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Y. Ito, F. Berry, M. A. Walter; Analysis of FOXC1 Direct Target Genes That Are Associated With Cellular Stress Response. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3631.
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FOXC1 mutations cause human Axenfeld-Rieger Syndrome (ARS), an autosomal-dominant disease that affects the anterior segment of the eye. ARS patients have an elevated risk to develop early-onset glaucoma. Correct expression of FOXC1 is essential for the normal development of the cardiac, skeletal, and urogenital systems, as well as ocular tissues. In addition, the FOXC1 transcription factor appears to have important functions in the adult eye, such as maintaining cell homeostasis by direct regulation of FOXO1. The purpose of this investigation was to examine molecular chaperones that are potential FOXC1 target genes and to determine whether FOXC1 plays a role in stress response of the cell.
Previous work in our laboratory using microarray technology has led to the identification of potential genes that are regulated by FOXC1. Several genes involved in cellular stress response were identified including heat shock protein 27 (HSP27) and heat shock protein 70 (HSP70). Experiments to validate the genes as bona fide target genes of FOXC1 were carried out by examining RNA and protein levels by northern and western analysis, respectively, chromatin immunoprecipitation (ChIP), and transactivation assays.
HSP27 was validated as an authentic direct target gene of FOXC1. In agreement with the microarray results which showed that HSP27 RNA levels decreased when FOXC1 was overexpressed, HSP27 RNA levels increased when FOXC1 was knocked down by siRNA. Interestingly, HSP27 protein levels increased when FOXC1 was knocked down by siRNA. ChIP analysis identified a bona fide FOXC1 binding site located in the upstream promoter region of the HSP27 gene.
Validation of HSP27 as a direct target gene of FOXC1 supports the idea that FOXC1 plays a role in responding to stress. Molecular chaperones such as HSP27 interact with unfolded or partially folded protein intermediates to promote efficient protein folding in vivo. Dysregulation of molecular chaperones by FOXC1 may disrupt the protein quality control system eventually overloading the protein degradation pathway. The resulting stress may lead to abnormal cell function and apoptosis, thus underlying part of the pathophysiology of ARS.
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