2164 Nucleic Acids Research, 2006, Vol. 34, No. 8 detection of HSIII in this region supports the previous conclusion that the two HNF1-binding sites in the proximal promoter are critical elements for hDBP activation. A comparison of the current studies with previous analyses of ALB and AFP genes highlights points of potential interest. Consistent with other numerous model systems, transgenic studies at the mouse ALB locus have revealed that the pro- moter region is insufﬁcient for robust and consistent transgene expression. These studies identiﬁed a transcriptional enhancer located 10 kb upstream of the transcription start site that is essential for full liver-speciﬁc expression (40). This element co-maps with a strong liver-speciﬁc HS (40,41) and in vitro studies have demonstrated that this enhancer can trigger the activation of a compacted chromatin template (42). Analysis of the mouse AFP gene has identiﬁed three enhancer elements at 2.5, 5.0 and 6.5 kb relative to the promoter (43,44). All three sites were required to achieve high-level expression of ALB in fetal liver (45). In contrast to these ﬁndings for the ALB and AFP genes, the current report reveals that the hDBP trans- gene is robustly activated in vivo in the absence of remote ﬂanking determinants. These data along with the HS mapping raise the possibility that HSIV, possibly in concert with the substantially weaker HSV, mediates enhancer functions com- parable to the distal enhancers far upstream of ALB or AFP promoter. In light of other studies in the literature, the intronic location of HSIV and HSV is fully compatible with their functioning as dominant locus control element(s) (46–51). The broad and multipartite structure of HSIV is consistent with multiple arrays of transcription factor-binding sites, a composition common among LCR elements (32,33). In addition to its expression in the liver, human and rodent DBPs are also expressed at substantially lower levels in several secondary sites. The most prominent and well described of these is in the kidney. The local expression of DBP in the kidney may be important in vitamin D recycling via interactions with the renal scavenger protein megalin (52–54). While the transcriptional initiation site for hDBP in the two tissues appears to be identical (4), it is likely that the complex of determinants driving expression in this secondary site differ from those in the liver. The 105kb-hDBP transgene paralleled the endogenous mDBP with respect to kidney expression; hDBP expression was observed at consid- erable levels ( 2–10% of liver) in the kidneys of all ﬁve 105kb-hDBP lines. In contrast, kidney expression of the more truncated 51kb-hDBP transgene was sporadic while liver expression remained robust and consistent. We have generated a single transgenic mouse line carrying a 123 kb insert of PAC clone 45P24 (Figure 6A). This transgene has the same 50 terminus as the 51kb-hDBP at 2.5 kb as well as a more extensive 78 kb 30-ﬂanking region. The tissue distribu- tion of hDBP expression from this transgene by Northern blots showed robust liver expression and an absence of expression in the kidney (data not shown). These results suggest that one or more important elements for kidney hDBP expression are located in the region between +37 kb and +2.5 kb upstream from the transcription start site. Whether these elements will constitute a kidney-speciﬁc element of the hDBP LCR remains to be determined. In conclusion, the present study demonstrates that elements critical to hepatic activation of the hDBP gene and establish- ment of a fully autonomous chromatin domain are located in close proximity to, and within the hDBP gene. The combina- tion of transgenic studies, HS mapping and phylogenetic sequence comparisons establish a model in which HSI, HSIII, HSIV, HSV and HSVII constitute components of a liver-speciﬁc LCR. How each element contributes to this activity can now be further explored. These data further estab- lish a functional independence of hDBP from the other mem- bers of its cluster. This suggests that critical elements that trigger the hepatic expression of these genes have duplicated during evolution and are represented at multiple sites within the cluster. It remains possible, that at some higher level of nuclear organization, these elements may interact to facilitate and coordinate their respective developmental proﬁles. ACKNOWLEDGEMENTS The authors would like to acknowledge funding from the NIH (Grant R01 GM32035 to N.E.C. and S.A.L.), and to thank Dr. Jean Richa and the Transgenic and Chimeric Mouse Core of the University of Pennsylvania (NIH P30 DK50306, P30 DK019525 and P30 CA016520) and the Center for Molecular Studies in Digestive and Liver Diseases for the generation of transgenic lines. Funding to pay the Open Access publication charges for this article was provided by Grant R01 GM32035. Conflict of interest statement. None declared. REFERENCES 1. Cooke,N.E. and Haddad,J.G. (1997) Vitamin D-binding protein. In Feldman,D., Glorieux,F.H. and Pike,J.W. (eds), Vitamin D. Academic Press, San Diego, CA, pp. 87–101. 2. Cooke,N.E. and Haddad,J.G. (1995) Vitamin D-binding protein (Gc-globulin). Endocr. Rev., 4, 125–128. 3. White,P. and Cooke,N.E. (2000) The multifunctional properties and characteristics of vitamin D-binding protein. Trends Endocrinol. Metab., 11, 320–327. 4. McLeod,J.M. and Cooke,N.E. 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