1: Invest Ophthalmol Vis Sci. 2004 Nov;45(11):3904-13. 

Identification of target genes regulated by FOXC1 using nickel agarose-based
chromatin enrichment.

Tamimi Y, Lines M, Coca-Prados M, Walter MA.

Department of Medical Genetics, University of Alberta, Edmonton, Canada.

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.

PMID: 15505035 [PubMed - indexed for MEDLINE]


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