VITAMIN D-BINDING PROTEIN GENE TRANSCRIPTION IS REGULATED BY THE RELATIVE ABUNDANCE OF HEPATOCYTE NUCLEAR FACTORS 1ALPHA AND 1BETA

Abstract 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 HNF1α and HNF1β 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 HNF1α. 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 HNF1α and HNF1β; HNF1α had a major stimulatory effect, whereas HNF1β acted as a trans-dominant inhibitor of HNF1α-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 HNF1α and HNF1β proteins in a cell may thus play a critical role in determining the pattern of gene expression.