Identification and analysis of vnd/NK-2 homeodomain binding sites in genomic DNA
- Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1654
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Contributed by Marshall Nirenberg, March 18, 2005
Abstract
Vnd/NK-2 homeodomain affinity column chromatography was used to purify Drosophila DNA fragments bound by the vnd/NK-2 homeodomain. Sequencing the selected genomic DNA fragments led to the identification of 77 Drosophila DNA fragments that were grouped into 42 vnd/NK-2 homeodomain-binding loci. Most loci were within upstream or intronic regions, especially first introns. Nineteen of the Drosophila DNA fragments cloned correspond to one locus, termed Clone A, which is 312 bp in length and contains five vnd/NK-2 homeodomain core consensus binding sites, 5′-AAGTG, and is part of the first intron of the Beadex gene. We further analyzed the interactions between Clone A and vnd/NK-2 homeodomain protein by mobility-shift assay, DNase I footprinting, methylation interference, and ethylation interference. The DNase I footprinting analysis of Clone A with vnd/NK-2 homeodomain protein revealed three strong binding sites and one weak binding site between 15 and 130 bp of Clone A. We also analyzed binding of the vnd/NK-2 homeodomain to the 5′-flanking sequence of vnd/NK-2 genomic DNA. The DNase I footprinting result showed that there are two strong binding sites and five weak binding sites in the fragment between -385 and -675 bp from the transcription start site of the vnd/NK-2 gene.
The Drosophila ventral nervous system defective (vnd)/NK-2 homeodomain (HD) protein contains a HD (1, 2) that recognizes the consensus nucleotide sequence 5′-T(T/C)AAGTG(G/C) with a core sequence of 5′-AAGTG (3). The vnd/NK-2 protein also contains repression and activation domains (4). The vnd/NK-2 gene initiates neural development in the ventral column of neuroectoderm that gives rise to part of the ventral nerve cord of embryos (5-8). During embryonic development, the vnd/NK-2 protein, directly or indirectly, activates achaete (9) and NK-6 (10), represses ind and msh (11), and autoregulates its own gene expression by positive feedback (6, 8, 12). The three-dimensional structure of the vnd/NK-2 HD and its interaction with a target oligodeoxynucleotide, which contains the sequence 5′-TCAAGTGG, has been reported (13-16). The interaction of tyrosine in position 54 (Y54) of the HD with DNA is the major specificity determinant of the vnd/NK-2 HD-DNA complex (15, 17). The Y54M mutant HD has a reduced affinity for the vnd/NK-2 target DNA sequence and does not repress ind or msh (18). The A35T mutant is embryonically lethal, and the mutant protein has reduced binding affinity for DNA (19, 20). The NK-2 HD has been highly conserved during evolution, and all members of the NK-2 class of HDs have Y54 in their HDs (5, 21). The DNA binding sites of many NK-2 family proteins that have been studied contain the core nucleotide sequence 5′-AAGTG (3). The NK-2 family of proteins plays important roles in the morphogenesis of different organs (3, 21).
There are gene mutations in the NK-2 family that cause human diseases. Mutations in human NKX2-1 result in a complex disease including neurological, thyroid, and respiratory problems (22). Nkx2.5 is required for the development of the ventricles of the heart (21). At least 11 disease-associated mutations in human NKX2.5 have been documented to date related to congenital heart defects (23, 24). Mutations in transcription factors TBX5 and GATA4, which directly interact with NKX2.5 to regulate downstream target genes during heart development, also caused congenital heart defects (reviewed in ref. 25). Characterization of the downstream targets of the NK-2 family of transcription factors should further clarify their roles in morphogenesis and disease.
Recently, at least four methods have been used for the genomewide identification of potential target genes for transcription factors. The first method used whole-genome microassay analysis to determine differential gene expression in three transgenic Drosophila lines containing varying amounts of Dorsal protein (26). The second method used computational identification of cis-regulatory modules, which contain unusually high concentrations of predicted binding sites for transcription factors (27, 28). The third method is chromatin profiling that used a fusion protein consisting of a transcription factor linked to a Dam methyltransferase in Drosophila cells to detect the in vivo binding patterns of transcription factors (29, 30). The fourth method is chromatin immunoprecipitation, which also detected in vivo binding patterns of transcription factors (31-34).
