In silico characterization of an Iroquois family-related homeodomain protein
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Homeobox genes have been demonstrated to play important roles during cancer differentiation and embryonic development. The subset of Iroquois-related homeobox genes (IRXs) have furthermore been demonstrated to be involved in several embryonic developmental processes such as patterning of the anterior-posterior and dorso-ventral axis, as well as specific regions of the central nervous system, and differentiation of the otic vesicle, branchial epithelium, and limbs. We have characterized a novel homeodomain protein and corresponding gene by means of computational biology. Since the protein sequence displayed high similarity to the human IRX proteins, the newly identified homeodomain protein was named Iroquois family related homeodomain protein (IFRX). The IFRX protein sequence was found to be highly conserved in vertebrates. The corresponding IFRX gene was located on chromosome 10p12.1 and is organized in seven exons. The protein is predicted to be localized to the nucleus, supporting evidence for a functional role as a transcription factor, as suggested by the existence of a homeodomain. Preliminary expression profiling by biocomputational means predicted a rather broad expression profile with expression in the bone marrow, brain, heart, kidney, liver, lung, pancreas, prostate, skeletal muscle, spinal cord, spleen and thymus. However, expression in several tissues seemed to be low. In addition, approximately one third of all available EST sequences were obtained from embryonic tissues, suggesting that IFRX has a role in embryonic development.Keywords:
EMX2
Homeobox A1
Homeobox protein Nkx-2.5
PAX4
Homeobox genes define a class of genes containing homeobox sequences and encoding proteins, that are identified as transcription regulator controlling cellular development and differentiation, and controlling patterns of gene expression. LIM homeobox genes contain both a homeobox and a conservative sequences (encoded LIM domain). The members of the LIM homeobox genes family which have recently been identified include lin-11, mec-3, apterous(ap), isl-1, LH-2, Rlim, lim-1, lmx-1, Xlim-1 and Xlim-3. Recent findings suggested that products expressed by LIM homeobox genes family play an important role in adjusting differentiation and developmental of the organism. The effects of LIM homeobox genes in the differentiation and development of neurons are currently attracting more and more attention.
Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
DLX5
EMX2
LIM domain
CDX2
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On the basis of the analysis of cDNA of three new homeobox-containing genes from human, chicken, and newt, a new class of homeobox genes Anf is characterized homologous to the Xanf-1 gene from Xenopus laevis, earlier cloned by us. These genes may be largely involved in the specification of embryonic subdivisions in the forebrain region of the embryo. The homeodomains of the proteins encoded by these genes differ greatly in the primary structure from all previously described homeobox genes. The high variability of the homeodomain sequences of the proteins of this class imply their rapid evolution.
Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
EMX2
DLX5
Cloning (programming)
Forebrain
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There was a highly conservative homeobox in nearly all homeobox gene family,which used for encoding transcription regulatory factors(homeobox protein) and generated the accurate regulating action in cells proliferation and differentiation.The mutation of homeobox gene not only resulted in development malformation,but also had a close relationship with the occurrence and development of some tumours.The structural features of homeobox gene and the effect in tumours were summarized.
Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
EMX2
DLX5
CDX2
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Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
CDX2
EMX2
DLX5
Cite
Citations (11)
Homeobox genes are involved in various aspects of the development of multicellular animals, including anterior-posterior patterning of the body plan. We performed a genomic survey of homeobox genes in the Japanese pearl oyster, Pinctada fucata, and annotated 92 homeobox-containing genes and five homeobox-less Pax genes. This species possesses 10 or 11 Hox genes. We annotated another homeobox genes that cover 77 out of the 111 gene families identified in the amphioxus genome. Investigation of these repertoires of homeobox genes will shed new light on the comparatively less well-understood lophotrochozoan development.
Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
EMX2
Pinctada fucata
CDX2
Body plan
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Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
EMX2
Cite
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Homeobox genes are regulatory genes encoding nuclear proteins that act as transcription factors, regulating aspects of morphogenesis and cell differentiation during normal embryonic development of several animals. Vertebrate homeobox genes can be divided in two subfamilies: clustered, or HOX genes, and nonclustered, or divergent, homeobox genes. During the last decades, several homeobox genes, clustered and nonclustered ones, were identified in normal tissue, in malignant cells, and in different diseases and metabolic alterations. Homeobox genes are involved in the normal teeth development and in familial teeth agenesis. Normal development and cancer have a great deal in common, as both processes involve shifts between cell proliferation and differentiation. The literature is accumulating evidences that homeobox genes play an important role in oncogenesis. Many cancers exhibit expression of or alteration in homeobox genes. Those include leukemias, colon, skin, prostate, breast and ovarian cancers, among others. This review is aimed at introducing readers to some of the homeobox family functions in normal tissues and especially in cancer.
Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
DLX5
CDX2
EMX2
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Homeobox genes, which are found in all eukaryotic organisms, encode transcriptional regulators involved in cell-type differentiation and development. Several homeobox genes encoding homeodomain proteins that bind and activate the insulin gene promoter have been described. In an attempt to identify additional beta-cell homeodomain proteins, we designed primers based on the sequences of beta-cell homeobox genes cdx3 and lmx1 and the Drosophila homeodomain protein Antennapedia and used these primers to amplify inserts by PCR from an insulinoma cDNA library. The resulting amplification products include sequences encoding 10 distinct homeodomain proteins; 3 of these proteins have not been described previously. In addition, an insert was obtained encoding a splice variant of engrailed-2, a homeodomain protein previously identified in the central nervous system. Northern analysis revealed a distinct pattern of expression for each homeobox gene. Interestingly, the PCR-derived clones do not represent a complete sampling of the beta-cell library because no inserts encoding cdx3 or lmx1 protein were obtained. Beta cells probably express additional homeobox genes. The abundance and diversity of homeodomain proteins found in beta cells illustrate the remarkable complexity and redundancy of the machinery controlling beta-cell development and differentiation.
