FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2012201162> ?p ?o ?g. }

• W2012201162 endingPage "24675" @default.
• W2012201162 startingPage "24670" @default.
• W2012201162 abstract "In the hindbrain of the mouse embryo, there is often coincident rhombomere-restricted expression of Eph receptor tyrosine kinases and Hox homeobox genes, raising the possibility of regulatory interactions. In this paper, we have identified cis-acting regulatory sequences of the EphA2(Eck) gene, which direct node and hindbrain-specific expression in transgenic embryos. An 8-kilobase region of mouse genomic DNA element was sufficient to drive rhombomere 4 (r4)-specific expression while conferring patchy expression in the node. Further analysis localized the rhombomere-specific enhancer to a 0.9-kilobase sequence. This element contains multiple Hox-Pbx consensus binding sites that bind to both HOXA1/Pbx1 and HOXB1/Pbx1 proteins in vitro. Co-expression of either HOXA1 or HOXB1 with Pbx1 transactivated EphA2 enhancer-dependent reporter gene expression. These results, together with observations of reduced EphA2 expression in hoxa1 and hoxb1 double mutant mice, suggest that expression of EphA2 gene in rhombomere 4 is directly regulated by Hoxa1 and Hoxb1 homeobox transcription factors. In the hindbrain of the mouse embryo, there is often coincident rhombomere-restricted expression of Eph receptor tyrosine kinases and Hox homeobox genes, raising the possibility of regulatory interactions. In this paper, we have identified cis-acting regulatory sequences of the EphA2(Eck) gene, which direct node and hindbrain-specific expression in transgenic embryos. An 8-kilobase region of mouse genomic DNA element was sufficient to drive rhombomere 4 (r4)-specific expression while conferring patchy expression in the node. Further analysis localized the rhombomere-specific enhancer to a 0.9-kilobase sequence. This element contains multiple Hox-Pbx consensus binding sites that bind to both HOXA1/Pbx1 and HOXB1/Pbx1 proteins in vitro. Co-expression of either HOXA1 or HOXB1 with Pbx1 transactivated EphA2 enhancer-dependent reporter gene expression. These results, together with observations of reduced EphA2 expression in hoxa1 and hoxb1 double mutant mice, suggest that expression of EphA2 gene in rhombomere 4 is directly regulated by Hoxa1 and Hoxb1 homeobox transcription factors. receptor tyrosine kinase kilobase(s) electrophoretic mobility shift assay long-terminal repeat autoregulatory element. Development of the vertebrate hindbrain involves specification of the neural epithelium into lineage-restricted compartments known as rhombomeres (r, r1 to r8 in mouse) (1Fraser S. Keynes R. Lumsden A. Nature. 1990; 344: 431-435Crossref PubMed Scopus (561) Google Scholar, 2Lumsden A. Keynes R. Nature. 1989; 337: 424-428Crossref PubMed Scopus (704) Google Scholar). Cells within each compartment can mix freely with each other but not with those of adjacent rhombomeres. After rhombomere formation, cells in each compartment acquire specific patterns of neuronal differentiation, axon outgrowth, and neural crest cell migration. Thus, the segmentation of the hindbrain into rhombomeres plays a critical role in generating specific neuronal cell types and regional architecture. Coupled to these morphogenetic events, signaling molecules, receptor tyrosine kinases, and transcription factors regulate multiple aspects of hindbrain development at the molecular level (reviewed in Ref. 3Lumsden A. Krumlauf R. Science. 1996; 274: 1109-1114Crossref PubMed Scopus (945) Google Scholar). Components of the regulatory networks include Hox homeobox genes and, more recently, the Eph receptor tyrosine kinases and their ligands, designated ephrins. Hox genes encode homeobox-containing transcription factors that are homologous to Drosophila homeotic genes. During hindbrain development, many members of the Hox gene family exhibit rhombomere-restricted expression patterns. Among the first expressed Hox genes, Hoxa1 and Hoxb1are activated during early gastrulation in the primitive streak; by the head fold stage, their expression domains reach a sharp anterior boundary in the neuroectoderm coinciding with the presumptive r3/r4 border. Whereas Hoxa1 is subsequently down-regulated,Hoxb1 expression becomes restricted to r4 and persists until the disappearance of rhombomere boundaries (4Frohman M.A. Boyle M. Martin G.R. Development. 1990; 110: 589-607Crossref PubMed Google Scholar, 5Frohman M.A. Martin G. Mech. Dev. 1992; 38: 55-67Crossref PubMed Scopus (36) Google Scholar, 6Hunt P. Gulisano M. Cook M. Sham M.H. Faiella A. Wilkinson D. Boncinelli E. Krumlauf R. Nature. 1991; 353: 861-864Crossref PubMed Scopus (422) Google Scholar, 7Murphy P. Davidson D.R. Hill R.E. Nature. 1989; 341: 156-159Crossref PubMed Scopus (143) Google Scholar, 8Murphy P. Hill R. Development. 1991; 111: 61-74Crossref PubMed Google Scholar). Genetic analysis of hox mutants revealed that precise anterior domains of Hox expression correlate with their functional roles in the hindbrain (reviewed in Ref. 9Krumlauf R. Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1730) Google Scholar). r4 is partially deleted in the absence of Hoxa1 but maintains its identity (10Carpenter E.M. Goddard J.M. Chisaka O. Manley N.R. Capecchi M.R. Development. 1993; 118: 1063-1075Crossref PubMed Google Scholar, 11Dolle P. Lufkin T. Krumlauf R. Mark M. Duboule D. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7666-7670Crossref PubMed Scopus (126) Google Scholar), whereas the absence of Hoxb1 causes r4 to lose identity without affecting its size (12Goddard J. Rossel M. Manley N. Capecchi M.R. Development. 1996; 122: 3217-3228PubMed Google Scholar, 13Studer M. Lumsden A. Ariza-McNaughton L. Bradley A. Krumlauf R. Nature. 1996; 384: 630-634Crossref PubMed Scopus (352) Google Scholar). Loss of both genes does not further reduce the size of r4 but creates an r4-like territory with unknown identity, suggesting synergistic roles between Hoxa1 and Hoxb1 in rhombomere patterning (14Studer M. Gavalas A. Marshall H. Ariza-McNaughton L. Rijli F.M. Chambon P. Krumlauf R. Development. 1998; 125: 1025-1036Crossref PubMed Google Scholar, 15Gavalas A. Studer M. Lumsden A. Rijli F. Krumlauf R. Chambon P. Development. 1998; 125: 1123-1136Crossref PubMed Google Scholar). Eph receptor tyrosine kinases (RTK)1 belong to a distinct class of RTKs that play important roles in many tissues, including the hindbrain. Structurally, each receptor of this family consists of a single polypeptide chain containing two fibronectin III repeats and a cysteine-rich region in the predicted extracellular domain. To date, at least 14 members of the Eph receptor family and a family of 8 ligands have been identified. Ligands of Eph family receptors are structurally related membrane-bound proteins that can be subdivided into two major subclasses (16Gale, N. W., Holland, S. J., Valenzuela, D. M., Flenniken, A., Pan, L., Ryan, T. E., Henkemeyer, M., Strebhardt, K., Hirai, H., Wilkinson, D. G., Pawson, T., and Yancopoulos, G. D. (1996) 17, 9–19Google Scholar), ephrin-A and ephrin-B. Ligands in the ephrin-A subclass, including the prototype family member ephrin-A1 (B61), are membrane associated through glycosylphosphatidylinositol linkages, whereas ephrin-B subclass consists of ligands with transmembrane domains. During hindbrain patterning, Eph receptors and their ligands exhibit complementary rhombomere-restricted expression patterns: Eph receptors EphA4 (Sek-1) (17Nieto M.A. Gilardi-Hebenstreit P. Charnay P. Wilkinson D.G. Development. 1992; 116: 1137-1150PubMed Google Scholar) and EphA2 (Eck/Sek-2) (18Becker N. Seitanidou T. Murphy P. Mattei M. Topilko P. Nieto M.A. Wilkinson D.G. Charnay P. Gilardi-Hebenstreit P. Mech. Dev. 1994; 47: 3-17Crossref PubMed Scopus (131) Google Scholar, 19Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624PubMed Google Scholar, 20Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Crossref PubMed Scopus (89) Google Scholar, 21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar, 22Flenniken A.M. Gale N.W. Yancopoulos G.D. Wilkinson D.G. Dev. Biol. 1996; 179: 382-401Crossref PubMed Scopus (190) Google Scholar) are up-regulated prior to rhombomere boundary formation in pre-r3/pre-r5 and in pre-r4, respectively, whereas EphB2 (Nuk/Sek-3) (18Becker N. Seitanidou T. Murphy P. Mattei M. Topilko P. Nieto M.A. Wilkinson D.G. Charnay P. Gilardi-Hebenstreit P. Mech. Dev. 1994; 47: 3-17Crossref PubMed Scopus (131) Google Scholar, 23Henkemeyer M. Marengere L.E.M. McGlade J. Olivier J.P. Conlon R.A. Holmyard D.P. Letwin K. Pawson T. Oncogene. 1994; 9: 1001-1014PubMed Google Scholar) and EphB3 (Sek-4) (18Becker N. Seitanidou T. Murphy P. Mattei M. Topilko P. Nieto M.A. Wilkinson D.G. Charnay P. Gilardi-Hebenstreit P. Mech. Dev. 1994; 47: 3-17Crossref PubMed Scopus (131) Google Scholar) are expressed in r3 and r5 after segmentation. All the ephrin ligands examined to date, Ephrin-B1 (Elk-L), Ephrin-B2 (Elf-2), and Ephrin-B3 (Elk-L3), are expressed in r2, r4, and r6 (22Flenniken A.M. Gale N.W. Yancopoulos G.D. Wilkinson D.G. Dev. Biol. 1996; 179: 382-401Crossref PubMed Scopus (190) Google Scholar) (for nomenclature see Ref. 40Eph Nomenclature Committee Cell. 1997; 90: 403-404Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar the name used in the original reference is in brackets). However, mice carrying null mutations in individual Eph receptor genes seem to have normal hindbrain development (21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar,28Henkemeyer M. Orioli D. Henderson J.T. Saxton T.M. Klein R. Cell. 1996; 86: 35-46Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar), 2J. C. Ruiz and E. J. Robertson, personal communication.2J. C. Ruiz and E. J. Robertson, personal communication. probably reflecting functional redundancy among the Eph family signaling pathways. Clues to the role of Eph receptors in the developing hindbrain came from the study of dominant negative EphA4 (sek-1) receptors. Injection of RNA encoding truncated EphA4 receptor into zebrafish and Xenopus embryos results in failure to establish sharp rhombomere boundaries (24Xu Q. Alldus G. Holder N. Wilkinson D.G. Development. 