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D. Bheeman, H. Strungaru, F. B. Berry, M. A. Walter; Discovery of Genes Directly Regulated by the Transcription Factors FOXC1 and PITX2. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1726.
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© ARVO (1962-2015); The Authors (2016-present)
Axenfeld-Rieger Syndrome (ARS) is an autosomal dominant disorder of the eye often associated with glaucoma. ARS can result from mutations of two transcription factor genes, FOXC1 and PITX2. In order to understand the involvement of FOXC1 and PITX2 in ARS and glaucomatous development, it is critical to study the genetic regulatory pathways controlled by FOXC1 and PITX2 in ocular tissues.
We utilized, in parallel, caged FOXC1 and PITX2 proteins that can be activated in the presence of protein synthesis inhibitors to identify genes directly regulated by the FOXC1 and PITX2 transcription factors. This expression system was coupled with microarray experiments to identify potential FOXC1- and PITX2-target genes. Microarray analysis of cells expressing either of the caged FOXC1 and PITX2 molecules were compared and resulted in the identification of 15 genes that were potentially differentially regulated by both FOXC1 and PITX2. Northern blot and/or quantitative RT-PCR were used to validate the altered expression of these genes in the presence of FOXC1 and PITX2 in human non-pigmented ciliary epithelium (NPCE) cells derived from the anterior segment of the eye.
Our analysis revealed that ITPR1 (inositol 1, 4, 5 - triphosphate receptor, type 1), SIVA and HSPA6 (heat shock 70kDa protein 6) are regulated by both FOXC1 and PITX2 in ocular cells. We have chosen to investigate SIVA as it is a proapoptotic and stress response gene. We have identified the consensus binding sites of both FOXC1 and PITX2 in the upstream promoter region of SIVA. Invivo promoter assays and EMSA are being done to see the transcriptional activity and specific binding of FOXC1 and PITX2 to SIVA gene.
Genes regulated by both FOXC1 and PITX2 in the eye are involved in apoptosis and stress pathways. Understanding the role of genes regulated by both FOXC1 and PITX2 will yield new insights into the gene regulatory pathways disrupted by these two transcription factors in ARS patients and in glaucoma pathogenesis.
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