May 2004
Volume 45, Issue 13
ARVO Annual Meeting Abstract  |   May 2004
Identification of FOXC1 targets genes utilizing a novel caged FOXC1 molecule and microarray analysis.
Author Affiliations & Notes
  • F.B. Berry
    Ophthalmology, University of Alberta, Edmonton, AB, Canada
  • Y. Fortin
    Genome Quebec Innovation Centre, McGill University, Montreal, PQ, Canada
  • T.J. Hudson
    Genome Quebec Innovation Centre, McGill University, Montreal, PQ, Canada
  • V. Raymond
    Molecular Endocrinology and Oncology, CHUL Research Center, Quebec, PQ, Canada
  • M.A. Walter
    Ophthalmology, University of Alberta, Edmonton, AB, Canada
  • Footnotes
    Commercial Relationships  F.B. Berry, None; Y. Fortin, None; T.J. Hudson, None; V. Raymond, None; M.A. Walter, None.
  • Footnotes
    Support  CIHR
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 2248. doi:
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      F.B. Berry, Y. Fortin, T.J. Hudson, V. Raymond, M.A. Walter; Identification of FOXC1 targets genes utilizing a novel caged FOXC1 molecule and microarray analysis. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2248.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Mutations in FOXC1 result in anterior segment dysgenesis and an increased susceptibility to develop glaucoma. Therefore it is imperative to identify the target genes regulated by FOXC1. Methods: A hormone inducible FOXC1 (FOXC1–HR) protein was constructed by inserting the progesterone ligand–binding domain between the FOXC1 DNA–binding domain and the C–terminal activation domain. This inactive, caged, FOXC1–HR protein is activated upon administration of mifepristone. Since the induction does not require new protein synthesis, cells can be treated with cycloheximade (CHX) to block translation of newly transcribed mRNAs, thus enriching for transcipts produced in direct response to FOXC1–HR. Non–pigmented ciliary epithelial (NPCE) cells were transfected with FOXC1–HR or with an empty expression vector as a control, then treated with CHX (100 ug/ml) and mifepristone (10–8 M) for 12 hours. RNA was harvested, reverse transcribed to cDNA and hybridized to the Affymetrix U133A gene chip. Data from 3 independent hormone induction and CHX treatments were analysed by two statistical methods, D–Chip and RMA analyses. Results:Of the 22,216 genes present on the Affymetrix U133A gene chip, 248 were differentially expressed in FOXC1–HR samples by both statistical analyses in all three experiments (p<0.05). Gene ontogeny profiling revealed key genes involved in a number of biological and molecular processes including cell growth, apoptosis, vesicle mediated transport, protein biosynthesis, transcription, signal transduction, cell structure and vision. The data set was further refined by identifying genes that had overlapping expression profiles as FOXC1, were expressed at high levels in the eye, and contained a FOXC1 core binding sequence 5'–TAAAYA–3' within 3 kb upstream of the 5' most exon. 44 genes met these criteria and were selected for further analysis. Conclusions: This novel experimental design allows a high throughput identification of primary transcripts produced in response FOXC1,thus likely candidates for FOXC1 transcriptional regulation. The diverse biological roles of the genes identified in this study indicate an involvement for FOXC1 in regulating developmental processes as well as maintenance of the differentiated cell state. These data suggest that ocular phenotypes resulting from FOXC1 mutations may arise from combinatorial misregulation of a number of FOXC1 targets rather than a single gene.

Keywords: anterior segment • transcription factors • gene microarray 

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