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• W1972959575 abstract "Three major PITX2 isoforms are differentially expressed in human, mice, zebrafish, chick, and frog tissues. To demonstrate differential regulation of gene expression by these isoforms we used three different promoters and three cell lines. Transient transfection of Chinese hamster ovary, HeLa, and LS-8 cell lines revealed differences in PITX2A and PITX2C activation of thePLOD1 and Dlx2 promoters, however, PITX2B is inactive. In contrast, PITX2B actives the pituitary-specificProlactin promoter at higher levels than either PITX2A or PITX2C. Interestingly, co-transfection of either PITX2A orPITX2C with PITX2B results in a synergistic activation of the PLOD1 and Dlx2 promoters. Furthermore, PITX2 isoforms have different transcriptional activity dependent upon the cells used for transfection analysis. We have isolated a fourth PITX2 isoform (PITX2D) expressed only in humans, which acts to suppress the transcriptional activity of the other PITX2 isoforms. Electrophoretic mobility shift assays and glutathione S-transferase pull-down experiments demonstrated that all isoforms interact with PITX2D and that PITX2B forms heterodimeric complexes with PITX2A and PITX2C. Our research provides a molecular basis for differential gene regulation through the expression of PITX2 isoforms. PITX2 isoform activities are both promoter- and cell-specific, and our data reveal new mechanisms for PITX2-regulated gene expression. Three major PITX2 isoforms are differentially expressed in human, mice, zebrafish, chick, and frog tissues. To demonstrate differential regulation of gene expression by these isoforms we used three different promoters and three cell lines. Transient transfection of Chinese hamster ovary, HeLa, and LS-8 cell lines revealed differences in PITX2A and PITX2C activation of thePLOD1 and Dlx2 promoters, however, PITX2B is inactive. In contrast, PITX2B actives the pituitary-specificProlactin promoter at higher levels than either PITX2A or PITX2C. Interestingly, co-transfection of either PITX2A orPITX2C with PITX2B results in a synergistic activation of the PLOD1 and Dlx2 promoters. Furthermore, PITX2 isoforms have different transcriptional activity dependent upon the cells used for transfection analysis. We have isolated a fourth PITX2 isoform (PITX2D) expressed only in humans, which acts to suppress the transcriptional activity of the other PITX2 isoforms. Electrophoretic mobility shift assays and glutathione S-transferase pull-down experiments demonstrated that all isoforms interact with PITX2D and that PITX2B forms heterodimeric complexes with PITX2A and PITX2C. Our research provides a molecular basis for differential gene regulation through the expression of PITX2 isoforms. PITX2 isoform activities are both promoter- and cell-specific, and our data reveal new mechanisms for PITX2-regulated gene expression. glutathioneS-transferase electrophoretic mobility shift assay cytomegalovirus Chinese hamster ovary cells The PITX genes are members of the bicoid class of the homeodomain proteins. These have a lysine residue at position nine of the third helix and are especially noteworthy for a role in both DNA and RNA binding (1Dubnau J. Struhl G. Nature. 