Transgenic Cdx2 induces endogenous Cdx1 in intestinal metaplasia of Cdx2-transgenic mouse stomach Hiroyuki Mutoh, Hiroko Hayakawa, Hirotsugu Sakamoto, Miho Sashikawa and Kentaro Sugano Department of Medicine, Division of Gastroenterology, Jichi Medical University, Tochigi, Japan Introduction In intestinal metaplasia of the human stomach, normal gastric mucosa is replaced by an intestinalized epithe- lium, and is mainly induced together with the progres- sion of Helicobacter pylori-infected chronic gastritis. Intestinal metaplasia of the human stomach has been extensively studied as a premalignant condition of gastric carcinoma [1]. The intestine-specific homeo- box genes Cdx1 and Cdx2 have been shown to be Keywords chromatin immunoprecipitation; luciferase reporter assay; methylation; RT-PCR; siRNA Correspondence H. Mutoh, Department of Medicine, Division of Gastroenterology, Jichi Medical University, Yakushiji 3311-1, Shimotsuke, Tochigi 329-0498, Japan Fax: +81 285 44 8297 Tel: +81 285 58 7348 E-mail: muto@jichi.ac.jp (Received 12 April 2009, revised 1 August 2009, accepted 6 August 2009) doi:10.1111/j.1742-4658.2009.07263.x Cdx1 and Cdx2, which are transcription factors regulating normal intesti- nal development, have been studied as potential key molecules in the pathogenesis of the precancerous intestinal metaplasia of the human stomach. However, the regulation of Cdx1 expression in the intestinal metaplasia is poorly understood. Cdx2-expressing gastric mucosa of Cdx2- transgenic mouse stomach was replaced by intestinal metaplastic mucosa. The aim of this study was to investigate the following: (a) Cdx1 expres- sion in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (b) the relationship between Cdx1 and Cdx2. A mouse model of intestinal metaplasia, the Cdx2-transgenic mouse, was used to investigate Cdx1 gene expression by RT-PCR. DNA methylation profile analysis was performed by bisulfite sequencing, and the interaction of Cdx2 with the Cdx1 promoter was examined by chromatin immunoprecip- itation assay, electrophoretic mobility shift assay, and luciferase reporter assays. Cdx2 mRNA was expressed in the Cdx2-transgenic mouse stom- ach. However, endogenous Cdx2 mRNA was not expressed in the intesti- nal metaplasia of the Cdx2-transgenic mouse stomach. On the other hand, endogenous Cdx1 mRNA and protein were expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. The Cdx1 promoter was unmethylated in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that Cdx2 was bound to the Cdx1 promoter region in the intestinal metaplasia and the normal intestine. Cdx2 upregu- lated and siRNA-Cdx2 downregulated the transcriptional activity of the Cdx1 gene in the human gastric carcinoma cell lines AGS, MKN45, and MKN74. In conclusion, transgenic Cdx2 induced endogenous Cdx1 through the binding of Cdx2 to the unmethylated Cdx1 promoter region in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. Abbreviations ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RA, retinoic acid; si, small interfering. FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS 5821 aberrantly expressed in human intestinal metaplasia. Cdx1 and Cdx2 are mammalian members of the cau- dal-related homeobox gene family. In adult mice and humans, expression is strictly confined to the gut, from the duodenum to the rectum. Normal stomach does not express the transcription factors Cdx1 and Cdx2. We and others have reported the presence of Cdx1 and Cdx2 in the intestinal metaplasia of the H. pylori- infected human stomach [2–4]. We have previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia [5,6]. Cdx2- transgenic mice specifically express Cdx2 in the gastric mucosa, and develop intestinal metaplasia in the stom- ach [5,6]. Gastric carcinoma spontaneously developed from intestinal metaplasia in all stomachs of Cdx2- transgenic mice examined [7]. In Barrett’s esophagus, normal squamous esopha- geal mucosa is also replaced by an intestinalized columnar epithelium in which Cdx2 is expressed [8]. Exposure to acid and ⁄ or bile acids has been reported to activate Cdx2 expression in human esophageal epi- thelial cells through promoter demethylation [9–11]. However, it is still unclear how Cdx1 is induced in intestinal metaplasia. Furthermore, the relationship between Cdx1 and Cdx2 in intestinal metaplasia has not been clarified as yet. To investigate these ques- tions, we focused on the induction of endogenous Cdx1 in Cdx2-induced intestinal metaplasia using Cdx2-transgenic mice. Results Expression of Cdx1 and Cdx2 in the intestinal metaplasia of the Cdx2-transgenic mouse stomach Cdx2-transgenic mice we generated showed