May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Identification of Novel Genes Regulated by FOXC1 using Chromatin Immunoprecipitation (ChIP)
Author Affiliations & Notes
  • Y. Tamimi
    Ophthalmology, University of Alberta, Edmonton, AB, Canada
  • M. Lines
    Ophthalmology, University of Alberta, Edmonton, AB, Canada
  • F. Berry
    Ophthalmology, University of Alberta, Edmonton, AB, Canada
  • M. Coca-Prados
    Ophthalmology, Yale University, New Haven, CT, United States
  • M. Walter
    Ophthalmology, Yale University, New Haven, CT, United States
  • Footnotes
    Commercial Relationships  Y. Tamimi, None; M. Lines, None; F. Berry, None; M. Coca-Prados, None; M. Walter, None.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1108. doi:
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      Y. Tamimi, M. Lines, F. Berry, M. Coca-Prados, M. Walter; Identification of Novel Genes Regulated by FOXC1 using Chromatin Immunoprecipitation (ChIP) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1108.

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

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Abstract

Abstract: : Purpose: FOXC1 gene, a member of the forkhead family, plays a key role in the proper formation of organs especially the eye and the heart. Mutations in FOXC1 are responsible for Axenfield Rieger malformations in the anterior segment of the eye and lead to an increase susceptibility of glaucoma. Our group has previously shown that the disease-causing missense mutations affect the FOXC1 transcription activation by decreasing FOXC1 activity (Saleem et al. 2001, Am. J. Hum. Genet. 68:627-41). The identity of the genes regulated by FOXC1 in normal development and adversely affected by FOXC1 mutations, are not known. Therefore, we hypothesize that the identification of such targets will shed light on our undestanding of the FOXC1 role in developmental diseases. Genes that are regulated by FOXC1 could be involved in glaucoma pathogenesis, and their identification could yield new targets for glaucoma therapy. Methods: Chromatin immunoprecipitation (ChIP), a chromatin enrichment method based on the pulling down of fixed complexes of chromatin-proteins of interest was performed as described (Hyalt et al 2001). Human ocular non pigmented celliary epithelium (NPCE) cells were transfected with a vector containing FOXC1 and subjected to ChIP analysis. Enriched DNA was cloned into pBluescript vector and sequenced prior to computer-based search analysis. Results: In preliminary experiments, one hundred twenty clones were chosen for analysis and several predicted unknown genes were found. However, twelve hits were detected in proximity to known genes. Expresssion analysis are under way to confirm positive/negative regulation by FOXC1 in eye tissues. To determine if the candidate genes isolated by the ChIP experiments are regulated by FOXC1, 5' elements of the candidate genes will be placed in a luciferase reporter plasmid and tested for regulation by FOXC1. Conclusion: These experiments will help define the role of FOXC1 in eye organogenesis and diseases. Understanding the mechanisms underlying FOXC1 regulation will be a major step toward efficient treatments for Axenfield Rieger syndrome and other diseases that might be related to genes involved in the pathway regulated by FOXC1.

Keywords: molecular biology • genetics • gene microarray 
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