The zinc-finger associated domain (ZAD) family is the largest transcription factor family in dipteran insects. Still, their functional significance is barely recognized in the literature due in part to their resistance to mutagenesis screens in genetic studies. Therefore, we employed in vitro techniques to identify the DNA-binding characteristics of several members of the Drosophila melanogaster ZAD family in an effort to study their target genes. In this comprehensive investigation, we constructed a panel of GST-Zinc finger (ZnF) array chimera from 21 selected ZAD proteins and used them to select binding sites from an oligonucleotide library by employing electrophoretic mobility shift assays (EMSA). Samples of the binding population were sequenced and used to derive DNA-binding consensus sequence for each member. These consensus sequences were tested for complex formation with their respective protein chimera and the specificity of binding ascertained by competition EMSA. Bioinformatics tools were used to identify potential genetic targets. The identified consensus sequences were distinct for each member and the putative genomic targets were clustered in the regulatory regions of specific genes. This appears to be consistent with a conservation of function between members and also suggests that the overlapping functions of ZAD proteins are the result of positive selection to maintain redundancy and not simply artifacts of recent expansion. Putative target genes suggest a major role of the ZAD family members in the regulation of several early developmental genes including homeobox transcription factors.
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