IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2004;45:3904-3913.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0628
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tamimi, Y.
Right arrow Articles by Walter, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tamimi, Y.
Right arrow Articles by Walter, M. A.

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

Yahya Tamimi,1 Matthew Lines,1 Miguel Coca-Prados,2 and Michael A. Walter1,3

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.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
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.

Eukaryotic chromatin is an organized structural hierarchy in which the smallest unit is the nucleosome, a complex of ~150 bp of DNA wrapped around two copies of each of four histones: H2A, -2B, -3, and -4.1 These histone-DNA complexes, associated with other components in the nucleus, are continuously interacting with one another to ensure the dynamic that characterizes living cells. Before transcription of target genes begins, transcription factors are gathered under complex interactions within the nucleus, as, simultaneously, the chromatin undergoes essential transformations (i.e., acetylation) to reach a relaxed form suitable for transcription initiation.2 However, the dynamic and structural complexity of chromatin usually hampers gene regulation studies. Chromatin immunoprecipitation (ChIP) is a powerful emerging technique designed to overcome this problem and to identify genes bound by transcription factors.3 4 5 The ChIP technique involves formaldehyde treatment of cells to cross-link transcription factors to their target elements on DNA in vivo. After cell lysis, the protein-DNA complexes are recovered by immunoprecipitation, using an antibody to the transcription factor of interest. After reversing the cross-link, the target element DNA is analyzed by different methods including PCR amplification. The resultant PCR product minilibraries can be sequenced and compared to public DNA sequence databases to discover the identity and likely function of nearby genes.6 Alternatively, the ChIP DNA pools can be labeled and hybridized to microarrays containing a large selection of expressed sequence tags (ESTs) or promoters.7 8

ChIP analyses have been applied successfully to identify targets of yeast and mammalian transcription factors.9 10 In the yeast Saccharomyces cerevisiae, ChIP was used to identify targets of the Gal4 and Ste12 transcription factors. Of the 10 Gal4 targets identified, 7 have been shown to be Gal4 targets, suggesting that ChIP analysis is robust and successful in obtaining bona fide target promoters.10 The Gal4 DNA target sequence was found in these promoters. In mammals, ChIP analyses revealed that 127 promoters involved in the cell cycle were bound by the E2F4 repressor and that a subset of these were also bound by the E2F1 activator.9

FOXC1 (formerly FREAC3 or FKHL7) is a member of the Forkhead box (Fox) family of transcription factor genes named for a well-conserved 100-amino-acid DNA-binding domain homologous to Drosophila forkhead. FOXC1 is a monoexonic gene of 1.6 kb encoding a protein of 553 amino acids11 12 located on chromosome 6 at p2513 14 and known to be expressed in the eye.15 Mutations in FOXC1 are responsible for Axenfeld-Rieger (AR) malformations mapped to 6p25.16 The most serious consequence for patients with AR is an approximately 50% risk of the development of glaucoma. How FOXC1 mutations lead to glaucoma is unknown. However, identification of potential candidate genes regulated by FOXC1 is a prerequisite for defining the role of FOXC1 in eye organogenesis and diseases. To address this question and to overcome the absence of an appropriate FOXC1 antibody, we modified the ChIP technique. Chromatin was affinity purified by using nickel agarose–based chromatin enrichment (NACE) to identify genes regulated by FOXC1 in the eye.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Cell Culture and Transient Transfection Assay
Nonpigmented ciliary epithelial (NPCE) cells17 were maintained and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (Gemini Bioproducts, Calabasas, CA), 50 U/mL penicillin, and 50 µg/mL streptomycin. Plates 100 mm in diameter containing 2 x 106 cells were transiently transfected, at 70% to 80% confluence, by 4 µg plasmid DNA (pcDNA4 His/Max FOXC1) through use of a transfection reagent (FuGene6; Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol. The NPCE cells were also transfected with an empty vector (pcDNA4 His/Max), a FOXC1 {Delta}Box control (FOXC1 lacking the forkhead domain), or a non-FOXC1 binding site vector, as negative controls. After 48 hours of incubation at 37°C in a humidified atmosphere containing 5% CO2, cells were subjected to NACE.

