Vitamin D-binding protein gene transcription is regulated by the relative abundance of hepatocyte nuclear factors 1alpha and 1beta.
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.
Departments of Medicine and Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Y H
YH
Song
K
K
Ray
S A
SA
Liebhaber
N E
NE
Cooke
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014809:Vitamin D-Binding Protein / Q000096:biosynthesis / Q000235:genetics*
D004268:DNA-Binding Proteins; D051538:Hepatocyte Nuclear Factor 1-alpha; C496489:Hnf1a protein, rat; D009687:Nuclear Proteins; D014157:Transcription Factors; D014809:Vitamin D-Binding Protein; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta