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• W2027150844 abstract "Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoidelement in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparentKD and only about a 2-fold reduction in theBmax. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome. Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoidelement in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparentKD and only about a 2-fold reduction in theBmax. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome. Rieger syndrome was defined as a genetic disorder in 1935 (1Rieger H. Z. Augenheilk. 1935; 86: 333Google Scholar, 2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar). It is an autosomal-dominant human disorder characterized by ocular anterior chamber anomalies causing glaucoma, dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities. Other features associated with Rieger syndrome include abnormal cardiac, limb, and pituitary development. Murray and co-workers (2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar) used a positional cloning strategy to find the Pitx2 gene and determined that it was a member of the homeobox gene family. The homeobox transcription gene family has been extensively studied and plays a fundamental role in development and evolution. Homeobox genes are involved in the genetic control of development, in particular in the specification of the body plan, pattern formation, and determination of cell fate (3Gehring W.J. Science. 1987; 236: 1245-1252Crossref PubMed Scopus (619) Google Scholar, 4Scott M.P. Tamkun J.W. Hartzel G.W. Biochim. Biophys. Acta. 1989; 989: 25-48Crossref PubMed Scopus (799) Google Scholar, 5McGinnis W. Krumlauf R. Cell. 1992; 68: 283-302Abstract Full Text PDF PubMed Scopus (2198) Google Scholar, 6Kornberg T.B. J. Biol. Chem. 1993; 268: 26813-26816Abstract Full Text PDF PubMed Google Scholar, 7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 8Lawrence P. Morata G. Cell. 1994; 78: 181-189Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 9Gruss P. Walther C. Cell. 1992; 69: 719-722Abstract Full Text PDF PubMed Scopus (351) Google Scholar, 10Kumar J. Moses K. Genes Dev. 1997; 11: 2023-2028Crossref PubMed Scopus (65) Google Scholar). In patients with Rieger syndrome five mutations were found to affect the homeobox region. Three of these mutations were missense causing nonconservative amino acid changes in the homeodomain and two splicing mutations in the intron dividing the homeobox sequence (2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar).The Pitx2 gene is a member of the Bicoid-like homeobox family (2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar). The defining characteristic is the lysine residue at position 9 of the third helix (11Hanes S.D. Brent R. Cell. 1989; 57: 1275-1283Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 12Jin Y. Hoskins R. Horvitz H.R. Nature. 1994; 372: 780-783Crossref PubMed Scopus (188) Google Scholar, 13Simeone A. Acampora D. Mallamaci A. Stornaiuolo A. D'Apice M.R. Nigro V. Boncinelli E. EMBO J. 1993; 12: 2735-2747Crossref PubMed Scopus (524) Google Scholar). The homeodomain of Pitx2 has a high degree of homology to P-OTX/Ptx1 and to a lesser extent to unc-30, Otx-1, Otx-2, otd and goosecoid (2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar). The Drosophila Bicoid (Bcd) protein binds to the DNA core sequence 5′-TAATCC-3′ (14Driever W. Nusslein-Volhard C. Nature. 1989; 337: 138-143Crossref PubMed Scopus (465) Google Scholar). Other investigators cloned the mouse Pitx2 gene from an adult pituitary and whole embryo cDNA library and termed the genePtx2 and Otlx2, respectively (15Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (190) Google Scholar, 16Mucchielli M. Martinez S. Pattyn A. Goridis C. Brunet J. Mol. Cell. Neurosci. 1996; 8: 258-271Crossref PubMed Scopus (106) Google Scholar). We now refer to this gene as Pitx2 as the recommended gene symbol (15Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (190) Google Scholar).We are studying the transcriptional regulation of Pitx2, its interaction with other factors and the role Pitx2 plays in vertebrate development. In this report, we have determined the DNA binding and transcriptional properties of Pitx2 and two Pitx2 homeodomain mutations found in Rieger syndrome patients. Interactions with another transcription factor, Pit-1, that is important in pituitary development are examined with both the wild type and mutant proteins. The results support a model in which Pitx2 DNA binding and transactivation