intestinal metaplasia in the stomach [5,6]. First, Cdx2 expression in the intestinal metaplasia of Cdx2-transgenic mouse stomachs was examined, using RT-PCR. Cdx2 mRNA was detected in normal intestine and in all of the intes- tinal metaplasia of the Cdx2-transgenic mouse stom- ach, but not in the normal mouse stomach (Fig. 1B). Cdx2 expression was detected using a primer pair for the Cdx2 coding region (Cdx2 coding-fw and Cdx2 coding-rv; Fig. 1A and Table 1). When Cdx2-trans- genic mice were generated, only the Cdx2 coding region, without the noncoding region, was used. To investigate whether endogenous Cdx2 was expressed in Cdx2-induced intestinal metaplasia, endogenous Cdx2 expression was detected using a primer pair for the coding region and the 3¢-noncoding region (Cdx2 cod- ing-fw and Cdx2 non-coding-rv; Fig. 1A and Table 1). Endogenous Cdx2 was expressed in the normal intes- tine, but in none of the intestinal metaplasia of the Cx2-transgenic mouse stomachs (Fig. 1C). Transgenic Cdx2 did not induce endogenous Cdx2 expression, indicating that Cdx2 is not autoregulated in intestinal metaplasia. Next, whether endogenous Cdx1 was expressed in Cdx2-induced intestinal metaplasia was investigated. Endogenous Cdx1 was detected in the normal intestine and in all of the Cdx2-induced intestinal metaplasia, but not in the normal stomach (Fig. 2A). Cdx1 gene expression was characterized by quantita- tive real-time RT-PCR (Fig. 2B). The Cdx1 mRNA level in the Cdx2-transgenic mouse stomach was almost same as that in the normal mouse small intes- tine (Fig. 2B). Cdx1 expression in the intestinal metaplasia of the Cdx2-transgenic mouse stomach was also investigated, using immunohistochemistry. Cdx1 was expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach (Fig. 2E) and normal intestine (Fig. 2D), but not in the normal stomach (Fig. 2C). The expression of Cdx1 mRNA and protein in the intestinal meta- plasia of the Cdx2-transgenic mouse stomach indicates that Cdx1 might be induced by Cdx2 in intestinal metaplasia. Cdx1 promoter methylation status We focused on epigenetic regulation of Cdx1 gene expression as a possible cause of Cdx1 activation in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. To investigate whether the differences in Cdx1 expression were under promoter methylation control, bisulfite sequencing was performed on DNA extracted from five normal stomachs, five normal intes- tines, and five intestinal metaplasias of Cdx2-transgenic mouse stomachs. All of the CpGs including the CpGs (located around the TATA box and indicated by the box in Fig. 3A) that appear to be critical for the con- trol of Cdx1 expression in colorectal carcinoma [12] were unmethylated in the Cdx1 promoter sequences from the five intestinal metaplasias, the five normal intestines and the five normal stomachs (Fig. 3A). These results made it clear that Cdx1 promoter meth- ylation status does not determine the expression of Cdx1 in the normal intestine and in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. Next, the methylation status of the Cdx2 promoter region was examined. All of the CpGs (shown in red in Fig. 3B) in the Cdx2 promoter sequences from five intestinal metaplasias, five normal intestines and five normal stomachs were unmethylated, except for one Cdx1 expression in intestinal metaplasia H. Mutoh et al. 5822 FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS CpG, indicated by the box in Fig. 3B, that was methy- lated in five normal intestines and five intestinal meta- plasias. These results indicate that Cdx2 promoter methylation status does not determine the expression of endogenous Cdx2 in the normal intestine and in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. Cdx2 binds directly to the Cdx1 promoter region in vivo The putative TATA-box (TATAAA) sequence at posi- tions )51 to )46 (relating to the transcription start site; GenBank number NM_009880) exhibits obvious sequence similarity with the consensus Cdx-binding site (C ⁄ TATAAAG ⁄ T) (Fig. 4A), whereas no additional putative Cdx-binding site could be found elsewhere in the Cdx1 promoter (at position )2000 from the tran- scription start site). To examine whether the expression of Cdx1 mRNA in the intestinal metaplasia of the Cdx2-transgenic mouse stomach is associated with the binding of Cdx2 to this TATAAA region, we per- formed chromatin immunoprecipitation (ChIP) assays, using an antibody against Cdx2. We cross-linked the protein and DNA in the intestinal metaplasia of Cdx2- transgenic mouse stomach as well as in the stomach and intestine of normal mice. The Cdx1 promoter region encompassing the TATAAA sequence at )51 to )46 was amplified by PCR with two sets of primers (Fig. 4B, Cdx1 promoter fw1 and Cdx1 promoter rv1; A Terminal codon Intron B C 345678 1 Stomach 2 Intestine Cdx2 stomach Marker β-actin Stomach Intestine Cdx2 stomach Marker 3456712 β-actin Fig. 1. RT-PCR analysis of Cdx2 expression. (A) Scheme of a part of the mouse Cdx2 mRNA, including the stop codon ‘tga’, which is shown in red. The primers used for detecting Cdx2 transcript are indicated by underlining and yellow shading. The exo- n 2–exon 3 boundary site is indicated by an arrow. (B) RT-PCR analysis of Cdx2 mRNA transcripts (primer pair; Cdx2 coding-fw and Cdx2 coding-rv) in normal mouse stomach (lane 1), normal mouse small intestine (lane 2), and Cdx2-transgenic mouse stom- ach (lanes 3–8). (C) RT-PCR analyses of endogenous Cdx2 mRNA transcripts (primer pair; Cdx2 coding-fw and Cdx2 non-coding- rv) in normal mouse stomach (lane 1), normal mouse small intestine (lane 2), and Cdx2-transgenic mouse stomach (lanes 3–7). The lower panels in (B) and (C) show standard RT-PCR conducted with primers designed to detect b-actin mRNA. H. Mutoh et al. Cdx1 expression in intestinal metaplasia FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS 5823 Fig. 4C, Cdx1 promoter fw2 and Cdx1 promoter rv1). Binding of Cdx2 to the promoter region of the Cdx1 gene, including the TATAAA sequence, was detected in the intestinal metaplasia of the Cdx2-transgenic mouse stomach and the normal intestine, but not in the normal stomach (Fig. 4B,C). Cdx2 binds to the TATAAA sequence We investigated Cdx2 binding to the TATAAA sequence, using the nuclear fractions extracted from Cdx2-expressing AGS cells (Fig. 4D). We found that nuclear extracts from AGS cells formed the Cdx2– DNA complex (Fig. 4D). The presence of Cdx2 in DNA–protein complexes was eliminated by using monoclonal antibody specific to Cdx2 (Fig. 4D, lane 3). With the use of a mutant probe, DNA–protein complexes were not formed (Fig. 4D, lane 1). These results indicate that Cdx2 binds to the TATAAA sequence. The Cdx1 promoter was activated in Cdx2-expressing human gastric carcinoma AGS, MKN45 and MKN74 cells The expression of Cdx1 in the intestinal metaplasia of the Cdx2-transgenic mouse stomach supports the hypothesis that Cdx2 could regulate Cdx1 transcrip- tion. Supporting this, the ChIP assay indicated that Cdx2 is bound to the region between )191 and +112 (relating to the transcription start site). The region between )191 and +112 contains the Cdx consensus sequence TATAAA ()51 to )46) (Fig. 4A). Further- more, electrophoretic mobility shift assay (EMSA) indicated that Cdx2 binds to the TATAAA sequence. We examined the Cdx1 transcriptional activity in Cdx2-expressing AGS, MKN45 and MKN74 cells (Fig. 5A), using pGL4.10[luc2]–Cdx1 deletion and mutation constructs. These cell lines (AGS, MKN45, and MKN74) also expressed Cdx1, which was detected by RT-PCR (Fig. 5A). The Cdx1 promoter reporter gene containing the region between )365 and +12 was activated, whereas the Cdx1 promoter reporter gene containing the region between )365 and )78 was not activated, in Cdx2-expressing AGS, MKN45 and MKN74 cells (Fig. 5B). This result suggests that the element between )77 and +12 in the Cdx1 promoter may be critical for Cdx1 gene transcriptional activity in Cdx2-expressing AGS, MKN45 and MKN74 cells. The sequence between )77 and +12 contains a poten- tial Cdx2-binding site (TATAAA, )51 and )46). Anal- ysis of a reporter construct with mutation of the Cdx2 consensus-binding element at )51 and )46 revealed that the element was critical for transcriptional activity of the Cdx1 reporter gene construct in AGS, MKN45 and MKN74 cells (Fig. 5B). Furthermore, we examined the effects of the transfec- tion of the Cdx2 expression plasmid or small interfering RNA targeting Cdx2 (siRNA-Cdx2) on the trans- criptional activities of the Cdx1 promoter luciferase Table 1. The sequences of oligonucleotide primers used in this study. Primers Sequence (5¢-to3¢) Primers used for mouse Cdx2 detection Cdx2-fw CGGCTGGAGCTGGAGAAGG Cdx2 coding-rv GACAGTGGAGTTTAAAACCC Cdx2 noncoding-rv GCCTGGGATTGCTGTGCCG Primers used for mouse Cdx1 detection Cdx1cDNAfw CCGAACCAAGGACAAGTACC Cdx1cDNArv GTTTACTTTGCGCTCCTTGG Primers used for mouse b-actin detection b-Actin-fw ATCTACGAGGGCTATGCTCT b-Actin-rv TACTCCTGCTTGCTGATCCA Primers used for human Cdx2 detection Cdx2-human-fw AGCCAAGTGAAAACCAGGAC Cdx2-human-rv ATTTCTTGAGGCCCCAAATC Primers used for human Cdx1 detection Cdx1-human-fw TCGGACCAAGGACAAGTACC Cdx1-human-rv TGTTGCTGCTGCTGTTTCTT Primers used for human GAPDH detection GAPDH-fw ACGGATTTGGTCGTATTGGG GAPDH-rv TGATTTTGGAGGGATCTCGC Primers used for Cdx1 