In this article, we demonstrate the use of vnd/NK-2 HD affinity column chromatography to isolate Drosophila DNA fragments that bound to the vnd/NK-2 HD, which may be potential vnd/NK-2 HD target sequences. Further analyses showed that the vnd/NK-2 HD binds strongly to a purified genomic DNA fragment (Clone A) and to the 5′-flanking sequence of vnd/NK-2 genomic DNA, which contain consensus vnd/NK-2 binding sites.
Materials and Methods
Isolation of Drosophila Genomic DNA Fragments Bound by the vnd/NK-2 HD. Drosophila genomic DNA was digested with TaqI, then ligated with a double-stranded TaqI adapter (5′-ACAGGATACTCTCGAGGATCCT-3′/3′-TGTCCTATGAGAGCTCCTAGGAGC-5′), which contained a TaqI cohensive end. Either strand of the adapter could be used as a primer for PCR. Isolation of target genomic sequences (Fig. 5, which is published as supporting information on the PNAS web site) was obtained by a modification of the protocol for selection of binding site oligonucleotides (3, 35). The TaqI adapter-DNA complex was amplified by PCR for 30 cycles. The amplified DNA was denatured and annealed to the primers. The 32P-labeled DNA (0.5-1 μg) then was prepared by extension of the primer catalyzed by the Klenow polymerase in the presence of unlabeled dCTP, dGTP, and TTP (500 μM each) and [α-32P]dATP [67 μM, 25 Ci/mmol (1 Ci = 37 GBq)]. 32P-labeled DNA (0.5 ml) in buffer V (20 mM Tris·HCl, pH 7.3/0.25 mM EDTA/1 mM DTT/10 μg/ml gelatin) containing 25 mM NaCl was loaded on a 1-ml preequilibrated vnd/NK-2 HD-Sepharose column. The vnd/NK-2 HD protein and vnd/NK-2 HD-Sepharose column were prepared as described by Wang et al. (3). The column was washed extensively with buffer V containing 25 mM NaCl and then eluted successively with buffer V containing 0.25, 0.4, and 1.0 M NaCl. The eluants were collected (1 ml per fraction), and the radioactivity of each fraction was monitored by Cerenkov counting. The 0.25-1.0 M NaCl eluant was amplified by PCR and then labeled with 32P after phenol-chloroform extraction and ethanol precipitation. The 32P-labeled DNA was applied to the column for a second round of selection, and then the 0.4-1.0 M NaCl eluant from this selection was applied to the column for a third round of selection. The 0.4-1.0 M NaCl fraction of DNA eluted was amplified by PCR, and 32P-labeled DNA then was applied to the column for a fourth round of selection. The 0.3-0.4 M NaCl eluant fractions and the 0.4-1.0 M eluant fractions were pooled separately. After labeling with 32P, the two DNA fractions were digested with TaqI and then applied separately to the column for a fifth round of selection. The DNA of the 0.4-1.0 M NaCl eluant fraction was cloned into Bluescript II SK+ (Stratagene) that had been cleaved with ClaI. The single-stranded DNAs were purified as described by the manufacturer (Stratagene) and then sequenced.
DNase I Footprinting. DNase I footprinting analysis was performed as described by Arcioni et al. (36) with modifications. In each 20-μl reaction mixture, 8-30 fmol of double-stranded DNA labeled at one end were incubated with a 200- to 500-fold excess (molar ratio) of vnd/NK-2 HD protein in buffer F (20 mM Tris·HCl, pH7.3/75 mM NaCl/1 mM EDTA/3 mM MgCl2/5% glycerol/50 μg/ml BSA/5 μg/ml poly d(I-C)/5 μg/ml Escherichia coli tRNA) at 25°C for 30 min. One microliter (0.05-0.12 units) of DNase I (Roche Molecular Biochemicals) then was added, and the reaction mixture was incubated for an additional 30 min at 25°C.