activities are modulated by protein-protein interactions. Thus, the Rieger mutations may affect these interactions during development. We provide evidence for the molecular/biochemical basis of Rieger syndrome.DISCUSSIONThis study represents a molecular and biochemical characterization of the newly discovered human homeodomain transcription factor, Pitx2. We compare the activities of wild type Pitx2 to homeodomain mutations in Pitx2 that cause Rieger syndrome. Pitx2 has a lysine residue at position 9 of helix 3 in the homeodomain, which is known to selectively recognize the 3′-CC dinucleotide adjacent to the TAAT core sequence (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar). Consistent with this phylogenetic relationship, we have demonstrated that Pitx2 can bind the DNA sequence 5′-TAATCC-3′, which is recognized by Bicoid protein (14Driever W. Nusslein-Volhard C. Nature. 1989; 337: 138-143Crossref PubMed Scopus (465) Google Scholar).Recently, a Pitx2-related murine gene product was identified by two independent researchers and termed Ptx1 and P-OTX(Pitx1 in new nomenclature) (28Lamonerie T. Tremblay J.J. Lanctot C. Therrien M. Gauthier Y. Drouin J. Genes Dev. 1996; 10: 1284-1295Crossref PubMed Scopus (352) Google Scholar, 29Szeto D.P. Ryan A.K. O'Connell S.M. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7706-7710Crossref PubMed Scopus (241) Google Scholar). The expression patterns of Pitx1 and Pitx2 are very similar, and expression in Rathke's pouch suggests that this new family may play an important role in pituitary gland development. Our transactivation data are similar to that reported for the P-OTX/Ptx1 activation of the prolactin promoter when transfected into nonpituitary cell lines (28Lamonerie T. Tremblay J.J. Lanctot C. Therrien M. Gauthier Y. Drouin J. Genes Dev. 1996; 10: 1284-1295Crossref PubMed Scopus (352) Google Scholar, 29Szeto D.P. Ryan A.K. O'Connell S.M. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7706-7710Crossref PubMed Scopus (241) Google Scholar). The POU homeodomain protein Pit-1 also binds to specific sites on the prolactin promoter to activate prolactin transcription. Pit-1 is an important transcription factor that regulates pituitary cell differentiation and expression of thyroid-stimulating hormone, growth hormone, and prolactin (27Ryan A.K. Rosenfeld M.G. Genes Dev. 1997; 11: 1207-1225Crossref PubMed Scopus (439) Google Scholar). Co-expression of Pit-1 and Pitx2 gave a 55-fold synergistic transactivation of the prolactin promoter. Consistent with this synergism, we demonstrated a direct interaction between Pitx2 and Pit-1in vitro. These results establish that Pitx2 transactivation activity is enhanced by interaction with another transcription factor.We demonstrate that binding of Pitx2 to the bicoid element is enhanced upon interacting with Pit-1. There is precedence for proteins interacting together to increase their binding activity (30Stark M.R. Johnson A.D. Nature. 1994; 371: 429-432Crossref PubMed Scopus (54) Google Scholar, 31Guichet A. Copeland J.W.R. Erdelyl M. Hlousek D. Zavorszky P. Ho J. Brown S. Percival-Smith A. Krause H.M. Ephrussl A. Nature ( Lond. ). 1997; 385: 548-552Crossref PubMed Scopus (158) Google Scholar, 32Yu Y. Li W. Su K. Yussa M. Han W. Perrimon N. Pick L. Nature. 1997; 385: 552-555Crossref PubMed Scopus (161) Google Scholar). It has been shown that Pbx can increase the binding of Hox proteins to their optimal DNA binding site (33Neuteboom S.T.C. Murre C. Mol. Cell. Biol. 1997; 17: 4696-4706Crossref PubMed Scopus (61) Google Scholar, 34Peltenburg L.T.C. Murre C. Development. 1997; 124: 1089-1098PubMed Google Scholar). Pbx can also interact with the Drosophila homeobox protein, Engrailed, to stimulate cooperative DNA binding (34Peltenburg L.T.C. Murre C. Development. 1997; 124: 1089-1098PubMed Google Scholar). The transcription factor Prospero, has been shown to increase the DNA binding of Dfd and Hoxa-5 (35Hassan B. Li L. Bremer K.A. Chang W. Pinsonneault J. Vaessin H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10991-10996Crossref PubMed Scopus (63) Google Scholar). They report that Prospero is not part of the Dfd-DNA complex and that Prospero interacts with Dfd to modify its DNA binding capacity. This enhancement of DNA binding activity without detectable ternary complexes has been reported previously with other homeodomain proteins (36Grueneberg D.A. Natesan S. Alexandre C. Gilman M.Z. Science. 1992; 257: 1089-1095Crossref PubMed Scopus (256) Google Scholar). This is similar to our finding that Pit-1 and Pitx2 physically interact, yet Pit-1 is apparently not part of the Pitx2-bicoid DNA complex. These results demonstrate that Pit-1 and Pitx2 interact to increase the binding capacity of Pitx2, which may facilitate transactivation of the prolactin promoter.In Rieger syndrome the T68P point mutation lies in helix 2 at position 30 of the homeodomain, whereas the L54Q point mutation is in helix 1 at position 16 of the homeodomain. To our knowledge there are no reports of amino acids in these positions affecting DNA binding specificity in homeodomain proteins. Comparison of the amino acid sequence of over 300 homeobox proteins reveals that position 30 of the homeodomain is not conserved, and several amino acids can be located at this position (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). Although, we have found no other proteins with a proline at position 30 it appears that this position can accommodate changes in amino acid identity without affecting DNA binding activity. In contrast, the amino acid residue at position 16 of the homeodomain is highly conserved (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). In the approximately 300 homeobox proteins analyzed, the residue at this position is a leucine, except for EgHbx4, ap, and LH-2, which contain a methionine, and Lmx-1, which has a phenylalanine (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). This strong conservation suggests that the leucine residue plays a fundamental role in the homeodomain. Our results support this prediction by demonstrating that a mutation of the leucine to a glutamine (L54Q) is detrimental for Pitx2 activity. Since this mutant protein could not be detected in transfected mammalian cells, this suggests that the leucine at position 16 is important for stability of the homeodomain.The binding specificity of homeodomains is dictated mostly by residues in the recognition helix and the N-terminal arm (11Hanes S.D. Brent R. Cell. 1989; 57: 1275-1283Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 38Damante G. Fabbro D. Pellizzari L. Civitareale D. Guazzi S. Schwartz M. Cauci S. Quadrifoglio F. Formisano S. Di Lauro R. Nucleic Acids Res. 1994; 22: 3075-3083Crossref PubMed Scopus (101) Google Scholar, 39Wilson D.S. Sheng G. Jun S. Desplan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6886-6891Crossref PubMed Scopus (119) Google Scholar, 40Percival-Smith A. Muller M. Affolter M. Gehring W.J. EMBO J. 1990; 9: 3967-3974Crossref PubMed Scopus (122) Google Scholar, 41Treisman J. Gonczy P. Vashishtha M. Harris E. Despian C. Cell. 1989; 59: 553-562Abstract Full Text PDF PubMed Scopus (397) Google Scholar). The side chains of amino acids at positions 25, 28, and 31 of the Antp homeodomain contact phosphate groups of the 5′-TAAT-3′ DNA binding element (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar). The amino acids located in positions 6 and 10 in helix 3 of the Drosophila Bicoid homeodomain have been shown to recognize the TAAT core sequence (42Treisman J. Harris E. Wilson D. Desplan C. BioEssays. 1992; 14: 145-150Crossref PubMed Scopus (100) Google Scholar). It has been demonstrated that the amino acid at position 50 of the Bicoid homeodomain is critical for recognizing the 3′-CC dinucleotide of the DNA binding sequence (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 39Wilson D.S. Sheng G. Jun S. Desplan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6886-6891Crossref PubMed Scopus (119) Google Scholar). Recently, the molecular basis of Boston-type craniosynostosis was determined to involve a point mutation in the N-terminal arm of the MSX2 homeodomain (43Ma L. Golden S. Wu L. Maxson R. Hum. Mol. Genet. 1996; 5: 1915-1920Crossref PubMed Scopus (147) Google Scholar). Similar to our results a mutation in the MSX2 homeodomain did not abolish DNA binding as one might expect. Overexpression of the wild type Msx2 gene can also produce craniosynostosis, therefore enhanced binding by the MSX2 mutant is implicated as the cause for this disorder. Pitx2 T68P binds DNA but this mutation results in slightly reduced binding specificity and capacity. This reduction in binding specificity might account for the loss of Pitx2 transactivation activity. However, although we cannot rule out this possibility, it seems unlikely that these changes are sufficient to yield no detectable transacting activity especially in the presence of Pit-1. A more plausible explanation for the mutant effect is that the point mutation affects a transactivation activity. We are currently mapping the transactivation and protein-protein interaction domains to further elucidate the biochemical activities of Pitx2. Rieger syndrome was defined as a genetic disorder in 1935 (1Rieger H. Z. Augenheilk. 1935; 86: 333Google Scholar, 2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar). It is an autosomal-dominant human disorder characterized by ocular anterior chamber anomalies causing glaucoma, dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities. Other features associated with Rieger syndrome include abnormal cardiac, limb, and pituitary development. Murray and co-workers (2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar) used a positional cloning strategy to find the Pitx2 gene and determined that it was a member of the homeobox gene family. The homeobox transcription gene family has been extensively studied and plays a fundamental role in development and evolution. Homeobox genes are involved in the genetic control of development, in particular in the specification of the body plan, pattern formation, and determination of cell fate (3Gehring W.J. Science. 1987; 236: 1245-1252Crossref PubMed Scopus (619) Google Scholar, 4Scott M.P. Tamkun J.W. Hartzel G.W. Biochim. Biophys. Acta. 1989; 989: 25-48Crossref PubMed Scopus (799) Google Scholar, 5McGinnis W. Krumlauf R. Cell. 1992; 68: 283-302Abstract Full Text PDF PubMed Scopus (2198) Google Scholar, 6Kornberg T.B. J. Biol. Chem. 1993; 268: 26813-26816Abstract Full Text PDF PubMed Google Scholar, 7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 8Lawrence P. Morata G. Cell. 1994; 78: 181-189Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 9Gruss P. Walther C. Cell. 1992; 69: 719-722Abstract Full Text PDF PubMed Scopus (351) Google Scholar, 10Kumar J. Moses K. Genes Dev. 1997; 11: 2023-2028Crossref PubMed Scopus (65) Google Scholar). In patients with Rieger syndrome five mutations were found to affect the homeobox region. Three of these mutations were missense causing nonconservative amino acid changes in the homeodomain and two splicing mutations in the intron dividing the homeobox sequence (2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar). The Pitx2 gene is a member of the Bicoid-like homeobox family (2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar). The defining characteristic is the lysine residue at position 9 of the third helix (11Hanes S.D. Brent R. Cell. 1989; 57: 1275-1283Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 12Jin Y. Hoskins R. Horvitz H.R. Nature. 1994; 372: 780-783Crossref PubMed Scopus (188) Google Scholar, 13Simeone A. Acampora D. Mallamaci A. Stornaiuolo A. D'Apice M.R. Nigro V. Boncinelli E. EMBO J. 1993; 12: 2735-2747Crossref PubMed Scopus (524) Google Scholar). The homeodomain of Pitx2 has a high degree of homology to P-OTX/Ptx1 and to a lesser extent to unc-30, Otx-1, Otx-2, otd and goosecoid (2Semina E.V. Reiter R. Leysens N.J. Alward L.M. Small K.W. Datson N.A. Siegel-Bartelt J. Bierke-Nelson D. Bitoun P. Zabel B.U. Carey J.C. Murray J.C. Nat. Genet. 1996; 14: 392-399Crossref PubMed Scopus (763) Google Scholar). The Drosophila Bicoid (Bcd) protein binds to the DNA core sequence 5′-TAATCC-3′ (14Driever W. Nusslein-Volhard C. Nature. 1989; 337: 138-143Crossref PubMed Scopus (465) Google Scholar). Other investigators cloned the mouse Pitx2 gene from an adult pituitary and whole embryo cDNA library and termed the genePtx2 and Otlx2, respectively (15Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (190) Google Scholar, 16Mucchielli M. Martinez S. Pattyn A. Goridis C. Brunet J. Mol. Cell. Neurosci. 1996; 8: 258-271Crossref PubMed Scopus (106) Google Scholar). We now refer to this gene as Pitx2 as the recommended gene symbol (15Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (190) Google Scholar). We are studying the transcriptional regulation of Pitx2, its interaction with other factors and the role Pitx2 plays in vertebrate development. In this report, we have determined the DNA binding and transcriptional properties of Pitx2 and two Pitx2 homeodomain mutations found in Rieger syndrome patients. Interactions with another transcription factor, Pit-1, that is important in pituitary development are examined with both the wild type and mutant proteins. The results support a model in which Pitx2 DNA binding and transactivation activities are modulated by protein-protein interactions. Thus, the Rieger mutations may affect these interactions during development. We provide evidence for the molecular/biochemical basis of Rieger syndrome. DISCUSSIONThis study represents a molecular and biochemical characterization of the newly discovered human homeodomain transcription factor, Pitx2. We compare the activities of wild type Pitx2 to homeodomain mutations in Pitx2 that cause Rieger syndrome. Pitx2 has a lysine residue at position 9 of helix 3 in the homeodomain, which is known to selectively recognize the 3′-CC dinucleotide adjacent to the TAAT core sequence (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar). Consistent with this phylogenetic relationship, we have demonstrated that Pitx2 can bind the DNA sequence 5′-TAATCC-3′, which is recognized by Bicoid protein (14Driever W. Nusslein-Volhard C. Nature. 