1995; 121: 4005-4016PubMed Google Scholar), indicating that EphA4 and its ligands restrict cell intermingling between odd- and even-numbered rhombomeres. These findings are consistent with a general role of the Eph family in mediating repulsive cell-cell interaction, as suggested by studies of axonal guidance (25Cheng H. Nakamoto M. Bergemann A.D. Flanagan J.G. Cell. 1995; 82: 371-381Abstract Full Text PDF PubMed Scopus (662) Google Scholar, 26Drescher U. Kremoser C. Handwerker C. Loschinger J. Noda M. Bonhoeffer F. Cell. 1995; 82: 359-370Abstract Full Text PDF PubMed Scopus (768) Google Scholar, 27Nakamoto M. Cheng H. Friedman G.C. McLaughlin T. Hansen M.J. Yoon C.H. O'Leary D.D.M. Flanagan J.G. Cell. 1996; 86: 755-766Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 28Henkemeyer M. Orioli D. Henderson J.T. Saxton T.M. Klein R. Cell. 1996; 86: 35-46Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar) and neural crest cell migration (29Krull C.E. Landsford R. Gale N.W. Marcelle C. Collazo A. Yancopoulos G.D. Fraser S.E. Bronner-Fraser M. Curr. Biol. 1997; 7: 571-580Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 30Smith A. Robinson V. Patel K. Wilkinson D.G. Curr. Biol. 1997; 7: 561-570Abstract Full Text Full Text PDF PubMed Google Scholar, 31Wang H.U. Anderson D.J. Neuron. 1997; 18: 383-396Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). The ordered and nested expression patterns of Eph receptors and their ligands along the neuraxis raise questions how these restricted domains of expression are achieved. The segment-restricted expression patterns of Eph receptors are reminiscent of those of Hox genes, which are expressed at similar developmental stages, raising the possibility of regulatory interactions. For example, likeHoxa1 and Hoxb1, EphA2 is expressed in the primitive streak during gastrulation and becomes restricted to prospective r4 during the head fold stage. At the 4–8 somite stage,EphA2 expression coincides with that of Hoxb1 in r4 and is subsequently down-regulated before Hoxb1 (19Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624PubMed Google Scholar, 20Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Crossref PubMed Scopus (89) Google Scholar, 21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). In contrast to extensive studies on Hox gene regulation, relatively little is known about the regulation of Eph receptors in the hindbrain. As a first step toward understanding the coupling between cell-cell interaction and transcriptional control in the hindbrain, we undertook an analysis of the regulatory elements of the murineEphA2 gene. Here, we report the identification of an enhancer element that drives EphA2 expression in r4 and show that human HOXA1 and HOXB1 can activate reporter gene expression through this enhancer element. These data suggest a direct mechanism for control of EphA2 expression by the HOX paralogy group 1. A 12-kb genomic DNA fragment (construct 1) upstream of the EphA2 gene was originally isolated from sequences surrounding an inserted U3βgeo (lacZ-neo) gene trap retrovirus (21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). For constructs 2–7, genomic DNA fragments isolated from EphA2 genomic clones were ligated into the SmaI site of ASShsplacZpA cassette (gift of Sasaki and Hogan (32Sasaki H. Hogan B.L. Genes Cells. 1996; 1: 59-72Crossref PubMed Scopus (86) Google Scholar)). Both strands of the 1.8-kb SmaI fragment (construct 5) were sequenced following cycle sequencing with fluorescent dideoxy chain terminators and analyzed on an ABI 377 DNA sequencer. NotI fragments containing the transgene expression cassette were isolated from agarose gels by the Gelase protocol (33Hogan B. Constantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar). DNA concentration was measured by either agarose gel electrophoresis or fluorometry. Transgenic embryos were produced by pronuclear injection as described (33Hogan B. Constantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar). Briefly, 6-week-old (C57BL/6 × DBA/2) F1 female mice were superovulated by intraperitoneal injection of 5 IU of gonadotropin from pregnant mare serum 48 h prior to injection of 5 IU of human chorionic gonadotropin. Female mice were then mated with (C57BL/6 × DBA/2) F1 male mice. The pronuclei of fertilized eggs were microinjected with transgene DNA at a concentration of 3–6 ng/μl. Injected eggs were transferred to pseudopregnant recipient ICR mice. The genotype of embryos was determined by polymerase chain reaction amplification of a lacZ sequence. Primers used were 5′-TTGCCGTCTGAATTTGACCTG and 5′-TCTGCTTCAATCTGCGTGCC. β-Galactosidase activity in dissected embryos was detected as described previously (21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). Briefly, embryos at day 8.5 postcoital (day of vaginal plug or retransfer was designated as day 0.5) were dissected away from extraembryonic tissues and fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in phosphate-buffered saline at 4 °C for 30 min. The embryos were then washed in phosphate-buffered saline for 1 h at 4 °C and stained at 37 °C overnight in phosphate-buffered saline, pH 7.2, 0.02% Nonidet P-40, 0.01% SDS, 2 mmMgCl2, 5 mm K3Fe(CN)6, 5 mm K4Fe(CN)6, and 1 mg/ml X-Gal. Pbx and HOX proteins were produced in vitro from the corresponding pCDNA3-derived expression vectors using a T7 polymerase-based coupled transcription-translation reticulocyte lysate system (Promega) according to manufacturer's instructions. HOX and Pbx proteins were translated separately in the presence of [35S]methionine and normalized for the methionine content of each protein. Electrophoretic mobility shift assays (EMSAs) were performed by pre-incubating 2–4 μl of in vitro synthesized protein for 30 min on ice in 15 μl of binding buffer (75 mm NaCl, 6% glycerol, 10 mm Tris-HCl, pH 7.6, 1 mm EDTA), together with 2 μl (0.5 ng, 5 × 104 cpm) of32P-labeled oligonucleotides. The incubation mixtures were resolved by electrophoresis on a non-denaturing 6% polyacrylamide gel in 0.25 × TBE at 10 V/cm. Gels were dried and exposed at −70 °C. Oligonucleotides used (only one strand shown) are as follows: repeat B, 5′-GGAAGGAAGGGTGGATGGATGGGTGTACAGA; repeat C, 5′-AGACAGATGGATGGATGGGC; repeat D, 5′-TGGATAGATGGATGGATGGG; repeat E, 5′-TTGCATGATGGATGGGCTGG; mutant repeat E, 5′-TTGCATGTCGACTGGGCTGG; and Hoxb-1 r4 enhancer repeat 3, 5′-GATCCGGGGGGTGATGGATGGGCGCTGGGA. COS7 and P19 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum or fetal bovine serum, respectively. COS7 cells were transfected by the standard DEAE-dextran method. In a typical transfection experiment, 8 μg of reporter construct, 4–8 μg of expression construct, and 0.4 μg of pCH110-β-gal as an internal control were used per 10-cm dish. P19 cells were transfected by LipofectAmine (Life Technologies, Inc.) reagent in a 6-well dish. Cells were harvested 48 h after transfection and assayed for luciferase and β-galactosidase activity. The expression of EphA2 during hindbrain patterning is very dynamic (19Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624PubMed Google Scholar, 20Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Crossref PubMed Scopus (89) Google Scholar, 21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). Initially, EphA2 is detected at the head fold stage in a narrow band corresponding to presumptive r4. As somitogenesis begins, EphA2 expression becomes restricted to the presumptive r4, which includes both dorsal neural ectoderm and the floor plate. By the 10-somite stage, EphA2 expression is down-regulated. In addition to its hindbrain expression, transcripts of EphA2 become restricted in the node during gastrulation. To define upstream signaling pathways and transcriptional events that control this dynamic expression pattern of EphA2, we mapped regulatory elements of the EphA2 gene in transgenic embryos. Our prior studies (21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar) have shown that integration of a gene trap provirus in the 5′ end of the EphA2 gene disrupted the expression of the EphA2 protein. Moreover, the expression of the reporter gene carried by the provirus recapitulated the endogenousEphA2 expression between 6.5 and 10.5 days of embryonic development (21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). Therefore, we reasoned that tissue-specific regulatory elements responsible for EphA2 gene expression might be in the regions flanking the provirus. To test this possibility, a 12-kb genomic DNA insert containing a single 4-kb provirus LTR and 8-kb flanking cellular DNA (Fig. 1 A, construct 1) was isolated from a genomic DNA library constructed fromepha2 i (eck i )heterozygous mice (21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). This 12-kb genomic DNA fragment carries a promoterless LacZ-neo fusion gene in the U3 region of the pro-viral LTR (viral enhancer deleted (34Scherer C.A. Chen J. Nachabeh A. Hopkins N. Ruley H.E. Cell Growth Differ. 1996; 7: 1393-1401PubMed Google Scholar)) and was microinjected to generate transgenic embryos. As shown in Fig. 1 B (construct 1), the spatial and temporal patterns of lacZ expression in transgenic embryos largely coincided with that of the endogenousEphA2 gene (19Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624PubMed Google Scholar, 20Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Crossref PubMed Scopus (89) Google Scholar, 21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). LacZ expression was detected in the node during gastrulation in E7.5 embryos (Fig. 1 B, left panel) and in the presumptive r4 in E8.5 embryos (Fig. 1 B, right panel). In E9.5 embryos,lacZ expression was down-regulated in r4 and regressed toward tail bud (data not shown). In the node, lacZ expression was only seen in some cells (data not shown), indicating that additional elements are required to recapitulate complete node expression of EphA2. Taken together, these results suggest that the 8-kbEphA2 genomic DNA flanking the provirus is sufficient to direct r4-specific expression of EphA2 gene during early embryogenesis. To further define cis-actingEphA2 regulatory elements, an 8-kb BglII fragment was isolated from a wild-type EphA2 genomic clone. Judging by restriction mapping, this 8-kb fragment is identical to the 12-kb fragment except that it does not contain the 4-kb viral LTR. Restriction fragments were isolated from the 8-kb BglII genomic DNA, placed upstream of an hsp68promoter-lacZ-poly(A) cassette (32Sasaki H. Hogan B.L. Genes Cells. 1996; 1: 59-72Crossref PubMed Scopus (86) Google Scholar), and tested for enhancer activity in transgenic embryos at E7.5 to E8.5, stages where EphA2 is expressed in presumptive r4. With such an enhancer assay, patterns and levels of gene expression may vary between transgenic embryos, depending on the integration site of the transgene. Therefore, only reproducible expression patterns reflecting enhancer activity of the test fragment were scored. Expression from the proximal 3.8-kb XhoI-BglII fragment (Fig. 2, construct 4) was similar to the original 12-kb fragment, i.e. expression in the rhombomere and spotty expression in the node. In contrast, the distal 5.2-kb XhoI-XhoI fragment (Fig. 2, construct 2) and the 4.5-kb BglII-XhoI fragment (Fig. 2, construct 3) gave only variable ectopic staining. Because the hsp68promoter-lacZ-poly(A) cassette is devoid of any enhancer sequences and, on its own, does not give a reproducible expression pattern in transgenic embryos (data not shown, see Ref. 32Sasaki H. Hogan B.L. Genes Cells. 1996; 1: 59-72Crossref PubMed Scopus (86) Google Scholar), these data indicate that the proximal 3.8-kb XhoI-BglII fragment (construct 4) contains sufficient enhancer activity to confer rhombomere-specific expression to a heterologous promoter and that the distal 5.2-kb XhoI-XhoI fragment (construct 2) and the 4.5-kb BglII-XhoI fragment (construct 3) do not contain any essential regulatory elements for expression in these domains. The 3.8-kb XhoI-BglII fragment was further divided into several fragments. Of these, a 1.8-kbSmaI fragment (Fig. 2, construct 5) recapitulated the expression pattern of the original 12-kb fragment, whereas the 1.5-kbSmaI-BglII fragment (Fig. 2, construct 6) gave only ectopic staining in embryos. Further division of the 1.8-kb fragment revealed that the 0.9-kb SacI-SmaI fragment (Fig. 2, construct 7) retained rhombomere-specific staining but did not give node staining. Taken together, these results demonstrated that the 0.9-kb SacI-SmaI fragment contains the rhombomere-specific enhancer and that enhancers for the rhombomere and the node reside in separate DNA elements. Localization of the EphA2 r4 enhancer element to a sequence of 0.9 kb enabled us to search for potential transcription factors that might mediate enhancer activity. As a first step, to examine whether any of the known transcription factor binding sites are present in the EphA2 enhancer regions, the 1.8-kbSmaI fragment (construct 5) was sequenced. Sequence analysis revealed that the 1.8-kb SmaI fragment contained many potential transcription factor binding sites, including the Hox-Pbx consensus binding repeat identified in the Hoxb1 r4 autoregulatory element (ARE) (35Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar). However, most binding sites are present in both r4-specific enhancer element (3′ half of the 1.8-kb fragment, construct 7) and nonspecific genomic DNA (5′ half of the 1.8-kb fragment). In contrast, five Hox-Pbx binding sites were found in the 0.9-kb r4-specific enhancer element (Fig. 3), whereas no such sites were present in the 5′ half of the 1.8-kb fragment. Because the up-regulation of EphA2 in r4 is very similar to the early expression patterns of Hoxa1 and Hoxb1 in the hindbrain, we focused our studies on the Hox-Pbx binding sites. These Hox-Pbx binding sites, 5′-(T/A)GAT(T/G)GA(T/A)G-3′, were shown to bind specifically to Hoxb1/Pbx1 and Hoxa1/Pbx1 complexes (35Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 36Rocco D.G. Fulvio M. Zappavigna V. EMBO J. 1997; 16: 3644-3654Crossref PubMed Scopus (97) Google Scholar). In addition, theHoxb1 r4 ARE contains three such sites (repeat 1 to repeat 3), which are necessary and sufficient to drive r4-specific expression in transgenic embryos (35Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar). These putative Hox-Pbx binding sites in theEphA2 enhancer have been named repeat A to repeat E. Four of these repeats are clustered in the 3′ end of the enhancer, whereas repeat A is located more centrally. All of the repeats share identical core nucleotides (bold in Fig. 3), with repeat D containing two overlapping repeats. These results, together with the similarity of expression patterns for EphA2, Hoxa1, and Hoxb1, raise the question whether transcriptional activation of EphA2 is directly regulated by Hoxb1 and/or its paralog Hoxa1. To investigate the possible involvement of Hoxa1 and Hoxb1 in mediating EphA2 enhancer activity, we tested the ability of HOXA1 and HOXB1 proteins to bind to a series of oligonucleotides, each of them spanning one repeat motif. Prior studies showed that specific interactions between Hox proteins and their target sequences require Pbx cofactors (35Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 36Rocco D.G. Fulvio M. Zappavigna V. EMBO J. 1997; 16: 3644-3654Crossref PubMed Scopus (97) Google Scholar, 37Mann R.S. Chan S.-K. Trends Genet. 1996; 12: 258-262Abstract Full Text PDF PubMed Scopus (390) Google Scholar). Therefore,in vitro translated Pbx1, HOXA1, and HOXB1 proteins were used in EMSAs. As repeats B, C, D, and E are clustered in the 3′ portion of the enhancer, we tested whether these four repeats can bind to HOXA1/Pbx1 or HOXB1/Pbx1 in vitro. The ability of HOX/Pbx protein to bind to repeats B–E (lanes 3–10) was also compared with binding to repeat 3 of the Hoxb-1 r4 ARE (lanes 1 and 2) (35Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar). As shown in Fig. 4 A (lanes 3–10), HOXA1/Pbx1 and HOXB1/Pbx1 complexes can bind to all four repeats, although apparently with different affinities. Repeat E showed the strongest binding to HOX/Pbx1 complexes (lanes 9 and 10), which is comparable with the binding of repeat 3 in the Hoxb1 r4 ARE (lanes 1 and 2). A close examination of these repeat sequences revealed that although all four repeats are identical in their core sequence 5′-GATGGA-3′, repeat E in EphA2 enhancer and repeat 3 in Hoxb1 ARE carried a nucleotide T flanking the 5′-G, which might contribute to the tighter binding of these repeats to HOX/Pbx complexes. EMSAs were also used to test whether cooperative interactions between HOXA1 or HOXB1 and Pbx1 are essential for binding to repeat E from theEphA2 enhancer region. As shown in Fig. 4 B, HOXA1 and HOXB1 alone did not bind to repeat E (lanes 2 and 3), whereas Pbx-1 alone bound only weakly (lane 4). However, under the same conditions, efficient binding was observed when Pbx1 was present together with either HOX protein (lanes 5 and 8). This binding was specific, as the complexes were competed by an excess of unlabeled normal (lanes 6 and 9) but not mutant (lanes 7 and 10) oligonucleotides containing repeat E. Similar results were obtained with repeat B and with Hoxb1 r4 ARE repeat 3 (data not shown; specific and cooperative binding to repeats C and D was not tested). These results showed that potential Hox-Pbx binding sites within the EphA2 enhancer can specifically bind to HOXA1/Pbx1 or HOXB1/Pbx1 proteins in vitro. In light of our binding results, the ability of the HOX/Pbx complexes to transactivate reporter gene expression through the EphA2 enhancer was tested in transient cotransfection experiments. Because the hsp68promoter-lacZ-poly(A) cassette gives rise to high background activity in cell lines, we subcloned a 200-base pair fragment (EphA2-r42B, Fig. 5 A) containing repeats B to E from the EphA2 enhancer into pML, a luciferase reporter vector that exhibits minimal background activity in both COS7 and P19 cells (36Rocco D.G. Fulvio M. Zappavigna V. EMBO J. 1997; 16: 3644-3654Crossref PubMed Scopus (97) Google Scholar). pML-ARE, a luciferase reporter under the control of the Hoxb-1 r4 ARE, was used as a positive control. As shown in Fig. 5 B, cotransfection of HOXA1 and Pbx1, or HOXB1 and Pbx1, with the pML-EphA2-r42B reporter led to significant transactivation of the reporter activity in COS7 cells, whereas cotransfection of pcDNA3 control expression vector with pML-EphA2-r42B reporter gave only background activity. This level of transactivation of pML-EphA2-r42B by HOX/Pbx proteins is comparable with that of the pML-ARE positive control reporter. Transactivation of pML-EphA2-r42B reporter activity by HOX/Pbx proteins was also observed in a different cell type, i.e. the murine embryonal carcinoma P19 cell line (Fig. 5 C). These data suggest that HOXA1 or HOXB1 in conjunction with Pbx1 protein can work in trans to activate enhancer-dependent expression of theEphA2 gene. Despite extensive investigation of the transcriptional regulation of Hox genes during hindbrain development, relatively few studies (38Theil T. Frain M. Gilardi-Hebenstreit P. Flenniken A. Charnay P. Development. 1998; 125: 443-452Crossref PubMed Google Scholar, 39Taneja R. Thisse B. Rijli F. Thisse C. Bouillet P. Dolle P. Chambon P. Dev. Biol. 1996; 177: 397-412Crossref PubMed Scopus (76) Google Scholar) have addressed rhombomere-specific expression of Eph receptors. Here, we report that an 8.0-kb genomic fragment 5′ of theEphA2 coding region directs reporter gene expression to r4 of transgenic mouse embryos. Further analysis showed that a 0.9-kb fragment within the region can recapitulate this r4-specific expression pattern. This activity is independent of the orientation of the 0.9-kb fragment, suggesting that it contains a transcriptional enhancer. r4-specific expression of the EphA2 gene is reminiscent of Hoxb1 and its paralog Hoxa1, raising the possibility that EphA2 expression is regulated by Hox genes. Here, we show that 1) the 0.9-kb enhancer of EphA2 contains multiple binding sites for HOXA1/Pbx1 and HOXB1/Pbx1 proteins, 2) oligonucleotides spanning the binding sites bind to these proteinsin vitro, and 3) co-expression of HOXA1 and Pbx1 or HOXB1 and Pbx1 proteins transactivates reporter gene expression from the EphA2-r42B enhancer. These results are consistent with recent genetic studies in which EphA2 expression is substantially decreased in hoxa1/hoxb1 double null mutant mice, although an r4-like territory with unknown identity is present in the mutant (14Studer M. Gavalas A. Marshall H. Ariza-McNaughton L. Rijli F.M. Chambon P. Krumlauf R. Development. 1998; 125: 1025-1036Crossref PubMed Google Scholar,15Gavalas A. Studer M. Lumsden A. Rijli F. Krumlauf R. Chambon P. Development. 1998; 125: 1123-1136Crossref PubMed Google Scholar). 3M. Rossel and M. Capecchi, personal communication. Taken together, these data suggest that expression of EphA2 in r4 is, at least in part, directly regulated by Hoxa1 and Hoxb1. r4 enhancer activity was first detected in sequences surrounding a U3βgeo gene trap retrovirus inserted into the EphA2gene. Although the provirus inserted over 8 kb upstream of the 5′ end of the previously published EphA2 cDNA sequence, the provirus completely abolished EphA2 protein expression, and expression of the inserted lacZ reporter recapitulated expression of the endogenous EphA2 gene (21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). These studies illustrate the utility of gene entrapment as a mean to identify functional elements involved in tissue-specific gene regulation. Although our evidence suggests a direct role of Hoxa1/Hoxb1 in regulating EphA2 expression,Hoxa1/Hoxb1 alone may not be sufficient to account for the complete dynamic temporal and spatial restricted expression pattern of EphA2 gene in r4 (19Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624PubMed Google Scholar, 20Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Crossref PubMed Scopus (89) Google Scholar, 21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar). For example, EphA2expression is substantially reduced, but not abolished, in thehoxa1/hoxb1 double null mutant, suggesting the existence of additional factors regulating residual EphA2 expression. Furthermore, EphA2 expression is down-regulated in r4 by embryonic day E8.75, whereas Hoxb-1 expression persists until at least E10.5 (8Murphy P. Hill R. Development. 1991; 111: 61-74Crossref PubMed Google Scholar). Although these findings suggest the existence of specific transcriptional factors, further studies will be required to identify such additional factors regulating EphA2expression in r4. Members of the Eph receptor family are known to mediate repulsive cell-cell interactions in axonal guidance during neural development (27Nakamoto M. Cheng H. Friedman G.C. McLaughlin T. Hansen M.J. Yoon C.H. O'Leary D.D.M. Flanagan J.G. Cell. 1996; 86: 755-766Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 28Henkemeyer M. Orioli D. Henderson J.T. Saxton T.M. Klein R. Cell. 1996; 86: 35-46Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Because of their segmental-restricted expression patterns in the hindbrain, this ligand/receptor family is likely to restrict cell intermingling between odd- and even-numbered rhombomeres. Experimental evidence to support this hypothesis came from a study involving truncated EphA4 receptors, in which injection of RNA encoding a dominant negative EphA4 into zebrafish and Xenopus embryos interfered with the establishment of sharp rhombomere boundaries (24Xu Q. Alldus G. Holder N. Wilkinson D.G. Development. 1995; 121: 4005-4016PubMed Google Scholar). The role of EphA2 in hindbrain is less clear. Mutant mice carrying mutations generated either by retroviral insertion (21Chen J. Nachabah A. Scherer C. Ganju P. Reith A. Bronson R. Ruley R. Oncogene. 1996; 12: 979-988PubMed Google Scholar) or by gene targeting2 seem to have normal hindbrain development, probably reflecting functional compensation by other receptors in the Eph family. The identification and characterization of thisEphA2 r4 enhancer element permit targeting the expression of trans-dominant inhibitors of Eph receptor, e.g. dominant negative receptors or receptor-Ig, to be expressed at the endogenous locus in transgenic mice, allowing functional characterization of EphA2 during hindbrain development. We are grateful to Dr. Brigid Hogan, without whose encouragement and support this manuscript would not be possible. We thank Drs. Mireille Rossel, Mario Capecchi, Joe Ruize, and Liz Robertson for communicating their unpublished results, Dr. Fulvio Mavilio for providing Pbx1 and HOX expression constructs and the pML luciferase reporter plasmid, Dr. Hiroshi Sasaki and Linda Harget for teaching microinjection techniques, Matthew McReynolds and Abudi Nachabah for technical assistance, and Drs. Brigid Hogan, Mark Boothby, and Justin Grindley for critical reading of the manuscript." @default.
• W2012201162 created "2016-06-24" @default.
• W2012201162 creator A5020742692 @default.
• W2012201162 creator A5072783861 @default.
• W2012201162 date "1998-09-01" @default.
• W2012201162 modified "2023-09-26" @default.