Interference Experiments. Alkylation interference assays were performed essentially as described in ref. 37. For methylation interference, the single end-labeled double-stranded DNA (≈2 × 106 cpm, 0.5-1.7 pmol) with 0.1 μg of poly d(I-C) in 200 μl of 50 mM sodium cacodylate, pH 8.0/1 mM EDTA was methylated with 0.5 μl of dimethyl sulfate (Aldrich) at 25°C for 2 min. For ethylation interference, the labeled DNA (≈1 × 106 cpm, 0.3-0.6 pmol) with 0.1 μg of poly d(I-C) in 100 μl of 50 mM sodium cacodylate, pH 7.0/1 mM EDTA was treated with 100 μl of ethanol saturated with N-ethyl-N-nitrosourea (Sigma, ≈0.3 g of N-ethyl-N-nitrosourea per ml of 95% ethanol, freshly made). The mixture was kept at 50°C for 1 h. Both modified DNA preparations were precipitated three times with ethanol and then used for the protein binding assay in buffer 8 (3) at 4°C for 30 min. For ethylation interference samples, phosphotriester bonds were cleaved by suspension of isolated, free or protein-bound DNA in 60 μl of 10 mM sodium phosphate, pH 7.0/1 mM EDTA, followed by the addition of 10 μl of 1 M NaOH. Then, the samples were incubated at 90°C for 30 min. For methylation interference samples, DNA was cleaved at methylated adenine and guanine residues by suspending DNA in 40 μl of 20 mM sodium phosphate, pH 7.0/1 mM EDTA, then incubating DNA at 90°C for 10 min. Four microliters of 1 M NaOH were added, and then the sample was incubated at 90°C for 5 min (38). The videoimages of autoradiograms were exported to a Macintosh computer by using quickcapture and analyzed with nih image computer software (developed by Wayne Rasban, National Institutes of Health). The peak areas were determined by optical density.
Results
Cloning of the 5′-Flanking Sequence of the vnd/NK-2 Gene. We cloned three Drosophila genomic DNA fragments: p411, NP-8, and NP6-1, which contain ≈7.9, 13, and 15 kb, respectively, of Drosophila genomic DNA fragments and extend approximately -2.2, -8, and -14 kb, respectively, from the transcription start site of the vnd/NK-2 gene. NP-8 has been used to find enhancer regions regulating vnd/NK-2 gene expression in neuroblasts. The enhancers are located between -5.3 and -2.8 kb from the transcription start site of the vnd/NK-2 gene (6, 8).
Isolation of Drosophila Genomic DNA Bound by the vnd/NK-2 HD. We designed a method called “genomic DNA purification” to select DNA fragments that bind to the vnd/NK-2 HD. Some of the DNA fragments found may be from genes that are regulated by the vnd/NK-2 HD protein. Computer analyses of these cloned DNA sequences were performed by using the blast algorithm of the National Center for Biotechnology Information (NCBI) of the National Institutes of Health and the gcg gap program (39). Eighty percent of the clones consisted only of a single genomic DNA fragment, and 20% consisted of two or three combined genomic DNA fragments. In all, 91 genomic DNA fragments purified by affinity column chromatography were cloned and sequenced. Seventy-seven (85%) were from Drosophila, five (6%) were from from yeast 26S rDNA, two (2%) were from from bacteria, and seven were unknown. Based on the matched genomic sequences from a blast search, we used the Entrez Nucleotide search engine of NCBI and the Drosophila Gene Region Map of FlyBase to identify the genomic positions. We also used the Drosophila Gene Report of FlyBase and the embryonic expression pattern database of the Berkeley Drosophila Genome Project to obtain information on gene ontology and expression data. Of the 77 Drosophila DNA fragments, 31 (40%) were within first introns, 5 (7%) were within second and later introns, 13 (17%) contained intron/exon boundaries, 18 (23%) were within upstream regions of genes, 6 (8%) were within repeat sequences, 1 (1%) was part of the genomic DNA encoding 18S ribosomal RNA, and only 3 (4%) were within exons (Fig. 6A, which is published as supporting information on the PNAS web site). Each of the six repeat sequences was isolated once. Some of the DNA sequences were cloned two or more times; therefore, the 91 isolated DNA fragments were grouped into 50 binding loci, including 42 (84%) from Drosophila, 1 (2%) from yeast, 2 (4%) from bacteria, and 5 (10%) unknown sequences. Of the 42 Drosophila binding loci, 30 loci (71%) were identified once, 11 loci (26%) were identified 2-4 times, and 1 locus (2%) was identified 19 times. Of the 42 Drosophila binding loci, 13 (31%) were within introns, 7 (17%) contained intron/exon boundaries, 13 (31%) were within upstream regions of genes, and only 2 (5%) were within exons (Fig. 6B).
