2154–2165 Nucleic Acids Research, 2006, Vol. 34, No. 8 doi:10.1093/nar/gkl174 The human vitamin D-binding protein gene contains locus control determinants sufficient for autonomous activation in hepatic chromatin Tomoko Hiroki, Young-Han Song, Stephen A. Liebhaber and Nancy E. Cooke* Departments of Medicine and Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA Received February 1, 2006; Revised March 8, 2006; Accepted March 20, 2006 ABSTRACT The human vitamin D-binding protein (hDBP) gene is a member of a cluster that includes albumin, a-fetoprotein and a-albumin genes. The common ori- gin, physical linkage and hepatic expression of these four genes predict shared regulatory element(s). However, separation of hDBP from the other three genes by 1.5 Mb argues that hDBP may be under auto- nomous control. To test for hDBP autonomy, mouse lines were generated with a transgene containing the hDBP gene along with extensive flanking sequences. Expression of this transgene was hepatic, robust and proportional to transgene copy number. DNase I hypersensitive site (HS) mapping revealed five liver-specific HS at the hDBP locus: HSI and HSIII at 2.1 kb and 0.13 kb upstream of the transcription initiation site, HSIV and HSV within intron 1 and HSVII located 30 to the poly(A) site. A second trans- gene with minimal flanking sequences confirmed the sufficiency of these gene-proximal determinants for hepatic activation. The hepatic-specific HS aligned with segments of phylogenetically conserved non- coding sequences. These data demonstrate the autonomy of the hDBP locus and suggest that this control is mediated by chromatin-based locus control determinants in close proximity to, and within the transcription unit. INTRODUCTION The vitamin D-binding protein (DBP) gene is robustly expressed in the livers of all mammalian species. In humans the hDBP protein (also known as Gc-globulin) is secreted from hepatocytes as a polymorphic glycoprotein that constitutes one of the most abundant serum proteins (232–464 mg/ml) (1). The major function of serum DBP is the binding and transport of 25-hydroxyvitamin D, the major circulating form of vitamin D and 1,25-dihydroxyvitamin D, the most active vitamin D metabolite. DBP also binds tightly to monomeric G-actin, blocking the formation of F-actin networks that may occlude the vasculature following cellular damage. Several studies suggest that covalent derivatives of DBP have pivotal roles in the innate immune response by serving as a macrophage-activating factor and by enhancing C5a-mediated chemotaxis for macrophages and neutrophils [reviewed in ref. (1–3)]. DBP is predominantly produced in hepatocytes, although secondary minor sites of synthesis, such as the kid- ney, have been identiﬁed (4). Due to its high-level of activity and its wide spectrum of functions, the DBP gene represents an important model for the analysis of hepatic gene expression. DBP is a member of a family of four robustly expressed liver-speciﬁc genes. The conserved intron/exon organizations along with primary and secondary structural conservation of encoded proteins indicate a paralogous relationship of DBP to albumin (ALB), a-fetoprotein (AFP) and a-albumin/afamin (AFM). These four genes, formed by local duplications of a common progenitor gene, remain physically linked in the human and rodent genomes (2,5–10). Structural comparisons among these genes suggest that DBP was the oldest and most divergent member in this family (11–13). The developmental proﬁles of these genes have been described. During rodent embryonic development, expression of ALB, AFP and DBP is induced in yolk sac and is maintained in fetal liver (14), whereas the hepatic expression of AFM is initiated in the perinatal period. AFP is selectively silenced at the end of the fetal period, whereas ALB, AFM and DBP maintain high constitutive levels of expression in adult liver (9). The molecular mechanisms for the transcriptional activation of these genes in the native hepatic chromatin environment, and for their respective developmental controls, have yet to be determined. *To whom correspondence should be addressed at 752b Clinical Research Building, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104, USA. Tel: +1 215 898 4425; Fax: +1 215 573 5809; Email: necooke@mail.med.upenn.edu Present address: Young-Han Song, Ilsong Institute of Life Science, Hallym University, Anyang 431-060, Korea The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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