methylation CpG-Cdx1-fw1 [)331 ⁄ )305] GAG TTAGTTTTTTTATTTGT AA TTTAG CpG-Cdx1-fw2 [)312 ⁄ )293] TAA TTTAGGGGTGGGTGGTG CpG-Cdx1-rv [+114 ⁄ +89] AAAAAATCCTTATCCAACAC ATA ACC Primers used for Cdx2 methylation CpG-Cdx2-fw1 [)234 ⁄ )212] AGTG TATTTAGGTTGGAAGGAG CpG-Cdx2-fw2 [)206 ⁄ )185] GTAG TTAGTAAGAAGGGTTTGA CpG-Cdx2-rv [+194 ⁄ +173] TA ACTAACTACACCTCAACCCA Primers used for ChIP assay Cdx1 promoter-fw1 CTAGGGTCATGCCACCACTC Cdx1 promoter-fw2 ATCCACCTCCCGCTTAGG Cdx1 promoter-rv2 GGAGTCCTTGTCCAGCACAT Primers used for Cdx1 promoter Cdx1 promoter-fw1-XhoI [)365 ⁄ )345] CTCGAGCTAGGGTCATGCCACCACTC Cdx1 promoter-rv1-HindIII [+12 ⁄ )7] AAGCTTACCAGCGACTGCTCACCT Cdx1 promoter-rv2-HindIII [)78 ⁄ )95] AAGCTTAAGCTTGGGCGGCTTTGC ATTTCA Cdx1-Csp45I-fw TTCGAAAGGCCGGGGTGGGGC Cdx1-Csp45I-rv TTCGAAGCCGCGGGCCGTCCGC Cdx1 expression in intestinal metaplasia H. Mutoh et al. 5824 FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS construct containing the region between )365 and +12 or the mutant Cdx1 reporter luciferase construct. Cotransfection with the Cdx2 expression plasmid increased the transcriptional activities of the intact Cdx1 reporter gene, but did not affect the transcriptional activities of the mutant Cdx1 reporter gene, in AGS, MKN45 and MKN74 cells (Fig. 5B). Cotransfection with siRNA-Cdx2 decreased the transcriptional activi- ties of the intact Cdx1 reporter gene, but did not affect the transcriptional activities of the mutant Cdx1 repor- ter gene, in AGS, MKN45 and MKN74 cells (Fig. 5B). Next, after transfection of Cdx2 expression plasmid or siRNA-Cdx2 into AGS, MKN45 and MKN74 cells, Cdx1 mRNA levels were measured using quanti- tative real-time RT-PCR. As compared with the transfection of a negative control, the transfection of the Cdx2 expression plasmid resulted in an increase in Cdx1 mRNA (Fig. 5C). As compared with the transfection of a negative control, the transfection of siRNA-Cdx2 resulted in a decrease in Cdx1 mRNA (Fig. 5C). Discussion Intestinal metaplasia has been extensively studied as a putative preneoplastic lesion in the human stomach [1]. In the present study, endogenous Cdx1, but not Cdx2, was induced by transgenic Cdx2 in the intestinal meta- plasia of the Cdx2-transgenic mouse stomach. Cdx1 is essential for anterior–posterior vertebral patterning of the body axis in the early embryonic per- iod [13], and its expression persists selectively in the intestinal epithelium from the later embryonic period to the adult [14]. In addition to its physiological expression, Cdx1 is ectopically expressed in the precan- cerous intestinal metaplasia of the stomach and Barrett’s esophagus. The regulatory mechanisms that modulate Cdx1 gene expression during development A Normal Normal Cdx2 3456781 stomach 2 intestine stomach Marker 9 β-actin B 1 0.6 0.8 0.2 0.4 Normal intestine Normal stomach Cdx2 stomach 0 CDE Cdx2 stomachNormal stomach Normal intestine Fig. 2. RT-PCR and immunohistochemical analysis of Cdx1 expression. (A) RT-PCR analysis of Cdx1 expression. RT-PCR analy- ses of Cdx1 mRNA transcripts in normal mouse stomach (lanes 1 and 2), normal mouse intestine (lanes 3 and 4) and Cdx2- transgenic mouse stomach (lanes 5–9) are shown. The lower panel in (A) shows standard RT-PCR conducted with primers designed to detect b-actin mRNA. (B) Cdx1 gene expression characterized by quantita- tive real-time RT-PCR. The Cdx1 mRNA level in Cdx2-transgenic mouse stomach was almost the same as that in normal mouse small intestine (B). (C–E) Immunohis- tochemical staining for Cdx1 in the normal stomach (C), the normal intestine (D) and the intestinal metaplasia of the Cdx2-trans- genic mouse stomach (E). H. Mutoh et al. Cdx1 expression in intestinal metaplasia FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS 5825 and in the normal intestinal epithelium have been gradually clarified. Cdx1 is a direct transcriptional tar- get of both retinoic acid (RA) and the Wnt ⁄ b-catenin signaling pathway during early embryogenesis [15,16]. The Wnt ⁄ b-catenin signaling pathway is also active in the crypt compartment [17]. Cdx1 regulation by RA and Wnt3a is mediated, respectively, through the RA response element and two LEF ⁄ TCF response ele- ments present on the Cdx1 promoter [17]. However, very little is known about the molecular mechanisms for induction of the ectopic expression of the Cdx1 gene in the intestinal metaplasia of the H. pylori- infected human stomach. In the present study, we focused on the initiation of Cdx1 gene transcription in the intestinal metaplasia through Cdx2-transgenic mouse studies. Unlike in normal regulation, ectopic expression of Cdx1 was upregulated by Cdx2. Cdx2 mRNA and protein