Identification and analysis of vnd/NK-2 homeodomain binding sites in genomic DNA

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.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.