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Molecular and Cellular Biology, November 2007, p. 7365-7380, Vol. 27, No. 21
0270-7306/07/$08.00+0     doi:10.1128/MCB.00331-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

An Intronic Locus Control Region Plays an Essential Role in the Establishment of an Autonomous Hepatic Chromatin Domain for the Human Vitamin D-Binding Protein Gene{triangledown}

Tomoko Hiroki, Stephen A. Liebhaber, and Nancy E. Cooke*

Departments of Medicine and Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received 23 February 2007/ Returned for modification 1 April 2007/ Accepted 27 August 2007

The human vitamin D-binding protein (hDBP) gene exists in a cluster of four liver-expressed genes. A minimal hDBP transgene, containing a defined set of liver-specific DNase I hypersensitive sites (HSs), is robustly expressed in mouse liver in a copy-number-dependent manner. Here we evaluate these HSs for function. Deletion of HSI, located 5' to the promoter (kb –2.1) had no significant effect on hDBP expression. In contrast, deletion of HSIV and HSV from intron 1 repressed hDBP expression and eliminated copy number dependency without a loss of liver specificity. Chromatin immunoprecipitation analysis revealed peaks of histone H3 and H4 acetylation coincident with HSIV in the intact hDBP locus. This region contains a conserved array of binding sites for the liver-enriched transcription factor C/EBP. In vitro studies revealed selective binding of C/EBP{alpha} to HSIV. In vivo occupancy of C/EBP{alpha} at HSIV was demonstrated in hepatic chromatin, and depletion of C/EBP{alpha} in a hepatic cell line decreased hDBP expression. A nonredundant role for C/EBP{alpha} was confirmed in vivo by demonstrating a reduction of hDBP expression in C/EBP{alpha}-null mice. Parallel studies revealed in vivo occupancy of the liver-enriched factor HNF1{alpha} at HSIII (at kb 0.13) within the hDBP promoter. These data demonstrate a critical role for elements within intron 1 in the establishment of an autonomous and productive hDBP chromatin locus and suggest that this function is dependent upon C/EBP{alpha}. Cooperative interactions between these intronic complexes and liver-restricted complexes within the target promoter are likely to underlie the consistency and liver specificity of the hDBP activation.

* Corresponding author. Mailing address: 547a Clinical Research Building, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104. Phone: (215) 898-4425. Fax: (215) 573-5157. E-mail: necooke{at}mail.med.upenn.edu

{triangledown} Published ahead of print on 4 September 2007.

Molecular and Cellular Biology, November 2007, p. 7365-7380, Vol. 27, No. 21
0270-7306/07/$08.00+0     doi:10.1128/MCB.00331-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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