA polymerase (Biotech International Ltd., constructed with fivefold (for ) or 10-fold (for ) Bentley, Australia). Each PCR cycle consisted of 941C for serial dilutions of the cDNA from the CDX2-transfected HT- p21 1 min, 60–631C for 2 min, and 721C for 1 min, followed by a 29cells, and the original mRNA concentrations of and GAPDH final extension at 721C for 10 min. All these primers were were arbitrarily designated as 125 and 1000, respectively. designed to be located in different exons to identify any genomic DNA contamination. The primers used for CDX2 amplification were the same as previously described (Bai et al., Acknowledgements 2000) and those for p21 were as follows, 50-CAAGCTC- We wish to thank Dr WG Kaelin Jr for providing the plasmid, TACCTTCCCACGG-30 (sense) and 50-GCCAGGGTATG and Drs H Yamamoto, Y Yanagisawa and Y Akiyama for TACATGAGG-30 (antisense). The GenBank accession num- valuable discussions. This study was supported in part by ber of the human p21 genomic DNA used in this study is Grants-in-Aid for Scientific Research on Priority Areas from 2276311. As an internal control for RT–PCR analysis, the Ministry of Education, Science, Sports and Culture of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tran- Japan.

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