Nickel Agarose–Based Chromatin Enrichment
Ni2+-NTA agarose beads (Qiagen; Valencia, CA) were used to isolate chromatin complexes containing the (His)-6-FOXC1 recombinant fusion protein from nuclear lysates. Figure 1 illustrates schematically the different steps of NACE protocol. Briefly, transfected NPCE cells were brought to room temperature (RT) and cross-linked by adding formaldehyde directly to tissue culture medium to a final concentration of 1% and left at RT for 15 minutes with gentle shaking. The cross-linking was stopped by adding glycine to a final concentration of 0.125 M at RT for 5 minutes on a shaking platform. Cross-linked cells were washed twice with cold 1x phosphate-buffered saline (137 mM NaCl, 10 mM sodium phosphate buffer [pH 7.4], and 2.7 mM KCl), scraped, and lysed at a density of 5 x 106 cells/mL for 10 minutes in ice-cold cell lysis buffer (5 mM piperazine-N-N'-bis(2-ethanesulfonic acid [PIPES; pH 8], 85 mM KCl, and 0.5% NP40 + protease inhibitors). Nuclei were pelleted by microcentrifugation at 6000 rpm, resuspended in 1x Ni2+-NTA resin binding buffer (50 mM NaH2PO4 [pH 8], 300 mM NaCl, and 10 mM imidazole) containing protease inhibitors (mammalian protease inhibitors; Sigma-Aldrich, St. Louis, MO) sonicated for 10 rounds of 20-second cycles, at 2-minute intervals, at 40% amplitude (Sonic Dismembrator, model 500; Fisher Scientific, Pittsburgh, PA). The sonicated chromatin (≤1.5 kb) was centrifuged at 4°C for 30 minutes at 12,000g and the supernatant transferred to a new tube containing 300 µL of charged nickel resin before overnight incubation in a cold room with gentle rocking. At this stage, a positive control (100% input, no Ni2+-NTA added to sample) and a negative control (mock, H2O plus Ni2+-NTA) were also included. Samples were centrifuged at 3000 rpm in 4°C, supernatant was removed, and the bead pellet was washed in 1 mL washing buffer (50 mM NaH2PO4 [pH 8], 300 mM NaCl, and 10 mM imidazole) for 10 minutes with gentle shaking. The supernatant from the 100% input was set aside and used as a positive control for PCR amplification (described later). The washing procedure was repeated four times, to eliminate a maximum of nonspecific binding. The chromatin was eluted from the beads four times by adding 200 µL of elution buffer (50 mM NaH2PO4 [pH 8], 300 mM NaCl, and 250 mM imidazole), for 15 minutes at RT with rotation; centrifuging at 3000 rpm; and removing the supernatant without disturbing the nickel resin, by using a 26-gauge syringe. Eluates were treated for 5 hours at 70°C with 1 µL of 10 mg/mL RNase A and 50 µL of 5 M NaCl, to reverse the cross-linking, and precipitated overnight at –20°C in a 2.5-volume of ethanol. Samples were then microfuged at 14,000g for 20 minutes, and pellets were dissolved in 100 µL TE (50 mM Tris-Cl [pH 7.5], 25 mM EDTA), 25 µL of 5x proteinase K buffer, and 1.5 µL of 40 µg/mL proteinase K and incubated for 2 hours at 45°C to remove protein. Finally, the DNA was purified (QIAQuick kit; Qiagen). At this stage, target DNA was enriched and ready for cloning or for PCR amplification.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 1. Schematic representation of the NACE procedure. DNA-binding proteins are cross-linked to DNA with formaldehyde in vivo. Stars: recombinant (His)-6-tagged FOXC1, hexagons represent other transcription factors present in the cells. After isolating the chromatin, the DNA along with bound proteins is sheared into small fragments by sonication. FOXC1 chromatin complexes are specifically isolated by nickel agarose (polygons). FOXC1-enriched chromatin is obtained after eluting and reversing the cross-linking to release the DNA and digest the proteins.

 
Cloning, Construct Preparation, and Sequencing
Three hundred nanograms of enriched chromatin was added to 5 µL of 10x end-filling buffer (50 mM Tris-HCl [pH 8] and 10 mM MgCl2), 10 µL of 0.25 mM dNTPs, and 3 U Klenow enzyme in a total volume of 50 µL. After an incubation of 1 hour at 37°C, the reaction was stopped at 75°C, and DNA was purified (Qiagen columns). One hundred fifty nanograms of the end-filled insert (enriched chromatin) was then added to 50 ng of EcoRV-digested DNA vector (pBluescript SKII; Stratagene, La Jolla, CA), 5 µL of 5x ligation buffer (60 mM Tris-HCl [pH 8], 20 mM MgCl2, and 20 mM dithiothreitol [DTT]), 5 µL of 1 M polyethylene glycol, and 5 U of T4 ligase enzyme. The reaction was finally incubated at 5°C for at least 15 hours. The ligation product was subsequently used for the transformation of XL-Blue cells. Transformed cells were plated, and putative positive clones, based on white/blue selection, were cultured overnight at 37°C in 5 mL Luria-Bertani (LB) medium before DNA extraction (Miniprep kit; Qiagen). The extracted DNA was subsequently digested with the BamH1 and HindIII restriction enzymes, to identify the clones containing the insert.

Fifty picomoles of forward and reverse M13 primers conjugated to chromophores emitting at different wavelengths (700 and 800 nm), were added to 10 µL of extracted DNA (1–3 µg) and subjected to a sequencing reaction with a fluorescence-labeled primer cycle sequencing kit with 7-deaza 2-dGTP (Thermo Sequenase; Amersham Pharmacia Biotech, Ltd., Amersham, UK).

PCR Analysis
Sequences of oligonucleotide primer pairs used for PCR amplification are listed in Table 1 and were designed for each gene (Primer-3 program; http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi; provided in the public domain by Massachusetts Institute of Technology, Cambridge, MA). The PCR reactions were performed in total volumes of 50 µL with a 50-second melting step at 95°C, an annealing step between 52 °C and 58°C (Tm – 5°C) for 50 seconds, and an elongation step at 72°C for 50 seconds. PCR reactions were preceded by a 5-minute melting period at 95°C and ended with an elongation period of 10 minutes at 72°C. The PCR products were resolved on 1% agarose gels, and bands were visualized by staining with ethidium bromide.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Sequences of the Oligonucleotide Used for PCR Amplification Assays

 
In Silico Analysis
Clones were sequenced and analyzed in silico using BLAST against the Ensembl database (http://www.ensembl.org).18 As well, expression analysis of candidate genes in different tissues was performed using the Source Web site (http://source.stanford.edu/cgi-bin/source/sourceSearch/ provided in the public domain by Stanford, University, Stanford, CA).


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
NACE Assay Specificity
To confirm the specificity of the enriched chromatin obtained by NACE, NPCE cells were transiently transfected with a FOXC1-pcDNA His/Max vector,19 which expresses 6xHis-Xpress–tagged FOXC1, along with a reporter gene (luciferase) containing upstream FOXC1 binding sites (pGL3-TK-FOXC1).19 The enriched chromatin was then analyzed by PCR amplification of the DNA flanking the FOXC1 binding site. The forward primer was designed in the pGL3-Basic vector upstream of the KpnI site, whereas the reverse primer was located in the TK promoter within the pGL3 vector. A product of 251 bp was obtained as expected in the positive control but not in samples without Ni2+-NTA or in the DNA mock samples (Fig. 2) , confirming the specificity of the NACE assays. Furthermore, sequence analysis confirmed the identity of the obtained bands.



View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 2. NACE specificity assay. NPCE cells were transfected with (His)-6-tagged FOXC1 and enriched by NACE. The enriched chromatin was subjected to PCR amplification with primers flanking the FOXC1 binding site. Bands of the expected sizes (arrow) were obtained specifically in FOXC1-transfected samples enriched by NACE. Chromatin enriched from NPCE cells transfected by an empty vector was used as the negative control along with water and nickel agarose beads (mock).

 
NPCE cells express FOXC1 endogenously (Fig. 3A) and therefore are an excellent reagent to use for FOXC1 target gene discovery. To isolate candidate target genes regulated by FOXC1, pcDNA FOXC1 was transfected into NPCE cells, as described earlier. One hundred fifty genomic DNA clones representing putative FOXC1 target sequences were randomly selected for sequence analysis using M13 forward and reverse primers. The size-range of the clones obtained varied from 500 to 1500 bp.



View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 3. Expression of selected genes in the NPCE cell line. Confluent NPCE cells were harvested and subjected to RNA extraction. Reverse transcription was performed with oligo dT primers and amplified to monitor the expression of FOXC1 (A) as well as the selected genes (BMP2K, DACH, FVT-1, PGER2, and SIX-1) validated in NACE products (B).

 
In silico analysis of the sequences revealed 104 clones yielding sequences of poor quality or containing only repetitive elements, precluding further analyses. However, 26 clones were uniquely mapped in proximity and 5' of known genes (Table 2) , and eight clones were mapped near predicted but uncharacterized genes. Four clones were chimeric, and eight were located within areas where no genes (known or predicted) have yet been mapped. According to published data and in silico analyses, 20 (76.9%) of the 26 known candidate genes are expressed in the eye and contain a potential FOX DNA binding site, consistent with their being targets of FOXC1 regulation of ocular gene pathways (Table 3) .


View this table:
[in this window]
[in a new window]
  		TABLE 2. NACE-Enriched Clones Matching Known Genes

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Known Gene Expression Distribution

 
Validation of Putative FOXC1 Target Genes Obtained from the NACE Microlibrary Screening
To determine whether the obtained potential targets would be consistently detected within the enriched chromatin, 5 of the 26 known candidate genes were subjected to PCR amplification to verify enrichment in the NACE eluate. Oligonucleotide primers were designed with the sequence of the 5' upstream elements. Outputs from two independent NACE experiments were used as the DNA template. All the five candidates selected for validation, (BMP2K, DACH, FVT-1, SIX-1, and PGE-2 receptor) were enriched in both NACE assays (Fig. 4) . PCR failed to amplify these genes in a negative control consisting of enriched chromatin derived from cells transfected by an empty vector. These results confirm the reliability of the NACE technique and are consistent with the hypothesis that these genes are regulated by FOXC1 in the eye.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 4. Validation of five NACE-enriched genes and the location of the 5' upstream elements. Schematic representation of the 5' upstream elements present in NACE-enriched target DNA for the following genes: (A) BMP2K, (B) DACH, (C) FVT-1, (D) SIX1, and (E) PTGER. Lane M: 100-bp DNA marker; lane 2: +Ni2+ enriched DNA lot1; lane 3: +Ni2+ enriched DNA lot2; and lane 4: +Ni2+ empty vector. Arrows, right: molecular sizes; arrows, left: locations of the NACE-enriched 5' upstream elements.

 
To test if these selected genes were endogenously expressed in NPCE cells, primers were designed in the coding region of each gene and used for RT-PCR to amplify the NPCE cell mRNA template. All five NACE-selected genes were expressed by NPCE cells (Fig. 3) , consistent with these genes’ being potential FOXC1 targets in NPCE cells.

Investigation of Selected Candidate FOXC1 Target Genes
The FOXC1-enriched chromatin was also used as a template for PCR amplification of nine candidate genes selected from the literature as potential ocular FOXC1 target genes, on the basis of their overlapping expression with FOXC1 and known role in the eye. Primers were designed within an ~1-kb area 5' upstream of the coding region of BMP4, BMP7, clathrin heavy chain, COX2, iNOS, SMAD2, TGF-B1, TYR, and WNT6 genes. Four (44.4%) genes (BMP7, SMAD2, TGF-B1, and WNT6) were detected in NACE-enriched chromatin consistently, in two independent lots. BMP4 displayed ambiguous expression, whereas TYR, clathrin heavy chain, COX2, and iNOS were not detected. Bands of the expected sizes were obtained exclusively in the enriched samples obtained from the nickel column, whereas no signal was detected in samples transfected with an empty vector or in the negative control samples (Fig. 5) .



View larger version (51K):
[in this window]
[in a new window]
  		FIGURE 5. NACE analysis of candidate FOXC1-regulated genes selected from the literature. PCR amplification of genes thought to play a role in eye development pathways. Oligonucleotide primers were designed in the 5' upstream region and used to amplify the two different lots of NACE-enriched chromatin. BMP7, SMAD2, TGF-B1, and WNT6 could be detected consistently in two different NACE-enriched samples.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
NACE for Enrichment of Chromatin Regulated by Transcription Factors
Previous identification of 5' upstream elements regulated by mammalian transcription factors has mainly relied on differential expression studies.42 43 Recently, ChIP assays, either alone6 or coupled to microarray analysis,7 8 have been used to elucidate the target sequences regulated by transcription factors. Little progress has been made, however, in the study of gene regulation involved in eye diseases, including glaucoma, using this robust technique. This is principally due to an absence of suitable antibodies to relevant transcription factors. This prompted us to develop an antibody-free affinity-enrichment scheme based on Ni2+-NTA affinity chromatography. In the current study, we used NACE to isolate candidate target sequences bound by recombinant FOXC1. The use of NACE has several advantages, including an active conformation due to the immobilized (His)-6-tagged biomolecules and optimal access of binding domains to potential interacting proteins.44 The tight-binding, 6xHis-tagged biomolecules-Ni2+ ions are not dependent on the three-dimensional conformation of the transcription factor and can be used under native or denaturing conditions, allowing stringent washing.44 Furthermore, the small size of the (His)-6 tag does not interfere with protein structure or function in most cases and can be detected by several antibodies.45 In addition to the use of NACE in situations in which an antibody is not available, a major advantage is the utility of the NACE method for high-throughput screening of multiple transcription factors.

However, like all sophisticated techniques, the NACE procedure has some limitations, mainly the potential of isolating, by means of transient transfection, false positives due to an overexpression of protein. Therefore, the choice of an appropriate cell line, in addition to strict controls, should be made carefully to avoid false results. To overcome these difficulties, we chose NPCE cells, a line that expresses FOXC1 endogenously. These cells were also used to validate our potential NACE-enriched targets. All the selected genes were expressed in NPCE cells, consistent with their being candidate targets of FOXC1 (Fig. 3) . Moreover, several controls were included to monitor the potential residual noise and to avoid harvesting nonspecific enriched chromatin. In addition to an empty-vector, negative control (Fig. 2) , we transfected cells with the FOXC1 vector lacking the forkhead box ({Delta}BOX)19 as well as a reporter without an upstream FOXC1 binding site to estimate residual noises. {Delta}BOX FOXC1 did not detect the FOXC1 binding site located upstream of the luciferase reporter gene (data not shown). Last, it is important to remember that NACE, like all chromatin immunoprecipitation-based protocols, is an enrichment method. All recovered candidate targets must be validated, including demonstration of reproducible enrichment, expression in the starting cell material, and detection of potential transcription factor binding sites in the recovered chromatin.

NACE was successful in isolating chromatin-containing recombinant (His)-6-Xpress FOXC1 in NPCE cells. Of the known genes isolated, 76.9% were expressed in eye tissues (Table 3) . Furthermore, all five selected candidate genes were found to be enriched by NACE in two independent experiments (Fig. 4) , confirming the consistency of this technique. In parallel, nine genes were selected from the literature as being potential FOXC1 targets based on their known function and expression in the eye. NACE failed to detect four of the selected genes (clathrin, COX2, iNOS, and TYR), suggesting that these genes are not regulated by FOXC1. Alternatively, the failure to detect these genes may be simply because the chosen primers did not cover the location of the FOXC1 regulatory elements. BMP4 systematically showed an ambiguous pattern, despite stringent conditions. However, among the selected genes, four (BMP7, SMAD2, TGF-B1, and WNT6) were detected consistently in two different lots of NACE-enriched chromatin (Fig. 5) . Putative FOX binding sites (AAAT/CA) were found in the detected 5' upstream regulatory elements. NACE recovery of these genes, together with their function, expression data, and putative FOX binding sites in their upstream regions, are consistent with the hypothesis that these genes are regulated by FOXC1 in the eye.

Implication of Roles for FOXC1 in Regulation of the Cell Cycle and Cancer
We identified a genomic DNA element immediately 5' of the follicular variant translocation protein gene (FVT-1) that was detected in two independent NACE experiments (Fig. 4C) . This represents the first evidence that FVT-1 may be subjected to regulation by FOXC1. The FVT-1 gene has been shown to be involved in cancer through chromosome translocation t(2;18) involving the 5' region of the BCL-2 gene located 10 kbp downstream of FVT-1 on chromosome 18.46 In a recent report, FOXC1 was shown to be upregulated by TGF-ß1 in several human cancer cell lines.47 In addition, homozygous deletion of FOXC1 was found in 6.7% of primary endometrial and ovarian cancers.47 As TGF-ß1 was also enriched in NACE eluates, this pathway could form an important feedback loop involving FOXC1 in cell regulation. Consistent with this hypothesis, BMP7 and SMAD2, known to be involved in the TGF-ß pathway,48 49 were also enriched in the NACE eluates. Further studies may clarify the role of FOXC1 in human oncogenes.

Role of FOXC1 in the Regulation of Intraocular Pressure
In two independent NACE experiments, we identified a NACE-enriched clone uniquely mapped to a region within the second intron of the prostaglandin E2 receptor (PTGER) gene (Fig. 4E) . PTGER is expressed in the ciliary body and plays a key role in modulating intraocular pressure (IOP) in humans.50 The corresponding ligand (PGE2) binds PTGER to initiate a cascade of events leading to IOP reduction in mice. Topical application of PGE2 in the mouse eye markedly reduces IOP and relaxes precontracted ciliary muscle.24 Prostaglandin has been approved for clinical use to increase drainage of intraocular fluid, probably through receptor E2 or E4, which through unknown mechanisms, promotes ciliary muscle relaxation and evacuation of the aqueous.28 It is tempting to speculate that failure of correct regulation of the PTGER gene by FOXC1 in patients with FOXC1 mutations may have a role in these patients’ elevated IOP and glaucoma. Additional experiments are needed to address this interesting question.

Role of FOXC1 in the Regulation of Ocular Development and Maintenance of Cell Identity
The enriched 5' upstream element of Dachshund homologue isoform A (DACH1) gene was approximately 18 kb upstream of the coding region and was consistently detected in NACE products (Fig. 4B) . DACH is expressed in several organs and malignant tissues, and particularly in the fetal eye, retinal fovea, macula, lens, eye anterior segment, and optic nerve.51 Dach1 is found in the developing eye and ear of both chick and mouse52 and has a role in retinal and pituitary precursor cell proliferation. It is also expressed in breast cancer cell lines, where it inhibits TGF-ß signaling through binding Smad4.34 Moreover, Dach, Six, and Eya act synergistically through Creb-binding protein to activate downstream target promoters.53 Upstream elements of SIX1, SMAD4, and TGF-B1 were also detected in the enriched chromatin, implying that FOXC1 is upstream in this gene regulatory pathway.

Consistent with this potential role of FOXC1 regulation of developing important genes, we also identified a 5' upstream element approximately 2 kb upstream of the initiation codon of Sin oculis homeobox homolog 1 (SIX1), which was detected consistently in two NACE products (Fig. 4D) . Six1 is required for the early organogenesis of mammalian kidney54 and has a synergistic effect with Dach2 and Eya2 on the regulation of vertebrate muscle development as well as on Drosophila eye formation.35

WNT6 was also reproducibly enriched by NACE from NPCE cells transfected with FOXC1, suggesting the potential regulation of WNT6 by FOXC1 in the eye. WNT6 is a member of a highly conserved family of developmental control genes, encoding secreted glycoproteins that are involved in signaling through Frizzled receptors.55 There is clear evidence that WNT genes are involved in the eye.56 Wnt5a, -5b, -7a, -7b, -8a, and -8b, as well as Frizzled receptors 1, 2, 3, 4, and 6, are detected in the lens,57 whereas Wnt2b controls retinal cell differentiation.58 Furthermore, it has been hypothesized that WNT6 also plays key roles in human carcinogenesis.59 The role that WNT6 has in the anterior chamber is not well understood at present. This is the first report showing a possible link of WNT6 to FOXC1, implying that FOXC1 has a role in regulating WNT6 expression to fulfill its developmental role in different organs, including the eye. Further experiments are in progress to investigate the interaction of these molecules in the regulation of target genes in the eye.

NACE: A Useful Technique to Isolate Genes Regulated by Transcription Factors
The present findings reveal the ability of the NACE method to isolate recombinant (His)-6-tagged FOXC1–enriched chromatin complexes. We propose that NACE can be an alternative to ChIP in cases in which suitable antibodies are unavailable.

Further experiments are needed to investigate these potential targets of FOXC1 by the use of reporter gene assays, gel shift, and gene modeling of knockout mice. With these methods, it would be fruitful to explore the potential roles of FOXC1 in IOP regulation through regulation of PTGER and in the regulation of eye development through control of key genes, including DACH1, SIX1, and WNT6. Our results are consistent with the hypothesis that FOXC1 is directly involved in the regulation of a wide range of genes expressed in the eye, reinforcing the crucial role of FOXC1 in proper ocular development and function.60 61


   Acknowledgements
 
The authors thank all members of Ocular Genetics Laboratory for help and discussions during the project, Fred Berry for help and discussion during the initiation of the project and for critical reading of the manuscript, Alan Underhill for comments during the preparation of the manuscript, Farideh Mirzayans and Tim Footz for technical help and support.


   Footnotes
 
Supported by the Alberta Heritage Foundation for Medical Research, the Canadian Institute for Health Research, and Novartis Ophthalmics.