were absent in the gastric-like heteroplasias arising spontaneously in the pericecal region and proximal colon of Cdx2 + ⁄ ) mice, and, in common with that of Cdx2, Cdx1 expression was also absent in the gastric-like heteroplasias [18]. The finding that the gastric-like heteroplasia, which does not express Cdx2, also shows a lack of Cdx1 expression is consistent with our present data showing that the stomach expressing Cdx2 generated endogenous Cdx1. Epigenetic inactivation, in particular aberrant DNA hypermethylation, is an important mechanism for gene silencing. In the majority of human colon cancer specimens and colorectal cancer cell lines, Cdx1 expression is lost due to active Cdx1 gene silencing by promoter hypermethylation [12,19]. However, in this study, we demonstrated that the Cdx1 promoter is unmethylated in the normal stomach, the normal intestine, and the intestinal metaplasia, indicating that loss of Cdx1 expression in the normal stomach is not associated with promoter hypermethylation. Cdx1 and Cdx2 proteins bind to a binding site in an AT-rich motif whose consensus sequence is C ⁄ TATAAAT ⁄ G in direct or reverse orientation [20]. In some instances, the Cdx-binding site presents high homology with the A Cdx1 promoter B Cdx2 promoter Fig. 3. Cdx1 (A) and Cdx2 (B) promoter bisulfite sequencing of the stomach and intestine of normal mice and the intestinal metaplasia of the Cdx2-transgenic mouse stomach. (A) A sequence of the 5¢-flanking region for the mouse Cdx1 gene, including the TATA box, transcrip- tion start site and initiation codon (ATG). The TATA box is highlighted in green, the transcription start site in blue, and the initiation codon (ATG) in red. Cdx1 promoter CpGs are shown in red. Base positions relative to the Cdx1 transcription start site are shown on the left of each line of sequence. All CpGs were unmethylated. CpGs enclosed by the box ()54 to )68) represent those suggested to be crucial for tran- scriptional control [12]. (B) A sequence of the 5¢-flanking region for the mouse Cdx2 gene, including the TATA box, transcription start site, and initiation codon (ATG). The TATA box and another AT-rich motif, designated DBS (downstream binding site) [28], are highlighted in green, the transcription start site in blue, and the initiation codon (ATG) in red. Cdx2 promoter CpGs are shown in red. Base positions rela- tive to the Cdx2 transcription start site are shown on the left of each line of sequence. All CpGs were unmethylated, except for the CpG enclosed by the box, which was methylated in the normal intestine and the intestinal metaplasia. Cdx1 expression in intestinal metaplasia H. Mutoh et al. 5826 FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS canonical TATA-box sequence, and, indeed, the Cdx1 and ⁄ or Cdx2 homeoproteins were found to be able to bind to the TATA-boxes of some intestinal genes, such as those of the calbindin-D9 gene [21], the clus- terin gene [22], and the glucose-6-phosphatase gene [23]. In the present study, CpGs in the 5 ¢-region of the TATAAAA sequence located )51 ⁄ )45 upstream of the transcription start site were also found to be unmethylated in the normal stomach, the normal intestine, and the intestinal metaplasia. The present results, including those from ChIP, EMSA and reporter gene analysis, indicate that Cdx2 is present on the Cdx1 promoter region containing the TATAAAA sequence located at )51 ⁄ )45. On the other hand, endogenous Cdx2 was not expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach, indicating that endogenous Cdx2 was not autoregulated. In the present study, we demonstrated that Cdx1 is expressed in the Cdx2-induced intestinal metaplasia of Cdx2-transgenic mice. This may coincide with our previous clinical data why the expression of Cdx2 pre- cedes that of Cdx1 during the progression of intestinal metaplasia [3]. These clinical data also suggest that Cdx2 might induce Cdx1 expression. In conclusion, we propose that the ectopic expres- sion of Cdx2 in the gastric epithelium is triggered first, and in turn Cdx1 is directly induced by Cdx2 in the intestinal metaplasia. The present results indicate that Cdx2 induces Cdx1 expression by directly binding to the Cdx2-consensus cis-regulatory element of the unmethylated Cdx1 promoter region. Experimental procedures Cdx2-transgenic mice The Cdx2-transgenic mice we generated had free access to standard food and drinking water and were maintained on a 12 h light ⁄ dark cycle. All experiments in this study were performed in accordance with the Jichi Medical University Guide for Laboratory Animals. A Cdx1 promoter-fw1 –400 –341 –281 Cdx1 promoter-fw2 TATA box –221 –161 –101 Cdx1 promoter-rv1 Initiation codon Transcription start site (+1) +20 +80 –41 B 312 45 C 312 45 D 312 Fig. 4. Cdx2 is present on the Cdx1 promoter region in vivo. (A) A sequence of the 5¢-flanking region for the mouse Cdx1 gene, including the TATA box, transcription start site, and initiation codon. The TATA box is highlighted in green, the transcription start site in blue, and the initiation codon in red. PCR fragments corresponding to the DNA sequences including the TATA box were designed for ChIP analysis. The sequences for the primers used for ChIP assays are underlined and highlighted in yellow. The base positions relative to the transcription start site for the mouse Cdx1 gene are shown on the left of each line of sequence. (B, C) ChIP assays that were performed using a Cdx2 antibody [26] or control IgG. The region of the Cdx1 promoter encompassing the TATA box sequence was amplified by PCR with the follow- ing primer pairs: (B) Cdx1 promoter-fw1 and Cdx1 promoter-rv1; (C) Cdx1 promoter-fw2 and Cdx1 promoter-rv1. Lane 1: normal stomach. Lane 2: normal intestine. Lane 3: Cdx2-transgenic mouse stomach. Lane 4: input. Lane 5: control IgG. (D) EMSA. A radiolabeled dsDNA probe (CCCGCGGCTATAAAAGGCCGGGGTGGGG) containing the TATAAA sequence in the Cdx1 promoter was incubated with nuclear extracts from AGS cells and separated on a 5% polyacrylamide gel (lane 2). Specificity was determined by addition of antibody for supershift (lane 3) and mutant probe (CCCGCGGCTTCGAAAGGCCGGGGTGGGG) (lane 1). H. Mutoh et al. Cdx1 expression in intestinal metaplasia FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS 5827 RNA isolation and RT-PCR Total RNA was extracted from the stomach (normal mice), small intestine (normal mice), intestinal metaplasia (Cdx2- transgenic mice), and human gastric cancer cell lines AGS, MKN45 and MKN74, using the guanidinium isothiocya- nate ⁄ phenol method (Isogen; Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. Total RNA (1 lg) was reverse-transcribed as previously described [24]. To compare endogenous Cdx1 expression, endogenous Cdx2 expression and total (endogenous and transgenic) Cdx2 expression in the stomach (normal mice), small intestine (normal mice), and intestinal metaplasia (Cdx2-transgenic mice), PCR amplification was performed using the primer pairs Cdx1cDNAfw and Cdx1cDNArv (for endogenous Cdx1), Cdx2-fw and Cdx2 coding-rv (for total Cdx2), and Cdx2-fw and Cdx2 noncoding-rv (for endogenous Cdx2) (Table 1), by incubation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR products were separated in 2% agarose gels. As an internal standard, RT-PCR was performed with primers hybridizing to the mRNA encoding b-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 1). Real-time RT-PCR One hundred nanograms of cDNA was used in each real- time PCR reaction. Expression levels for the Cdx1 gene were determined by real-time PCR using ready-to use Assay-on-Demand gene expression product (Applied Bio- systems, Foster City, CA, USA): Mm00438172_m1 for mouse Cdx1, and Hs00156451_m1 for human Cdx1. Each Assay-on-Demand gene expression product contains tar- get-specific primers and probes and a Taqman Gene Expression Master Mix containing AmpErase uracil-N-gly- cosylase (Applied Biosystems) to prevent reamplification of carryover PCR products. PCR amplification and fluores- cence data collection were performed with the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems), using the following conditions: 50 °C for A AGS MKN45 MKN74 Cdx2 GAPDH Cdx1 B Luciferase activity (Ratio of firefly to renilla luciferase) 024681012 1814 16 Luciferase (–) (–) (–) +Cdx2 +siCdx2 +Cdx2 (–) (–) –365 +12 +siCdx2 (–) +Cdx2 +siCdx2 +Cdx2 +siCdx2 +Cdx2 (–) (–) +siCdx2 (–) +Cdx2 +siCdx2 (–) (–) (–) –78–365 –365 +12 AGS MKN45 MKN74 TATAAA TTCGAA –51 –46 C 1 2 Relative Cdx1 expression 0 Cdx2 siRNA + + + + + + AGS MKN45 MKN74 Fig. 5. Activation of the Cdx1 promoter in Cdx2-expressing AGS, MKN45 and MKN74 cells. (A) Cdx2 and Cdx1 expression deter- mined by RT-PCR. Human gastric carcinoma AGS, MKN45 and MKN74 cells expressed both Cdx2 and Cdx1. The lower panel in (A) shows standard RT-PCR conducted with primers designed to detect GAPDH mRNA. (B) Cdx1 promoter reporter gene activities. AGS, MKN45 and MKN74 cells were transiently transfected with the different fragments of Cdx1 promoter fused to a luciferase reporter vector, pGL4.10[luc2], and pGL4.70[hRluc] vector. Lucifer- ase activities were normalized relative to the level of Renilla lucifer- ase activities. The lengths of the promoter fragments tested are indicated. The numbers correspond to the relative positions with respect to the transcription start site. The sequence of the pre- sumptive Cdx2-binding site (TATAAA) was changed to TTCGAA. Cdx1 promoter reporter plasmids were added to each plate with or without Cdx2 expression vector (pRC ⁄ CMV–Cdx2) or siRNA (Applied Biosystems, Silencer Select Pre-designed siRNA, #s2878; UUCUUGUUGAUUUUCCUCUcc). The luciferase activities of empty pGL4.10[luc2], which does not contain any Cdx1 promoter, were used as controls for AGS, MKN45 and MKN74 cells, respectively. Each bar represents the mean ± standard error. Transfections were performed in triplicate and repeated three times. (C) Cdx1 mRNA levels of AGS, MKN45 and MKN74 cells transfected with Cdx2 expression plasmid, Cdx2 siRNA, or negative control. At 24 h after transfection, total RNA was extracted. Cdx1 expression in intestinal metaplasia H. Mutoh et al. 5828 FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS 2 min, 95 ° C for 10 min, and 40 cycles for amplification (95 °C for 15 s, and 60 °C for 1 min). PCR reactions were performed in 96-well plates, using a final volume of 20 lL, and the Cdx1 gene was studied in triplicate. In order to normalize RNA transcript abundance for the Cdx1 gene, a housekeeping gene (the b-actin gene) (Pre-Developed Taq- man Assay Reagents; Applied biosystems) was used to cal- culate the DC T (DC T = C T target ⁄ C T actin ). The C t values for the b-actin gene for the normal stomach, the normal intestine and Cdx2-transgenic mouse stomach tissues fell in a close range, with no specific pattern of spatial or tem- poral variation (data not shown). A relative quantification approach was used in this study to describe the change in expression of the target gene in a test sample relative to a calibrator sample (reference). The relative RNA transcript abundance value was calculated as follows. First, the DC T for the normal stomach, normal small intestine and Cdx2- transgenic mouse stomach tissues was calculated. In the second step, differences between the normal and Cdx2- transgenic mouse stomach tissues were calculated as DDC T (DC T target ⁄ DC T reference ). The normal mouse small intestine was used as reference for Cdx1 expression. Finally, the fold difference (relative abundance) was calculated using the formula 2 )DDCT [25], and was plotted as means (n = 6). Immunohistochemistry Murine tissue sections were stained with the antibody for Cdx1 (1 : 40, rabbit polyclonal; Abcam, Cambridge, UK) after antigenicity was enhanced by autoclaving the sections, as previously described [24]. Bisulfite sequencing for Cdx1 and Cdx2 promoters The methylation status of gene promoter CpGs is best analyzed by using direct sequencing after sodium bisulfite modification of target DNA (bisulfite sequencing). DNA (1 lg of DNA per sample) was sodium bisulfite modified with the DNA modification kit (Zymo Research Intergen, Purchase, NY, USA), according to the manufacturer’s instructions. A 426 bp region of Cdx1 was amplified from bisulfite-modified genomic DNA by nested PCR using two sets of primers. Genomic DNAs were extracted from five stomachs and five intestines of five normal mice and five stomachs of five Cdx2-transgenic mice. The first PCR reaction was performed using the forward primer CpG- Cdx1-fw1[)331 ⁄ )305] and the reverse primer CpG-Cdx1- rv[+114 ⁄ +89] (Table 1). A second, nested, PCR was then performed on 1 lL of the amplificate, using the upstream (CpG-Cdx1-fw2[)312 ⁄ )293]) and downstream (CpG-Cdx1- rv[+114 ⁄ +89]) primers (Table 1). A 400 bp region of Cdx2 was amplified from bisulfite-modified genomic DNA by nested PCR, using two sets of primers. The first PCR reaction was performed using the forward primer CpG-Cdx2-fw1[)234 ⁄ )212] and reverse primer CpG-Cdx2- rv[+194 ⁄ +173] (Table 1). A second, nested, PCR was then performed on 1 lL of the amplificate, using the upstream (CpG-Cdx2-fw2[)206 ⁄ )185]) and downstream (CpG-Cdx2-rv[+194 ⁄ +173]) primers (Table 1). The pri- mer pairs were designed to bind sequences lacking any CpGs, therefore avoiding any preferential amplification of methylated or unmethylated DNA strands. The PCR products were purified (GenElute agarose spin column; Sigma, St Louis, MO, USA), and the purified product was used for cloning (Topo TA Cloning kit; Invitrogen, Carls- bad, CA, USA) and sequencing by using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems). ChIP assay The mucosae removed from the stomach (normal mice), the small intestine (normal mice) and the intestinal metaplasia (Cdx2-transgenic mice) were incubated with fixation solu- tion (1% formaldehyde, 4.5 mm Hepes, pH 8.0, 9 mm NaCl, 0.09 mm EDTA, 0.04 mm EGTA) in NaCl ⁄ P i for 30 min at 37 °C. The reaction