Information regarding 33 binding loci, which were within introns, across intron/exon boundaries, or in upstream regions of genes, is listed in Table 2, which is published as supporting information on the PNAS web site. Most of them are enriched in AAGT and/or TAAT sites. Potential target genes of 30 of 33 binding loci were expressed during embryonic development detected by microarray analysis from the Berkeley Drosophila Genome Project database. For some of the potential target genes, no information was available regarding biological process and molecular function in gene ontology of FlyBase. For other potential targets, known molecular functions include transcription factor activity, receptor activity, neurogenesis, wing morphogenesis, ectoderm and mesoderm development, transmission of nerve impulses, ion transport, and steroid metabolism. In situ hybridization data during embryonic development were available for seven genes, five were expressed in the nervous system, and one, the Mef2 gene, was expressed in muscle. Mef2 has been reported to be a target for Tinman, which is an NK family homeobox protein required for Drosophila heart development (40).
vnd/NK-2 HD Binding to Clone A-DNase I Footprinting. Nineteen clones were obtained of the DNA sequence termed Clone A. DNase I footprinting analysis of Clone A with the vnd/NK-2 HD protein (Fig. 1B) revealed three strongly protected sites (A1-A3) and one weakly protected site (a1′) clustered at the 5′ end of Clone A (Fig. 1 A). Strongly protected sites all contained one vnd/NK-2 consensus binding site, whereas sites A2 and A3 each had two vnd/NK-2 binding sites with slightly less affinity. The site a1′ contained three vnd/NK-2 putative binding sites with low affinity. Three double-stranded oligonucleotides containing the protected sequences and one oligonucleotide without a protected sequence were designed based on the footprinting data of Clone A. These oligonucleotides were used as substrates to detect specific DNA-protein complexes in gel mobility-shift assays. The K D values were determined as described by Wang et al. (3). The apparent K D values of oligos 13 (16-33 bp, contained the A1 site), 14 (62-79 bp, contained the A2 site), and 15 (96-113 bp, contained the A3 site) were 2.1, 3.5, and 3.4 × 10-10 M, respectively (Table 1). Oligo 18 (39-56 bp), located between the A1 and A2 sites, contained a putative vnd/NK-2 binding site with low affinity but was not protected by the vnd/NK-2 HD. The K D of oligo 18 was 1.1 × 10-9 M (Table 1). In Table 1, the letters in boldface and underlined indicate the core nucleotide sequence of the vnd/NK-2 HD binding sites. In competitive mobility gel-shift assays with a 200-fold excess of competitor, oligos 13, 14, and 15 competed with oligo 1; however, oligo 18 did not compete (data not shown).
DNase I footprinting of vnd/NK-2 HD binding sites in Clone A. (A) DNA sequence and footprinting sites of Clone A. Many vnd/NK-2 HD consensus binding sites, indicated with boxes, were found in these footprinting sites. The darker boxes represent higher binding affinity. The footprinting sites are indicated with lines. The solid lines labeled with uppercase and the dotted lines with lowercase represent strong and weak protection sites, respectively. The a1 protection site (complementary strand of a1′) was not determined because of the limitation of the DNA fragment used. (B) Autoradiographic analysis of DNase I footprinting of the vnd/NK-2 HD on both strands of Clone A DNA. The top strand corresponds to position 1 to ≈120, and the bottom corresponds to positions 10 to ≈180. + and -, DNA-binding reactions with and without the vnd/NK-2 HD, respectively. - Lanes G+A contain the products of Maxam-Gilbert chemical sequencing reactions for dG and dA residues that serve as sequence markers. The vertical boxes labeled with upper and lowercase letters correspond to strong and weak protection sites, respectively.
vnd/NK-2 HD Protein·DNA Contacts Within Clone A Monitored by Purine Methylation Interference and Phosphate Ethylation Interference. In Fig. 2A, the results of methylation interference obtained with the A1 site of Clone A are shown. Comparison of autoradiogram lanes U and B1 reveals methylation interference at G21, A22, A23, and G24 of the top strand and A19, A20, and A25 of the bottom strand (Fig. 2 B and E). A22 and A23 of the top strand and A19, A20, and A25 of the bottom strand have stronger interference than G21 and G24 of the top strand. This region (A1 site) contains a vnd/NK-2 consensus binding site. Comparison of lanes B1 and B2 reveals a second interference region (A5 site) that includes A10, A11, and G12 of the top strand and A8 and A9 of the bottom strand (Fig. 2 B and E). This region contains a TAAT, which is the core sequence recognized by a number of HD proteins. It also contains an AAG in the top strand of DNA, which is part of the vnd/NK-2 HD core consensus sequence. These results suggest that site A5 was bound by the vnd/NK-2 HD after site A1 was occupied. The sequence involved in methylation interference at positions 8-12 (in the A5 site) are similar to positions 19-24 (in the A1 site), except that the latter contains an extra G-C pair at position 21. In Fig. 2 C-E, ethylation interference results of the A1 site of Clone A are shown. The ethylated phosphates that interfered with vnd/NK-2 binding are 3′ to the bases at positions 20-24 of the top strand and 25-29 of the bottom strand and are located in the DNase I footprinting regions.
Methylation and ethylation interference analyses of vnd/NK-2 HD binding to the A1 site of Clone A. (A) Autoradiograph of a methylation interference analysis. In C, the cleaved products of the premethylated DNA are shown. U designates the cleavage products of unbound DNA isolated from a protein-DNA binding reaction. B1, B2, and B3 represent the results of one molecule of DNA bound to one, two, and three molecules of protein, respectively. Arrows indicate the modified bases that interfere with binding. (B) Image analysis of an autoradiograph of methylation interference assays. Lane B2, instead of B1, was used for the image tracing. Superimposed image tracings of bound (B, red) and unbound (U, black) patterns performed on the top and the bottom strands of DNA are shown in Top and Bottom, respectively. The bottom, horizontal line (red in each image tracing) is the baseline. The ratios of unbound to bound peak areas at each position are plotted as bar graphs. The ratios at no interference sites were normalized to 1. The positions of each base and bar were lined up with corresponding peaks. (C) Autoradiograph of an ethylation interference analysis of the A1 site of Clone A. The vertical bar represents the region of DNase I footprinting sites (A1 and A1′) from Fig. 1. G+A is the same as described in Fig. 1B. Other symbols are the same as described in A. (D) Image analysis of an autoradiograph of ethylation interference assays. Lane B2 was used for the image tracing. (E) Summary of methylation and ethylation interference results. Horizontal lines shown above and below the sequences are vnd/NK-2 HD-protected regions in the footprinting experiments. The box with the thick line indicates the consensus sequence for vnd/NK-2 HD binding. The box with the thin line indicates the TAAT site. Circles with solid lines represent methylated residues that interfere with vnd/NK-2 HD binding in B1, B2 and B3. Circles with dotted lines (black) represent extra methylated residues that interfere with vnd/NK-2 HD binding in B2 and B3 only. Red circles indicate stronger interference compared with black circles. Arrows indicate the modified phosphates interfering with binding.
vnd/NK-2 HD Binding to the 5′-Flanking Sequence of vnd/NK-2 Genomic DNA Shown by DNase I Footprinting. DNase I footprinting analysis was applied to two DNA segments that contain the consensus sequences for vnd/NK-2 HD binding. There are two strongly protected regions (N1 and N2) and five weakly protected sites (n1-n5) in the DNA fragment between -713 and -387 bp (Fig. 3) from the transcription initiation site of the vnd/NK-2 gene. The N1 site has four overlapping binding sites with high and low affinities, whereas the N2 site has only one strongly protected consensus binding site for the vnd/NK-2 HD. Among five weakly protected sites, only n2 contained a putative vnd/NK-2 HD core binding site. The weakly protected site n5 contained a TAAT core.
