PROTEIN PRODUCT OF THE SOMATIC-TYPE TRANSCRIPT OF THE HOXA-4 (HOX-14)GENE BINDS TO HOMEOBOX CONSENSUS BINDING-SITES IN ITS PROMOTER AND INTRON

The murine Hoxa-4 gene encodes a protein with a homeodomain closely re lated to those produced by the Antennapedia-like class of Drosophila g enes. Drosophila homeodomain proteins can function as transcription fa ctors, binding to several specific DNA sequences. One sequence that is frequently encountered contains a core ATTA motif within a larger con sensus sequence, such as CAATTAA. The in vitro synthesized protein pro duct 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 ATT A motif. Two regions contained sequences of the CAATTAA class and were located approximately 1 kb upstream from the putative somatic Hoxa-4 promoter and within the intron. Two additional binding sites containin g the consensus target sequence involved in autoregulation of Drosophi la Deformed gene were identified: one immediately downstream of the pu tative embryonic transcription start site and one within the intron, r espectively. Specific binding of the in vitro produced Hoxa-4 protein to oligonucleotides corresponding to these sequences was observed in g el retardation assays. The same results were obtained with Hoxa-4 prot ein produced in a Baculovirus expression system. Experiments using oli gonucleotides 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 tran sient co-transfection experiments revealed that the intact homeodomain was required for the binding. These results also suggested that the H oxa-4 gene has the potential to auto-regulate its expression by intera cting with the homeodomain binding sites present in the promoter as we ll as in the intron. (c) 1993 Wiley-Liss, Inc.