Wu K, Wolgemuth D J
Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032.
J Cell Biochem. 1993 Aug;52(4):449-62. doi: 10.1002/jcb.240520409.
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.
小鼠Hoxa - 4基因编码一种含有同源结构域的蛋白质，该同源结构域与果蝇触角足类基因所产生的同源结构域密切相关。果蝇同源结构域蛋白可作为转录因子，与几种特定的DNA序列结合。一种常见的序列在更大的共有序列（如CAATTAA）中包含一个核心ATTA基序。凝胶阻滞实验表明，体外合成的Hoxa - 4蛋白产物可与Hoxa - 4基因自身的一部分限制性片段结合。对Hoxa - 4蛋白结合片段的序列进行直接检测，发现有四个区域含有核心ATTA基序。两个区域含有CAATTAA类序列，分别位于推测的体细胞Hoxa - 4启动子上游约1 kb处和内含子内。另外还鉴定出两个含有参与果蝇变形基因自动调节的共有靶序列的结合位点：一个位于推测的胚胎转录起始位点下游紧邻处，另一个位于内含子内。在凝胶阻滞实验中观察到体外产生的Hoxa - 4蛋白与对应于这些序列的寡核苷酸特异性结合。在杆状病毒表达系统中产生的Hoxa - 4蛋白也得到了相同的结果。使用在CAATTAA序列中第1、3、4和5位含有碱基替换的寡核苷酸进行的实验表明，结合能力严重降低。在凝胶阻滞实验和瞬时共转染实验中使用截短的突变型Hoxa - 4蛋白表明，完整的同源结构域是结合所必需的。这些结果还表明，Hoxa - 4基因有可能通过与启动子以及内含子中存在的同源结构域结合位点相互作用来自动调节其表达。