DNase I footprinting of vnd/NK-2 HD in the 5′-flanking sequence of vnd/NK-2 genomic DNA. (A) Autoradiographic analysis of DNase I footprinting. (Left) The footprinting assays are from -570 to -387 bp. (Right) The footprinting experiments from -713 to -570 bp. Symbols are the same as described in Fig. 1B. (B) DNA sequence and footprinting sites of the 5′-flanking sequence of vnd/NK-2 genomic DNA. Symbols are the same as described in Fig. 1A.
vnd/NK-2 Protein·DNA Contacts Within the 5′-Flanking Sequence of vnd/NK-2 Genomic DNA Monitored by Purine Methylation Interference and Phosphate Ethylation Interference. In Fig. 4A, methylation interference results for the N2 site of the 5′-flanking sequence of vnd/NK-2 genomic DNA are shown. Comparing the ratios of unbound DNA to protein-bound DNA at each position between lane U (unbound) and lane B1, strong methylation interference occurred at A-637, A-636, and G-635 of the top DNA strand and A-634 and G-632 of the bottom strand, and weak interference occurred at G-633 and A-631 of the top strand and A-639 of the bottom strand (Fig. 4 B and E). Most of the interference occurred in the vnd/NK-2 consensus binding site (box, Fig. 4E). There was no discrete band for A-637 of the top DNA strand. This result might be due to the compressed bands of A-637 and A-636. That the space between G-638 and A-634 of the bottom strand was enough for three residues suggested the existence of A-637 in the top strand. A-637 was detected by DNA sequencing of the top strand of this region (data not shown). Contacts between the vnd/NK-2 HD protein and the DNA phosphodiester backbone were detected by ethylation interference. The ethylated phosphates that interfered with vnd/NK-2 binding to DNA were 3′ to the bases at positions -639 to -635 in the top strand and -635 to -631 in the bottom strand (Fig. 4 C-E). A schematic representation of the methylation and ethylation interference data of the N2 site and a simulated complex of the N2 site with the vnd/NK-2 HD were described in figures 2 and 5, respectively, in Gruschus et al. (15).
Methylation and ethylation interference analyses of vnd/NK-2 HD binding to the N2 site of the 5′-flanking sequence of vnd/NK-2 genomic DNA. (A) Autoradiograph of a methylation interference analysis. Symbols are the same as described in Fig. 2 A. (B) Image analysis of an autoradiograph of methylation interference assays. Symbols are the same as described in Fig. 2B. (C) Autoradiograph of an ethylation interference analysis of the 5′-flanking sequence of vnd/NK-2 genomic DNA. The vertical bar represents the region of DNase I footprinting sites (N2 and N2′) from Fig. 3. Other symbols are the same as described in Fig. 2C. (D) Image analysis of an autoradiograph of an ethylation interference assay. (E) Summary of methylation and ethylation interference results. The box indicates the consensus sequence for vnd/NK-2 HD binding. Circles represent methylated residues that interfere with vnd/NK-2 HD binding in B1. Other symbols are the same as described in Fig. 2E.
Discussion
With our in vitro genomic DNA purification method, Drosophila genomic DNA fragments that contain nucleotide sequences that bind to the vnd/NK-2 HD were purified by five rounds of vnd/NK-2 HD-Sepharose affinity column chromatography. Seventy-seven purified Drosophila DNA fragments were cloned and sequenced. Most DNA sequences were cloned once, but 11 sequences were cloned two to four times, and 19 clones were obtained of one sequence (Clone A). Forty-two different sequences (i.e., loci) were purified from Drosophila genomic DNA. We isolated six repeat fragments; however, they were within six different transposons. Therefore, our genomic DNA purification procedure resulted in an enrichment of DNA fragments that bind to the vnd/NK-2 HD with high affinity from single-copy genomic DNA. This approach should be applicable to many other transcription factors. To improve this approach, more purified DNA fragments need to be cloned and sequenced.