1996; 379: 694-699Crossref PubMed Scopus (294) Google Scholar, 2Lamonerie T. Tremblay J.J. Lanctot C. Therrien M. Gauthier Y. Drouin J. Genes Dev. 1996; 10: 1284-1295Crossref PubMed Scopus (360) Google Scholar, 3Amendt B.A. Sutherland L.B. Semina E. Russo A.F. J. Biol. Chem. 1998; 273: 20066-20072Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). PITX2 was identified by positional cloning of the 4q25 locus in patients with Axenfeld-Rieger syndrome (4Semina 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 (777) Google Scholar). Patients diagnosed with classical Rieger syndrome havePITX2 mutations, mostly clustered in the homeodomain (3Amendt B.A. Sutherland L.B. Semina E. Russo A.F. J. Biol. Chem. 1998; 273: 20066-20072Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 4Semina 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 (777) Google Scholar, 5Amendt B.A. Semina E.V. Alward W.L.M. Cell. Mol. Life Sci. 2000; 57: 1652-1666Crossref PubMed Scopus (95) Google Scholar). The mouse Pitx2 gene was subsequently cloned from a pituitary library (6Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (195) Google Scholar). This gene has been cloned by other groups and assigned various names (Ptx2, Otlx2,Brx1, and ARP1) (7Mucchielli M. Martinez S. Pattyn A. Goridis C. Brunet J. Mol. Cell. Neurosci. 1996; 8: 258-271Crossref PubMed Scopus (107) Google Scholar, 8Kitamura K. Miura H. Yanazawa M. Miyashita T. Kato K. Mech. Dev. 1997; 67: 83-96Crossref PubMed Scopus (122) Google Scholar, 9Arakawa H. Nakamura T. Zhadanov A.B. Fidanza V. Yano T. Bullrich F. Shimizu M. Blechman J. Mazo A. Canaani E. Croce C.M. Proc. Natl. Acad. Sci. 1998; 95: 4573-4578Crossref PubMed Scopus (97) Google Scholar). Pitx2 has been shown to be expressed in the brain, heart, pituitary, mandibular and maxillary regions, eye, and umbilicus (4Semina 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 (777) Google Scholar, 6Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (195) Google Scholar, 7Mucchielli M. Martinez S. Pattyn A. Goridis C. Brunet J. Mol. Cell. Neurosci. 1996; 8: 258-271Crossref PubMed Scopus (107) Google Scholar). Recent reports using genetic and epigenetic studies and Pitx2 knockout mice have demonstrated that this gene product is required for the proper development of the embryo (6Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (195) Google Scholar, 10Campione M. Steinbeisser H. Schweickert A. Deissler K. van Bebber F. Lowe L.A. Nowotschin S. Viebahn C. Haffter P. Kuehn M.R. Blum M. Dev. 1999; 126: 1225-1234PubMed Google Scholar, 11Yoshioka H. Meno C. Koshiba K. Sugihara M. Itoh H. Ishimaru Y. Inoue T. Ohuchi H. Semina E.V. Murray J.C. Hamada H. Noji S. Cell. 1998; 94: 299-305Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 12Semina E.V. Reiter R.S. Murray J.C. Hum. Mol. Genet. 1997; 6: 2109-2116Crossref PubMed Scopus (169) Google Scholar, 13Logan M. Pagan-Westphal S.M. Smith D.M. Paganessi L. Tabin C.J. Cell. 1998; 94: 307-317Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 14Piedra M.E. Icardo J.M. Albajar M. Rodriguez-Rey J.C. Ros M.A. Cell. 1998; 94: 319-324Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 15Ryan A.K. Blumberg B. Rodriguez-Esteban C. Yonei-Tamura S. Tamura K. Tsukui T. de la Pena J. Sabbagh W. Greenwald J. Choe S. Norris D.P. Robertson E.J. Evans R.M. Rosenfeld M.G. Belmonte J.C.I. Nature. 1998; 394: 545-551Crossref PubMed Scopus (448) Google Scholar, 16St. Amand T.R., Ra, J. Zhang Y., Hu, Y. Baber S.I. Qiu M. Chen Y.P. Biochem. Biophys. Res. Commun. 1998; 247: 100-105Crossref PubMed Scopus (101) Google Scholar, 17Gage P.J. Suh H. Camper S.A. Development. 1999; 126: 4643-4651Crossref PubMed Google Scholar, 18Lu M. Pressman C. Dyer R. Johnson R.L. Martin J.F. Nature. 1999; 401: 276-278Crossref PubMed Scopus (433) Google Scholar, 19Lin C.R. Kioussi C. O'Connell S. Briata P. Szeto D. Liu F. Izpisua-Belmonte J.C. Rosenfeld M.G. Nature. 1999; 401: 279-282Crossref PubMed Scopus (498) Google Scholar). Several laboratories have shown that Pitx2 is a mediator of left-right signaling in vertebrates. Epigenetic studies suggested a role for Pitx2in the determination of vertebrate heart and gut looping (10Campione M. Steinbeisser H. Schweickert A. Deissler K. van Bebber F. Lowe L.A. Nowotschin S. Viebahn C. Haffter P. Kuehn M.R. Blum M. Dev. 1999; 126: 1225-1234PubMed Google Scholar, 11Yoshioka H. Meno C. Koshiba K. Sugihara M. Itoh H. Ishimaru Y. Inoue T. Ohuchi H. Semina E.V. Murray J.C. Hamada H. Noji S. Cell. 1998; 94: 299-305Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar,13Logan M. Pagan-Westphal S.M. Smith D.M. Paganessi L. Tabin C.J. Cell. 1998; 94: 307-317Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 14Piedra M.E. Icardo J.M. Albajar M. Rodriguez-Rey J.C. Ros M.A. Cell. 1998; 94: 319-324Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 15Ryan A.K. Blumberg B. Rodriguez-Esteban C. Yonei-Tamura S. Tamura K. Tsukui T. de la Pena J. Sabbagh W. Greenwald J. Choe S. Norris D.P. Robertson E.J. Evans R.M. Rosenfeld M.G. Belmonte J.C.I. Nature. 1998; 394: 545-551Crossref PubMed Scopus (448) Google Scholar, 16St. Amand T.R., Ra, J. Zhang Y., Hu, Y. Baber S.I. Qiu M. Chen Y.P. Biochem. Biophys. Res. Commun. 1998; 247: 100-105Crossref PubMed Scopus (101) Google Scholar). The analysis of Pitx2 −/− homozygous knockout mice reveals that Pitx2 is required for normal heart morphogenesis, development of the mandibular and maxillary facial prominences, and normal tooth and pituitary development (17Gage P.J. Suh H. Camper S.A. Development. 1999; 126: 4643-4651Crossref PubMed Google Scholar, 18Lu M. Pressman C. Dyer R. Johnson R.L. Martin J.F. Nature. 1999; 401: 276-278Crossref PubMed Scopus (433) Google Scholar, 19Lin C.R. Kioussi C. O'Connell S. Briata P. Szeto D. Liu F. Izpisua-Belmonte J.C. Rosenfeld M.G. Nature. 1999; 401: 279-282Crossref PubMed Scopus (498) Google Scholar). However, Pitx2 −/+ heterozygous mice display certain defects in embryogenesis as seen with the homozygous mice (17Gage P.J. Suh H. Camper S.A. Development. 1999; 126: 4643-4651Crossref PubMed Google Scholar,19Lin C.R. Kioussi C. O'Connell S. Briata P. Szeto D. Liu F. Izpisua-Belmonte J.C. Rosenfeld M.G. Nature. 1999; 401: 279-282Crossref PubMed Scopus (498) Google Scholar). A small fraction of heterozygous mice exhibit anterior chamber defects of the eye and heart defects (17Gage P.J. Suh H. Camper S.A. Development. 1999; 126: 4643-4651Crossref PubMed Google Scholar). These mice also failed to close the ventral body wall consistent with omphalocele found in Rieger patients (17Gage P.J. Suh H. Camper S.A. Development. 1999; 126: 4643-4651Crossref PubMed Google Scholar). Rieger syndrome results from haploinsufficiency consistent with some of the defects seen inPitx2 −/+ heterozygous mice (17Gage P.J. Suh H. Camper S.A. Development. 1999; 126: 4643-4651Crossref PubMed Google Scholar, 20Flomen R.H. Gorman P.A. Vatcheva R. Groet J. Barisic I. Ligutic I. Sheer D. Nizetic D. J. Med. Genet. 1997; 34: 191-195Crossref PubMed Scopus (30) Google Scholar). We and others have identified three major PITX2 isoforms produced by alternative splicing and use of different promoters (4Semina 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 (777) Google Scholar, 6Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (195) Google Scholar,9Arakawa H. Nakamura T. Zhadanov A.B. Fidanza V. Yano T. Bullrich F. Shimizu M. Blechman J. Mazo A. Canaani E. Croce C.M. Proc. Natl. Acad. Sci. 1998; 95: 4573-4578Crossref PubMed Scopus (97) Google Scholar, 21Gage P.J. Suh H. Camper S.A. Dev. Biol. 1999; 210: 234Google Scholar, 22Kitamura K. Miura H. Miyagawa-Tomita S. Yanazawa M. Katoh-Fukui Y. Suzuki R. Ohuchi H. Suehiro A. Motegi Y. Nakahara Y. Kondo S. Yokoyama M. Development. 