Figure 7. 51kb-hDBP transgene expression is liver-specific and copy number dependent. (A) Tissue specificity of 51kb-hDBP transgene expression. A Northern blot tissue survey from a representative 51kb-hDBP transgenic mouse line (line 924B, transgene copy number 6) is shown. The probes used are indicated on the left of the autoradiograph. High-level liver-specific expression of the 51kb-hDBP transgene was observed. The table summarizes the relative quantification of transgene expression in various tissues of an adult male mouse from each of the four 51kb-hDBP lines; the expression of hDBP mRNA in the liver is defined as 100 in each line and expression in other tissues is related to this value. (B) Copy number dependency of 51kb-hDBP expression. Serum hDBP level per transgene copy of each 51kb-hDBP line was quantified. Means and SD were calculated from serum hDBP levels of at least eight mice in each line. Consistent and copy number dependent serum hDBP levels were observed. These levels were concordant with the levels in the 105kb-hDBP lines carrying the more extensive hDBP gene flanking regions. consistent and similar to that of DBP levels in human serum (Figure 2B). Thus, the 105kb-hDBP transgene was expressed in a site-of-insertion independent and copy number dependent manner, at full levels, and with appropriate tissue speciﬁcity. Nucleic Acids Research, 2006, Vol. 34, No. 8 2163 From these data we concluded that the determinants sufﬁcient to establish an autonomous and fully active chromatin locus capable of supporting full and accurate hDBP expression are within and/or closely linked to the hDBP gene. These deter- minants fulﬁll the operational deﬁnition of an LCR. The second phase of the study was to map the determinants involved in the activation of the hDBP gene in hepatic chro- matin. LCR determinants commonly map to sites of DNase I hypersensitivity (HS) in the chromatin of expressing cells. These HS reﬂect the localized displacement of histones or perturbation in chromatin structure by trans-acting factors binding at the site (37,38). Core elements that have been deﬁned at individual HS are usually composed of arrays of multiple tissue-speciﬁc and ubiquitous transcription factor- binding sites [reviewed in ref. (32,33)]. With this in mind, the 105kb-hDBP transgene locus was mapped for HS in hep- atic tissue as well as in brain, a non-expressing tissue. A set of ﬁve liver-speciﬁc HS was identiﬁed (Figure 4D). HSI and HSIII are located at 2.1 and 0.13 kb relative to the hDBP promoter, respectively, whereas HSIV and HSV are closely juxtaposed within a 1.9 kb region of intron 1 (10.3–10.9 kb and 12.2 kb, respectively). HSVII maps to a site 1.5 kb 30 to the poly(A) addition site. HSVI, although detected in liver, was also present in brain chromatin and thus not tissue-speciﬁc. To support the sufﬁciency of these elements to activate of hDBP, we assessed the expression of a second transgene with minimal ﬂanking sequences just long enough to encompass the full set of identiﬁed HS. High-level, copy number dependent and liver-speciﬁc expression of this shorter 51kb-hDBP transgene was observed in all four 51kb- hDBP transgenic lines. These results suggested that all deter- minants necessary for the establishment of a fully functional chromatin locus are present within the 51kb-hDBP transgene. These data support a model in which the set of determinants identiﬁed by the HS mapping constitute a liver-speciﬁc hDBP LCR and are sufﬁcient for hDBP activation in the transgenic setting. The exact structure and mode of action of the determinants at each of the HS remains to be established. Our prior assays in cell transfection systems had identiﬁed three functional HNF1-binding sites within the proximal 2 kb of the rat DBP gene (rDBP) 50-ﬂanking region (39). The region from 65 to 41 bp, the ‘A segment’, is the most proximal HNF1-binding site. Two more distal HNF1-binding sites, located in segments ‘B’ and ‘F-2’ (coordinates at 254 to 140 bp and 1844 to 1621 kb, respectively), function as classical enhancer elements. The B and F-2 segments both increase the activity of homologous and heterologous promoters when assayed in transfected HepG2 liver-derived cells. HNF1-a played a predominant role in the observed enhancer function. At the B and F-2 sites the HNF1b protein showed a trans-dominant negative effect on HNF1a- stimulated activity of the rDBP promoter suggesting that the relative levels of HNF1a and HNF1b in various tissues might contribute, in part, to tissue-speciﬁc expression of DBP in vivo (39). HSIII, detected in the present study, is located between the two HNF1-binding sites corresponding to the A and B binding sites described in the rDBP proximal promoter. This suggests that the formation of HSIII reﬂects local promoter complex assembly. HSIII is co-located to a highly conserved region among human, mouse and rat (Figure 5). The