Submitted for publication May 31, 2004; revised July 22 and July 27, 2004; accepted July 30, 2004.

Disclosure: Y. Tamimi, None; M. Lines, None; M. Coca-Prados, None; M.A. Walter, Novartis Ophthalmics (F)

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Michael A. Walter, Department of Ophthalmology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; mwalter{at}ualberta.ca.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Khorasanizadeh S. The nucleosome: from genomic organization to genomic regulation. Cell. 2004;116:259–272.[CrossRef][ISI][Medline][Order article via Infotrieve]
  2. Kobayashi H, Tan EM, Fleming SE. Acetylation of histones associated with the p21WAF1/CIP1 gene by butyrate is not sufficient for p21WAF1/CIP1 gene transcription in human colorectal adenocarcinoma cells. Int J Cancer. 2004;109:207–213.[CrossRef][ISI][Medline][Order article via Infotrieve]
  3. Zhou QP, Le TN, Qiu X, et al. Identification of a direct Dlx homeodomain target in the developing mouse forebrain and retina by optimization of chromatin immunoprecipitation. Nucleic Acids Res. 2004;32:884–892.[Abstract/Free Full Text]
  4. Weinmann AS, Farnham PJ. Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods. 2002;26:37–47.[CrossRef][ISI][Medline][Order article via Infotrieve]
  5. Johnson KD, Bresnick EH. Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation. Methods. 2002;26:27–36.[CrossRef][ISI][Medline][Order article via Infotrieve]
  6. Hjalt TA, Amendt BA, Murray JC. PITX2 regulates procollagen lysyl hydroxylase (PLOD) gene expression: implications for the pathology of Rieger syndrome. J Cell Biol. 2001;152:545–552.[Abstract/Free Full Text]
  7. Weinmann AS, Yan PS, Oberley MJ, Huang TH, Farnham PJ. Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis. Genes Dev. 2002;16:235–244.[Abstract/Free Full Text]
  8. Odom DT, Zizlsperger N, Gordon DB, et al. Control of pancreas and liver gene expression by HNF transcription factors. Science. 2004;303:1378–1381.[Abstract/Free Full Text]
  9. Ren B, Cam H, Takahashi Y, et al. E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints. Genes Dev. 2002;16:245–256.[Abstract/Free Full Text]
  10. Ren B, Robert F, Wyrick JJ, et al. Genome-wide location and function of DNA binding proteins. Science. 2000;290:2306–2309.[Abstract/Free Full Text]
  11. Gould DB, Mears AJ, Pearce WG, Walter MA. Autosomal dominant Axenfeld-Rieger anomaly maps to 6p25. Am J Hum Genet. 1997;61:765–768.[ISI][Medline][Order article via Infotrieve]
  12. Mirzayans F, Mears AJ, Guo SW, Pearce WG, Walter MA. Identification of the human chromosomal region containing the iridogoniodysgenesis anomaly locus by genomic-mismatch scanning. Am J Hum Genet. 1997;61:111–119.[ISI][Medline][Order article via Infotrieve]
  13. Mears AJ, Jordan T, Mirzayans F, et al. Mutations of the forkhead/winged-helix gene, FKHL7, in patients with Axenfeld-Rieger anomaly. Am J Hum Genet. 1998;63:1316–1328.[CrossRef][ISI][Medline][Order article via Infotrieve]
  14. Nishimura DY, Swiderski RE, Alward WL, et al. The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. Nat Genet. 1998;19:140–147.[CrossRef][ISI][Medline][Order article via Infotrieve]
  15. Kidson SH, Kume T, Deng K, Winfrey V, Hogan BL. The forkhead/winged-helix gene, Mf1, is necessary for the normal development of the cornea and formation of the anterior chamber in the mouse eye. Dev Biol. 1999;211:306–322.[CrossRef][ISI][Medline][Order article via Infotrieve]
  16. Lines MA, Kozlowski K, Walter MA. Molecular genetics of Axenfeld-Rieger malformations. Hum Mol Genet. 2002;11:1177–1184.[Abstract/Free Full Text]
  17. Coca-Prados M, Wax MB. Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors. Proc Natl Acad Sci USA. 1986;83:8754–8758.[Abstract/Free Full Text]
  18. Hubbard T, Barker D, Birney E, et al. The Ensembl genome database project. Nucleic Acids Res. 2002;30:38–41.[Abstract/Free Full Text]
  19. Saleem RA, Banerjee-Basu S, Berry FB, Baxevanis AD, Walter MA. Analyses of the effects that disease-causing missense mutations have on the structure and function of the winged-helix protein FOXC1. Am J Hum Genet. 2001;68:627–641.[CrossRef][ISI][Medline][Order article via Infotrieve]
  20. Hafner C, Schmitz G, Meyer S, et al. Differential gene expression of Eph receptors and ephrins in benign human tissues and cancers. Clin Chem. 2004;50:490–499.[Abstract/Free Full Text]
  21. Bach H, Feldheim DA, Flanagan JG, Scalia F. Persistence of graded EphA/Ephrin-A expression in the adult frog visual system. J Comp Neurol. 2003;467:549–565.[CrossRef][ISI][Medline][Order article via Infotrieve]
