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C.K. Birdi, M.W. Sharp, T. Footz, F. Mirzayans, M.A. Walter; Analyses of the Novel PITX2–Interacting Protein FIBULIN–4 as a Candidate Gene for Axenfeld–Rieger Malformations . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5444.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the homeobox transcription factor PITX2 cause Axenfeld–Rieger malformations (ARM) often leading to glaucoma. Understanding the function of PITX2 necessitates characterizing PITX2 interacting proteins. These interactions may modulate transcriptional activity of PITX2 and thus regulate gene expression in the eye. We therefore endeavored to isolate and characterize PITX2 interacting proteins. Genes encoding PITX2 interacting proteins are also candidates for ARM and glaucoma.
To identify PITX2 interacting proteins, we created a human trabecular meshwork yeast two–hybrid cDNA library and screened 300,000 clones for an interaction with PITX2. Clones supporting growth in four yeast reporter assays were selected and fourteen such clones corresponded to cDNAs encoding FIBULIN–4. This interaction was further analyzed by co–immunoprecipitation (Co–IP) assays. Co–transfected cells with FIBULIN–4 and PITX2 were used to determine the effect of their interaction on the cellular localization of each protein. Lastly, 55 ARM patients without mutations in known ARM genes were screened for possible mutations in FIBULIN–4.
Co–IP assays verified the PITX2–FIBULIN–4 interaction. Further analyses indicated that the PITX2–FIBULIN–4 interaction involves multiple domains, refinement of which is in progress. Co–transfected COS–7 cells revealed that FIBULIN–4 and PITX2 displayed a significant overlap in localization. PITX2 significantly relocated from the nucleus to the cytoplasm in the presence of FIBULIN–4. Simultaneously, FIBULIN–4 relocalized to a more nuclear localization in the presence of PITX2. Within the nucleus, the PITX2–FIBULIN–4 complex was localized to the euchromatic regions, consistent with a role for the complex in regulation of gene expression. Preliminary mutational screening revealed 6 alterations of FIBULIN–4 in ARM patients. Several alterations involved amino acid residues conserved in organisms from humans to zebrafish. Interestingly, Stone and colleagues, who recently screened FIBULIN–4 as a candidate for Age–related macular degeneration (AMD), did not observe these latter changes in 402 AMD patients and in 263 controls.
FIBULIN–4 is a PITX2 interacting protein. Discovery that the PITX2–EFEMP2 interaction appears to alter the localization of each protein suggests that the interaction has a functional consequence. The discovery of nucleotide alterations of FIBULIN–4 in ARM patients may indicate a direct role for FIBULIN–4 in ARM and glaucoma.
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