Based on DNase I footprinting results, there were two strong and five weak DNase I-protected regions located between -385 and -675 bp from the transcription initiation site of the vnd/NK-2 gene. The strong DNase I footprinting site N2, 14 nucleotides in length, contained one high-affinity vnd/NK-2 consensus binding site, 5′-T(T/C)AAGTG(G/C). The N1 site, 27 nucleotides in length, contained one high-affinity and three low-affinity binding sites. The N1 site may have more than one vnd/NK-2 HD molecule binding to this region. Among five weak footprinting sites, n2 contained a low-affinity vnd/NK-2 HD site, and n5 contained a TAAT core. The AAG of the n1 site and the GAG of the n4 site may be the nucleotides that are recognized in weak binding of the vnd/NK-2 HD to DNA. It has been shown that vnd/NK-2 genomic DNA between -410 and -750 bp from the transcription start site, which contains N1 and N2 as well as n2-n5 protected regions, is regulated by vnd/NK-2 protein in Drosophila S2 cells (4). Our footprinting data agree with the demonstrations that the vnd/NK-2 protein, directly or indirectly, regulates its own gene expression (6, 12) and that the DNase I footprinting regions containing the 5′-AAGTG core in the 5′ upstream region of the vnd/NK-2 gene may be functional (4). The DNase I footprinting analysis of Clone A with the vnd/NK-2 HD protein showed three strong and one weak protection sites clustered at the 5′ end of Clone A. Each strongly protected site contained one vnd/NK-2 consensus binding core.
Comparing methylation interference results of vnd/NK-2 binding to the 5′-flanking sequence of vnd/NK-2 genomic DNA (N2 site) and Clone A (A1 site), as well as the results of TTF-1 binding to site C (41), the invariant interference occurred at (+)A3, (+)A4, (+)G5, (-)A1 and (-)A6 (Fig. 7, which is published as supporting information on the PNAS web site). These invariant sites are consistent with the site AAGTG derived from the footprinting results and with the analysis of the vnd/NK-2 consensus binding site (3). These results demonstrate the importance of the AAGTG sequence for interactions between the vnd/NK-2 HD and DNA.
Other groups have used chromatin profiling to identify genomewide target sequences for the Drosophila GAGA factor and have found that protein binding occurs in intergenic DNA regions and introns and very few in exons (29, 30). A chromatin immunoprecipitation procedure also has been used to identify DNA binding sites; 203 Drosophila DNA fragments that bind the Engrailed protein were localized in intergenic (53%) or intronic (47%) regions (31). Among 85 isolated Krüppel-binding fragments of Drosophila genomic DNA, 42% corresponded to intergenic regions, 21% corresponded to introns, 22% overlapped intron/exon boundaries, and 15% corresponded to exons (32). Only 22% of binding sites for Sp1, cMyc, and p53 were located in upstream regions of genes, whereas 36% were within or were immediately 3′ to well characterized genes (34). Among the CREB 215 binding sites located in human chromosome 22, 22% were within 10 kb of the 5′ end of the gene, 4% were in exons, and 15% and 24% were in a first intron or other intron, respectively (33). Among 60 potential HD protein BARX2 target loci, 35% were located within introns, generally within the first or second intron, and 65% were in intergenic regions (42). Recently, the DNA elements located in the first intron have been shown to be functionally important for mouse phenotypic traits (43) and Drosophila neurogenesis (44). However, further work is needed to determine whether the purified DNA fragments that contain vnd/NK-2 HD binding sites and the candidate target genes listed in Table 2 are functional.
Acknowledgments
We thank Mrs. Vicky Guo for synthesizing oligonucleotides and sequencing DNA, Dr. Joseph Shiloach for growing the E. coli cells in large volume, and Dr. Wei-Mei Ching for determining the amino acid composition and sequence of the purified vnd/NK-2 HD.
Footnotes
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↵ * To whom correspondence should be addressed. E-mail: mnirenberg{at}nih.gov.
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Author contributions: L.-H.W. and M.N. designed research; L.-H.W., R.C., and D.T. performed research; L.-H.W. contributed new reagents/analytic tools; L.-H.W. and M.N. analyzed data; and L.-H.W. and M.N. wrote the paper.
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Abbreviations: HD, homeodomain; vnd/NK2, ventral nervous system defective NK-2.