  22. Moore KB, Mood K, Daar IO, Moody SA. Morphogenetic movements underlying eye field formation require interactions between the FGF and ephrinB1 signaling pathways. Dev Cell. 2004;6:55–67.[CrossRef][ISI][Medline][Order article via Infotrieve]
  23. Martemyanov KA, Lishko PV, Calero N, et al. The DEP domain determines subcellular targeting of the GTPase activating protein RGS9 in vivo. J Neurosci. 2003;23:10175–10181.[Abstract/Free Full Text]
  24. Takamatsu M, Hotehama Y, Goh Y, Mishima HK. Localization of prostaglandin E receptor subtypes in the ciliary body of mouse eye. Exp Eye Res. 2000;70:623–628.[CrossRef][ISI][Medline][Order article via Infotrieve]
  25. Wang Y, Jerdeva G, Yarber FA, et al. Cytoplasmic dynein participates in apically targeted stimulated secretory traffic in primary rabbit lacrimal acinar epithelial cells. J Cell Sci. 2003;116:2051–2065.[Abstract/Free Full Text]
  26. Martinez JM, Elfarissi H, De Velasco B, et al. Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas. Vis Neurosci. 2000;17:127–138.[CrossRef][ISI][Medline][Order article via Infotrieve]
  27. Hivert B, Liu Z, Chuang CY, Doherty P, Sundaresan V. Robo1 and Robo2 are homophilic binding molecules that promote axonal growth. Mol Cell Neurosci. 2002;21:534–545.[CrossRef][ISI][Medline][Order article via Infotrieve]
  28. Bradley JM, Kelley MJ, Rose A, Acott TS. Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch. Invest Ophthalmol Vis Sci. 2003;44:5174–5181.[Abstract/Free Full Text]
  29. Kim WJ, Mohan RR, Wilson SE. Effect of PDGF, IL-1alpha, and BMP2/4 on corneal fibroblast chemotaxis: expression of the platelet-derived growth factor system in the cornea. Invest Ophthalmol Vis Sci. 1999;40:1364–1372.[Abstract/Free Full Text]
  30. Kociok N, Esser P, Unfried K, et al. Upregulation of the RAS-GTPase activating protein (GAP)-binding protein (G3BP) in proliferating RPE cells. J Cell Biochem. 1999;74:194–201.[CrossRef][ISI][Medline][Order article via Infotrieve]
  31. Quignard JF, Harley EA, Duhault J, Vanhoutte PM, Feletou M. K+ channels in cultured bovine retinal pericytes: effects of beta-adrenergic stimulation. J Cardiovasc Pharmacol. 2003;42:379–388.[CrossRef][ISI][Medline][Order article via Infotrieve]
  32. Tian M, Zhao JW, Yang XL, Xie JX. Voltage-gated K(+) channel subunits on cholinergic and dopaminergic amacrine cells. Neuroreport. 2003;14:1763–1766.[CrossRef][ISI][Medline][Order article via Infotrieve]
  33. Tait S, Gunn-Moore F, Collinson JM, et al. An oligodendrocyte cell adhesion molecule at the site of assembly of the paranodal axo-glial junction. J Cell Biol. 2000;150:657–666.[Abstract/Free Full Text]
  34. Wu K, Yang Y, Wang C, et al. DACH1 inhibits transforming growth factor-beta signaling through binding Smad4. J Biol Chem. 2003;278:51673–51684.[Abstract/Free Full Text]
  35. Heanue TA, Reshef R, Davis RJ, et al. Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation. Genes Dev. 1999;13:3231–3243.[Abstract/Free Full Text]
  36. Ford HL, Landesman-Bollag E, Dacwag CS, Stukenberg PT, Pardee AB, Seldin DC. Cell cycle-regulated phosphorylation of the human SIX1 homeodomain protein. J Biol Chem. 2000;275:22245–22254.[Abstract/Free Full Text]
  37. Schmidt JT, Fleming MR, Leu B. Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: possible role in activity-driven sharpening. J Neurobiol. 2004;58:328–340.[CrossRef][ISI][Medline][Order article via Infotrieve]
  38. Azadi S, Zhang Y, Caffe AR, Holmqvist B, van Veen T. Thyroid-beta2 and the retinoid RAR-alpha, RXR-gamma and ROR-beta2 receptor mRNAs: expression profiles in mouse retina, retinal explants and neocortex. Neuroreport. 2002;13:745–750.[CrossRef][ISI][Medline][Order article via Infotrieve]
  39. Garriock RJ, Vokes SA, Small EM, Larson R, Krieg PA. Developmental expression of the Xenopus Iroquois-family homeobox genes, Irx4 and Irx5. Dev Genes Evol. 2001;211:257–260.[CrossRef][ISI][Medline][Order article via Infotrieve]
  40. Stohr H, Weber BH. Cloning and characterization of the human retina-specific gene MPP4, a novel member of the p55 subfamily of MAGUK proteins. Genomics. 2001;74:377–384.[CrossRef][ISI][Medline][Order article via Infotrieve]
  41. Ansari HR, Kaddour-Djebbar I, Abdel-Latif AA. Effects of prostaglandin F2alpha, latanoprost and carbachol on phosphoinositide turnover, MAP kinases, myosin light chain phosphorylation and contraction and functional existence and expression of FP receptors in bovine iris sphincter. Exp Eye Res. 2004;78:285–296.[CrossRef][ISI][Medline][Order article via Infotrieve]