• W2012201162 title "An Enhancer Element in the EphA2 (Eck) Gene Sufficient for Rhombomere-specific Expression Is Activated by HOXA1 and HOXB1 Homeobox Proteins" @default.
• W2012201162 cites W1784887600 @default.
• W2012201162 cites W1817142398 @default.
• W2012201162 cites W1977455576 @default.
• W2012201162 cites W1984755886 @default.
• W2012201162 cites W2004827507 @default.
• W2012201162 cites W2010055658 @default.
• W2012201162 cites W2014507351 @default.
• W2012201162 cites W2016686483 @default.
• W2012201162 cites W2021025315 @default.
• W2012201162 cites W2025921655 @default.
• W2012201162 cites W2034767141 @default.
• W2012201162 cites W2038894508 @default.
• W2012201162 cites W2046541575 @default.
• W2012201162 cites W2048249977 @default.
• W2012201162 cites W2057433337 @default.
• W2012201162 cites W2061191160 @default.
• W2012201162 cites W2067105964 @default.
• W2012201162 cites W2072325944 @default.
• W2012201162 cites W2074345277 @default.
• W2012201162 cites W2081748379 @default.
• W2012201162 cites W2082144494 @default.
• W2012201162 cites W2084158728 @default.
• W2012201162 cites W2085462828 @default.
• W2012201162 cites W2085532366 @default.
• W2012201162 cites W2092396168 @default.
• W2012201162 cites W2099715509 @default.
• W2012201162 cites W2125246072 @default.
• W2012201162 cites W2126247472 @default.
• W2012201162 cites W2127619526 @default.
• W2012201162 cites W2135980041 @default.
• W2012201162 cites W2137162173 @default.
• W2012201162 cites W2155048209 @default.
• W2012201162 cites W2188520922 @default.
• W2012201162 cites W4229680502 @default.
• W2012201162 cites W4229918205 @default.
• W2012201162 cites W4361805501 @default.
• W2012201162 doi "https://doi.org/10.1074/jbc.273.38.24670" @default.
• W2012201162 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9733765" @default.
• W2012201162 hasPublicationYear "1998" @default.
• W2012201162 type Work @default.
• W2012201162 sameAs 2012201162 @default.
• W2012201162 citedByCount "94" @default.
• W2012201162 countsByYear W20122011622012 @default.
• W2012201162 countsByYear W20122011622013 @default.
• W2012201162 countsByYear W20122011622014 @default.
• W2012201162 countsByYear W20122011622015 @default.
• W2012201162 countsByYear W20122011622016 @default.
• W2012201162 countsByYear W20122011622017 @default.
• W2012201162 countsByYear W20122011622018 @default.
• W2012201162 countsByYear W20122011622020 @default.
• W2012201162 countsByYear W20122011622021 @default.
• W2012201162 countsByYear W20122011622022 @default.
• W2012201162 countsByYear W20122011622023 @default.
• W2012201162 crossrefType "journal-article" @default.
• W2012201162 hasAuthorship W2012201162A5020742692 @default.
• W2012201162 hasAuthorship W2012201162A5072783861 @default.
• W2012201162 hasBestOaLocation W20122011621 @default.
• W2012201162 hasConcept C103652617 @default.
• W2012201162 hasConcept C104317684 @default.
• W2012201162 hasConcept C111936080 @default.
• W2012201162 hasConcept C121587040 @default.
• W2012201162 hasConcept C150194340 @default.
• W2012201162 hasConcept C153911025 @default.
• W2012201162 hasConcept C180504324 @default.
• W2012201162 hasConcept C191370860 @default.
• W2012201162 hasConcept C54355233 @default.
• W2012201162 hasConcept C61342607 @default.
• W2012201162 hasConcept C86803240 @default.
• W2012201162 hasConcept C95444343 @default.
• W2012201162 hasConceptScore W2012201162C103652617 @default.
• W2012201162 hasConceptScore W2012201162C104317684 @default.
• W2012201162 hasConceptScore W2012201162C111936080 @default.
• W2012201162 hasConceptScore W2012201162C121587040 @default.
• W2012201162 hasConceptScore W2012201162C150194340 @default.
• W2012201162 hasConceptScore W2012201162C153911025 @default.
• W2012201162 hasConceptScore W2012201162C180504324 @default.
• W2012201162 hasConceptScore W2012201162C191370860 @default.
• W2012201162 hasConceptScore W2012201162C54355233 @default.
• W2012201162 hasConceptScore W2012201162C61342607 @default.
• W2012201162 hasConceptScore W2012201162C86803240 @default.
• W2012201162 hasConceptScore W2012201162C95444343 @default.
• W2012201162 hasIssue "38" @default.
• W2012201162 hasLocation W20122011621 @default.
• W2012201162 hasOpenAccess W2012201162 @default.
• W2012201162 hasPrimaryLocation W20122011621 @default.
• W2012201162 hasRelatedWork W1970374717 @default.
• W2012201162 hasRelatedWork W1994194624 @default.
• W2012201162 hasRelatedWork W2012201162 @default.
• W2012201162 hasRelatedWork W2020713821 @default.
• W2012201162 hasRelatedWork W2042947886 @default.
• W2012201162 hasRelatedWork W2053078554 @default.

• next