1989; 337: 138-143Crossref PubMed Scopus (465) Google Scholar).Recently, a Pitx2-related murine gene product was identified by two independent researchers and termed Ptx1 and P-OTX(Pitx1 in new nomenclature) (28Lamonerie T. Tremblay J.J. Lanctot C. Therrien M. Gauthier Y. Drouin J. Genes Dev. 1996; 10: 1284-1295Crossref PubMed Scopus (352) Google Scholar, 29Szeto D.P. Ryan A.K. O'Connell S.M. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7706-7710Crossref PubMed Scopus (241) Google Scholar). The expression patterns of Pitx1 and Pitx2 are very similar, and expression in Rathke's pouch suggests that this new family may play an important role in pituitary gland development. Our transactivation data are similar to that reported for the P-OTX/Ptx1 activation of the prolactin promoter when transfected into nonpituitary cell lines (28Lamonerie T. Tremblay J.J. Lanctot C. Therrien M. Gauthier Y. Drouin J. Genes Dev. 1996; 10: 1284-1295Crossref PubMed Scopus (352) Google Scholar, 29Szeto D.P. Ryan A.K. O'Connell S.M. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7706-7710Crossref PubMed Scopus (241) Google Scholar). The POU homeodomain protein Pit-1 also binds to specific sites on the prolactin promoter to activate prolactin transcription. Pit-1 is an important transcription factor that regulates pituitary cell differentiation and expression of thyroid-stimulating hormone, growth hormone, and prolactin (27Ryan A.K. Rosenfeld M.G. Genes Dev. 1997; 11: 1207-1225Crossref PubMed Scopus (439) Google Scholar). Co-expression of Pit-1 and Pitx2 gave a 55-fold synergistic transactivation of the prolactin promoter. Consistent with this synergism, we demonstrated a direct interaction between Pitx2 and Pit-1in vitro. These results establish that Pitx2 transactivation activity is enhanced by interaction with another transcription factor.We demonstrate that binding of Pitx2 to the bicoid element is enhanced upon interacting with Pit-1. There is precedence for proteins interacting together to increase their binding activity (30Stark M.R. Johnson A.D. Nature. 1994; 371: 429-432Crossref PubMed Scopus (54) Google Scholar, 31Guichet A. Copeland J.W.R. Erdelyl M. Hlousek D. Zavorszky P. Ho J. Brown S. Percival-Smith A. Krause H.M. Ephrussl A. Nature ( Lond. ). 1997; 385: 548-552Crossref PubMed Scopus (158) Google Scholar, 32Yu Y. Li W. Su K. Yussa M. Han W. Perrimon N. Pick L. Nature. 1997; 385: 552-555Crossref PubMed Scopus (161) Google Scholar). It has been shown that Pbx can increase the binding of Hox proteins to their optimal DNA binding site (33Neuteboom S.T.C. Murre C. Mol. Cell. Biol. 1997; 17: 4696-4706Crossref PubMed Scopus (61) Google Scholar, 34Peltenburg L.T.C. Murre C. Development. 1997; 124: 1089-1098PubMed Google Scholar). Pbx can also interact with the Drosophila homeobox protein, Engrailed, to stimulate cooperative DNA binding (34Peltenburg L.T.C. Murre C. Development. 1997; 124: 1089-1098PubMed Google Scholar). The transcription factor Prospero, has been shown to increase the DNA binding of Dfd and Hoxa-5 (35Hassan B. Li L. Bremer K.A. Chang W. Pinsonneault J. Vaessin H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10991-10996Crossref PubMed Scopus (63) Google Scholar). They report that Prospero is not part of the Dfd-DNA complex and that Prospero interacts with Dfd to modify its DNA binding capacity. This enhancement of DNA binding activity without detectable ternary complexes has been reported previously with other homeodomain proteins (36Grueneberg D.A. Natesan S. Alexandre C. Gilman M.Z. Science. 