1999; 126: 5749-5758Crossref PubMed Google Scholar). PITX2A and PITX2B are generated by alternative splicing mechanisms, and PITX2C uses an alternative promoter located upstream of exon 4 (see Fig. 1 below). All isoforms contain dissimilar N-terminal domains, whereas the homeodomain and C-terminal domains are identical. The C-terminal domain contains a highly conserved 14-amino acid region described in the homeobox genesOtp and aristaless (4Semina 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 (777) Google Scholar, 6Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (195) Google Scholar), which is called the OAR (Otp and aristaless) domain. Recent reports have provided evidence of Pitx2 isoform regulation in left-right asymmetry (23Essner J.J. Branford W.W. Zhang J. Yost H.J. Development. 2000; 127: 1081-1093Crossref PubMed Google Scholar, 24Liu C. Liu W., Lu, M. Brown N.A. Martin J.F. Dev. 2001; 128: 2039-2048PubMed Google Scholar, 25Yu X., St. Amand T.R. Wang S., Li, G. Zhang Y., Hu, Y. Nguyen L. Qiu M. Chen Y. Development. 2001; 128: 1005-1013PubMed Google Scholar). Epigenetic and genetic studies reveal that tissue and organ developments are differentially regulated byPitx2a and Pitx2c isoforms. In the chick it appears that Pitx2c plays a crucial role in the left-right axis determination and rightward heart looping during chick embryogenesis (25Yu X., St. Amand T.R. Wang S., Li, G. Zhang Y., Hu, Y. Nguyen L. Qiu M. Chen Y. Development. 2001; 128: 1005-1013PubMed Google Scholar). However, these researchers were unable to detect the Pitx2b isoform in chicks. Zebrafish are somewhat different in that Pitx2a has a greater impact on cardiac symmetry than Pitx2c (23Essner J.J. Branford W.W. Zhang J. Yost H.J. Development. 2000; 127: 1081-1093Crossref PubMed Google Scholar). In Zebrafish Pitx2c is asymmetrically expressed in the left dorsal diencephalon and developing gut, whereas Pitx2a is seen in the left heart primordium. Eloquent experiments in mice that were defective in Pitx2aand Pitx2b expression demonstrate that different organs have distinct requirements for Pitx2c dosage (24Liu C. Liu W., Lu, M. Brown N.A. Martin J.F. Dev. 2001; 128: 2039-2048PubMed Google Scholar). These researchers have shown that lower levels of Pitx2cexpression were required for cardiac atria and higher levels for duodenum and lung development. In contrast, other investigators have reported expression of Pitx2c and Pitx2b but notPitx2a in mice and frogs (26Schweickert A. Campione M. Steinbeisser H. Blum M. Mech. Dev. 2000; 90: 41-51Crossref PubMed Scopus (137) Google Scholar). They report overlapping and distinct patterns of Pitx2 expression in the lateral plate mesoderm, heart, gut, cement gland, head mesenchyma, pituitary gland, branchial arches, myotome, and muscles. Pitx2c andPitx2b were expressed in the head region of mice, and overlapping expression patterns were seen in the brain of frogs. However, they report only Pitx2c expression was observed during heart development in both the mouse and frog (26Schweickert A. Campione M. Steinbeisser H. Blum M. Mech. Dev. 2000; 90: 41-51Crossref PubMed Scopus (137) Google Scholar). Other experiments in mice have shown Pitx2 expression in the odontogenic epithelium, and it is the first transcriptional marker of tooth development (27Mucchielli M.