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Identification of Target Genes Regulated by FOXC1 Using Nickel Agarose–Based Chromatin Enrichment

¹From the Departments of Medical Genetics and ³Ophthalmology, University of Alberta, Edmonton, Alberta, Canada; and the ²Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut.

PURPOSE. To overcome the problem of antibody availability, often encountered during chromatin immunoprecipitation (ChIP) assays, nickel agarose–based chromatin enrichment (NACE) was developed. Based on the affinity of (His)-₆-tagged proteins for the nickel ion, this modified form of ChIP allows the isolation of chromatin in the absence of specific antibodies.

METHODS. Nonpigmented ciliary epithelium cells were transfected with (His)-₆-tagged FOXC1. FOXC1-enriched chromatin complexes were isolated by using the tight electrostatic interaction between histidine residues of the recombinant FOXC1 protein and nickel. One hundred fifty NACE-enriched clones were sequenced and subjected to in silico and biochemical analyses.

RESULTS. Twenty-six clones were detected near known genes: Eight were near predicted but uncharacterized genes, eight were within areas where neither known nor predicted genes have yet been mapped, four were chimeric, and the rest were either repetitive (n = 81) or poor-quality (n = 23) sequences. Twenty of the 26 known genes were expressed in the eye. Five of the NACE-enriched clones (BMP2K, DACH, FVT-1, SIX-1, and PGE-2 receptor), as well as nine clones selected from the literature, were validated by PCR amplification in two independent lots of NACE-enriched chromatin. All five NACE-selected genes were detected in two independent assays, as well as four (BMP7, SMAD2, TGF-B1, and WNT6) of the nine genes selected from the literature, consistent with these genes’ being regulated by FOXC1.

CONCLUSIONS. NACE is a useful technique allowing specific chromatin enrichment in cases where antibodies are unavailable. Specific recovery of PTGER, DACH1, WNT6, and FVT-1 implicates FOXC1 in a variety of cellular events including modulation of intraocular pressure, cell cycle, ocular development, and oncogenesis.

This article has been cited by other articles:

				L. Huang, J. Chi, F. B. Berry, T. K. Footz, M. W. Sharp, and M. A. Walter Human p32 Is a Novel FOXC1-Interacting Protein That Regulates FOXC1 Transcriptional Activity in Ocular Cells Invest. Ophthalmol. Vis. Sci., December 1, 2008; 49(12): 5243 - 5249. [Abstract] [Full Text] [PDF]

				Y. Tamimi, J. M. Skarie, T. Footz, F. B. Berry, B. A. Link, and M. A. Walter FGF19 is a target for FOXC1 regulation in ciliary body-derived cells Hum. Mol. Genet., November 1, 2006; 15(21): 3229 - 3240. [Abstract] [Full Text] [PDF]