Mirmira R G, Watada H, German M S
Hormone Research Institute and Department of Medicine, University of California, San Francisco, California 94143, USA.
J Biol Chem. 2000 May 12;275(19):14743-51. doi: 10.1074/jbc.275.19.14743.
beta-Cell differentiation factor Nkx6.1 is a homeodomain protein expressed in developing and mature beta-cells in the pancreatic islets of Langerhans. To understand how it contributes to beta-cell development and function, we characterized its DNA binding and transactivation properties. A single copy of the homeodomain of Nkx6. 1 binds to a strictly conserved 8-base pair DNA consensus sequence, TTAATTAC; even minor variations to this consensus reduce DNA binding affinity significantly. Full-length Nkx6.1, however, has markedly reduced DNA binding affinity due to an acidic domain at the carboxyl end of the molecule that functions as a mobile binding interference domain capable of interrupting the interaction between DNA and DNA binding domains of the helix-turn-helix type. When expressed in fibroblast cell lines, Nkx6.1 represses transcription through isolated Nkx6.1 binding sites; in beta-cell lines, Nkx6.1 specifically represses the intact insulin promoter through TAAT-containing sequences. In Gal4 one-hybrid fusion studies, transcriptional repression maps to a discreet region within the amino terminus. Our findings suggest a model in which Nkx6.1, regulated by interactions through its carboxyl terminus, directs the repression of specific genes in developing and mature beta-cells.
β细胞分化因子Nkx6.1是一种同源结构域蛋白，在发育中的和成熟的胰岛β细胞中表达。为了了解它如何促进β细胞的发育和功能，我们对其DNA结合和反式激活特性进行了表征。Nkx6.1同源结构域的单拷贝与一个严格保守的8碱基对DNA共有序列TTAATTAC结合；即使对该共有序列有微小的改变也会显著降低DNA结合亲和力。然而，全长Nkx6.1的DNA结合亲和力明显降低，这是由于分子羧基末端的一个酸性结构域，该结构域作为一个可移动的结合干扰结构域，能够中断DNA与螺旋-转角-螺旋型DNA结合结构域之间的相互作用。当在成纤维细胞系中表达时，Nkx6.1通过分离的Nkx6.1结合位点抑制转录；在β细胞系中，Nkx6.1通过含TAAT的序列特异性抑制完整的胰岛素启动子。在Gal4单杂交融合研究中，转录抑制定位到氨基末端的一个离散区域。我们的研究结果提示了一个模型，其中Nkx6.1受其羧基末端相互作用的调控，在发育中的和成熟的β细胞中指导特定基因的抑制。