-L. Mitsiadis T.A. Raffo S. Brunet J.-F. Proust J.-P. Goridis C. Dev. Biol. 1997; 189: 275-284Crossref PubMed Scopus (132) Google Scholar). More recently, we have shown that Pitx2 protein is restricted to the developing dental epithelium (28Hjalt T.A. Semina E.V. Amendt B.A. Murray J.C. Dev. Dyn. 2000; 218: 195-200Crossref PubMed Scopus (117) Google Scholar). Altogether these data demonstrate that Pitx2 isoforms are required either separately or in overlapping domains and in different doses to regulate normal vertebrate heart, lung, brain, tooth, pituitary, and gut development. However, the biochemical/molecular mechanisms of these effects have not been determined. Target genes for PITX2 have been described for the pituitary; theProlactin gene is synergistically activated by Pit-1 and PITX2 (3Amendt B.A. Sutherland L.B. Semina E. Russo A.F. J. Biol. Chem. 1998; 273: 20066-20072Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Other pituitary-specific Pitx2 target genes have also been described (29Tremblay J.J. Goodyer C.G. Drouin J. Neuroendocrinology. 2000; 71: 277-286Crossref PubMed Scopus (85) Google Scholar). However, we have now identified two genes outside of the pituitary that are specifically regulated by PITX2. We have shown that PITX2 regulates procollagen lysyl hydroxylase (PLOD) and Dlx2 gene expression (30Hjalt T.A. Amendt B.A. Murray J.C. J. Cell Biol. 2001; 152: 545-552Crossref PubMed Scopus (73) Google Scholar, 31Green P.D. Hjalt T.A. Kirk D.E. Sutherland L.B. Thomas B.L. Sharpe P.T. Snead M.L. Murray J.C. Russo A.F. Amendt B.A. Gene Expr. 2001; 9: 265-281Crossref PubMed Scopus (59) Google Scholar). The PLOD1 gene encodes an enzyme responsible for hydroxylizing lysines in collagens that plays a role in specifying the extracellular matrix and provides a foundation for the morphogenesis of tissues and organs. TheDlx2 gene encodes a transcription factor expressed in the mesenchymal and epithelial cells of the mandibular and maxillary regions and expressed in the diencephalon. Dlx2, a member of the distal-less gene family, has been established as a regulator of branchial arch development (32Qiu M. Bulfone A. Martinez S. Meneses J.J. Shimamura K. Pedersen R.A. Rubenstein J.L.R. Genes Dev. 1995; 9: 2523-2538Crossref PubMed Scopus (337) Google Scholar, 33Thomas B.L. Liu J.K. Rubenstein J.L.R. Sharpe P.T. Development. 2000; 127: 217-224PubMed Google Scholar). Homozygous mutants ofDlx2 have abnormal development of forebrain cells and craniofacial abnormalities in developing neural tissue. Dlxgenes exhibit both sequential and overlapping expression, implying that temporo-spatial regulation of Dlx genes are tightly regulated (34Liu J.K. Ghattas I. Liu S. Chen S. Rubenstein J.L.R. Dev. Dyn. 1997; 210: 498-512Crossref PubMed Scopus (179) Google Scholar). Pitx2 and Dlx2 genes are expressed in the same tissues early during development. These reports establish that the PITX2 family ofbicoid-like homeodomain genes are key regulators of important development processes and are required to regulate specific genes during embryogenesis. Our studies demonstrate differential activation of the PLOD1 and Dlx2 promoters by PITX2 isoforms in several cell lines. We demonstrate synergism between PITX2 isoforms, and their activities appear to be promoter-dependent. We report the identification of a newPITX2 isoform (PITX2D) generated by thePITX2C alternative promoter and differential splicing (Fig.1). We have only observed this isoform expressed in humans, and it was identified from a human craniofacial library. The PITX2D isoform acts to down-regulate the transcriptional activities of PITX2A and PITX2C. All isoforms can form homodimers, and heterodimers are formed with PITX2B. We demonstrate new regulatory mechanisms for the fine-tuning of PITX2 transcriptional activity that is required for normal development. Our data provide a molecular/biochemical basis for the developmental regulation of organ and tissue development by PITX2 isoforms reported in humans, zebrafish, chicks, frogs, and mice. The PITX2A isoform was PCR-amplified from a cDNA clone as described (3Amendt B.A. Sutherland L.B. Semina E. Russo A.F. J. Biol. Chem. 1998; 273: 20066-20072Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The PITX2B, PITX2C, and PITX2D isoforms were PCR-amplified from cDNA clones provided by Drs. Elena Semina and Jeff Murray (Department of Pediatrics, University of Iowa). The 5′-primers all contained the initiation codon and a uniqueSalI site, whereas the 3′-antisense primer contained PITX2 sequences downstream of the stop codon and a unique NotI site (5′-GTACTGCAGATGCGGCCGCAGCATAATTCCCAGTC-3′) to facilitate cloning into the pGEX6P-2 GST1 vector (AmershamBiosciences) as previously described (3Amendt B.A. Sutherland L.B. Semina E. Russo A.F. J. Biol. Chem. 1998; 273: 20066-20072Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 35Amendt B.A. Sutherland L.B. Russo A.F. Mol. Cel. Biol. 1999; 19: 7001-7010Crossref PubMed Scopus (105) Google Scholar). The 5′-primers were unique for each isoform and consisted of the following: PITX2B (5′-CGTCGTCGACATGGAGACCAATTGTCGC-3′), PITX2C (5′-CGTCGTCGACATGAACTGCATGAAAGGC-3′), PITX2D (5′-CGTCGTCGACATGTCCACACGCGAAGAA-3′). All pGST-PITX2 plasmids were confirmed by DNA sequencing. The plasmids were transformed into BL21 cells. Proteins were isolated as described previously (3Amendt B.A. Sutherland L.B. Semina E. Russo A.F. J. Biol. Chem. 1998; 273: 20066-20072Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). PITX2 proteins were cleaved from the GST moiety using 80 units of PreScission Protease (Amersham Biosciences) per milliliter of glutathione-Sepharose. The cleaved proteins were analyzed on SDS-polyacrylamide gels and quantitated by the Bradford protein assay (Bio-Rad). Complementary oligonucleotides containing the Dlx2 bicoid andbicoid-like sites with flanking partial BamHI ends were annealed and filled with Klenow polymerase to generate32P-labeled probes for EMSAs, as described previously (31Green P.D. Hjalt T.A. Kirk D.E. Sutherland L.B. Thomas B.L. Sharpe P.T. Snead M.L. Murray J.C. Russo A.F. Amendt B.A. Gene Expr. 2001; 9: 265-281Crossref PubMed Scopus (59) Google Scholar). Standard binding assays were performed as previously described (35Amendt B.A. Sutherland L.B. Russo A.F. Mol. Cel. Biol. 1999; 19: 7001-7010Crossref PubMed Scopus (105) Google Scholar). The bacteria-expressed and -purified PITX2 proteins were used in the assays at the indicated amounts. The samples were electrophoresed, visualized, and quantitated as described previously, except quantitation of dried gels was performed on the Molecular Dynamics Storm PhosphorImager (Amersham Biosciences) (31Green P.D. Hjalt T.A. Kirk D.E. Sutherland L.B. Thomas B.L. Sharpe P.T. Snead M.L. Murray J.C. Russo A.F. Amendt B.A. Gene Expr. 2001; 9: 265-281Crossref PubMed Scopus (59) Google Scholar). Immobilized GST-PITX2D fusion protein was prepared as described above and suspended in binding buffer (20 mm Hepes, pH 7.5, 5% glycerol, 50 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, 1% milk, and 400 μg/ml ethidium bromide). Purified bacteria-expressed PITX2 proteins (200 ng) were added to 5 μg of immobilized GST-PITX2D fusion proteins or GST in a total volume of 100 μl and incubated for 30 min at 4 °C. The beads were pelleted and washed four times with 200 μl of binding buffer. The bound proteins were eluted by boiling in SDS-sample buffer and separated on a 12.5% SDS-polyacrylamide gel. Approximately 200 ng of purified PITX2 proteins were analyzed in separate Western blots. Following SDS-gel electrophoresis, the proteins were transferred to polyvinylidene difluoride filters (Millipore), immunoblotted, and detected using PITX2 antibody P2R10 (28Hjalt T.A. Semina E.V. Amendt B.A. Murray J.C. Dev. Dyn. 2000; 218: 195-200Crossref PubMed Scopus (117) Google Scholar, 31Green P.D. Hjalt T.A. Kirk D.E. Sutherland L.B. Thomas B.L. Sharpe P.T. Snead M.L. Murray J.C. Russo A.F. Amendt B.A. Gene Expr. 2001; 9: 265-281Crossref PubMed Scopus (59) Google Scholar) and ECL reagents from Amersham Biosciences. The homeobox sequence of thePITX2 gene was PCR-amplified using the primers, sense, 5′-caggggaagaatgaggacgt-3′, and antisense, 5′-gaagccattcttgcatagct-3′, and the PITX2A plasmid as a template (4Semina 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 (777) Google Scholar). The 175-bp fragment containing sequences of the first exon of the PITX2D isoform was PCR-amplified using the primers, sense, 5′-ctgagctgcggcaaggc-3′, and antisense, 5′-ggcagccctgacagagatg-3′, and the PITX2D plasmid as a template (this report). The PCR fragments were separated by electrophoresis in agarose gel and extracted from the gel, and DNA was cleaned using a Qiagen gel extraction kit and labeled with32P using a random-prime labeling kit (Roche Molecular Biochemicals) according to the manufacturer's protocols. The following cDNA libraries were screened: human craniofacial (constructed from mRNA derived from the craniofacial region of human embryos ranging from 42- to 53-day gestation (36Padanilam B.J. Stadler H.S. Mills K.A. McLeod L.B. Solursh M. Lee B. Ramirez F. Buetow K.H. Murray J.C. Hum. Mol. Genet. 1992; 1: 407-410Crossref PubMed Scopus (47) Google Scholar), mouse embryonic carcinoma (Stratagene), and mouse 15-day embryo (Novagene). The hybridization, washing, exposure, identification of the positive clones, excision, and sequencing procedures were performed as previously described (4Semina 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 (777) Google Scholar). The exon-intron boundaries were identified by comparison of the identified cDNA and the PITX2 genomic sequence. By using the 266-bp homeobox sequence of the PITX2 as a probe, we identified multiple positive clones that can be divided into seven groups: sequences of PITX2 isoform A, PITX2isoform B, PITX2 isoform C,PITX2 isoform D, partial PITX2sequences that can be attributed to any isoform, and various sequences belonging to either the PITX1 or PITX3genes. The PITX2 A–C isoforms were described before (4Semina 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 (777) Google Scholar, 6Gage P.J. Camper S.A. Hum. Mol. Genet. 1997; 6: 457-464Crossref PubMed Scopus (195) Google Scholar, 9Arakawa H. Nakamura T. Zhadanov A.B. Fidanza V. Yano T. Bullrich F. Shimizu M. Blechman J. Mazo A. Canaani E. Croce C.M. Proc. Natl. Acad. Sci. 1998; 95: 4573-4578Crossref PubMed Scopus (97) Google Scholar, 21Gage P.J. Suh H. Camper S.A. Dev. Biol. 1999; 210: 234Google Scholar). The PITX2D isoform was only identified from the human craniofacial library: two independent clones were isolated from the 3 × 106 clones examined. ThePITX2D sequence consists of three exons: the first exon (178 bp), which was found to be located 230-bp upstream of the first exon of the PITX2C isoform as it was identified from the human craniofacial library; the second exon (77 bp) representing a partial sequence of the PITX2 exon 5 lacking 129 of its 5′-nucleotides; and the third exon (1258 bp) that is equivalent to thePITX2 exon 6 (Fig. 1). By searching GenBankTM, we identified that the first exon of the PITX2D isoform was found to be a part of the first exon of the PITX2C isoform in two independent submissions: IMAGE 3937807 clone isolated from the lung library and ARP1C(PITX2C) cDNA. Sequences at the exon/intron junctions for the PITX2D were identified for the 5′-splice site, GGGCTGCCGC/gt, and for the 3′-splice site, cactttcc/AGAGGAACAGC. It is notable that the nucleotides at positions −1 and −2 of the 3′-splice site (CC) do not correspond with the conserved sequence identified as AG. The PITX2D isoform is predicted to encode the 205-amino acid protein with the initiation codon for methionine (ATG) located at the beginning of the second helix sequence of the homeobox. Additional screening of the above described libraries with the fragment containing the first exon sequence of the PITX2D isoform failed to identify any clones from the mouse cDNA libraries as well as any additional clones from the human craniofacial cDNA library. Expression plasmids containing the cytomegalovirus (CMV) promoter linked to the PITX2 DNA were constructed in pcDNA 3.1 MycHisC (Invitrogen) (35Amendt B.A. Sutherland L.B. Russo A.F. Mol. Cel. Biol. 1999; 19: 7001-7010Crossref PubMed Scopus (105) Google Scholar). Constructions of the Prolactin, Dlx2, andPLOD1 promoter plasmids have been previously described (3Amendt B.A. Sutherland L.B. Semina E. Russo A.F. J. Biol. Chem. 1998; 273: 20066-20072Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,30Hjalt T.A. Amendt B.A. Murray J.C. J. Cell Biol. 2001; 152: 545-552Crossref PubMed Scopus (73) Google Scholar, 31Green P.D. Hjalt T.A. Kirk D.E. Sutherland L.B. Thomas B.L. Sharpe P.T. Snead M.L. Murray J.C. Russo A.F. Amendt B.A. Gene Expr. 2001; 9: 265-281Crossref PubMed Scopus (59) Google Scholar). All constructs were confirmed by DNA sequencing. A CMV β-galactosidase reporter plasmid (CLONTECH) was co-transfected in all experiments as a control for transfection efficiency. CHO, HeLa, and LS-8 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum and penicillin/streptomycin in 60-mm dishes and transfected by electroporation. CHO, HeLa, and LS-8 cells were mixed with 2.5 μg of expression plasmids, 5 μg of reporter plasmid, and 0.5 μg of CMV β-galactosidase plasmid plated in 60-mm culture dishes and fed with 5% fetal bovine serum and Dulbecco's modified Eagle's medium. Electroporation of CHO cells was performed at 360 V and 950 microfarads (Bio-Rad), and electroporation of HeLa cells was at 220 V and 950 microfarads. The cells were fed 24 h prior to transfection. LS-8 cells were transfected by electroporation as previously described (31Green P.D. Hjalt T.A. Kirk D.E. Sutherland L.B. Thomas B.L. Sharpe P.T. Snead M.L. Murray J.C. Russo A.F. Amendt B.A. Gene Expr. 2001; 9: 265-281Crossref PubMed Scopus (59) Google Scholar). Transfected cells were incubated for 24 h then lysed and assayed for reporter activities and protein content by Bradford assay (Bio-Rad). Luciferase was measured using reagents from Promega. β-Galactosidase was measur" @default.
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• W1972959575 title "Differential Regulation of Gene Expression by PITX2 Isoforms" @default.
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