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Originally published In Press as doi:10.1074/jbc.M201737200 on April 10, 2002

J. Biol. Chem., Vol. 277, Issue 28, 25001-25010, July 12, 2002
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Differential Regulation of Gene Expression by PITX2 Isoforms*

Carol J. CoxDagger , Herbert M. EspinozaDagger , Bryan McWilliamsDagger , Kimberly ChappellDagger , Lisa MortonDagger , Tord A. Hjalt§, Elena V. Semina§, and Brad A. AmendtDagger

From the Dagger  Department of Biological Science, The University of Tulsa, Tulsa, Oklahoma 74104-3189 and the § Department of Pediatrics, The University of Iowa, Iowa City, Iowa 52242

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 the PLOD1 and Dlx2 promoters, however, PITX2B is inactive. In contrast, PITX2B actives the pituitary-specific Prolactin promoter at higher levels than either PITX2A or PITX2C. Interestingly, co-transfection of either PITX2A or PITX2C 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.

* This work was supported by Grant 1-RO1-DE13941 from the NIDCR, National Institutes of Health (to B. A. A.) and by the Fight For Sight Research Division of Prevent Blindness America (Postdoctoral Grant PD99018 to T. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biological Sciences, The University of Tulsa, 600 S. College Ave., Tulsa, OK 74104-3189. Tel.: 918-631-3328; Fax: 918-631-2762; E-mail: brad-amendt@utulsa.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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