  42. Kim Y, Fischer SM. Transcriptional regulation of cyclooxygenase-2 in mouse skin carcinoma cells: regulatory role of CCAAT/enhancer-binding proteins in the differential expression of cyclooxygenase-2 in normal and neoplastic tissues. J Biol Chem. 1998;273:27686–27694.[Abstract/Free Full Text]
  43. Johnson AD, Owens GK. Differential activation of the SMalphaA promoter in smooth vs. skeletal muscle cells by bHLH factors. Am J Physiol. 1999;276:C1420–C1431.
  44. Schmitt J, Hess H, Stunnenberg HG. Affinity purification of histidine-tagged proteins. Mol Biol Rep. 1993;18:223–230.[CrossRef][ISI][Medline][Order article via Infotrieve]
  45. Muller KM, Arndt KM, Bauer K, Pluckthun A. Tandem immobilized metal-ion affinity chromatography/immunoaffinity purification of His-tagged proteins–evaluation of two anti-His-tag monoclonal antibodies. Anal Biochem. 1998;259:54–61.[CrossRef][ISI][Medline][Order article via Infotrieve]
  46. Rimokh R, Gadoux M, Bertheas MF, et al. FVT-1, a novel human transcription unit affected by variant translocation t(2;18)(p11;q21) of follicular lymphoma. Blood. 1993;81:136–142.[Abstract/Free Full Text]
  47. Zhou Y, Kato H, Asanoma K, et al. Identification of FOXC1 as a TGF-beta1 responsive gene and its involvement in negative regulation of cell growth. Genomics. 2002;80:465–472.[CrossRef][ISI][Medline][Order article via Infotrieve]
  48. Xanthos JB, Kofron M, Tao Q, Schaible K, Wylie C, Heasman J. The roles of three signaling pathways in the formation and function of the Spemann Organizer. Development. 2002;129:4027–4043.[Abstract/Free Full Text]
  49. Lange D, Persson U, Wollina U, et al. Expression of TGF-beta related Smad proteins in human epithelial skin tumors. Int J Oncol. 1999;14:1049–1056.[ISI][Medline][Order article via Infotrieve]
  50. Matsuo T, Cynader MS. Localisation of prostaglandin F2 alpha and E2 binding sites in the human eye. Br J Ophthalmol. 1992;76:210–213.[Abstract/Free Full Text]
  51. Diehn M, Sherlock G, Binkley G, et al. SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data. Nucleic Acids Res. 2003;31:219–223.[Abstract/Free Full Text]
  52. Heanue TA, Davis RJ, Rowitch DH, et al. Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes. Mech Dev. 2002;111:75–87.[CrossRef][ISI][Medline][Order article via Infotrieve]
  53. Ikeda K, Watanabe Y, Ohto H, Kawakami K. Molecular interaction and synergistic activation of a promoter by Six, Eya, and Dach proteins mediated through CREB binding protein. Mol Cell Biol. 2002;22:6759–6766.[Abstract/Free Full Text]
  54. Xu PX, Zheng W, Huang L, Maire P, Laclef C, Silvius D. Six1 is required for the early organogenesis of mammalian kidney. Development. 2003;130:3085–3094.[Abstract/Free Full Text]
  55. Lu D, Zhao Y, Tawatao R, et al. Activation of the Wnt signaling pathway in chronic lymphocytic leukemia. Proc Natl Acad Sci USA. 2004;101:3118–3123.[Abstract/Free Full Text]
  56. Jin EJ, Burrus LW, Erickson CA. The expression patterns of Wnts and their antagonists during avian eye development. Mech Dev. 2002;116:173–176.[CrossRef][ISI][Medline][Order article via Infotrieve]
  57. Stump RJ, Ang S, Chen Y, et al. A role for Wnt/beta-catenin signaling in lens epithelial differentiation. Dev Biol. 2003;259:48–61.[CrossRef][ISI][Medline][Order article via Infotrieve]
  58. Kubo F, Takeichi M, Nakagawa S. Wnt2b controls retinal cell differentiation at the ciliary marginal zone. Development. 2003;130:587–598.[Abstract/Free Full Text]
  59. Kirikoshi H, Sekihara H, Katoh M. WNT10A and WNT6, clustered in human chromosome 2q35 region with head-to-tail manner, are strongly coexpressed in SW480 cells. Biochem Biophys Res Commun. 2001;283:798–805.[CrossRef][ISI][Medline][Order article via Infotrieve]
  60. Kume T, Jiang H, Topczewska JM, Hogan BL. The murine winged helix transcription factors, Foxc1 and Foxc2, are both required for cardiovascular development and somitogenesis. Genes Dev. 2001;15:2470–2482.[Abstract/Free Full Text]
  61. Kume T, Deng KY, Winfrey V, Gould DB, Walter MA, Hogan BL. The forkhead/winged helix gene Mf1 is disrupted in the pleiotropic mouse mutation congenital hydrocephalus. Cell. 1998;93:985–996.[CrossRef][ISI][Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 IOVSHome page
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]

Home page
 Hum Mol GenetHome page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tamimi, Y.
Right arrow Articles by Walter, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tamimi, Y.
Right arrow Articles by Walter, M. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS