DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1.
DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1.
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DOI：
10.1093/nar/28.12.2389
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发表时间：
2000-06
影响因子：
14.9
通讯作者：
David J. Steadman;Domenica Giuffrida;E. P. Gelmann
中科院分区：
文献类型：
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作者：
David J. Steadman;Domenica Giuffrida;E. P. Gelmann
NKX3.1是NK类同源结构域蛋白的成员，与果蝇NK-3最密切相关。NKX3.1在成年人中主要具有前列腺特异性表达。以前的研究表明，NKX3.1对前列腺上皮细胞具有生长抑制作用，并控制分化的腺体功能。使用与重组NKX3.1蛋白的结合位点选择测定，我们鉴定了TAAGTA共有结合序列，其尚未报道用于任何其他NK类同源异型蛋白。通过电迁移率变动分析，我们证明NKX3.1优先结合TAAGTA序列，而不是Nkx2.1（CAAGTG）或Msx 1（TAATTG）的结合位点。在竞争性凝胶迁移试验中使用突变的结合位点，我们分析了TAAGTA共有序列中对NKX3.1结合重要的核苷酸。天然存在的多态性NKX3.1蛋白的共有结合位点与野生型结合序列相同，其中第52位的精氨酸被半胱氨酸取代。野生型和多态性NKX3.1对TAAGTA共有位点的结合亲和力非常相似，分别为20和22 nM。野生型和多态性NKX3.1特异性抑制来自报告载体的荧光素酶的转录，该报告载体具有三个拷贝的NKX3.1结合位点，位于胸苷激酶启动子上游。数据显示，在NK家族蛋白中，NKX3.1结合一种新的DNA序列，并可作为体外转录抑制因子。
NKX3.1 is a member of the NK class of homeodomain proteins and is most closely related to Drosophila NK-3. NKX3.1 has predominantly prostate-specific expression in the adult human. Previous studies suggested that NKX3.1 exerts a growth-suppressive effect on prostatic epithelial cells and controls differentiated glandular functions. Using a binding site selection assay with recombinant NKX3.1 protein we identified a TAAGTA consensus binding sequence that has not been reported for any other NK class homeoprotein. By electromobility shift assay we demonstrated that NKX3.1 preferentially binds the TAAGTA sequence rather than the binding site for Nkx2.1 (CAAGTG) or Msx1 (TAATTG). Using mutated binding sites in competitive gel shift assays, we analyzed the nucleotides in the TAAGTA consensus sequence that are important for NKX3.1 binding. The consensus binding site of a naturally occurring polymorphic NKX3.1 protein with arginine replaced by cysteine at position 52 was identical to the wild-type binding sequence. The binding affinities of wild-type and polymorphic NKX3.1 for the TAAGTA consensus site were very similar, with values of 20 and 22 nM, respectively. Wild-type and polymorphic NKX3.1 specifically repressed transcription of luciferase from a reporter vector with three copies of the NKX3.1-binding site upstream from a thymidine kinase promoter. The data show that among NK family proteins NKX3.1 binds a novel DNA sequence and can behave as an in vitro transcriptional repressor.