EMX2
Homeobox A1
PAX4
Homeobox protein Nkx-2.5
HNF1B
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The caudal homeobox gene Cdx-2 is a transcriptional activator for approximately a dozen genes specifically expressed in pancreatic islets and intestinal cells. It is also involved in preventing the development of colorectal tumors. Studies using “knockout” approaches demonstrated that Cdx-2 is haplo-insufficient in certain tissues including the intestines but not the pancreatic islets. The mechanisms, especially transcription factors, which regulate Cdx-2 expression, are virtually unknown. We found previously that Cdx-2 expression could be autoregulated in a cell type-specific manner. In this study, we located an octamer (OCT) binding site within the mouse Cdx-2 gene promoter. This site, designated as Cdx-2POCT, is involved in the expression of the Cdx-2 promoter. Both pancreatic and intestinal cell lines were found to express a number of POU (OCT binding) homeodomain proteins examined by electrophoretic mobility shift assay. However, it appears that Cdx-2POCT interacts only with OCT1 in the nuclear extracts of the intestinal cell lines examined, although it interacts with OCT1 and at least two other POU proteins that are to be identified in the pancreatic InR1-G9 cell nuclear extract. Co-transfecting OCT1 cDNA but not five other POU gene cDNAs activates the Cdx-2 promoter in the pancreatic InR1-G9 and the intestinal Caco-2 cell lines. In contrast, Cdx-2POCT cannot act as an enhancer element if it is fused to a thymidine kinase promoter. Furthermore, Cdx-2POCT-thymidine kinase fusion promoters cannot be activated by OCT1 co-transfection. Cell type-specific expression, cell type-specific binding affinity of POU proteins to thecis-element Cdx-2POCT, and the DNA content-dependent activation of Cdx-2 promoter via Cdx-2POCT by OCT1 suggest that POU proteins play important and complicated roles in modulating Cdx-2 expression in cell type-specific manners. The caudal homeobox gene Cdx-2 is a transcriptional activator for approximately a dozen genes specifically expressed in pancreatic islets and intestinal cells. It is also involved in preventing the development of colorectal tumors. Studies using “knockout” approaches demonstrated that Cdx-2 is haplo-insufficient in certain tissues including the intestines but not the pancreatic islets. The mechanisms, especially transcription factors, which regulate Cdx-2 expression, are virtually unknown. We found previously that Cdx-2 expression could be autoregulated in a cell type-specific manner. In this study, we located an octamer (OCT) binding site within the mouse Cdx-2 gene promoter. This site, designated as Cdx-2POCT, is involved in the expression of the Cdx-2 promoter. Both pancreatic and intestinal cell lines were found to express a number of POU (OCT binding) homeodomain proteins examined by electrophoretic mobility shift assay. However, it appears that Cdx-2POCT interacts only with OCT1 in the nuclear extracts of the intestinal cell lines examined, although it interacts with OCT1 and at least two other POU proteins that are to be identified in the pancreatic InR1-G9 cell nuclear extract. Co-transfecting OCT1 cDNA but not five other POU gene cDNAs activates the Cdx-2 promoter in the pancreatic InR1-G9 and the intestinal Caco-2 cell lines. In contrast, Cdx-2POCT cannot act as an enhancer element if it is fused to a thymidine kinase promoter. Furthermore, Cdx-2POCT-thymidine kinase fusion promoters cannot be activated by OCT1 co-transfection. Cell type-specific expression, cell type-specific binding affinity of POU proteins to thecis-element Cdx-2POCT, and the DNA content-dependent activation of Cdx-2 promoter via Cdx-2POCT by OCT1 suggest that POU proteins play important and complicated roles in modulating Cdx-2 expression in cell type-specific manners. homeodomain polymerase chain reaction antisense baby hamster kidney luciferase electrophoretic mobility shift assay base pair(s) thymidine kinase Transcription factors are almost never expressed as specific as their downstream target genes (for review see Ref. 1Graef I.A. Crabtree G.R. Science. 1997; 277: 193-194Crossref PubMed Scopus (11) Google Scholar). A given transcription factor is always able to exert multiple biological functions via regulating the expression of different downstream target genes in tissue/cell type-specific manners. This is mainly attributed to the temporal and spatial expression property of a given transcription factor, its ability to bind tocis-elements within different downstream target promoters, and more importantly, its ability to recruit other transcription factors or nuclear co-activators and/or repressors (1Graef I.A. Crabtree G.R. Science. 1997; 277: 193-194Crossref PubMed Scopus (11) Google Scholar, 2Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 3Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577Crossref PubMed Google Scholar). Cdx-2 (CDX2 for humans) is one of the three caudal-related homeodomain (HD)1 proteins identified in mammals (4James R. Kazenwadel J. J. Biol. Chem. 1991; 266: 3246-3251Abstract Full Text PDF PubMed Google Scholar, 5German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes Dev. 1992; 6: 2165-2176Crossref PubMed Scopus (362) Google Scholar, 6James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Abstract Full Text PDF PubMed Google Scholar, 7Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (384) Google Scholar). Post-embryogenesis, Cdx-2 is expressed in pancreatic islets, differentiated intestinal epithelia including the proglucagon-producing endocrine L cells (6James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Abstract Full Text PDF PubMed Google Scholar, 7Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (384) Google Scholar, 8Jin T. Drucker D.J. Mol. Cell. Biol. 1996; 16: 19-28Crossref PubMed Scopus (116) Google Scholar, 9Laser B. Meda P. Constant I. Philippe J. J. Biol. Chem. 1996; 271: 28984-28994Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Cdx-2 mull mutant mice have been generated recently (10Chawengsaksophak K. James R. Hammond V.E. Kontgen F. Beck F. Nature. 1997; 386: 84-87Crossref PubMed Scopus (567) Google Scholar, 11Tamai Y. Nakajima R. Ishikawa T. Takaku K. Seldin M.F. Taketo M.M. Cancer Res. 1999; 59: 2965-2970PubMed Google Scholar).Cdx-2−/− mutants die between 3.5 and 5.5 days postcoitum, whereas Cdx-2 +/− mutants show multiple malfunctions including the formation of multiple intestinal adenomatous polyps. However, malfunctions in the pancreata have not been detected in the Cdx-2 heterozygotic mice. Therefore, Cdx-2 is haplo-insufficient for selected tissues including the intestines but not the pancreas. Although the physiological and pathological significance of Cdx-2 has been well recognized, the mechanisms, especially transcription factors, which regulate the expression of Cdx-2, remain virtually unknown. We have isolated the mouse Cdx-2 promoter and examined the transcriptional properties of this promoter in pancreatic and intestinal cell lines. We have found that Cdx-2 activates the expression of its own promoter in a cell type-specific manner (12Xu F. Li H. Jin T. J. Biol. Chem. 1999; 274: 34310-34316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We have dissected the Cdx-2 gene promoter and demonstrated that Cdx-2 binds to two separated AT-rich motifs, namely the TATA and DBS sites, within a proximal region of the Cdx-2 promoter. The DBS site is critical for autoactivation, whereas the TATA site may serve as an attenuating component for the autoregulatory loop (12Xu F. Li H. Jin T. J. Biol. Chem. 1999; 274: 34310-34316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Members of the POU HD protein family share a common feature of harboring a bipartite DNA binding domain consisting of a POU-specific domain and a POU homeodomain joined by a short variable linker (13Herr W. Cleary M.A. Genes Dev. 1995; 9: 1679-1693Crossref PubMed Scopus (346) Google Scholar). Many POU proteins act as transcriptional regulators via recognizing a conserved 8-base pair octamer (OCT) binding element (13Herr W. Cleary M.A. Genes Dev. 1995; 9: 1679-1693Crossref PubMed Scopus (346) Google Scholar). POU proteins are expressed in early embryogenesis and in diverse organs and/or systems and play fundamentally important roles in the organ development, cell type specification, and growth and differentiation of cells (14Treier M. Rosenfeld M.G. Curr. Opin. Cell Biol. 1996; 8: 833-843Crossref PubMed Scopus (125) Google Scholar, 15Ryan A.K. Rosenfeld M.G. Genes Dev. 1997; 11: 1207-1225Crossref PubMed Scopus (444) Google Scholar). In this study, we located an OCT binding site in the mouse, Cdx-2 promoter, designated as Cdx-2POCT. We demonstrate here that Cdx-2POCT is critical for Cdx-2 promoter expression. OCT1 and other to-be-identified POU proteins are expressed in pancreatic islet, and intestinal cell lines examined. It appears that Cdx-2POCT physically interacts with only OCT1 expressed in intestinal cell lines, whereas Cdx-2POCT interacts with OCT1 and at least two other yet to-be-identified POU proteins expressed in a pancreatic A cell line. Although OCT1 was found to bind to and activate Cdx-2 promoter in pancreatic and intestinal cell lines, Cdx-2POCT itself cannot act as an enhancer element when it is fused to a heterologous promoter. In addition, the fusion promoters cannot be activated by OCT1 co-transfection. We conclude that OCT1 and other to-be-identified POU proteins play important and complicated roles in modulating Cdx-2 expression in cell type-specific manners. Radioisotopes were obtained from Amersham Pharmacia Biotech. Oligonucleotides were provided by ACGT Co. (Toronto, Canada). Restriction enzymes and DNA modification enzymes were of molecular biology grade and were purchased from several sources. Construction of the mouse Cdx-2 promoter and the luciferase fusion gene plasmid and its deletion constructs have been described previously (12Xu F. Li H. Jin T. J. Biol. Chem. 1999; 274: 34310-34316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Cdx-2-LUC (−138 to +137) and its mutant were constructed by subcloning the correspondent polymerase chain reaction products into a promoter-less luciferase reporter gene plasmid. The forward primers used in polymerase chain reaction are Cdx-2pOCT and Cdx-2pOCT(M) as indicated in Fig. 1 B. The reverse primer represents the mouse Cdx-2 DNA sequence from +119 to +137 (GAGCTTCTGCCTCCCGGG). OCT1, OCT2A, and OCT2B expression plasmids were kindly provided by Dr. Winship Herr (16Cleary M.A. Stern S. Tanaka M. Herr W. Genes Dev. 1993; 7: 72-83Crossref PubMed Scopus (82) Google Scholar), OCT3A was a gift from Dr. Graeme Bell (17Takeda J. Seino S. Bell G.I. Nucleic Acids Res. 1992; 20: 4613-4620Crossref PubMed Scopus (260) Google Scholar), and the Skn-1a and Skn-Ii were gifts of Dr. Bogi Andersen (18Andersen B. Schonemann M.D Flynn S., E. Pearse R.V., II Singh H. Rosenfeld M.G. Science. 1993; 260: 78-82Crossref PubMed Scopus (104) Google Scholar). Construction of Cdx-2 expression plasmid and its antisense counterpart (Cdx-2(AS)) has been described previously (19Jin T. Drucker D.J. Mol. Endocrinol. 1995; 9: 1306-1320Crossref PubMed Google Scholar). OCT1(AS) was generated by flipping the 1.1-kb EcoRI fragment in the pCG-OCT1 expression vector (16Cleary M.A. Stern S. Tanaka M. Herr W. Genes Dev. 1993; 7: 72-83Crossref PubMed Scopus (82) Google Scholar). Human colon cancer cell lines HT-29, COLO-205, SW480, and Caco-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to instruction. Cultivation of the baby hamster kidney (BHK) fibroblast, pancreatic islet cell lines InR1-G9, In111, RIN-56, and HIT, and the intestinal endocrine L cell lines GLUTag and STC-1 have been described previously (8Jin T. Drucker D.J. Mol. Cell. Biol. 1996; 16: 19-28Crossref PubMed Scopus (116) Google Scholar, 12Xu F. Li H. Jin T. J. Biol. Chem. 1999; 274: 34310-34316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 19Jin T. Drucker D.J. Mol. Endocrinol. 1995; 9: 1306-1320Crossref PubMed Google Scholar, 20Drucker D.J. Jin T. Asa S. Young T. Brubaker P. Mol. Endocrinol. 1994; 8: 1646-1655Crossref PubMed Google Scholar). BHK, InR1-G9, and Caco-2 cell lines were transfected by a calcium phosphate precipitation method (12Xu F. Li H. Jin T. J. Biol. Chem. 1999; 274: 34310-34316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The cells were harvested for the luciferase (LUC) reporter gene analysis 16 h after the transfection. Nuclear proteins from the cultivated cell lines were prepared as described by Schreiber et al. (21Schreiber E. Matias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3921) Google Scholar). EMSA and supershifting analysis were performed as described previously (19Jin T. Drucker D.J. Mol. Endocrinol. 1995; 9: 1306-1320Crossref PubMed Google Scholar). We located an OCT binding site within the proximal portion of the mouse Cdx-2 promoter, which we designated as Cdx-2POCT (Fig.1 A). To examine the contribution of this OCT binding site to the expression and autoregulation of the Cdx-2 promoter, we generated a pair of Cdx-2-LUC fusion gene constructs, Cdx-2-LUC (−138 to +137 wild type) and Cdx-2-LUC (−138 to +137 mutant). These two fusion genes are identical with the exception that Cdx-2-LUC (−138 to +137 mutant) carries a mutated OCT binding site (Fig. 1 B). We then transfected these two fusion genes into the pancreatic InR1-G9 cell line (Fig.2 A) and the intestinal Caco-2 cell line (Fig. 2 B). The mutated promoter was found to activate the reporter gene in both cell lines ∼30% compared with the wild type promoter indicating that this OCT binding site is critical for Cdx-2 expression. The wild type promoter could be activated >10-fold by Cdx-2 cDNA co-transfection, whereas the mutant promoter could be activated <5-fold. This result suggests that Cdx-2POCT could also be implicated in Cdx-2 autoregulation (12Xu F. Li H. Jin T. J. Biol. Chem. 1999; 274: 34310-34316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In addition, the wild type Cdx-2 promoter could be activated approximately 5-fold by OCT1 cDNA co-transfection, whereas the mutant promoter has no response to OCT1 co-transfection (Fig. 2). Cdx-2 (AS) and OCT1 (AS), which contain Cdx-2 or OCT1 cDNA, respectively, in the antisense orientation, cannot activate Cdx-2 promoter (Fig.2). We then conducted an examination of the expression of nuclear proteins that are able to physically interact with Cdx-2POCT in pancreatic and intestinal cell lines by EMSA. When the pancreatic islet A cell line InR1-G9 was examined, four DNA protein complexes were obtained designated as C1–C4 (Fig. 3, lanes 2 and 10). The formation of these four complexes could be effectively competed by unlabeled native Cdx-2POCT probe (lanes 3 and 4) but not by an unlabeled nonspecific probe (lanes 5 and 6). Therefore, these complexes all represent specific DNA binding events. Complexes C1, C2, and C3 could be effectively competed by another OCT binding probe (22Jin T. Branch D.R Zhang X. Qi S. Youngson B. Goss P.E. Int. J. Cancer. 1999; 81: 104-112Crossref PubMed Scopus (96) Google Scholar) (lanes 7 and 8, see below), indicating the likelihood that these three complexes all represent POU protein binding events. When the mouse large intestinal endocrine L cell line GLUTag was examined, only one complex, C3, was clearly observable (lane 11). When the human colon cancer cell line Caco-2 was examined, the only observable complex was C1 (lane 16). The C1 complex was determined to represent the ubiquitously expressed POU protein OCT1 by a supershifting analysis (compare lane 14with 15 and lane 16 with 17). The above results would suggest that either the intestinal cell lines express fewer number of POU proteins or most POU proteins expressed in intestinal cell lines cannot bind to Cdx-2pOCT. To examine these two possibilities, we then conducted EMSA to examine four human colon cancer cell lines using a synthetic OCT probe (22Jin T. Branch D.R Zhang X. Qi S. Youngson B. Goss P.E. Int. J. Cancer. 1999; 81: 104-112Crossref PubMed Scopus (96) Google Scholar). DNA sequence of the synthetic OCT probe is shown in Fig. 1 B. This artificial probe has been used successfully previously by us to examine the expression of POU proteins in normal and malignant human mammary gland epithelial cell lines (22Jin T. Branch D.R Zhang X. Qi S. Youngson B. Goss P.E. Int. J. Cancer. 1999; 81: 104-112Crossref PubMed Scopus (96) Google Scholar). When this probe was used, nuclear extracts from four intestinal cell lines were found to form a number of complexes (Fig. 4 A). Among these complexes, C1, C2, and C4 were observable for all four colon cancer cell lines examined (lanes 1–4). The formation of C1, C2, and C3 in the Caco-2 cell line was completely competed by the unlabeled OCT probe (lanes 7 and 8) but not by the mutant OCT probe (lanes 9 and 10), indicating that they represent POU protein binding. The native Cdx-2pOCT probe was found to inhibit the formation of C1 and C4 but not C2 and C3 (lane 6). This result would suggest that the POU proteins in C2 and C3, expressed by the Caco-2 cell line, are unable to bind to Cdx-2pOCT. To further examine this observation, we conducted EMSA using the native Cdx-2POCT probe against four colon cancer cell lines. As shown in Fig.4 B, the nuclear extracts from all these cell lines examined were found to form only one major complex, C1 (Fig. 4 B). Taken together, our results suggest that with the exception of OCT1, the other to-be-identified POU proteins are also expressed in the intestinal cell lines examined in this study. However, they are not capable of binding to Cdx-2POCT. When the synthetic OCT probe was applied to the pancreatic islet cell line InR1-G9, three complexes from C1 to C3 were obtained (Fig.5). Supershifting analyses further revealed that C1 represents OCT1 (lane 2). The identities of C2 and C3 cannot be determined using antibodies against two other POU proteins, namely OCT2 and OCT11 (Skn-1a/Skn-1i) (lanes 3–5). We also cannot detect OCT2 and OCT11 expression in the pancreatic and intestinal cell lines used in this study by Western blot analysis (data not shown). Our observations indicated that OCT1 is expressed in most pancreatic and intestinal Cdx-2-expressing cell lines, and OCT1 physically interacts with the Cdx-2 promoter. We then examined if co-transfection of OCT1 and other POU gene cDNAs would activate Cdx-2 promoter. We found that OCT1 activated Cdx-2 promoter nearly 6-fold in the pancreatic InR1-G9 cell line (Figs. 2 A and 6A) and ∼4-fold in the intestinal Caco-2 cell line (Figs. 2 B and 6B). Among the other five POU genes examined, only OCT2A showed a moderate activation on the Cdx-2 promoter. Because OCT2 is not expressed in any of the Cdx-2-expressing cell lines examined, such a moderate activation may have no biological significance. To examine whether the 31-bp native Cdx-2POCT itself could act as an enhancer element, we fused this element with the thymidine kinase (TK) promoter (19Jin T. Drucker D.J. Mol. Endocrinol. 1995; 9: 1306-1320Crossref PubMed Google Scholar) in either 5′ to 3′ (forward) or 3′ to 5′ (reverse) orientations. The Cdx-2POCT-TK-LUC fusion genes together with the wild type TK-LUC were then transfected into the pancreatic InR1-G9 and the BHK fibroblast cell lines. As shown in Fig.7 A, these fusion promoters show a 40–70% activity compared with that of wild type TK promoter when transfected into the InR1-G9 cell line. Therefore, the 31-bp Cdx-2POCT itself cannot act as an enhancer element for this Cdx-2-producing cell line. In contrast, these two fusion promoters are 10–20-fold more active compared with the parental TK promoter when they were transfected into the BHK fibroblasts (Fig. 7 B). Furthermore, these two fusion promoters showed a negative response to OCT1 co-transfection in both InR1-G9 and BHK cell lines examined. These results suggest that OCT1 activates Cdx-2 promoter through binding to Cdx-2POCT in a DNA content-dependent manner. Figure 7Cdx-2POCT cannot act as an enhancer element when fused to a heterologous promoter. TK-LUC, a reporter gene construct in which the luciferase reporter gene expression is driven by a thymidine kinase gene promoter;OCT-TK-LUC(F) and OCT-TK-LUC(R), the 31-bp Cdx-2POCT was inserted in front of the TK promoter in 5′-3′ (forward) or 3′ to 5′ (reverse) orientation. Pancreatic InR1-G9 cells (A) or BHK fibroblasts (B) were transfected by the combination of the indicated reporter gene and the cDNA plasmids (5 μg). Luciferase activity was measured 16 h after transfection. The data are expressed as mean relative luciferase activity (n = 3) ± S.E. normalized to: 1) the activity obtained after transfection of the promoter-less SK-LUC in the same experiment, and 2) total amount of protein utilized in each assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In 1991, James and Kazenwadel (4James R. Kazenwadel J. J. Biol. Chem. 1991; 266: 3246-3251Abstract Full Text PDF PubMed Google Scholar) examined the expression of Hox and related homeobox genes in mouse intestines by reverse transcription-polymerase chain reaction using degenerate primers. With the exception of the detection of Cdx-1 (23Duprey P. Chowdhury K. Dressler G.R. Balling R. Simon D. Guenet J.-L. Gruss P. Genes Dev. 1988; 2: 1647-1654Crossref PubMed Scopus (177) Google Scholar), they also obtained a portion of a novel caudal cDNA, which they named Cdx-2 (4James R. Kazenwadel J. J. Biol. Chem. 1991; 266: 3246-3251Abstract Full Text PDF PubMed Google Scholar). Complete cDNA sequences for mouse Cdx-2 were reported in 1994 (6James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Abstract Full Text PDF PubMed Google Scholar,7Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (384) Google Scholar). However, in 1992 German et al. (5German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes Dev. 1992; 6: 2165-2176Crossref PubMed Scopus (362) Google Scholar) isolated a complete caudal cDNA from a hamster insulinoma cell line, HIT. This cDNA was named Cdx-3. Cdx-2 and Cdx-3 are now considered to be the same gene in different rodents. We and others (8Jin T. Drucker D.J. Mol. Cell. Biol. 1996; 16: 19-28Crossref PubMed Scopus (116) Google Scholar, 9Laser B. Meda P. Constant I. Philippe J. J. Biol. Chem. 1996; 271: 28984-28994Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Xu F. Li H. Jin T. J. Biol. Chem. 1999; 274: 34310-34316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) have used the term Cdx-2/3 previously. To follow the nomenclature regulation for HOX/Hox genes (24Scott M.P. Cell. 1992; 71: 551-553Abstract Full Text PDF PubMed Scopus (397) Google Scholar), we now use the term CDX2 for this gene in humans and the term Cdx-2 in rodent species. Cdx-2 is expressed in intestinal epithelia along the entire crypt-villus axis of the small intestine and in crypts of the colon (7Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (384) Google Scholar). It is also expressed in pancreatic A and B cell lines (5German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes Dev. 1992; 6: 2165-2176Crossref PubMed Scopus (362) Google Scholar, 8Jin T. Drucker D.J. Mol. Cell. Biol. 1996; 16: 19-28Crossref PubMed Scopus (116) Google Scholar, 9Laser B. Meda P. Constant I. Philippe J. J. Biol. Chem. 1996; 271: 28984-28994Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and in the large and small intestinal endocrine L cell lines (8Jin T. Drucker D.J. Mol. Cell. Biol. 1996; 16: 19-28Crossref PubMed Scopus (116) Google Scholar). More recently, the Cdx-2 expression in human gastric cancer cell lines has been reported (25Bai Y.Q. Akiyama Y. Nagasaki H. Yagi O.K. Kikuchi Y. Saito N. Takeshita K. Iwai T. Yuasa Y. Mol. Carcinog. 2000; 28: 184-188Crossref PubMed Scopus (52) Google Scholar). CDX2 expression was found to be ceased in the colon cancer cells of patients at later stages, and mutations in the CDX2-coding region has been found in both colon cancer patients and colon cancer cell lines (26Ee H.E. Erler T. Bhathal P.S. Yound G.P. Jame R.J. Am. J. Physiol. 1995; 273: G3-G6Google Scholar, 27Wicking C. Simms L.A. Evans T. Walsh M. Chawengsaksophak K. Beck F. Chenevix-Trench G. Young J. Jass J. Leggett B. Wainwright B. Oncogene. 1998; 17: 657-659Crossref PubMed Scopus (93) Google Scholar, 28da Costa L.T. He T.C., Yu, J. Sparks A.B. Morin P.J. Polyak K. Laken S. Vogelstein B. Kinzler K.W. Oncogene. 1999; 18: 5010-5014Crossref PubMed Scopus (120) Google Scholar). 2T. Jin and H. Li, unpublished data. More importantly,Cdx-2 +/− mice were found to develop multiple malfunctions including a homeotic shift on vertebrae, stunted growth, abnormalities on tails, and the development of multiple intestinal neoplasia within three months of their birth. However, no observable malfunction has been found in the pancreata of these heterozygotic mice (10Chawengsaksophak K. James R. Hammond V.E. Kontgen F. Beck F. Nature. 1997; 386: 84-87Crossref PubMed Scopus (567) Google Scholar), and proglucagon gene expression appears normal in theCdx-2 +/−mice. 3D. Drucker, personal communication. Such extraordinary multiple malfunctions observed in the heterozygotic null mutants suggest that 1) Cdx-2 is haplo-insufficient for selected tissues, and normal cellular phenotype in these tissues requires the expression of both alleles, 2) Cdx-2 has a broad spectrum of downstream target genes, and 3) Cdx-2 requires co-factors to achieve its diverse and cell type-specific biological functions. Indeed, about a dozen potential downstream target genes of Cdx-2 have been identified including insulin (5German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes Dev. 1992; 6: 2165-2176Crossref PubMed Scopus (362) Google Scholar), proglucagon (8Jin T. Drucker D.J. Mol. Cell. Biol. 1996; 16: 19-28Crossref PubMed Scopus (116) Google Scholar, 9Laser B. Meda P. Constant I. Philippe J. J. Biol. Chem. 1996; 271: 28984-28994Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), sucrase-isomaltase (7Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (384) Google Scholar), phospholipase A/lysophospholipase (29Taylor J.K. Boll W. Levy T. Suh E. Siang S. Mantei N. Traber P.G. DNA Cell Biol. 1997; 16: 1419-1428Crossref PubMed Scopus (60) Google Scholar), lactase-phlorizin hydrolase (30Troelsen J.T. Mitchelmore C. Spodsberg N. Jensen A.M. Noren O. Sjostrom H. Biochem. J. 1997; 322: 833-838Crossref PubMed Scopus (146) Google Scholar), carbonic anhydrase 1 (31Drummond F. Sowden J. Morrison K. Edwards Y.H. Eur. J. Biochem. 1996; 236: 670-681Crossref PubMed Scopus (68) Google Scholar), calbindin-d9K (32Colnot S. Romagnolo B. Lambert M. Cluzeaud F. Porteu A. Vandewalle A. Thomasset M. Kahn A. Perret C. J. Biol. Chem. 1998; 273: 31939-31946Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), vitamin D receptor (33Yamamoto H. Miyamoto K. Li B. Taketani Y. Kitano M. Inoue Y. Morita K. Pike J.W. Takeda E.J. Bone Miner. 1999; 14: 240-247Crossref Scopus (156) Google Scholar), and the homeobox gene Hoxc-8 (34Taylor J.K. Levy T. Suh E.R. Traber P.G. Nucleic Acids Res. 1997; 12: 2293-2300Crossref Scopus (68) Google Scholar). Cdx-2, therefore, provides a very good model system for addressing a fundamentally important biological question: How can a single transcription factor exert its multiple biological functions in tissue/cell type-specific manners? One of the essential steps for understanding tissue-specific biological functions of Cdx-2 is to investigate mechanisms controlling its expression in different types of cells. For this purpose, we isolated the mouse Cdx-2 promoter and examined the transcriptional properties of this promoter in pancreatic islet, intestinal endocrine, and human colon cancer cell lines. We demonstrated that Cdx-2 is able to activate it own promoter in pancreatic and intestinal cell lines that express CDX2/Cdx-2, but repress its own promoter in fibroblasts (12Xu F. Li H. Jin T. J. Biol. Chem. 1999; 274: 34310-34316Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This observation, on one hand, provided an explanation for why one wild type Cdx-2 allele is sufficient for certain tissue such as pancreas. On the other hand, it raised a question why one functional allele is insufficient for other tissues including intestines. We proposed that, except for Cdx-2 itself, other transcription factors and signals might determine whether Cdx-2 expression in a given cell reaches the threshold for triggering the autoregulatory loop. Some of these factors or signals may function only, or more effectively, in one type of cells such as pancreatic cells. If this hypothesis is correct, it would provide an explanation as to why various abnormalities observed amongCdx-2 +/− mice vary, in degree, time, and location, from one mouse to another. In this study, we assessed the contribution of Cdx-2POCT in Cdx-2 expression. Because the mutant promoter shows ∼30% activity compared with that of the wild type, we conclude that this site is important for Cdx-2 expression. In addition, because the mutant promoter loses more than a 2-fold response to Cdx-2 activation, we suggest that this binding site might be also involved in the autoregulation process. To examine POU proteins that are expressed and capable of binding to Cdx-2POCT, we conducted EMSA using both the typical synthetic OCT binding probe (22Jin T. Branch D.R Zhang X. Qi S. Youngson B. Goss P.E. Int. J. Cancer. 1999; 81: 104-112Crossref PubMed Scopus (96) Google Scholar) and the native 31-bp Cdx-2POCT probe. Our results indicated that although Cdx-2POCT forms four specific DNA-protein complexes with the pancreatic InR1-G9 cell line, it forms only one complex, C3, with the intestinal endocrine cell line GLUTag and one complex, C1, with the colon cancer cell line Caco-2. Because Cdx-2 haplo-insufficient occurs in intestines but not pancreata, our results would suggest that pancreatic islet cells might express more POU proteins as transcriptional activators for Cdx-2. To further examine this, we need to identify each individual complex formed between the InR1-G9 cell line and the Cdx-2POCT probe. The C1 complex was found to represent the ubiquitously expressed POU protein OCT1. However, four colon cancer cell lines examined do express OCT-binding proteins other than OCT1 detected by EMSA using the synthetic OCT binding probe. These observations would suggest that, except for OCT1, other POU proteins expressed in pancreatic and intestinal cell lines have different binding affinity to the 31-bpcis-element Cdx-2POCT. This could be due to the expression of different profiles of POU proteins by pancreatic islets and intestine cells. Alternatively, the same POU protein(s) expressed in pancreatic and intestine cells may have different binding affinities. The latter hypothesis is plausible because the binding ability of POU proteins could be influenced by posttranslational events including phosphorylation status (35Grenfell S.J. Latchman D.S. Thomas N.S. Biochem. J. 1996; 315: 889-893Crossref PubMed Scopus (33) Google Scholar, 36Inamoto S. Segil N. Pan Z.Q. Kimura M. Roeder R.G. J. Biol. Chem. 1997; 272: 29852-29858Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 37Zhang H. Shepherd A.T. Eason D.D. Wei S. Diaz J.I. Djeu J.Y. Wu G.D. Blanck G. Cell Growth Differ. 1999; 10: 457-465PubMed Google Scholar, 38O'Reilly D. Hanscombe O. O'Hare P. EMBO J. 1997; 16: 2420-2430Crossref PubMed Scopus (40) Google Scholar). Grenfell et al.(35Grenfell S.J. Latchman D.S. Thomas N.S. Biochem. J. 1996; 315: 889-893Crossref PubMed Scopus (33) Google Scholar) have demonstrated that Oct-1 and Oct-2 bind differentially to three octamer binding sequences, the one from the H2B promoter (ATGCTAATAA, similar to the synthetic OCT probe utilized in this study), a TAATGARAT motif, and a perfect consensus overlapping octamer/TAATGARAT motif (ATGCTAATGAGAT). The binding activity of these octamer probes could be modulated by PKA, PKC, and casein kinase 2 (35Grenfell S.J. Latchman D.S. Thomas N.S. Biochem. J. 1996; 315: 889-893Crossref PubMed Scopus (33) Google Scholar). Inamoto et al. (36Inamoto S. Segil N. Pan Z.Q. Kimura M. Roeder R.G. J. Biol. Chem. 1997; 272: 29852-29858Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) found that MAT1 is able to target cyclin-dependent kinase-activating kinase to Oct factors and lead to their phosphorylation. This may play a role in the recruitment of transcription factor IIH to the preinitiation complex or in subsequent initiation and elongation reactions (36Inamoto S. Segil N. Pan Z.Q. Kimura M. Roeder R.G. J. Biol. Chem. 1997; 272: 29852-29858Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Zhang et al. (37Zhang H. Shepherd A.T. Eason D.D. Wei S. Diaz J.I. Djeu J.Y. Wu G.D. Blanck G. Cell Growth Differ. 1999; 10: 457-465PubMed Google Scholar), however, shown that Oct-1 is a repressor for HLA-DRA. Expression of the tumor suppressor gene Rb leads to the phosphorylation of Oct-1 relieving its repressive effect on HLA-DRA. To assess the overall contribution of POU proteins in regulating Cdx-2 expression in different types of cells, it is necessary to examine the phosphorylation status of these POU proteins in each type of cells in both physiological and pathological conditions. The expression of POU genes in the gastrointestinal tract has not been actively examined. Hussain et al. (39Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 7: 7186-7194Crossref Google Scholar), however, demonstrated that both InR1-G9 and GLUTag cell lines do express Brn-4, a class III POU gene that acts as a regulator for proglucagon gene expression. Because the C3 complex is observed for both InR1-G9 and GLUTag cell lines (Fig. 3, lanes 10 and 11), it is possible that this complex represents Brn-4. This complex was not observed for the four colon cancer cell lines examined. It will be interesting to examine whether Brn-4 or the protein component for the complex C3 is a tissue-specific transcriptional activator for Cdx-2 in proglucagon producing cells. Another interesting observation is that although OCT1 activates Cdx-2 promoter via binding to Cdx-2POCT, this 31-bpcis-element itself cannot act as an enhancer element for the heterologous TK promoter. Furthermore, the Cdx-2POCT-TK fusion promoters do not respond to OCT1 activation. Therefore, OCT1 activates Cdx-2 promoter in a DNA content-dependent manner. Because OCT1 activates both the −769 to + 137 and the −138 to +137 Cdx-2 gene promoters, the cis-element(s) critical for this content-dependent activation should be downstream of the Cdx-2POCT itself. The to-be-definedcis-element(s) may overlap with Cdx-2 binding sites and the area with which components for general transcription machinery might interact. It is possible that OCT1 and other to-be-identified POU proteins may interact with other transcription factors that bind to yet-to-be-defined cis-element(s) downstream of Cdx-2POCT for activating Cdx-2 expression. This contact may promote further protein-protein interactions and recruitment events that interface more effectively with the transcriptional machinery. Indeed, it has been shown previously that OCT1 requires different co-factors such as OCA-B (40Schubart D.B. Rolink A. Kosco-Vilbois M.H. Botteri F. Matthias P. Nature. 1996; 383: 538-542Crossref PubMed Scopus (248) Google Scholar), proximal sequence element-binding transcription factor (41Murphy S. Yoon J.B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (150) Google Scholar), and VP-16 (42Stern S. Herr W. Genes Dev. 1991; 5: 2555-2566Crossref PubMed Scopus (99) Google Scholar) for its diverse biological functions. OCT1 is also able to interact with MAT1, a subunit of cyclin-dependent kinase-activating kinase (36Inamoto S. Segil N. Pan Z.Q. Kimura M. Roeder R.G. J. Biol. Chem. 1997; 272: 29852-29858Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Furthermore, it has been shown that OCT1 and the glucocorticoid receptor interact synergistically to activate the transcription of mouse mammary tumor virus and many other cellular genes (43Chandran U.R. Warren B.S. Baumann C.T. Hager G.L. DeFranco D.B. J. Biol. Chem. 1999; 274: 2372-2378Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 44Prefontaine G.G. Walther R. Giffin W. Lemieux M.E. Pope L. Hache R.J. J. Biol. Chem. 1999; 274: 26713-26719Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 45Prefontaine G.G. Lemieux M.E. Giffin W. Schild-Poulter C. Pope L. LaCasse E. Walker P. Hache R.J. Mol. Cell. Biol. 1998; 18: 3416-3430Crossref PubMed Scopus (87) Google Scholar). Other steroid hormone receptors including progesterone receptor and androgen receptor, were also found to be able to efficiently recruit POU proteins to adjacent octamer motifs in the cell (45Prefontaine G.G. Lemieux M.E. Giffin W. Schild-Poulter C. Pope L. LaCasse E. Walker P. Hache R.J. Mol. Cell. Biol. 1998; 18: 3416-3430Crossref PubMed Scopus (87) Google Scholar) or to interact with OCT1 independently of DNA (46Song C.S. Jung M.H. Kim S.C. Hassan T. Roy A.K. Chatterjee B. J. Biol. Chem. 1998; 273: 21856-21866Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To identify co-factor(s) that are essential for OCT1 to activate Cdx-2 expression will add more to our understanding of cell type-specific autoregulation and cell type-specific functions of Cdx-2. In summary, we demonstrated in this study the complexity of the role of the 31-bp cis-element Cdx-2POCT in regulating Cdx-2 expression and autoregulation. The complexity has been shown at three different levels: 1) expression of POU proteins may occur cell type specifically, 2) binding affinity of POU proteins to Cdx-2POCT also shows cell type specificity, 3) activation of Cdx-2 promoter via Cdx-2POCT by OCT1 depends upon its native DNA content. We suggest that POU proteins play important and sophisticated roles in modulating Cdx-2 expression in a cell type-specific manner. We thank Drs. Winship Herr, Graeme Bell, and Bogi Andersen for providing cDNAs of OCT1, OCT2A, OCT2B, OCT3A, Skn-1a, and Skn-1I and Dr. Vincent Giguere for the TK-LUC luciferase reporter gene.
POU domain
PAX4
CDX2
PDX1
Homeobox A1
HNF1B
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Abstract: Any list of past and recent findings on vertebrate brain prenatal development would have to include the fundamental roles of homeobox genes, the genes encoding the nuclear regulatory homeodomain proteins. The discovery of homeobox genes and their involvement as master regulatory elements in programing the development of an embryo into a complete adult organism has provided a key to our understanding of ontogenesis. Also, the correlation of mouse developmental mutants and their corresponding human syndromes with mutations in homeobox genes has provided further evidence for the fundamental role of homeobox genes during the vertebrate brain embryonic development. Here, we review the expression patterns and the phenotypes of gene mutations that implicate a large repertoire of mouse homeobox genes in the specification of neuronal functions during brain embryogenesis.
Homeobox A1
HNF1B
Homeobox protein Nkx-2.5
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Citations (42)