1992; 257: 1089-1095Crossref PubMed Scopus (256) Google Scholar). This is similar to our finding that Pit-1 and Pitx2 physically interact, yet Pit-1 is apparently not part of the Pitx2-bicoid DNA complex. These results demonstrate that Pit-1 and Pitx2 interact to increase the binding capacity of Pitx2, which may facilitate transactivation of the prolactin promoter.In Rieger syndrome the T68P point mutation lies in helix 2 at position 30 of the homeodomain, whereas the L54Q point mutation is in helix 1 at position 16 of the homeodomain. To our knowledge there are no reports of amino acids in these positions affecting DNA binding specificity in homeodomain proteins. Comparison of the amino acid sequence of over 300 homeobox proteins reveals that position 30 of the homeodomain is not conserved, and several amino acids can be located at this position (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). Although, we have found no other proteins with a proline at position 30 it appears that this position can accommodate changes in amino acid identity without affecting DNA binding activity. In contrast, the amino acid residue at position 16 of the homeodomain is highly conserved (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). In the approximately 300 homeobox proteins analyzed, the residue at this position is a leucine, except for EgHbx4, ap, and LH-2, which contain a methionine, and Lmx-1, which has a phenylalanine (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). This strong conservation suggests that the leucine residue plays a fundamental role in the homeodomain. Our results support this prediction by demonstrating that a mutation of the leucine to a glutamine (L54Q) is detrimental for Pitx2 activity. Since this mutant protein could not be detected in transfected mammalian cells, this suggests that the leucine at position 16 is important for stability of the homeodomain.The binding specificity of homeodomains is dictated mostly by residues in the recognition helix and the N-terminal arm (11Hanes S.D. Brent R. Cell. 1989; 57: 1275-1283Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 38Damante G. Fabbro D. Pellizzari L. Civitareale D. Guazzi S. Schwartz M. Cauci S. Quadrifoglio F. Formisano S. Di Lauro R. Nucleic Acids Res. 1994; 22: 3075-3083Crossref PubMed Scopus (101) Google Scholar, 39Wilson D.S. Sheng G. Jun S. Desplan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6886-6891Crossref PubMed Scopus (119) Google Scholar, 40Percival-Smith A. Muller M. Affolter M. Gehring W.J. EMBO J. 1990; 9: 3967-3974Crossref PubMed Scopus (122) Google Scholar, 41Treisman J. Gonczy P. Vashishtha M. Harris E. Despian C. Cell. 1989; 59: 553-562Abstract Full Text PDF PubMed Scopus (397) Google Scholar). The side chains of amino acids at positions 25, 28, and 31 of the Antp homeodomain contact phosphate groups of the 5′-TAAT-3′ DNA binding element (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar). The amino acids located in positions 6 and 10 in helix 3 of the Drosophila Bicoid homeodomain have been shown to recognize the TAAT core sequence (42Treisman J. Harris E. Wilson D. Desplan C. BioEssays. 1992; 14: 145-150Crossref PubMed Scopus (100) Google Scholar). It has been demonstrated that the amino acid at position 50 of the Bicoid homeodomain is critical for recognizing the 3′-CC dinucleotide of the DNA binding sequence (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 39Wilson D.S. Sheng G. Jun S. Desplan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6886-6891Crossref PubMed Scopus (119) Google Scholar). Recently, the molecular basis of Boston-type craniosynostosis was determined to involve a point mutation in the N-terminal arm of the MSX2 homeodomain (43Ma L. Golden S. Wu L. Maxson R. Hum. Mol. Genet. 1996; 5: 1915-1920Crossref PubMed Scopus (147) Google Scholar). Similar to our results a mutation in the MSX2 homeodomain did not abolish DNA binding as one might expect. Overexpression of the wild type Msx2 gene can also produce craniosynostosis, therefore enhanced binding by the MSX2 mutant is implicated as the cause for this disorder. Pitx2 T68P binds DNA but this mutation results in slightly reduced binding specificity and capacity. This reduction in binding specificity might account for the loss of Pitx2 transactivation activity. However, although we cannot rule out this possibility, it seems unlikely that these changes are sufficient to yield no detectable transacting activity especially in the presence of Pit-1. A more plausible explanation for the mutant effect is that the point mutation affects a transactivation activity. We are currently mapping the transactivation and protein-protein interaction domains to further elucidate the biochemical activities of Pitx2. This study represents a molecular and biochemical characterization of the newly discovered human homeodomain transcription factor, Pitx2. We compare the activities of wild type Pitx2 to homeodomain mutations in Pitx2 that cause Rieger syndrome. Pitx2 has a lysine residue at position 9 of helix 3 in the homeodomain, which is known to selectively recognize the 3′-CC dinucleotide adjacent to the TAAT core sequence (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar). Consistent with this phylogenetic relationship, we have demonstrated that Pitx2 can bind the DNA sequence 5′-TAATCC-3′, which is recognized by Bicoid protein (14Driever W. Nusslein-Volhard C. Nature. 1989; 337: 138-143Crossref PubMed Scopus (465) Google Scholar). Recently, a Pitx2-related murine gene product was identified by two independent researchers and termed Ptx1 and P-OTX(Pitx1 in new nomenclature) (28Lamonerie T. Tremblay J.J. Lanctot C. Therrien M. Gauthier Y. Drouin J. Genes Dev. 1996; 10: 1284-1295Crossref PubMed Scopus (352) Google Scholar, 29Szeto D.P. Ryan A.K. O'Connell S.M. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7706-7710Crossref PubMed Scopus (241) Google Scholar). The expression patterns of Pitx1 and Pitx2 are very similar, and expression in Rathke's pouch suggests that this new family may play an important role in pituitary gland development. Our transactivation data are similar to that reported for the P-OTX/Ptx1 activation of the prolactin promoter when transfected into nonpituitary cell lines (28Lamonerie T. Tremblay J.J. Lanctot C. Therrien M. Gauthier Y. Drouin J. Genes Dev. 1996; 10: 1284-1295Crossref PubMed Scopus (352) Google Scholar, 29Szeto D.P. Ryan A.K. O'Connell S.M. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7706-7710Crossref PubMed Scopus (241) Google Scholar). The POU homeodomain protein Pit-1 also binds to specific sites on the prolactin promoter to activate prolactin transcription. Pit-1 is an important transcription factor that regulates pituitary cell differentiation and expression of thyroid-stimulating hormone, growth hormone, and prolactin (27Ryan A.K. Rosenfeld M.G. Genes Dev. 1997; 11: 1207-1225Crossref PubMed Scopus (439) Google Scholar). Co-expression of Pit-1 and Pitx2 gave a 55-fold synergistic transactivation of the prolactin promoter. Consistent with this synergism, we demonstrated a direct interaction between Pitx2 and Pit-1in vitro. These results establish that Pitx2 transactivation activity is enhanced by interaction with another transcription factor. We demonstrate that binding of Pitx2 to the bicoid element is enhanced upon interacting with Pit-1. There is precedence for proteins interacting together to increase their binding activity (30Stark M.R. Johnson A.D. Nature. 1994; 371: 429-432Crossref PubMed Scopus (54) Google Scholar, 31Guichet A. Copeland J.W.R. Erdelyl M. Hlousek D. Zavorszky P. Ho J. Brown S. Percival-Smith A. Krause H.M. Ephrussl A. Nature ( Lond. ). 1997; 385: 548-552Crossref PubMed Scopus (158) Google Scholar, 32Yu Y. Li W. Su K. Yussa M. Han W. Perrimon N. Pick L. Nature. 1997; 385: 552-555Crossref PubMed Scopus (161) Google Scholar). It has been shown that Pbx can increase the binding of Hox proteins to their optimal DNA binding site (33Neuteboom S.T.C. Murre C. Mol. Cell. Biol. 1997; 17: 4696-4706Crossref PubMed Scopus (61) Google Scholar, 34Peltenburg L.T.C. Murre C. Development. 1997; 124: 1089-1098PubMed Google Scholar). Pbx can also interact with the Drosophila homeobox protein, Engrailed, to stimulate cooperative DNA binding (34Peltenburg L.T.C. Murre C. Development. 1997; 124: 1089-1098PubMed Google Scholar). The transcription factor Prospero, has been shown to increase the DNA binding of Dfd and Hoxa-5 (35Hassan B. Li L. Bremer K.A. Chang W. Pinsonneault J. Vaessin H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10991-10996Crossref PubMed Scopus (63) Google Scholar). They report that Prospero is not part of the Dfd-DNA complex and that Prospero interacts with Dfd to modify its DNA binding capacity. This enhancement of DNA binding activity without detectable ternary complexes has been reported previously with other homeodomain proteins (36Grueneberg D.A. Natesan S. Alexandre C. Gilman M.Z. Science. 1992; 257: 1089-1095Crossref PubMed Scopus (256) Google Scholar). This is similar to our finding that Pit-1 and Pitx2 physically interact, yet Pit-1 is apparently not part of the Pitx2-bicoid DNA complex. These results demonstrate that Pit-1 and Pitx2 interact to increase the binding capacity of Pitx2, which may facilitate transactivation of the prolactin promoter. In Rieger syndrome the T68P point mutation lies in helix 2 at position 30 of the homeodomain, whereas the L54Q point mutation is in helix 1 at position 16 of the homeodomain. To our knowledge there are no reports of amino acids in these positions affecting DNA binding specificity in homeodomain proteins. Comparison of the amino acid sequence of over 300 homeobox proteins reveals that position 30 of the homeodomain is not conserved, and several amino acids can be located at this position (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). Although, we have found no other proteins with a proline at position 30 it appears that this position can accommodate changes in amino acid identity without affecting DNA binding activity. In contrast, the amino acid residue at position 16 of the homeodomain is highly conserved (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). In the approximately 300 homeobox proteins analyzed, the residue at this position is a leucine, except for EgHbx4, ap, and LH-2, which contain a methionine, and Lmx-1, which has a phenylalanine (37Duboule D. Guidebook to the Homeobox Genes. Oxford University Press, New York1994Google Scholar). This strong conservation suggests that the leucine residue plays a fundamental role in the homeodomain. Our results support this prediction by demonstrating that a mutation of the leucine to a glutamine (L54Q) is detrimental for Pitx2 activity. Since this mutant protein could not be detected in transfected mammalian cells, this suggests that the leucine at position 16 is important for stability of the homeodomain. The binding specificity of homeodomains is dictated mostly by residues in the recognition helix and the N-terminal arm (11Hanes S.D. Brent R. Cell. 1989; 57: 1275-1283Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 38Damante G. Fabbro D. Pellizzari L. Civitareale D. Guazzi S. Schwartz M. Cauci S. Quadrifoglio F. Formisano S. Di Lauro R. Nucleic Acids Res. 1994; 22: 3075-3083Crossref PubMed Scopus (101) Google Scholar, 39Wilson D.S. Sheng G. Jun S. Desplan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6886-6891Crossref PubMed Scopus (119) Google Scholar, 40Percival-Smith A. Muller M. Affolter M. Gehring W.J. EMBO J. 1990; 9: 3967-3974Crossref PubMed Scopus (122) Google Scholar, 41Treisman J. Gonczy P. Vashishtha M. Harris E. Despian C. Cell. 1989; 59: 553-562Abstract Full Text PDF PubMed Scopus (397) Google Scholar). The side chains of amino acids at positions 25, 28, and 31 of the Antp homeodomain contact phosphate groups of the 5′-TAAT-3′ DNA binding element (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar). The amino acids located in positions 6 and 10 in helix 3 of the Drosophila Bicoid homeodomain have been shown to recognize the TAAT core sequence (42Treisman J. Harris E. Wilson D. Desplan C. BioEssays. 1992; 14: 145-150Crossref PubMed Scopus (100) Google Scholar). It has been demonstrated that the amino acid at position 50 of the Bicoid homeodomain is critical for recognizing the 3′-CC dinucleotide of the DNA binding sequence (7Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wuthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 39Wilson D.S. Sheng G. Jun S. Desplan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6886-6891Crossref PubMed Scopus (119) Google Scholar). Recently, the molecular basis of Boston-type craniosynostosis was determined to involve a point mutation in the N-terminal arm of the MSX2 homeodomain (43Ma L. Golden S. Wu L. Maxson R. Hum. Mol. Genet. 1996; 5: 1915-1920Crossref PubMed Scopus (147) Google Scholar). Similar to our results a mutation in the MSX2 homeodomain did not abolish DNA binding as one might expect. Overexpression of the wild type Msx2 gene can also produce craniosynostosis, therefore enhanced binding by the MSX2 mutant is implicated as the cause for this disorder. Pitx2 T68P binds DNA but this mutation results in slightly reduced binding specificity and capacity. This reduction in binding specificity might account for the loss of Pitx2 transactivation activity. However, although we cannot rule out this possibility, it seems unlikely that these changes are sufficient to yield no detectable transacting activity especially in the presence of Pit-1. A more plausible explanation for the mutant effect is that the point mutation affects a transactivation activity. We are currently mapping the transactivation and protein-protein interaction domains to further elucidate the biochemical activities of Pitx2. We thank Ms. Christina Anderson and Wendy Giles for their excellent technical assistance and Drs. J. Murray, and W. Johnson for providing reagents and helpful discussions." @default.
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