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

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.