The close relationship among the four genes in the DBP-ALB cluster, and the fact that they are all expressed predominantly in liver, suggests that they may share one or more common cis-regulatory determinants. The possibility of shared regulatory control among these genes is supported by the identiﬁcation of intergenic enhancers located between ALB and AFP that have the potential to regulate both gene promoters (15–18). Whether shared transcriptional control elements are more widely involved in this four-gene family has not been explored. Models of regulatory elements controlling multi-gene fami- lies can be found in the locus control regions (LCRs) for the human and mouse b-globin gene clusters (19,20) and the human growth hormone gene cluster (21). In these cases, sets of regulatory elements, located quite distant from target promoters, appear to act in concert to generate ‘open’ chro- matin structures or chromatin ‘hubs’ (22) conducive to local promoter access and transcription initiation. In some cases, the genomic regions harboring regulatory elements can be located as much as 1 Mb from the target promoter (23). Such remote determinants can be located 50 or 30 of the target gene and in some cases can reside within the introns of unrelated neigh- boring gene(s) [reviewed ref. (24)]. The mechanistic basis for such long-range control is presently under study. Whether the DBP-ALB gene family is controlled by a common set of chro- matin regulatory elements remains unknown. With the recent clariﬁcation of the genomic organization of the DBP-ALB multi-gene cluster, these questions can now be directly addressed. A recently reﬁned physical and meiotic map of the 4q11–q13 region in the human genome reveals the organiza- tion of the cluster: centromere-30-DBP-50-50-ALB-30-50-AFP- 30-50-AFM-30-telomere (Figure 1A). Of particular note is the inverse transcriptional orientation and wide (>1.5 Mb) separa- tion of the DBP gene from the other three more tightly clus- tered genes in the family (25) (GenBanK accession no. NT_006216). The rat DBP (rDBP) multi-gene cluster is located at 14p21–p22 in chromosome14, a region syntenic to human chromosome 4, and the order and transcriptional orientation of each gene correspond to the human cluster. The distance between the rDBP gene and the remaining family members is also quite large (1.0 Mb) in rat (GenBank acces- sion no. NW_047424). These data demonstrate that the organ- ization, transcriptional orientation and expression patterns of the DBP multi-gene clusters in rat and human are highly conserved. Whereas the close juxtaposition of ALB, AFP and AFM is consistent with the presence of common transcrip- tional determinants, the substantial separation of DBP from the other members suggests an alternative model of autonomous control. In the present report we test for autonomy of the DBP locus using two sets of transgenic mouse models. We further begin to explore its mechanistic basis by probing hepatic chromatin by DNase I mapping for sites of potential transfactor interac- tions. These studies indicate that determinants sufﬁcient for establishment of an autonomous hepatic chromatin environ- ment that supports robust hDBP expression in vivo are located in close proximity to, and within, the hDBP gene itself. In our transgenic setting, these locus control determinants are res- ponsible for copy number dependent, site-of-integration independent, and robust tissue-speciﬁc expression of the Nucleic Acids Research, 2006, Vol. 34, No. 8 2155 hDBP locus. These data allow us to propose a model in which the hDBP gene is independently regulated by a dedicated LCR. It further implies that the DBP-ALB gene cluster has evolved into at least two separable chromatin units that maintain common liver speciﬁcity. MATERIALS AND METHODS Materials Restriction and modiﬁcation enzymes were purchased from New England Biolabs (Beverly, MA), Life Technologies (Rockville, MD), and Roche Molecular Biochemicals (Indianapolis, IN). [a-32P]dCTP and MicroSpin G-50 Columns were purchased from Amersham Biosciences (Piscataway, NJ). Random-primed DNA labeling kits and Taq DNA polymerase were from Roche Molecular Bio- chemicals. QIAEX II Kit was from QIAGEN and the GENE- CLEAN II Kit was from BIO 101, Inc (La Jolla, CA). Elutip columns were from Schleicher and Schuell (Keene, NH), and RNAzol B RNA isolation solvent was from TEL-TEST Inc. (Friendswood, TX). Zetabind membranes were from CUNO Inc. (Meriden, CT). Oligonucleotides The various oligonucleotides listed in Table 1, were synthe- sized by Life Technologies, Inc. or by the DNA Sequencing Facility of University of Pennsylvania. Preparation of DNA probes The DNA probes for genomic Southern blots and northern blots were released as EcoRI fragments from the rat (6) and human (26) DBP cDNA plasmids. The MX probe, which detects the unique sequence 30-ﬂanking region of the mz-globin gene, was released as a 1.3 kb BamHI fragment from the pMX plasmid (27). The mouse ribosomal protein L32 (mrpL32) cDNA probe was released as 0.32 kb EcoRI and HindIII fragment from the mrpL32 plasmid (28). The probes used for DNase I mapping were generated by PCR using Taq DNA polymerase. The templates for the PCR were PAC clones, 231M2 or 45P24 (25). Each fragment was recovered using a QIAEX II kit from an agarose gel, and labeled by [a-32P] dCTP using a random-primed DNA labeling kit. Frag- ments were then puriﬁed on MicroSpin G-50 columns. Generation and analysis of transgenic mice The 105kb-hDBP fragment was released from vector sequences by NotI digestion of PAC clone, 231M2 plasmid (25) and the 51kb-hDBP fragment by double-digestion of PAC clone, 45P24 plasmid (25) with NotI and FspI. Fragments were separated by ﬁeld-inversion gel electrophoresis (FIGE) using 1% SeaKem LE agarose gels (FMC BioProducts, Rockland, MA). Fragments were recovered with the GENECLEAN II Kit or by electroelution. Each fragment was puriﬁed by Elutip, diluted to 2 ng/ml in 10 mM Tris–HCl (pH 7.6), 0.1 mM EDTA and then microinjected into fertilized mouse oocytes (University of Pennsylvania Transgenic and Chimeric Mouse Core). Positive founders were detected by dot-blot analysis of tail DNAs using hDBP cDNA 771 bp EcoRI