Song Y H, Ray K, Liebhaber S A, Cooke N E
Departments of Medicine and Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1998 Oct 23;273(43):28408-18. doi: 10.1074/jbc.273.43.28408.
Vitamin D-binding protein (DBP)/Gc-globulin, the major carrier of vitamin D and its metabolites in blood, is synthesized predominantly in the liver in a developmentally regulated fashion. By transient transfection analysis, we identified three regions in the 5'-flanking region of the rat DBP gene, segments F-2, B, and A, that contain tissue-specific transcriptional determinants. Gel mobility shift and DNase I footprinting analyses showed that all three regions contained binding sites for the hepatocyte nuclear factor 1 (HNF1), a transcriptional regulator composed of HNF1alpha and HNF1beta hetero- and homodimers. The activity of the most proximal segment A (coordinates -141 to -43) was DBP promoter-specific, position-dependent, and positively controlled by HNF1alpha. In contrast, the two more distal determinants (segments F-2 and B; coordinates -1844 to -1621 and -254 to -140, respectively) acted as classical enhancers in transfected hepatocyte-derived HepG2 cells; their activities were promoter- and orientation-independent, and disruption of their respective HNF1-binding sites resulted in marked loss of DBP gene expression. Remarkably, the activities of these two distal elements depended upon the relative levels of HNF1alpha and HNF1beta; HNF1alpha had a major stimulatory effect, whereas HNF1beta acted as a trans-dominant inhibitor of HNF1alpha-mediated enhancer activity. These results suggested that the net expression of the DBP gene reflected a balance between the two major HNF1 species; the relative abundance of HNF1alpha and HNF1beta proteins in a cell may thus play a critical role in determining the pattern of gene expression.
维生素D结合蛋白（DBP）/Gc球蛋白是血液中维生素D及其代谢产物的主要载体，主要在肝脏中以发育调控的方式合成。通过瞬时转染分析，我们在大鼠DBP基因的5'侧翼区域鉴定出三个区域，即F-2、B和A片段，它们包含组织特异性转录决定因素。凝胶迁移率变动分析和DNase I足迹分析表明，所有这三个区域都含有肝细胞核因子1（HNF1）的结合位点，HNF1是一种由HNF1α和HNF1β异源二聚体和同源二聚体组成的转录调节因子。最靠近近端的A片段（坐标为-141至-43）的活性是DBP启动子特异性的、位置依赖性的，并受到HNF1α的正向调控。相比之下，另外两个更远端的决定因素（F-2和B片段；坐标分别为-1844至-1621和-254至-140）在转染的肝细胞来源的HepG2细胞中作为经典增强子发挥作用；它们的活性与启动子和方向无关，并且其各自HNF1结合位点的破坏导致DBP基因表达明显丧失。值得注意的是，这两个远端元件的活性取决于HNF1α和HNF1β的相对水平；HNF1α具有主要的刺激作用，而HNF1β作为HNF1α介导的增强子活性的反式显性抑制剂。这些结果表明，DBP基因的净表达反映了两种主要HNF1种类之间的平衡；因此，细胞中HNF1α和HNF1β蛋白的相对丰度可能在决定基因表达模式中起关键作用。