was terminated by the addi- tion of glycine to a final concentration of 150 mm. After being washed in NaCl/P i containing protease inhibitors (Protease inhibitor cocktail; Sigma), the samples were soni- cated in SDS lysis buffer (50 mm Tris ⁄ HCl, pH 8.0, 10 mm EDTA, pH 8.0, 1% SDS, 0.5 mm phenylmethanesulfonyl fluoride), when the DNA size of samples was 200–500 bp. The solubilized chromatin was incubated with anti-Cdx2 IgG (BioGenex, San Ramon, CA, USA) [26] or control IgG for 90 min at 4 °C. Beads were washed five times with IP buffer (50 mm Hepes, pH 7.5, 150 mm KCl, 5 mm MgCl 2 ,10lm ZnSO 4 , 1% Triton X-100, 0.05% SDS), and then incubated with elution buffer (50 mm Tris ⁄ HCl, pH 8.0, 1% SDS, 10 mm EDTA) for 30 min at 65 °C. The supernatant was collected and coimmunoprecipitated DNA was recovered. Primer sequences used for the ChIP assays are listed in Table 1. All ChIP assays were repeated at least twice, and representative data are presented. EMSA Nuclear fractions were extracted for EMSA from AGS cells. To extract nuclear fractions for EMSA studies, AGS cells were washed in NaCl ⁄ P i , and subjected to swelling in 400 lL of hypotonic buffer A (10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dith- iothreitol) supplemented with protease inhibitor cocktail (Sigma Chemical Co.), and lysed [27]. Then, 25 lL of 10% Nonidet P-40 solution were added, and nuclear fractions were collected by sedimentation for 5 min at 500 g. Super- natants were discarded, and precipitated nuclei were resus- pended in 100 lL of buffer C (20 mm Hepes, pH 7.9, 400 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 1 mm H. Mutoh et al. Cdx1 expression in intestinal metaplasia FEBS Journal 276 (2009) 5821–5831 ª 2009 The Authors Journal compilation ª 2009 FEBS 5829 EGTA, and protease inhibitor cocktail) and centrifuged for 5 min at 14 000 g. Supernatants containing nuclear proteins were collected, and tested for their ability to bind labeled nucleotides corresponding to the Cdx1 promoter. All DNA–protein binding reaction protocols were those of the manufacturer (Promega, Madison, WI, USA). The dsDNA probes used in the gel mobility shift assays were as follows: wild-type sequence, CCCGCGGCTATAAAAGGCCGGG GTGGGG; mutant sequence, CCCGCGGCTTCGAAAG GCCGGGGTGGGG. Briefly, 0.5 ng of 32 P-labeled probe was incubated for 20 min at 4 °C with 5 lg of nuclear extracts in the presence of 1 · gel shift buffer (Promega). Subsequently, 1.5 lLof10· loading buffer were added to the reaction, and this was followed by separation by elec- trophoresis on 5% nondenaturing polyacrylamide gel until free probe was close to the bottom of the gel. Luciferase assays To construct the luciferase reporter vector pGL4.10[luc2]– Cdx1, 377 bp ()365 to +12) and 288 bp ()365 to )78) fragments, located at 5¢-region of the mouse Cdx1 coding sequence, were amplified by PCR with specific primers (Table 1) from 500 ng of mouse genomic DNA. The ampli- fied fragments for the Cdx1 promoter were directly cloned into the TA cloning vector pCRII (Invitrogen), to yield the plasmid pCRII ⁄ Cdx1 promoter. Each pCRII ⁄ Cdx1 pro- moter was digested with XhoI and HindIII (sites underlined in the primers in Table 1), and the resulting fragments were subcloned into the XhoI and HindIII restriction sites of the pGL4.10[luc2] vector (Promega) and confirmed by sequence analysis. The sequence of the presumptive Cdx2-binding site (TATAAA) was changed to TTCGAA (underlined in the primers) by using Cdx1-Csp45I-fw and Cdx1-Csp45I-rv primers (Table 1). AGS, MKN45 and MKN74 cells were seeded at 2 · 10 5 cells per well in Nunc 24-well dishes 18–24 h before transfection. Transient transfections were performed using Lipofectamine 2000 (Invitrogen). One hundred nanograms of a Cdx1 promoter reporter plasmid with or without 800 ng of Cdx2 expression vector (pRC ⁄ CMV–Cdx2) or 2.5 pmol of siRNA (Applied Biosystems, Silencer Select Pre-designed siRNA, #s2878; UUCUUGUUGAUUUUC CUCUcc) were added to each plate, together with 50 ng of the Renilla luciferase control reporter plasmid (pGL4.70[hRluc]; Promega) as a control for the transfection efficiency. At 24 h after transfection, the cells were lysed in lysis buffer (Promega), and the firefly and Renilla luciferase activities were measured, using the Dual-Luciferase Repor- ter Assay System (Promega) in a luminometer. The relative firefly luciferase activities were calculated by normalizing the transfection efficiency according to the Renilla luciferase activities produced by the internal control plasmid pGL4.70[hRluc]. Three separate experiments were per- formed in triplicate. 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