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.
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 approximately 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 retardation 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.
Wu
K
K
Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Wolgemuth
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051717:Fushi Tarazu Transcription Factors; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D007301:Insect Hormones / Q000378:metabolism; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; C484111:Hoxa4 protein, mouse; D007301:Insect Hormones; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA