Protein product of the somatic‐type transcript of the Hoxa‐4 (Hox‐1.4) gene binds to homeobox consensus binding sites in its promoter and intron

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Abstract
The murine Hoxa‐4 gene encodes a protein with a homeodomain closely related to those produced by the Antennapedia‐like class of Drosophila genes. Drosophila homeodomain proteins can function as transcription factors, binding to several specific DNA sequences. One sequence that is frequently encountered contains a core ATTA motif within a larger consensus sequence, such as CAATTAA. The in vitro synthesized protein product of Hoxa‐4 was shown to bind to a subset of restriction fragments of the Hoxa‐4 gene itself as determined by gel retardation experiments. Direct examination of the sequences of the fragments bound by Hoxa‐4 protein revealed the presence of four regions containing the core ATTA motif. Two regions contained sequences of the CAATTAA class and were located ∼ 1 kb upstream from the putative somatic Hoxa‐4 promoter and within the intron. Two additional binding sites containing the consensus target sequence involved in autoregulation of Drosophila Deformed gene were identified: one immediately downstream of the putative embryonic transcription start site and one within the intron, respectively. Specific binding of the in vitro produced Hoxa‐4 protein to oligonucleotides corresponding to these sequences was observed in gel retardatin assays. The same results were obtained with Hoxa‐4 protein produced in a Baculovirus expression system. Experiments using oligonucleotides containing base substitutions in positions 1, 3, 4, and 5 in the sequence CAATTAA showed severely reduced binding. The use of truncated mutant Hoxa‐4 proteins in gel retardation assays and in transient co‐transfection experiments revealed that the intact homeodomain was required for the binding. These results also suggested that the Hoxa‐4 gene has the potential to auto‐regulate its expression by interacting with the homeodomain binding sites present in the promoter as well as in the intron.